CA1217718A - Plasminogen activator derivatives - Google Patents
Plasminogen activator derivativesInfo
- Publication number
- CA1217718A CA1217718A CA000443230A CA443230A CA1217718A CA 1217718 A CA1217718 A CA 1217718A CA 000443230 A CA000443230 A CA 000443230A CA 443230 A CA443230 A CA 443230A CA 1217718 A CA1217718 A CA 1217718A
- Authority
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- Canada
- Prior art keywords
- urokinase
- peg
- molecular weight
- triazine
- polyethylene glycol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
ABSTRACT OF THE DISCLOSURE
Derivatives of a nonimmunogenic plasminogen activator which comprises at least one polyalkylene glycol group chemically bonded with at least one coupling agent to amino acid side chains of said plasminogen activator, wherein said polyalkylene glycol has a molecular weight of about 200-20,000 and is unsubstituted or is substituted with one or more alkyl, alkoxy or alkanoyl groups or a mixture thereof.
The plasminogen activator derivatives have an extended circulating life in the mammalian bloodstream and also inhibit the formation of thrombus in the same.
Derivatives of a nonimmunogenic plasminogen activator which comprises at least one polyalkylene glycol group chemically bonded with at least one coupling agent to amino acid side chains of said plasminogen activator, wherein said polyalkylene glycol has a molecular weight of about 200-20,000 and is unsubstituted or is substituted with one or more alkyl, alkoxy or alkanoyl groups or a mixture thereof.
The plasminogen activator derivatives have an extended circulating life in the mammalian bloodstream and also inhibit the formation of thrombus in the same.
Description
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BACKGROUND OF THE INVENTION
Field of the Invention ~ his invention relates to derivatives of human-originated non-immunogenic plasminogen activators, and more particularly to such a derivative comprising at least one polyalkylene glycol attached with at least one coupling agent to amino acid side chains of a plasminogen activator of the type described above, the polyalkylene glycol having a molecular weight in the range of 200 -10 20,000 and optionally containing one or more alkyl, alkoxy and/oralkanoyl groups as substituents. Further, the inveniion is concerned with a process for producing such deri-vativesand with a thrombolytic agent containing such derivativeS .
Description of the Prior Art It is known that human tissues contain a variety of substances which activate plasminogen into a fibrinoly-tic enzyme or plasmin. Among such known substances, 'he most representative is a plasminogen activator, i.e.
20 urokinase, which is formed in the kidney tissue and excre~d into urine. Urokinase may be obtained by isolation and purification from human urine, tissue culture or genetic engineering. As fibrinolytic enzyme activators which hav~
nowadays found widespread commercial utility, there exist proteins originated from hemolytic Streptococcus and urokinase which is an enzyme originated from human urine. In view of its immug~nic behavior towards humans,urokinase re~ulting from such source is favorably employed for clinical application.
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BACKGROUND OF THE INVENTION
Field of the Invention ~ his invention relates to derivatives of human-originated non-immunogenic plasminogen activators, and more particularly to such a derivative comprising at least one polyalkylene glycol attached with at least one coupling agent to amino acid side chains of a plasminogen activator of the type described above, the polyalkylene glycol having a molecular weight in the range of 200 -10 20,000 and optionally containing one or more alkyl, alkoxy and/oralkanoyl groups as substituents. Further, the inveniion is concerned with a process for producing such deri-vativesand with a thrombolytic agent containing such derivativeS .
Description of the Prior Art It is known that human tissues contain a variety of substances which activate plasminogen into a fibrinoly-tic enzyme or plasmin. Among such known substances, 'he most representative is a plasminogen activator, i.e.
20 urokinase, which is formed in the kidney tissue and excre~d into urine. Urokinase may be obtained by isolation and purification from human urine, tissue culture or genetic engineering. As fibrinolytic enzyme activators which hav~
nowadays found widespread commercial utility, there exist proteins originated from hemolytic Streptococcus and urokinase which is an enzyme originated from human urine. In view of its immug~nic behavior towards humans,urokinase re~ulting from such source is favorably employed for clinical application.
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-2-Urokinase originated from human urine is believed to con-tain both high molecular weight urokinase (molecular weight: 54,000) and low molecular weight urokinase (molecu-lar weight: 33,000). Uroklnase has been used, in recent years, as a thrombolytic agent or an adiuvant for carcino-static substances, and its consumption for clinical application`is increasing year by year.
However, urokinase is unstable under certain conditions since it is an enzyme and loses its enzymatic activity, for example, in the course of extraction, isolation and purification from a urokinase-bearing raw material, for example, urine; during the lyophilization processing in preparing dosable formulations; during tne heat treatment for deactivating viruses, or when it is placed in a diluted state in a dripping bottle and kept for a prolonged time period in such a diluted state at room temperature for clinical application. This physically unstable nature of urokinase has created a serious problem in preparing and formulating urokinase on an industrial scale or in actually using the same for clinical purposes.
Human albumin has been employed as an additive to urokinas~
so as to improve its stability. However, this can be by no means a break-through solution to the problem just dis-cussed because pure albumin, i.e. a globulin fraction, is difficult to obtain without immunogenic contamination;
pure albumin is expensive; albumin and urokinase form a complex of a high molecular weight under virus deacti-~ Z~7 7~
vating conditions in which urokinase is subjected to heattreatment at 60C for 10 hours together with albumin added to stabilize urokinase; and such stabilizer if added may be effective to a certain extent for protecting urokinase from losing its enzymatic activity upon the lyophilization but cannotPreVentits loss of activity upon actual clinical use.
The physiological activity of urokinase when admi-nistered intravenously to living bodies is promptly retarded by protease inhibitors present in blood (~2-macroglobulin, and a2-plasmin inhibitors and the like), and the metabolic rate of urokinase per se is ve~y hig;~, -es~l.-ing in extremely shortened half-life which does not exceed even several minutes. Nothing has been heretofore propos-d to solve the problem of s~ort half-life of urokinase in blood. - -_ The present inventors have carried out extensive research with a view toward developing derivatives of human-oriyinated non-immunogenic plasminogen activators which will overcome the above-noted drawbac~s of the prior art techniques. As a result, they have succeeded in ind-ing plasminogen activator derivatives whicn are stable and hardly retarded by inhibitors present in blood and hence achi2ve prolonged half-life in blood, thereby leading to the present discovery.
::' SUMMARY OF T~E INVENTION
-An object of the present invention is to provide a derivative of a human-originated non-immunogenic ~ .
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plasminogen activator which is stable and exhibits prolong-ed fibrinolytic activity when administered to living bodies.
Another object of the invention is to provide a process for preparing the novel plasminogen activator derivative.
A further object of the invention is to provide a therapeutically acceptable thrombolytic agent comprising the novel plasminogen activator derivative.
These and other objects and advantages of the inven-tion can be attained by the provision of a derivative of a human-originated ncn-immunogeniC plasminogen activator, comprising at least one polyalkylene glycol attached with at least one coupling agent to amino ~cid side chains of the plasminogen activator, the polyalkylene glycol having a molecular weight in the range of 200 -20,000 and option-ally containing one or more alkyl,alkoxy and/or alkanoyl groups as substituents.
l.Z~7~3 -4a-Thus in one aspect the present invention provides a composition for inhibiting the formation of thrombus in a mammalian bloodstream, which composition comprises an effective amount of a nonimmunogenic plasminogen activator comprising at least one polyalkylene glycol moiety chemically bonded with at least one coupling agent to the amino acid side chains of said plasminogen activator, wherein the polyalkylene glycol has a molecular weight in the range of 200-20,000 and is unsubstituted or is substituted with one or more alkyl, alkoxy or alkanoyl groups or a mixture thereof, together with a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DR~WINGS
A more complete appreciation of the present inven-tion and many of the attendant advantages thereof will be readily obtained as the invention becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIG. 1 shows the optimum pH ranges of modified high molecular weight urokinase (PEG-DCI-UX) and unmodified high molecular weight urokinase (molecular weight: 54,000) as lZ~7~
_5_ measured in terms of the amidase activity with a synthetic substrate (S-2444), where the open circles correspond to PEG-DCT-UX and the closed circles to unmodified urokinase;
FIG. 2 shows the optimum pH ranges of modified low molecular weight urokinase (PEG-DCT-L-UK) and unmodified low molecular weight urokinase (molecular weight: 33,000) as measured in terms of the amidase activity with S-2444, where the open circles correspond to PEG-DCT-L-UK and the closed circles to unmodified urokinase;
FIG. 3 diagrammatically shows the stability of PET-DCT-UK and unmodified uro~inase at room temperature in either physiological saline or a Ringer solution, where the solid and broken lines indicate the physiological saline and the Ringer solution, respectively, and where the open circles correspond to PEG-DCT-UK and the closed circles to unmodi-fied urokinase; - _ FIG. 4 shows the stability of PEG-DCT-L-UK and un-modified urokinase (molecular weight: 33,000) against freezing, followed by thawing operation repeated 2, 4 and 6 times, where the open circles correspond toPEG-DCT-L-VK
and the closed circles to unmodified urokinase;
FIG. S shows the varied amounts, as a function of time, of a plasmin inhibitor in a plasma fraction Fl of each of two groups of healthy rabbits, one group adminis-tered with PEG-DCT-UK and the other group with unmodified urokinase (molecular weight: 54,000), where the open cir-cles correspond to the PEG-DCT-UK-administered group and the closed circles to the unmodified urokinase-administered group;
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FIG. 6 shows the varied amounts, as a function of time, of plasminogen in a plasma fraction F3 of each of two groups of healthy rabbits, one group administered with PEG-DCT-UK and the other group with unmodified urokinase (mole-cular weight: 54,000), where the open circles correspond to the PEG-DCT-UK-administered group and the closed circles to the unmodified urokinase-administered group; and FIG. 7 shows the varied amounts, as a function of time, of plasminogen in a plasma fraction F3 of each of two groups of healthy rabbits, which plasma was treated w1th an acid and then with a base to inactivate a plasmin inhibitor, one group administered with PEG-DCT-UK and the other grou~
with unmodified urokinase (molecular weight: 54,000), where the open circles correspond to the PEG-~CT-UK-administered group and the closed circles to the unmodified urokinase-administered group.
Figure 8 illustrates the substrate specificity of PEG-Modified UK's. _ _ Figure 9a illustrates the stability of modified Urokinase to UK-Inhibi~ors in Human Plasma.
Figure 9b illustrates the effect of Placental UK-Inhibitor on the Urokinase Activity.
Figure 10 illustrates UK activity in Plasma after UX
injection.
Figure 11 illustrates the relative radioactivity in Blood (%1 for 125I-M-Md and 125I-Native-UK.
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Figure 12a illustrates a comparison of the immunogenicity of M-Md with that of native UK by Schultz-Dale test.
Figure 12b illustrates a comparison of the immunogenicity o~ M-Md with that of native UK by Passive Cutaneous Anaphylaxis (PCA) reaction.
Figure 13a illustrates a compaxison of the Plasmin Inhibitor in Fl from Citrated Dog Plasma at certain time intervals after injection of Native UK or PEG-UK into a dog.
Figure 13b illustrates a comparison of the Plasminogen in F3 from Citrated Dog Plasma at certain time interYals after injection of Native UK or PEG-UK into a dog.
Figure 13c illustrates a compari50n of the FDP in Dog Serum at certain time intervals after i.v. Injection of Urokinase or PEG-UK in~o a dog.
Figure 14 illustrates a comparison of FDP levels in dogs bearing artificial thrombus in A. femoralis. Dog A had native UK injected into a proximal branch of the thrombus, while Dog B had PEG-UK injected into the same.
- DESCRIPTION OF THE PREFERRED E~ODIMENTS
By the term "human-originated non-immunogenic plas-minogen activator" as used herein are encompassed not on;y urokinase but also tissue plasminogen activators obtained from human tissues such as uterine, tumor and the like.
These human tissue plasminogen activators also contain those obtained by tissue culture or genetic engineerins.
It should be noted that no limitations are imposed on the molecular weishts of these activators so long as they are obtained in the above-described manner. For example, as urokinase which is a plasminogen activator origina.ed from human urine, high molecular weight urokinase (molecular .
7~3 weight: S4,000) and low molecular weight urokinase (molecu-lar weight: 33,000) may be used solely or in combination.
Suitable polyalkylene glycols which may be used in the invention include a polyethylene glycol and a polypro-pylene glycol. In the case of the polypropylene glycol, both straight-chain polypropylene glycols such as those represented by ~O~CH(CH3)CH2O]nH and branched-chain poly-propylene glycols such as those represented by CH3CH2C-{CH20~cH2cH(cH3)o~nH}3 or HIOCH(CH3)CH2~nOCH{cH2~ocH2cH-(C-~3)]nOH}2.
The molecular weights of the polyalkylene glycols may range from 200 to 20,000. Particularly preferred molecular weights are in the range of 500 - 10,000.
The polyalkylene glycols each may o?tionally contain one or more alkyl, alkoxy and/or alkanoyl groups as substituent groups. Typical example,s of the alkyl groups are methyl, ethyl, propyl,stearyl and the like. Typical examples of the alkanoyl groups are acetyl, propibnyl, stearoyl and the like. As a preferred polyalkylene glycol, an unsubsti-tuted or methyl-substituted polyalkylene glycol is useful.
However, of particular interest is the use of methoxy-polyethylene glycol as the polyalkylene glycol. Chemical modification of urokinase(UK) with activated methoxypoly-ethylene glycol(PEG) of MW 5,000 increased the stability of the urokinase and imparted a markedly extended circulation life in rabbits and rats. Also of interest is the surprising fact that PEG-UK has a superior thrombolytic ability as compared to that ~`;
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of native UK. It appears likely that the superiority of PEG-UK to native UK with respect to the fibrinolytic activation is from the PEG chains which protect the UK
molecule from interation with inhibitors, thus extending its circulation life.
Suitable coupling agents which may be used in the invention and are adapted to attach a polyalkylene glycol to a non-immunogenic plasminogen activator, for example, urokinase, include those capable of reacting with amino acid side chains of the protein to be modifiedand forming chemical bonds therebetween, for example,.acyl azide, cyanuric halides, p-diazoniumbenzyl ether, 3-(p-diazonium-phenoxy)-2-hydroxypropyl ether, dihalogenosuccinic anhydride and the like. The following partial formulae may be given as examples of the coupling structures between a polyalkylene glycol and urokinase through these coupling agents.
-~ -UK
X
_o- ~ -UK
OH
-O-CH2- ~ N2-UK
-o-cH2cH(OH)cH2 ~ -r~2-UK
wherein X is a halogen atom, and UK is a residual part of the urokinase ~olecule.
The novel urokinase derivative according to the invention can ~e prepared by reacting a coupled product of at least one corresponding polyalkylene glycol and at least one coupling agent with urokinase, the polyalkylene glycol having a molecular weisht in the range of 2Q0 -20,000 and optionally containing one or more alkyl , alkoxy and~or alkanoyl groups as substituents.
Typical examples of the polyalkylene ~lycol-cou~lins agent coupled product include oolyalkylene glycol-~,6-.~2~
dichloro-1,3,5-triazine, polyalkylene glycol-4,6-difluoro-1,3,5-triazine, polyalkylene glycol-4-chloro-6-hydroxy~
1,3,5-triazine, polyalkylene glycol-~-(bro~ocarbonyl)-monopropionate, polyalkylene glycol-azidocarbonyl methyl etller, polyalkylene glycol-(p-diazoniumbenzyl) ether, polyalkylene glycol-3-(p-diazoniumphenoxy)-2-hydroxypropyl ether and the like.
When reacting the polyalkylene glycol-coupling agent coupled product with uroki~ase, it is necessary to choose such reaction conditions that the enzymatic acti-vity is held to a minimum loss. Namely, it is desirable to carry out the reaction at low temperatures, for example, at a temperature of 0C to room temperature in an a~ueous solution such as a buffer. Preferred reaction time may range from several minutes to 5 hours. The pH of the buffer is preferably within such a range that the enzymatic activity of urokinase is not lowered, namely, 2 - 10, preferably 5 - 9. However, the preferred pH range may vary depending on the reactivity of each coupling agent employed and/or the nature of an amino acid residue. The modification degreesof amino acid side chains of the plasminogen activator can be controlled by changing the concentration of the polyalkylene glycol activated witn the coupling agent in a reaction medium.
By wày of illustration, monomethyl ether polyethylene glycol-4,6-dicnloro-1,3,5-triazine having an average molecular weight of 5,000 was reacted at pH 7.0 with hig~
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molecular weight urokinase to obtain novel urokinase derivatives, while changing the concentration of the former reactant to 0.4, 4.0 and 6.0 mM, respectively. Unmodified -amino groups of lysine of the resultant urokinase deri-vatives were quantitatively determined using sodium ~,4,6-trinitrobenzenesulfonate. Their modification degrees were investigated on the basis of the results obtained by the quantitative analyses. The modification percentages of the ~-amino groups of lysine, which were reactive with sodium 2,4,6-trinitrobenzenesulfonate, were 6-7% at 0.4~M, about 40% at 4.0 mM and about 60~ at 6.0 mM. In addition, the molecular weights of these reaction products were determined by SDS polyacryl amide gel electrophoresis.
The average molecular weights of the reaction products were about 60,000 at 0.4 mM and about 120,000 at 4.0 m~1. This finding is substantially in conformity with the results obtained above by the quantitative analyses of the E-amino groups of lyslne. Accordingly, it is necessary to conduct the reaction between the polyalkylene glycol activated with the coupling agent and the plasminogen activator at a high pH level and at a high concentration of tne former reagent when increased modification degrees of the plasminogen activator are desired. On the other hand, where decreased modification degrees are preferable, tne xeaction should be effected at a relatively low pH level and at a low con-centration of the coupled product. The modification degrees .. . .
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may of course be changed by controlling the reaction time.
By suitably combining these reaction conditions, it is possible to obtain the intended novel plasminogen activator derivatives which are stable and have prolongedfibrinolytic activities.
The polyalkylene glycol activated with the coupling agent can be obtained in the following manner. A terminal-substituted polyalkylene glycol-4,6-dihalogeno-1,3,5-triazine is obtained by reacting its corresponding mono-substituted polyalkylene glycol with a cyanuric halide in an anhydrous solvent and in the presenc,e of a base.
A polyalkylene glycol-4-halogeno-6-hydroxy-1,3,5-triazine is formed by first reacting its corresponding polyalkyler,e glycol with cyanuric halide and then treating the resultant reaction product with water. A polyalkylene glycol-acetoazide is formed by reacting an-ani~n of its corre-sponding polyalkylene glycol with ethyl chloroacetate, followed by treating the reaction product with hydrazine, and finally activating the resulting hydrazide with nitrous acid. A polyalkylene glycol-p-diazoniumbenzyl ether or a polyalkylene glycol-3-(p-diazoniumphenoxy)-2-hydroxypropyl ether is obtained by reacting its corresponding polyalky-lene glycol with p-nitrobenzyl chloride or p-nitrophenyl glyceryl ether and, after reducing the nitro group into an amino group, diazotizing the resultant product with nitrous,acid.
After completion of the reaction of the . . .
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polyalkylene glycol activated with the coupling agent and the plasminogen activator, the isolation and purification of the reaction product may be effected in a biochemical manner known per se in the art, for example, by using singly or in combination gel filtration, dialysis, ion-exchange chromatography, affinity chromatography and the - like. Preferably, the conjugate is kept as a solution containing a buffer or a physiological salt, by freezins the solution below -20C, or by lyophilizing the solution.
The optimum pH levels for the novel urokinase derivatives prepared above vary depending on the molecular weights and types of polyalkylene glycols emplo~ed, the types of coupling agents employed, the modification degrees of urokinase, the modification conditions and the measure-ment conditions such as substrates used. ~ihen measured in terms of the amidase activity using a synthetic substrate S-2444, the optimum pH level is within the range of about 8 to 9. In the case of nigh molecular weight urokinase modified by reaction with monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine having an average molecular weight of 5,000 at pH 7.0 and at a temperature of 0C for 3 hou-s, while using the latter reactant at a concentration of 4.0 mM (hereinafter abbreviated as PEG-DC~-UK), the optimum pH as measured in terms of the amidase activity is 8.2 as shown in FIG. 1. The optimum pH of low molecular weight urokinase modified under the same conditions (hereinarter abbreviated as PEG-DCT-L-UK) is, as measured in terms of `
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the amidase activity, 8~2 as shown in FIG. 2.
The novel urokinase derivatives according to the invention have extremely enhanced stability compared with unmodified urokinase. For instance, FIG. 3 shows the results of the residual urokinase activity obtained relative to the passage of time when urokinase was dilu~ed with a Ringer solution or a physiological solution to sucn a concentration as used by dripping administration for clinical application and then allowed to stand at room temperature. As a result, it has been found that PEG-DCT-UK dissolved at a concentration of 105.3 iu/ml in the Rinser solution maintains an activity of 73.4~ at the initiation of the experiment even after 6 hours. In addition, PEG-DCT~UK retains an initial activity of 79.4~ in the physio-logical saline. On the other hand, unmodified urokinase dissolved at a concentration of 76.I iu~l in a Ringer solution keeps only 33.3% in its initial activity even after 2 hours. In a physiological saline, the residual activity is as low as 26.3%.
FIG. 4 illustrates the results obtained by comparing the stability of PEG-DCT-L-UK and unmodified low moiecula~
weight urokinase, both in the course of freezing, followed by thawing operation. The results confirm th~t th2 supe-b stability of PEG-DCT-L-UK is attained even under such conditions.
Furthermore, the novel urokinase derivatives according to the invention have prolonged urokinase ac~i-lZ:~`7~3 vities in blood as compared with unmodified urokinase.For example, the effectiveness of urokinase was investigated by administering 8,000 iu/kg of each of PEG-DCT-UK and unmodified urokinase to healthy rabbits by intravenous injection, sampling blood periodically before the dosage and after the lapse of 4 hours, and measurins the amounts of the plasmin inhibitor and plasminogen present in the citrated plasma. The results are shown in FIGS. 5, 6 and 7. From these results, it has been con-firmed that PEG-DCT-UK alters to a substantial extent biochemical parameters such as a plasmin inhibitor and plasminogen and maintains the parameters at lowered values for an extended period of time as compared with unmodified urokinase.
Therefore, the ~;~ urokinase derivatives accorâing to the invention are excellent plasminog~n activators which, while retaining plasminogen-activating potency as unmodified urokinase does, have surmounted the shortcomings of urokinase such as poor stability and short half-life in blood.
The urokinase derivatives according to the invention may be used as pharmaceutical products for the treatment of a variety of diseases stenlmed from hyper-coagulability of blood such as arterial and venous throm-boses, coronary artery clotting, myocardial infarction, intracerebral infarction, pulmonary embolism, nephritis and the like. These urokinase derivatives can be suitAbly . . .
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administered by intravenous injection or dripping or by an oral route. The intravenous injection is particulary preferred. As dosable forms, preferably usable is a lyophilized form. When supplied eithex in a neat state or with a physiological salt such as a salt of a Ringer solution or sodium chloride, the lyophilized products may be used for clinical application by dissolution with sterile distilled water or further dilution with sterile distilled water in order to adjust their osmotic pressure, prior to the actual use. Since the novel urokinase de-i-vatives according to the invention are stable,they do not require a stabilizer such as albumin. However, the addi-tion of such a stabilizer does not cause any problem or inconvenience. An excipient may also be added in subjec~-ing the novel urokinase derivatives to lyophilization.
The above description genera-lly ~escribes the present invention. A more complete understanding can be obtained by reference to the following examples which are provided for purposes of illustration only and are not intended to be limiting. The modification procedure may be carried out in any one of the preparation steps of plasminogen activators.
Example 1 Monomethyl ether polyethylene glycol-4,6-dicnloro-1,3,5-triazine Polyethylene glycol monomethyl ether having an average molecular weight of 5,000 (25.0 g; O.OS mole) was ~ 77:~
dissolved with warming in dry benzene (200 ml). After cooling the resultant solution to room temperature, anhydrous sodium carbonate (5.0 g) and cyanuric chloride (2.75 g; 0.015 mole) were added, and the mixture was stirred overnight at room temperature. After com?letion of the reaction, the reaction mixture was filtered, and petroleum ether t600 m1) was added to the filtrate. The resulting precipitate was collected by suction filtration and washed with a small amount of petroleum ether. The precipitate was purified by being reprecipitated three times from dry benzene and petroleum ether to remove excess cyanuric chloride, thereby stoichiometrically obtaining monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine as white powder. The product after hydrolyzed showed qualitative reaction characteristics of chlorine ions (AgNO3).
- Similarly, polyethylene glycol monomethyl ethers having average molecular weights of 550, 700, 2,000 and 20,000, respectively, were each reacted with cyanuric chloride to stoichiometrically obtain monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines having the corresponding average molecular weights.
Example 2 Monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines (average molecular weights o~ their polyethylene glycol moieties: 10,000 and 15,000) Polyethylene glycol monomethyl ether having an ~77~
average molecular weight of 10,000 (25.0 g; 0.025 mole) was taken up with warming in dry benzene (200 ml~. After cooling the resultant solution, anhydrous sodium carbonate (2.5 g) and cyanuric chloride (1.38g; 0.0075 mole) were added. The resultant mixture was stirred overnight at 33C. After completion of the reaction, undissolved matter was removed by filtration. The filtrate was added with n-hexane (about 600 ml) to induce reprecipitation. Dry benzene (100 ml) was added to the precipitate which was then warmed and dissolved. n-~exane (500 rll) was then added to induce reprecipitation. This procedure was repeated three times.- The thus obtained precipitate was dried overnight at 50C in vacuo, thereby stoichiometri-cally obtaining monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: lO,~OOO)~as while powder.
Polyethylene glycol monomethyl ether having an average molecular weight of 15,000 was similarly reacted with cyanuric chloride to stoichiometrically obtain monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety:
15,000).
Example 3 Monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine Polyethylene glycol monostearyl ether having an average molecular weight of 3,200 (lo .0 g; 0.005 mole) was 77~
dissolved in dry benzene (200 ml), followed by the addi-tion of anhydrous sodium carbonate (5.0 g) and cyanuri~
chloride (2.75 g; 0.015 mole) under stirring. The resul-tant mixture was stirred overnight at room temperature.
After completion of the reaction, undissolved matter was removed by filtration. The filtrate was added with n-hexane (about 600 ml) to induce reprecipitation. Dry benzene (100 ml) was added to the precipitate to dissolve the latter. n-Hexane (500 ml) was added to induce reprecipitation. This procedure was repeated three times, thereby stoichiometrically obtaining monostearly ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety:
However, urokinase is unstable under certain conditions since it is an enzyme and loses its enzymatic activity, for example, in the course of extraction, isolation and purification from a urokinase-bearing raw material, for example, urine; during the lyophilization processing in preparing dosable formulations; during tne heat treatment for deactivating viruses, or when it is placed in a diluted state in a dripping bottle and kept for a prolonged time period in such a diluted state at room temperature for clinical application. This physically unstable nature of urokinase has created a serious problem in preparing and formulating urokinase on an industrial scale or in actually using the same for clinical purposes.
Human albumin has been employed as an additive to urokinas~
so as to improve its stability. However, this can be by no means a break-through solution to the problem just dis-cussed because pure albumin, i.e. a globulin fraction, is difficult to obtain without immunogenic contamination;
pure albumin is expensive; albumin and urokinase form a complex of a high molecular weight under virus deacti-~ Z~7 7~
vating conditions in which urokinase is subjected to heattreatment at 60C for 10 hours together with albumin added to stabilize urokinase; and such stabilizer if added may be effective to a certain extent for protecting urokinase from losing its enzymatic activity upon the lyophilization but cannotPreVentits loss of activity upon actual clinical use.
The physiological activity of urokinase when admi-nistered intravenously to living bodies is promptly retarded by protease inhibitors present in blood (~2-macroglobulin, and a2-plasmin inhibitors and the like), and the metabolic rate of urokinase per se is ve~y hig;~, -es~l.-ing in extremely shortened half-life which does not exceed even several minutes. Nothing has been heretofore propos-d to solve the problem of s~ort half-life of urokinase in blood. - -_ The present inventors have carried out extensive research with a view toward developing derivatives of human-oriyinated non-immunogenic plasminogen activators which will overcome the above-noted drawbac~s of the prior art techniques. As a result, they have succeeded in ind-ing plasminogen activator derivatives whicn are stable and hardly retarded by inhibitors present in blood and hence achi2ve prolonged half-life in blood, thereby leading to the present discovery.
::' SUMMARY OF T~E INVENTION
-An object of the present invention is to provide a derivative of a human-originated non-immunogenic ~ .
7~
plasminogen activator which is stable and exhibits prolong-ed fibrinolytic activity when administered to living bodies.
Another object of the invention is to provide a process for preparing the novel plasminogen activator derivative.
A further object of the invention is to provide a therapeutically acceptable thrombolytic agent comprising the novel plasminogen activator derivative.
These and other objects and advantages of the inven-tion can be attained by the provision of a derivative of a human-originated ncn-immunogeniC plasminogen activator, comprising at least one polyalkylene glycol attached with at least one coupling agent to amino ~cid side chains of the plasminogen activator, the polyalkylene glycol having a molecular weight in the range of 200 -20,000 and option-ally containing one or more alkyl,alkoxy and/or alkanoyl groups as substituents.
l.Z~7~3 -4a-Thus in one aspect the present invention provides a composition for inhibiting the formation of thrombus in a mammalian bloodstream, which composition comprises an effective amount of a nonimmunogenic plasminogen activator comprising at least one polyalkylene glycol moiety chemically bonded with at least one coupling agent to the amino acid side chains of said plasminogen activator, wherein the polyalkylene glycol has a molecular weight in the range of 200-20,000 and is unsubstituted or is substituted with one or more alkyl, alkoxy or alkanoyl groups or a mixture thereof, together with a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DR~WINGS
A more complete appreciation of the present inven-tion and many of the attendant advantages thereof will be readily obtained as the invention becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIG. 1 shows the optimum pH ranges of modified high molecular weight urokinase (PEG-DCI-UX) and unmodified high molecular weight urokinase (molecular weight: 54,000) as lZ~7~
_5_ measured in terms of the amidase activity with a synthetic substrate (S-2444), where the open circles correspond to PEG-DCT-UX and the closed circles to unmodified urokinase;
FIG. 2 shows the optimum pH ranges of modified low molecular weight urokinase (PEG-DCT-L-UK) and unmodified low molecular weight urokinase (molecular weight: 33,000) as measured in terms of the amidase activity with S-2444, where the open circles correspond to PEG-DCT-L-UK and the closed circles to unmodified urokinase;
FIG. 3 diagrammatically shows the stability of PET-DCT-UK and unmodified uro~inase at room temperature in either physiological saline or a Ringer solution, where the solid and broken lines indicate the physiological saline and the Ringer solution, respectively, and where the open circles correspond to PEG-DCT-UK and the closed circles to unmodi-fied urokinase; - _ FIG. 4 shows the stability of PEG-DCT-L-UK and un-modified urokinase (molecular weight: 33,000) against freezing, followed by thawing operation repeated 2, 4 and 6 times, where the open circles correspond toPEG-DCT-L-VK
and the closed circles to unmodified urokinase;
FIG. S shows the varied amounts, as a function of time, of a plasmin inhibitor in a plasma fraction Fl of each of two groups of healthy rabbits, one group adminis-tered with PEG-DCT-UK and the other group with unmodified urokinase (molecular weight: 54,000), where the open cir-cles correspond to the PEG-DCT-UK-administered group and the closed circles to the unmodified urokinase-administered group;
-6- 1~7~
FIG. 6 shows the varied amounts, as a function of time, of plasminogen in a plasma fraction F3 of each of two groups of healthy rabbits, one group administered with PEG-DCT-UK and the other group with unmodified urokinase (mole-cular weight: 54,000), where the open circles correspond to the PEG-DCT-UK-administered group and the closed circles to the unmodified urokinase-administered group; and FIG. 7 shows the varied amounts, as a function of time, of plasminogen in a plasma fraction F3 of each of two groups of healthy rabbits, which plasma was treated w1th an acid and then with a base to inactivate a plasmin inhibitor, one group administered with PEG-DCT-UK and the other grou~
with unmodified urokinase (molecular weight: 54,000), where the open circles correspond to the PEG-~CT-UK-administered group and the closed circles to the unmodified urokinase-administered group.
Figure 8 illustrates the substrate specificity of PEG-Modified UK's. _ _ Figure 9a illustrates the stability of modified Urokinase to UK-Inhibi~ors in Human Plasma.
Figure 9b illustrates the effect of Placental UK-Inhibitor on the Urokinase Activity.
Figure 10 illustrates UK activity in Plasma after UX
injection.
Figure 11 illustrates the relative radioactivity in Blood (%1 for 125I-M-Md and 125I-Native-UK.
:
J ~'~
~7- lZ~77~
Figure 12a illustrates a comparison of the immunogenicity of M-Md with that of native UK by Schultz-Dale test.
Figure 12b illustrates a comparison of the immunogenicity o~ M-Md with that of native UK by Passive Cutaneous Anaphylaxis (PCA) reaction.
Figure 13a illustrates a compaxison of the Plasmin Inhibitor in Fl from Citrated Dog Plasma at certain time intervals after injection of Native UK or PEG-UK into a dog.
Figure 13b illustrates a comparison of the Plasminogen in F3 from Citrated Dog Plasma at certain time interYals after injection of Native UK or PEG-UK into a dog.
Figure 13c illustrates a compari50n of the FDP in Dog Serum at certain time intervals after i.v. Injection of Urokinase or PEG-UK in~o a dog.
Figure 14 illustrates a comparison of FDP levels in dogs bearing artificial thrombus in A. femoralis. Dog A had native UK injected into a proximal branch of the thrombus, while Dog B had PEG-UK injected into the same.
- DESCRIPTION OF THE PREFERRED E~ODIMENTS
By the term "human-originated non-immunogenic plas-minogen activator" as used herein are encompassed not on;y urokinase but also tissue plasminogen activators obtained from human tissues such as uterine, tumor and the like.
These human tissue plasminogen activators also contain those obtained by tissue culture or genetic engineerins.
It should be noted that no limitations are imposed on the molecular weishts of these activators so long as they are obtained in the above-described manner. For example, as urokinase which is a plasminogen activator origina.ed from human urine, high molecular weight urokinase (molecular .
7~3 weight: S4,000) and low molecular weight urokinase (molecu-lar weight: 33,000) may be used solely or in combination.
Suitable polyalkylene glycols which may be used in the invention include a polyethylene glycol and a polypro-pylene glycol. In the case of the polypropylene glycol, both straight-chain polypropylene glycols such as those represented by ~O~CH(CH3)CH2O]nH and branched-chain poly-propylene glycols such as those represented by CH3CH2C-{CH20~cH2cH(cH3)o~nH}3 or HIOCH(CH3)CH2~nOCH{cH2~ocH2cH-(C-~3)]nOH}2.
The molecular weights of the polyalkylene glycols may range from 200 to 20,000. Particularly preferred molecular weights are in the range of 500 - 10,000.
The polyalkylene glycols each may o?tionally contain one or more alkyl, alkoxy and/or alkanoyl groups as substituent groups. Typical example,s of the alkyl groups are methyl, ethyl, propyl,stearyl and the like. Typical examples of the alkanoyl groups are acetyl, propibnyl, stearoyl and the like. As a preferred polyalkylene glycol, an unsubsti-tuted or methyl-substituted polyalkylene glycol is useful.
However, of particular interest is the use of methoxy-polyethylene glycol as the polyalkylene glycol. Chemical modification of urokinase(UK) with activated methoxypoly-ethylene glycol(PEG) of MW 5,000 increased the stability of the urokinase and imparted a markedly extended circulation life in rabbits and rats. Also of interest is the surprising fact that PEG-UK has a superior thrombolytic ability as compared to that ~`;
:~Z~77~
g .
of native UK. It appears likely that the superiority of PEG-UK to native UK with respect to the fibrinolytic activation is from the PEG chains which protect the UK
molecule from interation with inhibitors, thus extending its circulation life.
Suitable coupling agents which may be used in the invention and are adapted to attach a polyalkylene glycol to a non-immunogenic plasminogen activator, for example, urokinase, include those capable of reacting with amino acid side chains of the protein to be modifiedand forming chemical bonds therebetween, for example,.acyl azide, cyanuric halides, p-diazoniumbenzyl ether, 3-(p-diazonium-phenoxy)-2-hydroxypropyl ether, dihalogenosuccinic anhydride and the like. The following partial formulae may be given as examples of the coupling structures between a polyalkylene glycol and urokinase through these coupling agents.
-~ -UK
X
_o- ~ -UK
OH
-O-CH2- ~ N2-UK
-o-cH2cH(OH)cH2 ~ -r~2-UK
wherein X is a halogen atom, and UK is a residual part of the urokinase ~olecule.
The novel urokinase derivative according to the invention can ~e prepared by reacting a coupled product of at least one corresponding polyalkylene glycol and at least one coupling agent with urokinase, the polyalkylene glycol having a molecular weisht in the range of 2Q0 -20,000 and optionally containing one or more alkyl , alkoxy and~or alkanoyl groups as substituents.
Typical examples of the polyalkylene ~lycol-cou~lins agent coupled product include oolyalkylene glycol-~,6-.~2~
dichloro-1,3,5-triazine, polyalkylene glycol-4,6-difluoro-1,3,5-triazine, polyalkylene glycol-4-chloro-6-hydroxy~
1,3,5-triazine, polyalkylene glycol-~-(bro~ocarbonyl)-monopropionate, polyalkylene glycol-azidocarbonyl methyl etller, polyalkylene glycol-(p-diazoniumbenzyl) ether, polyalkylene glycol-3-(p-diazoniumphenoxy)-2-hydroxypropyl ether and the like.
When reacting the polyalkylene glycol-coupling agent coupled product with uroki~ase, it is necessary to choose such reaction conditions that the enzymatic acti-vity is held to a minimum loss. Namely, it is desirable to carry out the reaction at low temperatures, for example, at a temperature of 0C to room temperature in an a~ueous solution such as a buffer. Preferred reaction time may range from several minutes to 5 hours. The pH of the buffer is preferably within such a range that the enzymatic activity of urokinase is not lowered, namely, 2 - 10, preferably 5 - 9. However, the preferred pH range may vary depending on the reactivity of each coupling agent employed and/or the nature of an amino acid residue. The modification degreesof amino acid side chains of the plasminogen activator can be controlled by changing the concentration of the polyalkylene glycol activated witn the coupling agent in a reaction medium.
By wày of illustration, monomethyl ether polyethylene glycol-4,6-dicnloro-1,3,5-triazine having an average molecular weight of 5,000 was reacted at pH 7.0 with hig~
lZ~7~
molecular weight urokinase to obtain novel urokinase derivatives, while changing the concentration of the former reactant to 0.4, 4.0 and 6.0 mM, respectively. Unmodified -amino groups of lysine of the resultant urokinase deri-vatives were quantitatively determined using sodium ~,4,6-trinitrobenzenesulfonate. Their modification degrees were investigated on the basis of the results obtained by the quantitative analyses. The modification percentages of the ~-amino groups of lysine, which were reactive with sodium 2,4,6-trinitrobenzenesulfonate, were 6-7% at 0.4~M, about 40% at 4.0 mM and about 60~ at 6.0 mM. In addition, the molecular weights of these reaction products were determined by SDS polyacryl amide gel electrophoresis.
The average molecular weights of the reaction products were about 60,000 at 0.4 mM and about 120,000 at 4.0 m~1. This finding is substantially in conformity with the results obtained above by the quantitative analyses of the E-amino groups of lyslne. Accordingly, it is necessary to conduct the reaction between the polyalkylene glycol activated with the coupling agent and the plasminogen activator at a high pH level and at a high concentration of tne former reagent when increased modification degrees of the plasminogen activator are desired. On the other hand, where decreased modification degrees are preferable, tne xeaction should be effected at a relatively low pH level and at a low con-centration of the coupled product. The modification degrees .. . .
i~`77 ~
may of course be changed by controlling the reaction time.
By suitably combining these reaction conditions, it is possible to obtain the intended novel plasminogen activator derivatives which are stable and have prolongedfibrinolytic activities.
The polyalkylene glycol activated with the coupling agent can be obtained in the following manner. A terminal-substituted polyalkylene glycol-4,6-dihalogeno-1,3,5-triazine is obtained by reacting its corresponding mono-substituted polyalkylene glycol with a cyanuric halide in an anhydrous solvent and in the presenc,e of a base.
A polyalkylene glycol-4-halogeno-6-hydroxy-1,3,5-triazine is formed by first reacting its corresponding polyalkyler,e glycol with cyanuric halide and then treating the resultant reaction product with water. A polyalkylene glycol-acetoazide is formed by reacting an-ani~n of its corre-sponding polyalkylene glycol with ethyl chloroacetate, followed by treating the reaction product with hydrazine, and finally activating the resulting hydrazide with nitrous acid. A polyalkylene glycol-p-diazoniumbenzyl ether or a polyalkylene glycol-3-(p-diazoniumphenoxy)-2-hydroxypropyl ether is obtained by reacting its corresponding polyalky-lene glycol with p-nitrobenzyl chloride or p-nitrophenyl glyceryl ether and, after reducing the nitro group into an amino group, diazotizing the resultant product with nitrous,acid.
After completion of the reaction of the . . .
lZ177~
polyalkylene glycol activated with the coupling agent and the plasminogen activator, the isolation and purification of the reaction product may be effected in a biochemical manner known per se in the art, for example, by using singly or in combination gel filtration, dialysis, ion-exchange chromatography, affinity chromatography and the - like. Preferably, the conjugate is kept as a solution containing a buffer or a physiological salt, by freezins the solution below -20C, or by lyophilizing the solution.
The optimum pH levels for the novel urokinase derivatives prepared above vary depending on the molecular weights and types of polyalkylene glycols emplo~ed, the types of coupling agents employed, the modification degrees of urokinase, the modification conditions and the measure-ment conditions such as substrates used. ~ihen measured in terms of the amidase activity using a synthetic substrate S-2444, the optimum pH level is within the range of about 8 to 9. In the case of nigh molecular weight urokinase modified by reaction with monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine having an average molecular weight of 5,000 at pH 7.0 and at a temperature of 0C for 3 hou-s, while using the latter reactant at a concentration of 4.0 mM (hereinafter abbreviated as PEG-DC~-UK), the optimum pH as measured in terms of the amidase activity is 8.2 as shown in FIG. 1. The optimum pH of low molecular weight urokinase modified under the same conditions (hereinarter abbreviated as PEG-DCT-L-UK) is, as measured in terms of `
lZ;~ 7~
the amidase activity, 8~2 as shown in FIG. 2.
The novel urokinase derivatives according to the invention have extremely enhanced stability compared with unmodified urokinase. For instance, FIG. 3 shows the results of the residual urokinase activity obtained relative to the passage of time when urokinase was dilu~ed with a Ringer solution or a physiological solution to sucn a concentration as used by dripping administration for clinical application and then allowed to stand at room temperature. As a result, it has been found that PEG-DCT-UK dissolved at a concentration of 105.3 iu/ml in the Rinser solution maintains an activity of 73.4~ at the initiation of the experiment even after 6 hours. In addition, PEG-DCT~UK retains an initial activity of 79.4~ in the physio-logical saline. On the other hand, unmodified urokinase dissolved at a concentration of 76.I iu~l in a Ringer solution keeps only 33.3% in its initial activity even after 2 hours. In a physiological saline, the residual activity is as low as 26.3%.
FIG. 4 illustrates the results obtained by comparing the stability of PEG-DCT-L-UK and unmodified low moiecula~
weight urokinase, both in the course of freezing, followed by thawing operation. The results confirm th~t th2 supe-b stability of PEG-DCT-L-UK is attained even under such conditions.
Furthermore, the novel urokinase derivatives according to the invention have prolonged urokinase ac~i-lZ:~`7~3 vities in blood as compared with unmodified urokinase.For example, the effectiveness of urokinase was investigated by administering 8,000 iu/kg of each of PEG-DCT-UK and unmodified urokinase to healthy rabbits by intravenous injection, sampling blood periodically before the dosage and after the lapse of 4 hours, and measurins the amounts of the plasmin inhibitor and plasminogen present in the citrated plasma. The results are shown in FIGS. 5, 6 and 7. From these results, it has been con-firmed that PEG-DCT-UK alters to a substantial extent biochemical parameters such as a plasmin inhibitor and plasminogen and maintains the parameters at lowered values for an extended period of time as compared with unmodified urokinase.
Therefore, the ~;~ urokinase derivatives accorâing to the invention are excellent plasminog~n activators which, while retaining plasminogen-activating potency as unmodified urokinase does, have surmounted the shortcomings of urokinase such as poor stability and short half-life in blood.
The urokinase derivatives according to the invention may be used as pharmaceutical products for the treatment of a variety of diseases stenlmed from hyper-coagulability of blood such as arterial and venous throm-boses, coronary artery clotting, myocardial infarction, intracerebral infarction, pulmonary embolism, nephritis and the like. These urokinase derivatives can be suitAbly . . .
L77~
administered by intravenous injection or dripping or by an oral route. The intravenous injection is particulary preferred. As dosable forms, preferably usable is a lyophilized form. When supplied eithex in a neat state or with a physiological salt such as a salt of a Ringer solution or sodium chloride, the lyophilized products may be used for clinical application by dissolution with sterile distilled water or further dilution with sterile distilled water in order to adjust their osmotic pressure, prior to the actual use. Since the novel urokinase de-i-vatives according to the invention are stable,they do not require a stabilizer such as albumin. However, the addi-tion of such a stabilizer does not cause any problem or inconvenience. An excipient may also be added in subjec~-ing the novel urokinase derivatives to lyophilization.
The above description genera-lly ~escribes the present invention. A more complete understanding can be obtained by reference to the following examples which are provided for purposes of illustration only and are not intended to be limiting. The modification procedure may be carried out in any one of the preparation steps of plasminogen activators.
Example 1 Monomethyl ether polyethylene glycol-4,6-dicnloro-1,3,5-triazine Polyethylene glycol monomethyl ether having an average molecular weight of 5,000 (25.0 g; O.OS mole) was ~ 77:~
dissolved with warming in dry benzene (200 ml). After cooling the resultant solution to room temperature, anhydrous sodium carbonate (5.0 g) and cyanuric chloride (2.75 g; 0.015 mole) were added, and the mixture was stirred overnight at room temperature. After com?letion of the reaction, the reaction mixture was filtered, and petroleum ether t600 m1) was added to the filtrate. The resulting precipitate was collected by suction filtration and washed with a small amount of petroleum ether. The precipitate was purified by being reprecipitated three times from dry benzene and petroleum ether to remove excess cyanuric chloride, thereby stoichiometrically obtaining monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine as white powder. The product after hydrolyzed showed qualitative reaction characteristics of chlorine ions (AgNO3).
- Similarly, polyethylene glycol monomethyl ethers having average molecular weights of 550, 700, 2,000 and 20,000, respectively, were each reacted with cyanuric chloride to stoichiometrically obtain monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines having the corresponding average molecular weights.
Example 2 Monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines (average molecular weights o~ their polyethylene glycol moieties: 10,000 and 15,000) Polyethylene glycol monomethyl ether having an ~77~
average molecular weight of 10,000 (25.0 g; 0.025 mole) was taken up with warming in dry benzene (200 ml~. After cooling the resultant solution, anhydrous sodium carbonate (2.5 g) and cyanuric chloride (1.38g; 0.0075 mole) were added. The resultant mixture was stirred overnight at 33C. After completion of the reaction, undissolved matter was removed by filtration. The filtrate was added with n-hexane (about 600 ml) to induce reprecipitation. Dry benzene (100 ml) was added to the precipitate which was then warmed and dissolved. n-~exane (500 rll) was then added to induce reprecipitation. This procedure was repeated three times.- The thus obtained precipitate was dried overnight at 50C in vacuo, thereby stoichiometri-cally obtaining monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: lO,~OOO)~as while powder.
Polyethylene glycol monomethyl ether having an average molecular weight of 15,000 was similarly reacted with cyanuric chloride to stoichiometrically obtain monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety:
15,000).
Example 3 Monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine Polyethylene glycol monostearyl ether having an average molecular weight of 3,200 (lo .0 g; 0.005 mole) was 77~
dissolved in dry benzene (200 ml), followed by the addi-tion of anhydrous sodium carbonate (5.0 g) and cyanuri~
chloride (2.75 g; 0.015 mole) under stirring. The resul-tant mixture was stirred overnight at room temperature.
After completion of the reaction, undissolved matter was removed by filtration. The filtrate was added with n-hexane (about 600 ml) to induce reprecipitation. Dry benzene (100 ml) was added to the precipitate to dissolve the latter. n-Hexane (500 ml) was added to induce reprecipitation. This procedure was repeated three times, thereby stoichiometrically obtaining monostearly ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety:
3,200) as white powder.
Example 4 Polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Polyethylene glycol having an average molecular weight of 6,000 (33.6 g) was dissolved with warming in dry benzene (150 ml). After cooling the resultant solution, anhydrous sodium carbonate (1.6 g) and cyanuric chloride (0.74 g) were added, and the resultant mixture was stirred overnight at room temperature. Thereafter, water (1.0 ml) was added, followed by stirring the mi~ture at room tem-perature for 6 hours and then at 40C for an overnight period. Undissolved matter was removed by centrifuge (2,000 ppm; 10 minutes), and the supernatant was subjected 7 7~
to condensation under reduced pressure. The residue was taken up with warming in dry benzene, and the soIvent w~s then evaporated. This procedure was further repeated twice. The residue was dried under reduced pressure, thereby obtaining polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 6,000).
Polyethylene glycols having different average molecular weights of 1,000 and 4,000 were each reacted in the same manner as above with cyanuric chloride and water, resulting in the stoichiometric formation of polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazines having their correspondins average molecular weishts.
Example 5 Stearoylpolyethylene glycol-4,6-dichloro-1,3,5-triazine - Polyethylene glycol monostearate having an average molecular weight of 2,700 (6.765 g) was dissolved in anhydrous benzene (100 ml), followed by the addition of anhydrous sodium carbonate (2.5 g). While stirring the resultant mixture, cyanuric chloride (1.38 g~ was further added. The resulting mixture was stirred overnight at room temperature and then filtered. The filtrate was concentrated under reduced pressure, and the residue was dried under reduced pressure, thereby stoichiometrically obtaining stearoylpolyethylene glycol-4,6-dlchloro-1,3,5-~riazine (the average molecular weight of the polyethylere 77~
glycol moiety: 2,700) as a white waxy substance.
Example 6 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Polypropylene glycol having an average molecular weight of 1,000 (4.0 g) was taken up in anhydrous benzene (50 ml), followed ~y the addition of anhydrous sodium carbonate ~1.27 g). Cyanuxic chloride (0.552 g) was further added with stirring. After stirring the resultant mixture overnight at room temperature, water (1 ml) was added. The mixture was stirred at room temperature for further 6 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure.
The residue was added with anhydrous benzene and anhydrous sodium sulfate. The mixture was thereafter stirred at room temperature for 10 minutes. ~ter~~iltration of the mixture, the solvent was evaporated from the filtrate.
The residue was dried under reduced pressure, thereby stoichiometrically obtaining polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 1,000) as colorless viscous oil.
Following the same procedure as described above, there were obtained a polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 4,000) and anothe~ poly-propylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the ~z~
average molecular weight of the polypropylene glycol moiety: 10,000).
The polypropylene glycols employed in the above examples were of a straight-chain type, namely, those respresented by the formula HO[CH(C~3)CH2O]nH.
Example 7 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Following the same procedure as in Example 1, poly-propylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 4,000) was stoichiometrically obtained as a white substance from polypropylene glycol having an average molecular weihgt of A,000 ~8.0 g), anhydrous benzene t80 ml), anhydrous sodium carbonate (0.636 g) and cyanuric chloride (0.368 g).
. The polypropylene glycol used in this example was of a branched-chain type, namely, those re~resented by the formula CH3CH2C{CH2O[CH2CH(CH3)O]nH}3-Furthermore, using polypropylene glycol representedby the formula, CH2O[CH2cH(cH3)O]nH
CHO[CH2CH(CH3)O]nH
CH2O[CH2CH(cH3)O~nH
and naving an average molecular weight of 3,000, there was obtained polypropylene glycol-4-chloro-6-hydroxy-1,3 "-triazine (the average molecular weight of the polypropylene ~2~7~
glycol moiety: 3,000).
Example 8 ~lonomethyl ether polyethylene glycol methoxycarbohydrazide Polyethylene glycol monomethyl ether having an average molecular weight of 5,000 (13.3 g; 0.0027 mole) was taken up in anhydrous tetrahydrofuran (400 ml) under nitrogen. A small amount of diphenylacetic acid was added as an indicator, and n-butyl lithium was dropped under ice-cooling until the reaction solution turned to pale yellow.
Thereafter, ethyl chloroacetate (5 ml; 0.0~7 mole) was dropped at room temperature, and the resultant solution was stirred overnight and then refluxed for one hour.
After completion of the reaction, the solvent was driven off under reduced pressure,and the residue was taken up in aqueous acetone(200 ml). The resul~nt solution was treated with charcoal. After filtration and subsequent concentration of the filtrate, benzene was added. After removing water by azeotropic distillation, the residue was reprecipitated from benzene and n-hexane to obtain yellowish powder. The thus obtained powder was dissolved in methanol (150 ml) and tnen added with hydrazine hydrate (15 ml?. The mixture was heated overnight under reflux conditions. After driving off the solvent under reduced pressure, water (150 ml) was added, and excess hydrazine was removed by azeotropic distillation. Water, still remaining in the residue, was removed azeotropically 7~
together with benzene. The residue was again dissolved in benzene and dried with anhydrous sodium sulfa~e.
Thereafter, the solvent was driven off, and the residue was dissolved in warm benzene. The benzene solution was treated with charcoal and then concentrated. The reaction product was reprecipitated twice from benzene and n-hexane, treated with charcoal again and with silica gel and then reprecipitated from benzene and n-hexane, thereby obtain-ing monomethyl ether polyethylene glycol methoxycarbo-hydrazide as white powder. In the same manner, a variety of hydrazides were obtained using polyethylene glycol monomethyl ethers havlng average molecular weights of 550, 700, 2,000, 10,000, 15,000 and 20,000, respectively, as starting materials.
Example 9 Monomethyl ether polyethylene glYCo1-4, 6-dichloro-1,3,5-triazine-modified urokinase (PEG-DCT-UK) A 0.1 M phosphate buffer of pH 7.0 (2.0 ml) W2S
added under ice-cooling to 0. 61 ml of a urokinase solution (molecular weight: 54,000; 66,300 iu/ml). Thereafter, monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 5,000) was further added in such an amount as to bring the concentration of the triazine to 4 mM.
The mixture was reacted under ice-cooling for 3 hours.
After completion of the reaction; the reaction solution i;~177~
was transferred into a dialyzing tube, and excess triazine derivative was removed by dialysis. The dialysis was carrled out under ice-cooling for 3 hours against a 0.1 M
phosphate buffer (pH 7.2) and then for further one hour against physiological saline. The content in the tube ~7as then filled up to 4 ml and stored in a frozen state at -80C. The urokinase activity of the thus obtained mono-methyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase (PEG-DCT-U~) was determined to be 4,550 iu/ml by the fibrin plate method. Therefore, the total activity was 13,200 iu. Since the activity of the starting urokinase was 40,443 iu, an activity drop of 55% was recognized.
The above procedure was repeated except that the monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight~~of the polyethylene glycol moiety: 5,000) was replaced by monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines (the average molecular weights of the polyethylene glycol moieties: 550, 700 and 2,000, respectively), thereby obtaining modified urokinases having urokinase activities ....
Of 9,800, 10JOOO and 6,500 iu/ml as determined by the fi~rin plate method.
Example 10 Monomethyl ether polyethylene ~lycol-4,6-dichloro-1,3,5-triazine-modified low molecular ~ .
weight urokinase (PEG-DCT-L-U~) A 0.1 M phosphate buffer of pH 7.0 (4.7 ml) was ~ .
.
1~77~
added under ice-cooling to 0.2 ml of a low molecular weight urokinase solution (molecular weight: 33,000; 567,828 iu/
ml). Thereafter, monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 5,000) was further added in such an amount as to bring the concentration of the triazine derivative to 4 mM. The mixture was reacted under ice-cooling for 3 hours. After completion of the reaction, the reaction solution was transferred into a dialyzing tube, and excess triazine derivative was removec by dialysis. The dialysis was carried out under ice-cooling for 3 hours against a 0.1 M phosphate buffer (pH
7.2) and then for further one hoùr against physiological saline. After the dialysis, the content was filled u? to 8 ml and stored in a frozen state at -80C. The urokinase activity of the thus obtained monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified low molecular weight urokinase (PEG-DCT-L-UK) was determined to be 10,300 iu/ml by the fibrin plate method. Thus, the total activity was 82,400 iu. Since the activity of the starting urokinase was 113,566 iu, an activity drop of 27.4~ was recognized.
Example 11 Monomethyl ether polyeth~lene ~lycol-4,6-dichloro-1,3,5-triazine-modified urokinase By repeating the procedure of Example 9 except t~at the concentration of each monomethyl ether polyethvlene .
~77~
glycol-4,6-dichloro-1,3,5-triazi~e was changed to 0.4 mM, there were obtained, with modification degrees different from that achieved in Example 9, monomethyl ether poly-ethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinases (the average molecular weights of the poly-ethylene glycol moieties: 550, 700, 2,000 and 5,000, respectively). Their urokinase activities were 8,670, 8,900, 6,770 and 8,670 iu/ml as measured by the fibrin plate method.
Example 12 Monomethyl ether polyethylene carbomethylazide-modified urokinase (1) To 200 mg of the monomethyl ether polyethylene glycol methoxycarbohydrazide prepared in Example 8 (the average molecular weight of the polyethylene glycol moiety:
5,000) were added under ice-coolin~ 1 ~_hydrochloric acid (2 ml) and then a 0.008 N aqueous solution of sodium nitrite (1 ml). The resultant mixture was stirred at room temperature for 20 minutes, and a 1 N aqueous solution of sodium hydroxide (2 ml) was further added to neutralize the mixture. The resultant solution of monomethyl ether polyethylene glycol carboxymethylazide was stored at 0C.
(2) A 0.1 M phosphate buffer of pH 8.0 (3.656 ml) was added to 1 ml of a urokinase solution (molecular weight: 33,000; 45,600 iu/ml). Thereafter, 0.435 ml of the monomethyl ether polyethylene glycol carbomethylazide solution prepared in the procedure (1) above was added .. .. .
~Z~7~3 -under mild stirring. The mixture was reacted at room temperature for 2 hours. The resulting reaction solution was then transferred into a dialyzing tube and dialyzed under ice-cooling for 4 hours against a 0.1 M phosphate buffer (pH 7.2). The content was filled up to 8 ml. The resultant solution was stored in a frozen state at -80C.
The urokinase activity of the thus obtained monomethyl ether polyethylene glycol carbomethylazide-modified uroki-nase was determined to be 7,300 iu/ml by the fibrin plate method. Compared with unmodified urokinase, an activity increase of 28% was recognized.
E~ample 13 Monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase To 0.61 ml of a urokinase solution (molecular weight: 54,000; 101,167 iu/ml) were added under ice-cool`ng -a 0.1 M phosphate buffer of pH 7.0 (2.0 ml) and then under mild stirring monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 10,000). The triazine derivative was added in such an amount as to bring the concentration to 0.1 mM. The mixture was reacted for 3 hours under ice-cooling. After completion of the reaction, the reaction solution was transferred into a dialyzing tube, and excess triazine derivative was removed by dialy-sis. The dialysis was carried out under ice-cooling for 3 hours against a 0.1 M phosphoric acid bùffer added wi-n . . .
~Z~'77~3 .
0.035 v/v % of ethyl amine (pH8.0) and then for further 2 nours against a O.lM phosphate buffer (pH 7.2). The con-tent was adde~ with 3 w/v % bovine serum albumin (0.1 ml) and then filled up to 4.0ml with a 0.1 M phosphate buffer (pH 7.0~. The resultant solution was stored in a frozen state at -80C. The activity of the thus obtained mono-methyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase was determined to be 1~,0~8 iu/ml by the fibrin plate method. Therefore, the total activity was 40,11~ iu, and the activity drop was 35%. Similar modi-fied urokinases were obtained by changing the concentration of the monometnyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the poly-ethylene glycol moiety: lO,OOO)to 0.4and 1.0 ~1, respectively.
Their urokinase activities were 8,7~4 and 6,634 iu/ml.
Furthermore, monomethyl ethe~ por~ethylene glycol-
Example 4 Polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Polyethylene glycol having an average molecular weight of 6,000 (33.6 g) was dissolved with warming in dry benzene (150 ml). After cooling the resultant solution, anhydrous sodium carbonate (1.6 g) and cyanuric chloride (0.74 g) were added, and the resultant mixture was stirred overnight at room temperature. Thereafter, water (1.0 ml) was added, followed by stirring the mi~ture at room tem-perature for 6 hours and then at 40C for an overnight period. Undissolved matter was removed by centrifuge (2,000 ppm; 10 minutes), and the supernatant was subjected 7 7~
to condensation under reduced pressure. The residue was taken up with warming in dry benzene, and the soIvent w~s then evaporated. This procedure was further repeated twice. The residue was dried under reduced pressure, thereby obtaining polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 6,000).
Polyethylene glycols having different average molecular weights of 1,000 and 4,000 were each reacted in the same manner as above with cyanuric chloride and water, resulting in the stoichiometric formation of polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazines having their correspondins average molecular weishts.
Example 5 Stearoylpolyethylene glycol-4,6-dichloro-1,3,5-triazine - Polyethylene glycol monostearate having an average molecular weight of 2,700 (6.765 g) was dissolved in anhydrous benzene (100 ml), followed by the addition of anhydrous sodium carbonate (2.5 g). While stirring the resultant mixture, cyanuric chloride (1.38 g~ was further added. The resulting mixture was stirred overnight at room temperature and then filtered. The filtrate was concentrated under reduced pressure, and the residue was dried under reduced pressure, thereby stoichiometrically obtaining stearoylpolyethylene glycol-4,6-dlchloro-1,3,5-~riazine (the average molecular weight of the polyethylere 77~
glycol moiety: 2,700) as a white waxy substance.
Example 6 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Polypropylene glycol having an average molecular weight of 1,000 (4.0 g) was taken up in anhydrous benzene (50 ml), followed ~y the addition of anhydrous sodium carbonate ~1.27 g). Cyanuxic chloride (0.552 g) was further added with stirring. After stirring the resultant mixture overnight at room temperature, water (1 ml) was added. The mixture was stirred at room temperature for further 6 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure.
The residue was added with anhydrous benzene and anhydrous sodium sulfate. The mixture was thereafter stirred at room temperature for 10 minutes. ~ter~~iltration of the mixture, the solvent was evaporated from the filtrate.
The residue was dried under reduced pressure, thereby stoichiometrically obtaining polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 1,000) as colorless viscous oil.
Following the same procedure as described above, there were obtained a polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 4,000) and anothe~ poly-propylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the ~z~
average molecular weight of the polypropylene glycol moiety: 10,000).
The polypropylene glycols employed in the above examples were of a straight-chain type, namely, those respresented by the formula HO[CH(C~3)CH2O]nH.
Example 7 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine Following the same procedure as in Example 1, poly-propylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (the average molecular weight of the polypropylene glycol moiety: 4,000) was stoichiometrically obtained as a white substance from polypropylene glycol having an average molecular weihgt of A,000 ~8.0 g), anhydrous benzene t80 ml), anhydrous sodium carbonate (0.636 g) and cyanuric chloride (0.368 g).
. The polypropylene glycol used in this example was of a branched-chain type, namely, those re~resented by the formula CH3CH2C{CH2O[CH2CH(CH3)O]nH}3-Furthermore, using polypropylene glycol representedby the formula, CH2O[CH2cH(cH3)O]nH
CHO[CH2CH(CH3)O]nH
CH2O[CH2CH(cH3)O~nH
and naving an average molecular weight of 3,000, there was obtained polypropylene glycol-4-chloro-6-hydroxy-1,3 "-triazine (the average molecular weight of the polypropylene ~2~7~
glycol moiety: 3,000).
Example 8 ~lonomethyl ether polyethylene glycol methoxycarbohydrazide Polyethylene glycol monomethyl ether having an average molecular weight of 5,000 (13.3 g; 0.0027 mole) was taken up in anhydrous tetrahydrofuran (400 ml) under nitrogen. A small amount of diphenylacetic acid was added as an indicator, and n-butyl lithium was dropped under ice-cooling until the reaction solution turned to pale yellow.
Thereafter, ethyl chloroacetate (5 ml; 0.0~7 mole) was dropped at room temperature, and the resultant solution was stirred overnight and then refluxed for one hour.
After completion of the reaction, the solvent was driven off under reduced pressure,and the residue was taken up in aqueous acetone(200 ml). The resul~nt solution was treated with charcoal. After filtration and subsequent concentration of the filtrate, benzene was added. After removing water by azeotropic distillation, the residue was reprecipitated from benzene and n-hexane to obtain yellowish powder. The thus obtained powder was dissolved in methanol (150 ml) and tnen added with hydrazine hydrate (15 ml?. The mixture was heated overnight under reflux conditions. After driving off the solvent under reduced pressure, water (150 ml) was added, and excess hydrazine was removed by azeotropic distillation. Water, still remaining in the residue, was removed azeotropically 7~
together with benzene. The residue was again dissolved in benzene and dried with anhydrous sodium sulfa~e.
Thereafter, the solvent was driven off, and the residue was dissolved in warm benzene. The benzene solution was treated with charcoal and then concentrated. The reaction product was reprecipitated twice from benzene and n-hexane, treated with charcoal again and with silica gel and then reprecipitated from benzene and n-hexane, thereby obtain-ing monomethyl ether polyethylene glycol methoxycarbo-hydrazide as white powder. In the same manner, a variety of hydrazides were obtained using polyethylene glycol monomethyl ethers havlng average molecular weights of 550, 700, 2,000, 10,000, 15,000 and 20,000, respectively, as starting materials.
Example 9 Monomethyl ether polyethylene glYCo1-4, 6-dichloro-1,3,5-triazine-modified urokinase (PEG-DCT-UK) A 0.1 M phosphate buffer of pH 7.0 (2.0 ml) W2S
added under ice-cooling to 0. 61 ml of a urokinase solution (molecular weight: 54,000; 66,300 iu/ml). Thereafter, monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 5,000) was further added in such an amount as to bring the concentration of the triazine to 4 mM.
The mixture was reacted under ice-cooling for 3 hours.
After completion of the reaction; the reaction solution i;~177~
was transferred into a dialyzing tube, and excess triazine derivative was removed by dialysis. The dialysis was carrled out under ice-cooling for 3 hours against a 0.1 M
phosphate buffer (pH 7.2) and then for further one hour against physiological saline. The content in the tube ~7as then filled up to 4 ml and stored in a frozen state at -80C. The urokinase activity of the thus obtained mono-methyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase (PEG-DCT-U~) was determined to be 4,550 iu/ml by the fibrin plate method. Therefore, the total activity was 13,200 iu. Since the activity of the starting urokinase was 40,443 iu, an activity drop of 55% was recognized.
The above procedure was repeated except that the monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight~~of the polyethylene glycol moiety: 5,000) was replaced by monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazines (the average molecular weights of the polyethylene glycol moieties: 550, 700 and 2,000, respectively), thereby obtaining modified urokinases having urokinase activities ....
Of 9,800, 10JOOO and 6,500 iu/ml as determined by the fi~rin plate method.
Example 10 Monomethyl ether polyethylene ~lycol-4,6-dichloro-1,3,5-triazine-modified low molecular ~ .
weight urokinase (PEG-DCT-L-U~) A 0.1 M phosphate buffer of pH 7.0 (4.7 ml) was ~ .
.
1~77~
added under ice-cooling to 0.2 ml of a low molecular weight urokinase solution (molecular weight: 33,000; 567,828 iu/
ml). Thereafter, monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 5,000) was further added in such an amount as to bring the concentration of the triazine derivative to 4 mM. The mixture was reacted under ice-cooling for 3 hours. After completion of the reaction, the reaction solution was transferred into a dialyzing tube, and excess triazine derivative was removec by dialysis. The dialysis was carried out under ice-cooling for 3 hours against a 0.1 M phosphate buffer (pH
7.2) and then for further one hoùr against physiological saline. After the dialysis, the content was filled u? to 8 ml and stored in a frozen state at -80C. The urokinase activity of the thus obtained monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified low molecular weight urokinase (PEG-DCT-L-UK) was determined to be 10,300 iu/ml by the fibrin plate method. Thus, the total activity was 82,400 iu. Since the activity of the starting urokinase was 113,566 iu, an activity drop of 27.4~ was recognized.
Example 11 Monomethyl ether polyeth~lene ~lycol-4,6-dichloro-1,3,5-triazine-modified urokinase By repeating the procedure of Example 9 except t~at the concentration of each monomethyl ether polyethvlene .
~77~
glycol-4,6-dichloro-1,3,5-triazi~e was changed to 0.4 mM, there were obtained, with modification degrees different from that achieved in Example 9, monomethyl ether poly-ethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinases (the average molecular weights of the poly-ethylene glycol moieties: 550, 700, 2,000 and 5,000, respectively). Their urokinase activities were 8,670, 8,900, 6,770 and 8,670 iu/ml as measured by the fibrin plate method.
Example 12 Monomethyl ether polyethylene carbomethylazide-modified urokinase (1) To 200 mg of the monomethyl ether polyethylene glycol methoxycarbohydrazide prepared in Example 8 (the average molecular weight of the polyethylene glycol moiety:
5,000) were added under ice-coolin~ 1 ~_hydrochloric acid (2 ml) and then a 0.008 N aqueous solution of sodium nitrite (1 ml). The resultant mixture was stirred at room temperature for 20 minutes, and a 1 N aqueous solution of sodium hydroxide (2 ml) was further added to neutralize the mixture. The resultant solution of monomethyl ether polyethylene glycol carboxymethylazide was stored at 0C.
(2) A 0.1 M phosphate buffer of pH 8.0 (3.656 ml) was added to 1 ml of a urokinase solution (molecular weight: 33,000; 45,600 iu/ml). Thereafter, 0.435 ml of the monomethyl ether polyethylene glycol carbomethylazide solution prepared in the procedure (1) above was added .. .. .
~Z~7~3 -under mild stirring. The mixture was reacted at room temperature for 2 hours. The resulting reaction solution was then transferred into a dialyzing tube and dialyzed under ice-cooling for 4 hours against a 0.1 M phosphate buffer (pH 7.2). The content was filled up to 8 ml. The resultant solution was stored in a frozen state at -80C.
The urokinase activity of the thus obtained monomethyl ether polyethylene glycol carbomethylazide-modified uroki-nase was determined to be 7,300 iu/ml by the fibrin plate method. Compared with unmodified urokinase, an activity increase of 28% was recognized.
E~ample 13 Monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase To 0.61 ml of a urokinase solution (molecular weight: 54,000; 101,167 iu/ml) were added under ice-cool`ng -a 0.1 M phosphate buffer of pH 7.0 (2.0 ml) and then under mild stirring monomethyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 10,000). The triazine derivative was added in such an amount as to bring the concentration to 0.1 mM. The mixture was reacted for 3 hours under ice-cooling. After completion of the reaction, the reaction solution was transferred into a dialyzing tube, and excess triazine derivative was removed by dialy-sis. The dialysis was carried out under ice-cooling for 3 hours against a 0.1 M phosphoric acid bùffer added wi-n . . .
~Z~'77~3 .
0.035 v/v % of ethyl amine (pH8.0) and then for further 2 nours against a O.lM phosphate buffer (pH 7.2). The con-tent was adde~ with 3 w/v % bovine serum albumin (0.1 ml) and then filled up to 4.0ml with a 0.1 M phosphate buffer (pH 7.0~. The resultant solution was stored in a frozen state at -80C. The activity of the thus obtained mono-methyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase was determined to be 1~,0~8 iu/ml by the fibrin plate method. Therefore, the total activity was 40,11~ iu, and the activity drop was 35%. Similar modi-fied urokinases were obtained by changing the concentration of the monometnyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine (the average molecular weight of the poly-ethylene glycol moiety: lO,OOO)to 0.4and 1.0 ~1, respectively.
Their urokinase activities were 8,7~4 and 6,634 iu/ml.
Furthermore, monomethyl ethe~ por~ethylene glycol-
4,6-dichloro~1,3,5-triazine (the average molecular weight of the polyethylene glycol moiety: 15,000~ and urokinase were similarly reacted, thereby obtaining monomethyl ether polyethylene glycol-4,6-dLchloro-1,3,5-triazine-modified urokinàse (the average molecular weight of the polyethylene glycol moiety: 15,000). The activities of the modified urokinases obtained by changing the concentration of the triazine derivative to 0.1, 0.4 and 1.0 ~I, respectively, were 11,388, ~,580 and 7,176 iu/ml.
'771~
Example 14 Monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase To 0.2 ml of a urokinase solution (molecular weight:
54,000; 101,167 iu/ml) were added under ice-cooling a 0.1 M
phosphate buffer of pH 7.0 (0.66 ml) and then monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-txiazine (the average molecular weight of the polyethylene glycol moiety:
3,200). The triazine derivative was added in such an amount as to bring the concentration to 2 mM. The mixture was reacted under ice-cooling for 3 hours. After comple-tion of the reaction,~the reaction solution was transferred into a dializing tube and subjected to dialysis to remove excess triazine derivative. The dialysis was carried ou~
under ice-cooling for 4 hours against a 0.1 M phosphate buffer of pH 7.2. The content was ~dde~ with a 3.0% aque-ous solution of bovine serum albumin (0.1 ml) and then filled up to 4.0 m~ with a 0.1 M phosphate buffer of pH
7Ø The resultant solution was stored in a frozen state at -80C. The urokinase activity of the thus prepared monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase (the average molecular weight of the polyethylene glycol moiety: 3,200) was determined to be 2,826 iu/ml by the fibrin plate method. Since the total activity was 11,304 iu, the activity of the thus modified urokinas~e was 55.9% of that of t~e starting urokinase. The activities of tle modified urokinases 7 ~
obtained by changing tne concentration of the monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine to 4, 6 and 8 mM, respectively, were 2,351, 1,430 and 1,059 iu/ml.
Example 15 .
Polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase To 1 ml of a urokinase solution (molecular weight:
54,000; 45,600 iu/ml) were added under ice-cooling a 0.05 M
phosphate buffer of pH 9.2 (4.0 ml) and then polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (2.34 mg). The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 6,000. The mixture was reacted under ice-cooling for 3 hours. After completion of the reaction, the reaction solution was transferred into a dialyzin~ tube, and excess txiaz~ne derivative was removed by dialysis. The dialysis was carried out under ice-cooling for one hour against a 0.05 M phosphate buffer of pH 9.2 and then for further 3 hours against a 0.1 M
phosphate buffer of pH 7.2. The content was filled up to 8.0 ml with a 0.1 M phosphate buffer of pH 7.2. The resultant solution was stored in a frozen state at -80C.
The urokinase activity of the thus obtained polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase (the average molecular weight of the polyethylene glycol moiety: 6,000) was determined to be 460 iu/ml by the fibrin plate method.
~'77~'~
Similarly, there were obtained other polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases (the average molecular weights of the polyethylene glycol moieties: 4,000 and 1,000, respectively).
Example 16 Stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase .
- To 0.5 ml of a uro~inase solution (molecular weisht:
54,000; 101,167 iu/ml) were added under ice-cooling a 0.1 phosphate buffer of pH 7.0 (1.5 ml) and then 0~05 ml of a dioxane solution of stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine (270 mg/ml) (the average molecular weight of the polyethylene glycol moiety: 2,700). The mixture was reacted under ice-cooling for 3 hours. ~fter completion of the reaction, the reactio~ solution was transferred into a dia~yzing ~ube~and-~ùbjected to dialysis to remove excess triazine derivative. The dialysis was carried out under ice-cooling for 4 hours against a 0.1 M
phosphate buffer of pH 7.2. The content was filled up to ~ ml with a pnosphate buffer. The resultant solution was stored in a frozen state at -80C.
The thus obtained stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase was found, a result of the urokinase activity measurement by the fibrin plate method, to have an activity of la6% of that of the starting urokinase.
~Z177~
Other stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinases having different modi-fication degrees were obtained by reaction with urokinase using a dioxane solution of stéaroyl polyethylene glycol-4,6-dichloro-1,3,5-triazine (270 mg/ml) in amounts of 0.1 and 0.2 ml, respectively. The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 2,700. The thus prepared modified urokinases had activities of 106 and 101% of that of the starting urokinase.
Example 17 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase To 0. 5 ml of a urokinase solution (molecular weight:
54,000; 101,167 iu/ml) were added under ice-cooling a Ool M phosphate buffer of pH 7.0 (I.5 m~) and then 0.05 ml of a dioxane solution of the polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (1-00 mg/ml) obtained in Example 6. The average molecular weight of the polypro-pylene glycol moiety of the triazine derivative was 1,000.
The mixture was reacted under ice-cooling for 3 hours.
Ater completion of the reaction, the reaction solution was transferred into a dialyzingtube and subjected to dialysis to remove excess triazine derivative. The dialy-sis was carried out under ice-cooling for 4 hours against a 0.1 M phosphate buffer of pH 7.2. The content was filled up to 5 ml with a phosphate buffer and then stored lZ~77~
in a frozen state at -80C. The thus obtained polypro-pylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase was found to retain an activity of 96.4~ of that of the starting urokinase by the fibrin plate method.
Other polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases having different modi-fication degrees were similarly obtained by using a dioxane solution of polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (100 mg/ml) in amounts of 0.1 and 0.2 ml, respectively. The average molecular weight of the polypropylene glycol moiety of the triazine deriva-tive was 1,000. The modified urokinases showed activities of 99.3 and 106.8% in comparison with that of the starting unmodified urokinase.
ExamPle 18 ;
Polypropylene glycol-4-cnloro-6-~droxy-- . 1,3,5-triazine-modified urokinase Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases having different modification degrees were obtained exactly in the same manner as in Example 3 with use of a dioxane solution containing 400 mg/
ml of the polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine obtained in Example 7 in amounts of 0.05, 0.1 and 0.2 ml, respectively. The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 4,000. Their urokinase activities were found to be Rg,7, 100.0 and 98.9% of the that of the 7 ~
starting urokinase by the fibrin pla~e method.
Example 19 Optimum pH for modified urokinase as measured in terms of amidase activity PEG-DCT-UK obtained in Example 9 and unmodified urokinase were each diluted with physiological saline containing 0.1% human albumin to obtain two solutions of 167 iu/ml. Each of the solutions were divided into por-tions of 0.3 ml which were added with 50 mM tris-HCl buffers of different pH levels (each 1.0 ml) and tnen with a synthetic substrate S-2444 (pyrGlu-Gly-Arg-p-nitro anilide made by Kabi Corporation). The resultant mi:~ture;
were incubated at 37C for 10 minutes. The reactions were then stopped by the addition of 30% acetic acid. Their absorbance was measured at 405 nm. As a result, the optimum pH levels for PEG-DCT-UK and unmodified urokinase were 8.2 and 8.5, respectively, as measured in terms of the amidase activity when the synthetic substrate S-2444 was employed. The results are shown in FIG. 1.
The optimum pH levels for PEG-DCT-L-UK obtained in Example 4 and unmodified low molecular weight urokinase were determined similarly in terms of the amidase activity and we~e found to be 8.2 and 8.4, respectively. The results are shown in FIG. 2.
~2~7'71~
Example 20 Stabilit of modified urokinase at room Y
temperature PEG-DCT-UK obtained in Example 9 was diluted with either a Ringer solution or physiological saline to a concentration of 105.3 iu/ml. For the sake of comparison, unmodified urokinase was also diluted with either a Ringer solution or physiological saline to a concentration of 76.1 iu/ml. 3.5 ml of each of the thus diluted solutions was allowed to stand at room temperature (27~C) for 6 hours. The residual urokinase activity was periodically measured by the fibrin pla~e method until such time tha~
6 hours elapsed. The extent of activity loss of the modified urokinase according to the invention was by far smaller than that of unmodified urokinase. The results are shown in FIG. 3.
-Example 21 -Stability of modified urokinase against freezing and thawing processing PEG-DCT-L-UK obtained in Example 10 was diluted with physiological saline to a concentration of 2Q0 iu/ml.
For the sake of comparison, unmodified low molecular weight urokinase was diluted with physiological saline to a concentration of 200 iu/ml. Each of the thus diluted solutions was frozen to -80C and subsequently thawed at room temperature. The freezing and thawing operations were repeated 0, 2, 4 and 6 times. The residual urokinase 1;Z~'77;~
activity was measured using a synthetic substrate S-244~
(made by Kabi Corporation). The extent of activity loss of the modified urokinase according to the invention was extremely low as compared with that of unmodified low molecular weight urokinase. The results are shown in FIG. 4.
Example 22 Eight healthy male rabbits (Japanese white) each having a body weight of 2.6 - 3.1 kg were divided into two groups. One group was dosed with 8,000 iu/kg of PEG-DCT-UK and the other with 8,000 iu/kg of unmodified urokinase, both through the ear veins. Blood was collec-ted from the ear veins before the dosage and at the time of 1, 2 and 4 hours after the dosage. Blood samples W21-e added with a sodium citrate solution to separate plasma.
After wash-out of the modified and unmo~ified urokinases for 18 days, the two groups were crossed with each other, and similar experiments were carried out to take plasma samples. A part of each of the plasma samples thus obtained was separated and subjected to affinity column chromatography using Lysine Sepharose 4B, thereby obtain-ing a plasmin inhibitor fraction (fraction Fl) and a plasmin and plasminogen fraction (fraction F3). Chan~es in the amount of the plasmin inhibitor in the fraction Flalong the passage of time were determined by adding plasmin to the fraction Fl and measuring the amount of residual plasmin which was not adversely affected b~ the inhibitor, with a svnthe'ic ,tl 7-.lB
substrate S-2251 (made by Kabi Corporation). The plasmin inhibitor from the PEG-DCT-UK-administered group showed its apparent decrease and slower recovexy. In the unmodi-fied urokinase-administered group, the decrease in the plasmin inhibitor was rather less, and its time course changes were not made clear. The results are shown in FIG. 5.
On the other hand, the amount of plasminogen in the fraction F3 was determined by adding urokinase to the fraction F3 to produce plasmin and measuring the amount of the thus produced plasmin by a synthetic substrate S 2251. In the PEG-DCT-UK-administered group, plasminogen decreased to about 50% after 2 hours and thereafter re-covered gradually. However, in the unmodified urokinase-administered group, plasminogen decreased up to about 65~
after one hour but did not proceed ~to decrease any fur.her.
-There was observed a tendency of plasminogen being recov~
ered even 2 hours after the dosage. The results are illustrated in FIG. 6.
Furthermore, a part of each plasma was sampled ana treated with an acid to pH 5.2 so as to deactivate the inhibitor therein. It was then neutrali~ed, and the a~ount of plasminogen present in the acid treated plasma was measured by adding urokinase to the acid treated plasma to produce plasmin and measuring the amount of the thus produced plasmin using a synthetlc SuDstrate S-2251.
In the PEG-DCT-UK-administered gro~lp, plasminogen decreased to about 70% after 2 hours but showed its gradual recovery thereafter. In the unmodified urokinase-administered group, however, the amount of plasminogen was decreased to about 84% after one hour but was not decreased any further.
Thereafter, the amount of plasminogen was increased gra-dually. The results are also shown in FIG. 6. Incidental-ly, no plasmin activity was detected from the fraction F3.
Example 23 Lyophilized_product suitable for the preparation of injectable formulations The PEG-DCT-UK solution obtained in Example 9 was concentrated by a membrane filter (Amicon ~el: trademark~
and then added with physiological saline and a O.OS M
phosphate buffer. The resultant mixture was filtered aseptically using a membrane filter. The filtrate was poured in portions into sterilized vials and then lyophi-lized. The fibrinolytic activity of the lyophilized product of PEG-DCT-UK suitable for the preparation of injectable formulations was found to be 57,000 iu/vial by the fibrin plate method.
Example 24 L o hilized product suitable for tne preparation . Y P
of injectable formulations The PEG-DCT-UK solution obtained in Example 9 was concentrated by ~ membrane filter (~micon Gel: trademark) and then added with physiological saline. The resultant mixture was filtered aseptically using a membrane filter.
; 7 ~r~
- The filtrate was poured in portions into sterilized vials and then lyophilized. The fibrinolytic activity of ~he thus obtained lyophilized product of PEG-DCT-UK suitable for the preparation of injectable formulations was found to be 67,000 iu/vial by the fibrin plate method.
In the following experiments Urokinase (MW 54,000) was obtained from Japan Chemical Research Co., Ltd. PEG
#5,000 (MN = 4,700, MW/MN = l.08) was obtained from Nishio Industry Co., Ltd. Cyanuric chloride was obtained from Kanto Chemical Co., Inc. Fibrinogen and thrombin fcr fibrin plate was obtained from Sigma Chemical Co. and Mochida Pharmaceutical Co., Ltd.
respectively. S-2251 and S-2444 were obtained from Kabi Diagnostica. TNBS (trinitrobenzene sulfonic acid sodium salt) was obtained from Wako Pure Chemical Industry, Ltd. Lysine-Sepharose 4B and Sephadex G-200 were obtained from Pharmacia Fine Chemicals. Nal25I
was obtained from Amersham. GGA-MCA (L-glutaryl-glycyl-L-argir.ine-4-methylcoumarin-7-amide) was obtained from Protein Research Founda~ion.
Urokinase dissolved in O.lM phosphate buffer (P~), pH 7.0, was reacted with PEG #5,000 activated with cyanuric chloride by the method of A~uchowski et al at * trade mark.
3~2~i7~'~
4 for 4 hr. Concentration of PEG was adjusted to 1.7 mM for L-Md, 2 mM for M-Md and 5 mM for H-Md which represent low, medium and high degrees of modification, respectively. Dialysis against 0.05 m PB, pH 5.0, containing 0.9% ~aCl followed by concentration and gel filtration using Sephadex G-200 afforded modified UR's. After adjusting pH to 7.0, UK'5 were lyophilized in small portions for experimental convenience. Lysine residues in M-Md were determined with TNBS by the method of Habeeb. UK's were assayed either by the standard fibrin plate method or by the synthetic substrate method. Plasma was fractioned into Fl, F2 and F3 with Lysine-Sepharose 4B by the method of Igarashi et al. Native UK and M-Md were labelled with 12SI by the procedure of Hunter and Greenwood.
Immunological properties of native UK and M-Md were compared according to the procedures of Arai et al.
Example 2S - SUBSTRATE SPECIFICITY OF PEG-MODIFIED UK's Activation of PEG #5,000 with cyanuric chloride followed by reaction with native UK caused a covalent attachment of PEG's on the lysine residues of UK
protein. Three types of modified UK's, L-Md, M-Md and H-Md, were prepared by changing PEG concentration during the reaction. Their enzymatic activitie~ depend on the assay method which sugges~s that PEG chains ~Z~ 7~
presen~ steric hindrance to bulky substrat~s such as plasminogen in fibrin plate while not to the low molecular weight ones such as GGA-MCA.
Attention was specifically focused on M-Md and the pH Optimum of M-Md was found to be 8.16 and that of native UK to be 8.51 using S-2444 as a substrate in 0.05M Tris HCI buffer at 37C. The molecular weight (MW) of M-Md was calculated to be about 120,000 daltons from the result of lysine residue determination with TNBS. As PEG is a kind of neutral detergent the apparent MW obtained from SDS-PAGE of gel filtration does not reflect the true MW. FIGURE 8 illustrates the substrte specificity of PEG-Modified UK's.
Example 26 - EFFECT OF UK INHIBITORS ON THE ACTIVITY OF
~ATIVE UK AND L-MD.
It is known that in human plasma, protease inhibitors play an important role in retaining blood fluidity. Physiological activity of native UK when administered intraveneously is rapidly retarded not only by the interaction with these inhibitors but also by the fragmentation. The effect of UK inhibitors on the UK activity was examined using Fl, inhibitor fraction from human plasma, and placental UK
inhibitor. In the first experiment, U~s were incubated wieh the former at 37 then residual UK was assayed with S-2444. In the next experiment, placental UK
-:^
771~
inhibitor of different dilution was added to UKs then residual UK was assayed with a combination of plasminogen and S-2251.
In both cases, L-Md retained its activity better than native UK did. Neutral and chemically inert PEG
chains seem to protect UK from the action of UK
inhibitors. FIGURE 9 shows the stability of modified Urokinase to UK-Inhibitors in Human Plasma. ~IGURE 9b shows the effect of placental UK-Inhibitor on the Urokinase activity.
Example 27 - EXTENSION OF CIRCULATING LIVES OF UKs IN RABBITS
The short half-life (~/2) of native UK in the circulatory system often becomes a limitation of UK
therapy. As the ~20f native UK does not exceed several minutes, prolonged drop infusion is necessary to maintain the high plasmin level. Half lives of native and modified UKs were measured in rabbits by an injection of one of them followed by the determination of UK activity in the plasma drawn, periodically, from an ear vein.
As a re~ult, the half-life of native UK in the first phase was calculated as 4-5 minutes, L-Md as 30-40 minutes, M-Md as 80-100 minutes and H-Md as 110-150 minutes. ~20f modified UKs were extended about 10 to 30 times that of the native UK depending upon the 77~
respective degree of modification. In the case of M-Md or H-Md, a one compartment model is approximate enough to describe the pharmacokinetics, which suggests tha~
distribution to a second compartment is suppressed.
FIGURE 10 shows the UK activity of L-Md, M-Md and H-Md in Plasma after UK injection.
Example 28 - BEHAVIOR O~ 125I-NA'rIVE UK AND 125I-M-MD
IN RATS
Native UK and M-Md were labeled with 125I. Then, their ~2in the circulatory system and their distribution among organs were investigated in rats.
Extension of ~2f UK by PEG modification was also observed and was more prominent in these experiments.
Rapid decay of radioactivity in blood of rats injected with 125I-native UK was observed. On the contrary, radioactivity was retained well with 125I-M-Md. In the first phase, ~20f native UK was 1.1 min. while that of M-Md was 89.9 min.
Native UK administered intravenously accumulates in the liver and kidneys and other organs resulting in a short ~/2~ With M-Md, accumulation in these organs was suppressed probably due to less interaction of the M-Md molecule~ with organ cells because of the neutral and inert PEG chains on UK.
Tables 1 and 2 ~how the relative radioactivity in various tissues after 125I-native UK and 125I-M-Md ~L77~
injection, respectively. FIGURE 11 illustrates the relative radioactivity in Blood (~) for 125I-M-Md and 125I-native UK-RELATIVE RADIOACTIVITY AF~ER
125I-NATIVE UK INJ_CTIO~
Time Organ 5 min.10 min.20 min. 40 min.12 hr Brain 0.050.03 0.04 0.010.01 Lung 0.470.62 0.31 0.100.01 Thymus 0.030.02 0.03 0.030.01 Liver 27.8932.60 23.06 2.590.46 Heart 0.140.26 0.17 0.170.10 Kidney 3.686.82 2.40 0.400.25 Adrenal 0.05 -0.07 0.05 0.020.00 Spleen 0.39 1.32 0.67 0.050.02 RELATIVE RADIOACTIVITY AFTER
125I_M_MD INJECTION __ Time organ 5 min. 10 min.20 min~40 min.12 hr Brain 0.17 0.20 0.20 0.220.11 Lung 1.24 2.30 1.31 1.370.71 Thymus 0.15 0.21 0.15 0.240.11 Liver 6.84 8.7511.55 7.472.91 Heart 0.93 1.73 1.38 1.020.53 Kidney 2.21 3~10 3.83 3.270.68 Adrenal 0.06 0.07 0.08 0.060.04 Spleen 0.45 0.40 0.63 0.510.21 EXamP1e 29 - IMMUNOGENICITY AND TOXICITY OF M-MD
The changes in immunogenicity ~y chemical modification of proteins are of interest.
7~3 Immunogenicity of M-Md was compared with that of native UK by ASA reaction, Schultz-Dale test, PCA reaction and Ouchterlony method. Both native UK and M-Md were immunogenic for guinea pigs by ASA reaction after sensitization of them with the respective UK and FCA.
However, the immunological response to M-Md was weaker than that of native UK in the other three tests.
The results summarized in FIGURES 12a and 12b suggest that immunological determinants in M-Md are common with those in native UK. Furthermore, antigen production and/or reactivity with antigen are/is suppressed in M-Md compared with native UK. As native UK itself does not cause immunological response in man, M-Md is not expected to either.
The toxicity of M-Md was found to be extremely low. None of the mice died even after a shot of M-MD
at a dosage of 1,000,000 ~/kg nor afte~ repetitive injection of 500,000 ~/kg/day of M-Md.
FIGURE 12a shows a comparison of the immunogenicity of M-Md with that oE native UK by Schultz-Dale test~ FIGURE 12b shows a comparison of the immunogenicity of M-Md with that of native UK by Passive Cu~aneous Anaphylaxis (PCA) reaction.
In ~ummary, the instability of native UK has created a serious problem in formulating it for clinical purposes. The foregoing examples illustrate that covalent attachment of PEG chains to native UK
greatly increases resistance to inhibition of the UK by UK inhibitors. Moreover, covalent attachment of PEG
chains to native UK also prolonged ~2f the UK in the circulatory system. It seems likely that the PEG
chains may surround the UK molecule to form, in effect an inert capsule to protect the UK molecule from inhibitors and other proteases.
In the next set of examples attention was focused on _ vivo experiments using dogs, where the thrombolytic ability of PEG-modified UK (PEG-UK) was compared wlth that of native UK. In these examples, the PEG-UK used is the same as M-Md in Examples 25-29. The unit for UK activity was determined by the standard fibrin plate (F.P.) method. Plasmin was obtained from Sigma Chemical Co. FDP latex for dog FDP
was obtained from MBL Co., Ltd. Terufus~on~, a blood transfusion set type 1, obtained from Terumo Co., Ltd.
was connected with a three-way stopcock of type PX2-50 from Top Co., Ltd. using ATOM 6 Fr extension tubing.
Other materials used in the following examples are the same as used in Examples 25-29.
T~e haemostatic index for plasmin inhibitor, Fl, was determined by an addition of plasmin to the Fl fraction followed by the assay of residual pla~min with S-2251. F3, plasminogen, was determined with a ~Z~,t - 7 ~
combination of UK and S-2251. FDP was determined wi~h latex sensiti~ed with antigen to dog fibrinogen.
For the shun~ preparation, a dog was anaesthetized with ketamine hydrochloride then A. femoralis and V.
femoralis were exposed in the inguinal region. A
saline solution-filled shunt with the three-way stopcock was connected between them. Blood was allowed to run by opening the stopcock while monitoring the flow with a magnetic bloodflow meter. Either native UK
or PEG-UK dissolved in saline solution was administered from the stopcock. To maintain the anaesthesia, sodium barbiturate was injected.
For the preparation of artifical thrombus, A.
fermoralis of an anaesthetized dog was exposed by the same procedure. A transfusion catheter was fixed at a branch of A. femoralis, then the distal portion of the A. femoralis was clamped with two b~lldo~ clamps at a distance of 2 cm from each other and was emptied, then washed with saline solution. Dry oxygen was passed therethrough at a rate of 2 l/min for 10 minutes to injure intima. After injection of a fibrinogen solution followed by thrombin solution, the formed thrombus was allowed to age for 10 minutes. Then, the bulldog clamps were removed. Thrombus formation was confirmed angiographically by an injection of 10 ml of 60% Urografin~ from the catheter. UK dissolved in 16~
- ~o-saline was injected from the same catheter and angiographies were taken periodically thereafter.
Example 30 - CHANGES IN Fl, F3 AND FDP AFTER UK
INJECTION
Changes in the haemostatic indices are good indications of UK treatment. Often used are Fl (plasmin inhibi~or), F3 (plasminogen) and FDP (fibrin and/or fibrinogen degradation product). Native UK or PEG-UK was injected into a dog at the superficial vein of the fore leg at dosages shown in FIGURES 13a, b and c. Blood was sampled from the opposite side at certain time intervals and the Fl, F3 and FDP contents of the samples were determined.
A decrease in both Fl and F3 levels is a direct proof of plasminogen activation. The consumption of F
and F3 indicates that plasmin activated by UK from plasminogen (F3) was deactivated by its inhibitor (Fl) and was removed from the circulatory system. Another proof is a prominent increase in FDP. In a dog injected with PEG-UK, the FDP level was kept higher for several hours although its enæymatic activity to activate plasminogen was lower than that of native UK. These findings reveal clearly that chemical modification with PEG amplifies the physiological activity of UK.
7'~
FIGURE 13a illustrates a comparison of the Plasmin Inhibitor in Fl from Citrated Dog Plasma at certain time intervals after injection of native UK or PEG-UK
into a dog.
FIGURE 13b illustrates a comparison of the Plasminogen in F3 from Citrated Dog Plasma at certain time intervals after injection of native UK or PEG-UK
in~o a dog.
FIGURE 13c illustrates a comparison of the FDP in Dog Serum at certain time intervals after i.v.
injection of UK or PEG-UK into a dog.
Example 31 - THROMBOLYTIC EFFECT OF PEG-UK I~
EXPERIMENTAL THROMBOSIS
Thrombolytic activities of native UK and PEG-UK
were compared in dogs with experimental thrombosis. A
shunt having a fine filter was fixed between A.
femoralis and V. femoralis and then blood was run through it. After a complete stop of flow (several minutes) either native UK or PEG-UK was administered through the three-way stopcock at a dose of 10,000 ~/kg. As a result, the flow of blood in the dog with PEG-UK recovered while not in the dog having native UK administered even after repeated flushes of saline ~olution.
1' ;LI'i7.~3 -~2-Example 32 - INHIBITION OF THROMBUS FORMATION WITH
PEG-UK
The inhibitory effect of PEG-UK on the thrombus formation was investigated as compared with native UK
with a saline solution-filled shunt prepared by the same procedures de~cribed above. In this experiment either native UK or P~G-UK was administered prior to the blood circulation. The dose was 20,000 ~/kg in both cases. Results obtained are as follows:
1) In the dog without UK, the blood flow stopped within 5 minutes.
2) In the dog with native UK, the blood flow stopped within 5 minutes.
3) In the dog with PEG-UK, the blood flow continued during the experiment for >1 hour.
At this stage, the three filters were washed with saline. Complete inhibition of thrombus formation was observed in PEG-UK, which is probably due to the thrombin inhibition by FDP.
Example 33 - LYSIS OF ARTIFICIAL THROM~US WITH PEG-UK
USING ANGIOGRAM
The thombolytic effect of PEG-UK was evaluated in dogs bearing artifical thrombus in A. femoralis. After confirmation of thrombus formation, either native UK
(dog "A" ) or PEG-UK (dog "B") was injected from the transfusion catheter fixed on a proximal branch of the ~7~
thrombus at a dose of 10,00~ ~/kg. Angiographies were taken periodically to observe ~he recovery of blood flow. It is concluded from these angiographies that PEG-UK dissolved the artifical ~hrombus formed in A.
femoralis while native UK was unable to do so. FIGURE
14 illustrates a comparison of FDP levels in dogs bearing artifical thrombus in A. femoralis. Dog A had native UK injected into a proximal branch of the thrombus, while dog B had PEG-UK injected into the same.
Example 34 - INHIBITORY EFFECT ON THROMBUS FORMATION
Using a dog, a shunt was established under anesthesia by a blood transfu~ion filter ("Terufusion"
blood transfusion Set Type-l) between A. femoralis and V. femoralis and a blood flowmeter ("MF-26", rectangular wave electromagnetic bl~od f~-owmeter manufactured by Nippon Koden K.K.) was connected at the vein side. The filter had in advance been filled with physiological saline and, after administration of a drug, the blood was caused to flow through the shunt by opening a three-way stopcock. Without administration of the drug, thrombus was formed in the filter within 4-5 minutes after the blood had started flowing through the filter, whereby any further flow of the blood ~hrough the shunt was preven~ed. The time period until 1~ o 7 ~
the stoppage of the blood flow due to the formation of thrombus was not prolonged at all even when 200,000 units of unmodified urokinase were administered. On the contrary, thrombus was not formed nor was the blood flow rate reduced at all even after 40 minutes after the initiation of the blood flow through the shunt when PEG-DCT-UK, which pertains to the present invention, had been administered.
In summary, Examples 30-34 dealt with the thrombolysis by PEG-UK wherein the thrombolytic ability thereof was compared with that of native UK using two thrombosis models. The superiority of PEG-UK to native UK with respect to fibrinolytic activation is due to the PEG chains which appear to protect the UK molecule from deactivating interactions with inhibitors. This protection serves to extend the UK circulating life.
Having fully described the pre5ent-~nvention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.
'771~
Example 14 Monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase To 0.2 ml of a urokinase solution (molecular weight:
54,000; 101,167 iu/ml) were added under ice-cooling a 0.1 M
phosphate buffer of pH 7.0 (0.66 ml) and then monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-txiazine (the average molecular weight of the polyethylene glycol moiety:
3,200). The triazine derivative was added in such an amount as to bring the concentration to 2 mM. The mixture was reacted under ice-cooling for 3 hours. After comple-tion of the reaction,~the reaction solution was transferred into a dializing tube and subjected to dialysis to remove excess triazine derivative. The dialysis was carried ou~
under ice-cooling for 4 hours against a 0.1 M phosphate buffer of pH 7.2. The content was ~dde~ with a 3.0% aque-ous solution of bovine serum albumin (0.1 ml) and then filled up to 4.0 m~ with a 0.1 M phosphate buffer of pH
7Ø The resultant solution was stored in a frozen state at -80C. The urokinase activity of the thus prepared monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase (the average molecular weight of the polyethylene glycol moiety: 3,200) was determined to be 2,826 iu/ml by the fibrin plate method. Since the total activity was 11,304 iu, the activity of the thus modified urokinas~e was 55.9% of that of t~e starting urokinase. The activities of tle modified urokinases 7 ~
obtained by changing tne concentration of the monostearyl ether polyethylene glycol-4,6-dichloro-1,3,5-triazine to 4, 6 and 8 mM, respectively, were 2,351, 1,430 and 1,059 iu/ml.
Example 15 .
Polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase To 1 ml of a urokinase solution (molecular weight:
54,000; 45,600 iu/ml) were added under ice-cooling a 0.05 M
phosphate buffer of pH 9.2 (4.0 ml) and then polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (2.34 mg). The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 6,000. The mixture was reacted under ice-cooling for 3 hours. After completion of the reaction, the reaction solution was transferred into a dialyzin~ tube, and excess txiaz~ne derivative was removed by dialysis. The dialysis was carried out under ice-cooling for one hour against a 0.05 M phosphate buffer of pH 9.2 and then for further 3 hours against a 0.1 M
phosphate buffer of pH 7.2. The content was filled up to 8.0 ml with a 0.1 M phosphate buffer of pH 7.2. The resultant solution was stored in a frozen state at -80C.
The urokinase activity of the thus obtained polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase (the average molecular weight of the polyethylene glycol moiety: 6,000) was determined to be 460 iu/ml by the fibrin plate method.
~'77~'~
Similarly, there were obtained other polyethylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases (the average molecular weights of the polyethylene glycol moieties: 4,000 and 1,000, respectively).
Example 16 Stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase .
- To 0.5 ml of a uro~inase solution (molecular weisht:
54,000; 101,167 iu/ml) were added under ice-cooling a 0.1 phosphate buffer of pH 7.0 (1.5 ml) and then 0~05 ml of a dioxane solution of stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine (270 mg/ml) (the average molecular weight of the polyethylene glycol moiety: 2,700). The mixture was reacted under ice-cooling for 3 hours. ~fter completion of the reaction, the reactio~ solution was transferred into a dia~yzing ~ube~and-~ùbjected to dialysis to remove excess triazine derivative. The dialysis was carried out under ice-cooling for 4 hours against a 0.1 M
phosphate buffer of pH 7.2. The content was filled up to ~ ml with a pnosphate buffer. The resultant solution was stored in a frozen state at -80C.
The thus obtained stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinase was found, a result of the urokinase activity measurement by the fibrin plate method, to have an activity of la6% of that of the starting urokinase.
~Z177~
Other stearoyl polyethylene glycol-4,6-dichloro-1,3,5-triazine-modified urokinases having different modi-fication degrees were obtained by reaction with urokinase using a dioxane solution of stéaroyl polyethylene glycol-4,6-dichloro-1,3,5-triazine (270 mg/ml) in amounts of 0.1 and 0.2 ml, respectively. The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 2,700. The thus prepared modified urokinases had activities of 106 and 101% of that of the starting urokinase.
Example 17 Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase To 0. 5 ml of a urokinase solution (molecular weight:
54,000; 101,167 iu/ml) were added under ice-cooling a Ool M phosphate buffer of pH 7.0 (I.5 m~) and then 0.05 ml of a dioxane solution of the polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (1-00 mg/ml) obtained in Example 6. The average molecular weight of the polypro-pylene glycol moiety of the triazine derivative was 1,000.
The mixture was reacted under ice-cooling for 3 hours.
Ater completion of the reaction, the reaction solution was transferred into a dialyzingtube and subjected to dialysis to remove excess triazine derivative. The dialy-sis was carried out under ice-cooling for 4 hours against a 0.1 M phosphate buffer of pH 7.2. The content was filled up to 5 ml with a phosphate buffer and then stored lZ~77~
in a frozen state at -80C. The thus obtained polypro-pylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinase was found to retain an activity of 96.4~ of that of the starting urokinase by the fibrin plate method.
Other polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases having different modi-fication degrees were similarly obtained by using a dioxane solution of polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine (100 mg/ml) in amounts of 0.1 and 0.2 ml, respectively. The average molecular weight of the polypropylene glycol moiety of the triazine deriva-tive was 1,000. The modified urokinases showed activities of 99.3 and 106.8% in comparison with that of the starting unmodified urokinase.
ExamPle 18 ;
Polypropylene glycol-4-cnloro-6-~droxy-- . 1,3,5-triazine-modified urokinase Polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine-modified urokinases having different modification degrees were obtained exactly in the same manner as in Example 3 with use of a dioxane solution containing 400 mg/
ml of the polypropylene glycol-4-chloro-6-hydroxy-1,3,5-triazine obtained in Example 7 in amounts of 0.05, 0.1 and 0.2 ml, respectively. The average molecular weight of the polyethylene glycol moiety of the triazine derivative was 4,000. Their urokinase activities were found to be Rg,7, 100.0 and 98.9% of the that of the 7 ~
starting urokinase by the fibrin pla~e method.
Example 19 Optimum pH for modified urokinase as measured in terms of amidase activity PEG-DCT-UK obtained in Example 9 and unmodified urokinase were each diluted with physiological saline containing 0.1% human albumin to obtain two solutions of 167 iu/ml. Each of the solutions were divided into por-tions of 0.3 ml which were added with 50 mM tris-HCl buffers of different pH levels (each 1.0 ml) and tnen with a synthetic substrate S-2444 (pyrGlu-Gly-Arg-p-nitro anilide made by Kabi Corporation). The resultant mi:~ture;
were incubated at 37C for 10 minutes. The reactions were then stopped by the addition of 30% acetic acid. Their absorbance was measured at 405 nm. As a result, the optimum pH levels for PEG-DCT-UK and unmodified urokinase were 8.2 and 8.5, respectively, as measured in terms of the amidase activity when the synthetic substrate S-2444 was employed. The results are shown in FIG. 1.
The optimum pH levels for PEG-DCT-L-UK obtained in Example 4 and unmodified low molecular weight urokinase were determined similarly in terms of the amidase activity and we~e found to be 8.2 and 8.4, respectively. The results are shown in FIG. 2.
~2~7'71~
Example 20 Stabilit of modified urokinase at room Y
temperature PEG-DCT-UK obtained in Example 9 was diluted with either a Ringer solution or physiological saline to a concentration of 105.3 iu/ml. For the sake of comparison, unmodified urokinase was also diluted with either a Ringer solution or physiological saline to a concentration of 76.1 iu/ml. 3.5 ml of each of the thus diluted solutions was allowed to stand at room temperature (27~C) for 6 hours. The residual urokinase activity was periodically measured by the fibrin pla~e method until such time tha~
6 hours elapsed. The extent of activity loss of the modified urokinase according to the invention was by far smaller than that of unmodified urokinase. The results are shown in FIG. 3.
-Example 21 -Stability of modified urokinase against freezing and thawing processing PEG-DCT-L-UK obtained in Example 10 was diluted with physiological saline to a concentration of 2Q0 iu/ml.
For the sake of comparison, unmodified low molecular weight urokinase was diluted with physiological saline to a concentration of 200 iu/ml. Each of the thus diluted solutions was frozen to -80C and subsequently thawed at room temperature. The freezing and thawing operations were repeated 0, 2, 4 and 6 times. The residual urokinase 1;Z~'77;~
activity was measured using a synthetic substrate S-244~
(made by Kabi Corporation). The extent of activity loss of the modified urokinase according to the invention was extremely low as compared with that of unmodified low molecular weight urokinase. The results are shown in FIG. 4.
Example 22 Eight healthy male rabbits (Japanese white) each having a body weight of 2.6 - 3.1 kg were divided into two groups. One group was dosed with 8,000 iu/kg of PEG-DCT-UK and the other with 8,000 iu/kg of unmodified urokinase, both through the ear veins. Blood was collec-ted from the ear veins before the dosage and at the time of 1, 2 and 4 hours after the dosage. Blood samples W21-e added with a sodium citrate solution to separate plasma.
After wash-out of the modified and unmo~ified urokinases for 18 days, the two groups were crossed with each other, and similar experiments were carried out to take plasma samples. A part of each of the plasma samples thus obtained was separated and subjected to affinity column chromatography using Lysine Sepharose 4B, thereby obtain-ing a plasmin inhibitor fraction (fraction Fl) and a plasmin and plasminogen fraction (fraction F3). Chan~es in the amount of the plasmin inhibitor in the fraction Flalong the passage of time were determined by adding plasmin to the fraction Fl and measuring the amount of residual plasmin which was not adversely affected b~ the inhibitor, with a svnthe'ic ,tl 7-.lB
substrate S-2251 (made by Kabi Corporation). The plasmin inhibitor from the PEG-DCT-UK-administered group showed its apparent decrease and slower recovexy. In the unmodi-fied urokinase-administered group, the decrease in the plasmin inhibitor was rather less, and its time course changes were not made clear. The results are shown in FIG. 5.
On the other hand, the amount of plasminogen in the fraction F3 was determined by adding urokinase to the fraction F3 to produce plasmin and measuring the amount of the thus produced plasmin by a synthetic substrate S 2251. In the PEG-DCT-UK-administered group, plasminogen decreased to about 50% after 2 hours and thereafter re-covered gradually. However, in the unmodified urokinase-administered group, plasminogen decreased up to about 65~
after one hour but did not proceed ~to decrease any fur.her.
-There was observed a tendency of plasminogen being recov~
ered even 2 hours after the dosage. The results are illustrated in FIG. 6.
Furthermore, a part of each plasma was sampled ana treated with an acid to pH 5.2 so as to deactivate the inhibitor therein. It was then neutrali~ed, and the a~ount of plasminogen present in the acid treated plasma was measured by adding urokinase to the acid treated plasma to produce plasmin and measuring the amount of the thus produced plasmin using a synthetlc SuDstrate S-2251.
In the PEG-DCT-UK-administered gro~lp, plasminogen decreased to about 70% after 2 hours but showed its gradual recovery thereafter. In the unmodified urokinase-administered group, however, the amount of plasminogen was decreased to about 84% after one hour but was not decreased any further.
Thereafter, the amount of plasminogen was increased gra-dually. The results are also shown in FIG. 6. Incidental-ly, no plasmin activity was detected from the fraction F3.
Example 23 Lyophilized_product suitable for the preparation of injectable formulations The PEG-DCT-UK solution obtained in Example 9 was concentrated by a membrane filter (Amicon ~el: trademark~
and then added with physiological saline and a O.OS M
phosphate buffer. The resultant mixture was filtered aseptically using a membrane filter. The filtrate was poured in portions into sterilized vials and then lyophi-lized. The fibrinolytic activity of the lyophilized product of PEG-DCT-UK suitable for the preparation of injectable formulations was found to be 57,000 iu/vial by the fibrin plate method.
Example 24 L o hilized product suitable for tne preparation . Y P
of injectable formulations The PEG-DCT-UK solution obtained in Example 9 was concentrated by ~ membrane filter (~micon Gel: trademark) and then added with physiological saline. The resultant mixture was filtered aseptically using a membrane filter.
; 7 ~r~
- The filtrate was poured in portions into sterilized vials and then lyophilized. The fibrinolytic activity of ~he thus obtained lyophilized product of PEG-DCT-UK suitable for the preparation of injectable formulations was found to be 67,000 iu/vial by the fibrin plate method.
In the following experiments Urokinase (MW 54,000) was obtained from Japan Chemical Research Co., Ltd. PEG
#5,000 (MN = 4,700, MW/MN = l.08) was obtained from Nishio Industry Co., Ltd. Cyanuric chloride was obtained from Kanto Chemical Co., Inc. Fibrinogen and thrombin fcr fibrin plate was obtained from Sigma Chemical Co. and Mochida Pharmaceutical Co., Ltd.
respectively. S-2251 and S-2444 were obtained from Kabi Diagnostica. TNBS (trinitrobenzene sulfonic acid sodium salt) was obtained from Wako Pure Chemical Industry, Ltd. Lysine-Sepharose 4B and Sephadex G-200 were obtained from Pharmacia Fine Chemicals. Nal25I
was obtained from Amersham. GGA-MCA (L-glutaryl-glycyl-L-argir.ine-4-methylcoumarin-7-amide) was obtained from Protein Research Founda~ion.
Urokinase dissolved in O.lM phosphate buffer (P~), pH 7.0, was reacted with PEG #5,000 activated with cyanuric chloride by the method of A~uchowski et al at * trade mark.
3~2~i7~'~
4 for 4 hr. Concentration of PEG was adjusted to 1.7 mM for L-Md, 2 mM for M-Md and 5 mM for H-Md which represent low, medium and high degrees of modification, respectively. Dialysis against 0.05 m PB, pH 5.0, containing 0.9% ~aCl followed by concentration and gel filtration using Sephadex G-200 afforded modified UR's. After adjusting pH to 7.0, UK'5 were lyophilized in small portions for experimental convenience. Lysine residues in M-Md were determined with TNBS by the method of Habeeb. UK's were assayed either by the standard fibrin plate method or by the synthetic substrate method. Plasma was fractioned into Fl, F2 and F3 with Lysine-Sepharose 4B by the method of Igarashi et al. Native UK and M-Md were labelled with 12SI by the procedure of Hunter and Greenwood.
Immunological properties of native UK and M-Md were compared according to the procedures of Arai et al.
Example 2S - SUBSTRATE SPECIFICITY OF PEG-MODIFIED UK's Activation of PEG #5,000 with cyanuric chloride followed by reaction with native UK caused a covalent attachment of PEG's on the lysine residues of UK
protein. Three types of modified UK's, L-Md, M-Md and H-Md, were prepared by changing PEG concentration during the reaction. Their enzymatic activitie~ depend on the assay method which sugges~s that PEG chains ~Z~ 7~
presen~ steric hindrance to bulky substrat~s such as plasminogen in fibrin plate while not to the low molecular weight ones such as GGA-MCA.
Attention was specifically focused on M-Md and the pH Optimum of M-Md was found to be 8.16 and that of native UK to be 8.51 using S-2444 as a substrate in 0.05M Tris HCI buffer at 37C. The molecular weight (MW) of M-Md was calculated to be about 120,000 daltons from the result of lysine residue determination with TNBS. As PEG is a kind of neutral detergent the apparent MW obtained from SDS-PAGE of gel filtration does not reflect the true MW. FIGURE 8 illustrates the substrte specificity of PEG-Modified UK's.
Example 26 - EFFECT OF UK INHIBITORS ON THE ACTIVITY OF
~ATIVE UK AND L-MD.
It is known that in human plasma, protease inhibitors play an important role in retaining blood fluidity. Physiological activity of native UK when administered intraveneously is rapidly retarded not only by the interaction with these inhibitors but also by the fragmentation. The effect of UK inhibitors on the UK activity was examined using Fl, inhibitor fraction from human plasma, and placental UK
inhibitor. In the first experiment, U~s were incubated wieh the former at 37 then residual UK was assayed with S-2444. In the next experiment, placental UK
-:^
771~
inhibitor of different dilution was added to UKs then residual UK was assayed with a combination of plasminogen and S-2251.
In both cases, L-Md retained its activity better than native UK did. Neutral and chemically inert PEG
chains seem to protect UK from the action of UK
inhibitors. FIGURE 9 shows the stability of modified Urokinase to UK-Inhibitors in Human Plasma. ~IGURE 9b shows the effect of placental UK-Inhibitor on the Urokinase activity.
Example 27 - EXTENSION OF CIRCULATING LIVES OF UKs IN RABBITS
The short half-life (~/2) of native UK in the circulatory system often becomes a limitation of UK
therapy. As the ~20f native UK does not exceed several minutes, prolonged drop infusion is necessary to maintain the high plasmin level. Half lives of native and modified UKs were measured in rabbits by an injection of one of them followed by the determination of UK activity in the plasma drawn, periodically, from an ear vein.
As a re~ult, the half-life of native UK in the first phase was calculated as 4-5 minutes, L-Md as 30-40 minutes, M-Md as 80-100 minutes and H-Md as 110-150 minutes. ~20f modified UKs were extended about 10 to 30 times that of the native UK depending upon the 77~
respective degree of modification. In the case of M-Md or H-Md, a one compartment model is approximate enough to describe the pharmacokinetics, which suggests tha~
distribution to a second compartment is suppressed.
FIGURE 10 shows the UK activity of L-Md, M-Md and H-Md in Plasma after UK injection.
Example 28 - BEHAVIOR O~ 125I-NA'rIVE UK AND 125I-M-MD
IN RATS
Native UK and M-Md were labeled with 125I. Then, their ~2in the circulatory system and their distribution among organs were investigated in rats.
Extension of ~2f UK by PEG modification was also observed and was more prominent in these experiments.
Rapid decay of radioactivity in blood of rats injected with 125I-native UK was observed. On the contrary, radioactivity was retained well with 125I-M-Md. In the first phase, ~20f native UK was 1.1 min. while that of M-Md was 89.9 min.
Native UK administered intravenously accumulates in the liver and kidneys and other organs resulting in a short ~/2~ With M-Md, accumulation in these organs was suppressed probably due to less interaction of the M-Md molecule~ with organ cells because of the neutral and inert PEG chains on UK.
Tables 1 and 2 ~how the relative radioactivity in various tissues after 125I-native UK and 125I-M-Md ~L77~
injection, respectively. FIGURE 11 illustrates the relative radioactivity in Blood (~) for 125I-M-Md and 125I-native UK-RELATIVE RADIOACTIVITY AF~ER
125I-NATIVE UK INJ_CTIO~
Time Organ 5 min.10 min.20 min. 40 min.12 hr Brain 0.050.03 0.04 0.010.01 Lung 0.470.62 0.31 0.100.01 Thymus 0.030.02 0.03 0.030.01 Liver 27.8932.60 23.06 2.590.46 Heart 0.140.26 0.17 0.170.10 Kidney 3.686.82 2.40 0.400.25 Adrenal 0.05 -0.07 0.05 0.020.00 Spleen 0.39 1.32 0.67 0.050.02 RELATIVE RADIOACTIVITY AFTER
125I_M_MD INJECTION __ Time organ 5 min. 10 min.20 min~40 min.12 hr Brain 0.17 0.20 0.20 0.220.11 Lung 1.24 2.30 1.31 1.370.71 Thymus 0.15 0.21 0.15 0.240.11 Liver 6.84 8.7511.55 7.472.91 Heart 0.93 1.73 1.38 1.020.53 Kidney 2.21 3~10 3.83 3.270.68 Adrenal 0.06 0.07 0.08 0.060.04 Spleen 0.45 0.40 0.63 0.510.21 EXamP1e 29 - IMMUNOGENICITY AND TOXICITY OF M-MD
The changes in immunogenicity ~y chemical modification of proteins are of interest.
7~3 Immunogenicity of M-Md was compared with that of native UK by ASA reaction, Schultz-Dale test, PCA reaction and Ouchterlony method. Both native UK and M-Md were immunogenic for guinea pigs by ASA reaction after sensitization of them with the respective UK and FCA.
However, the immunological response to M-Md was weaker than that of native UK in the other three tests.
The results summarized in FIGURES 12a and 12b suggest that immunological determinants in M-Md are common with those in native UK. Furthermore, antigen production and/or reactivity with antigen are/is suppressed in M-Md compared with native UK. As native UK itself does not cause immunological response in man, M-Md is not expected to either.
The toxicity of M-Md was found to be extremely low. None of the mice died even after a shot of M-MD
at a dosage of 1,000,000 ~/kg nor afte~ repetitive injection of 500,000 ~/kg/day of M-Md.
FIGURE 12a shows a comparison of the immunogenicity of M-Md with that oE native UK by Schultz-Dale test~ FIGURE 12b shows a comparison of the immunogenicity of M-Md with that of native UK by Passive Cu~aneous Anaphylaxis (PCA) reaction.
In ~ummary, the instability of native UK has created a serious problem in formulating it for clinical purposes. The foregoing examples illustrate that covalent attachment of PEG chains to native UK
greatly increases resistance to inhibition of the UK by UK inhibitors. Moreover, covalent attachment of PEG
chains to native UK also prolonged ~2f the UK in the circulatory system. It seems likely that the PEG
chains may surround the UK molecule to form, in effect an inert capsule to protect the UK molecule from inhibitors and other proteases.
In the next set of examples attention was focused on _ vivo experiments using dogs, where the thrombolytic ability of PEG-modified UK (PEG-UK) was compared wlth that of native UK. In these examples, the PEG-UK used is the same as M-Md in Examples 25-29. The unit for UK activity was determined by the standard fibrin plate (F.P.) method. Plasmin was obtained from Sigma Chemical Co. FDP latex for dog FDP
was obtained from MBL Co., Ltd. Terufus~on~, a blood transfusion set type 1, obtained from Terumo Co., Ltd.
was connected with a three-way stopcock of type PX2-50 from Top Co., Ltd. using ATOM 6 Fr extension tubing.
Other materials used in the following examples are the same as used in Examples 25-29.
T~e haemostatic index for plasmin inhibitor, Fl, was determined by an addition of plasmin to the Fl fraction followed by the assay of residual pla~min with S-2251. F3, plasminogen, was determined with a ~Z~,t - 7 ~
combination of UK and S-2251. FDP was determined wi~h latex sensiti~ed with antigen to dog fibrinogen.
For the shun~ preparation, a dog was anaesthetized with ketamine hydrochloride then A. femoralis and V.
femoralis were exposed in the inguinal region. A
saline solution-filled shunt with the three-way stopcock was connected between them. Blood was allowed to run by opening the stopcock while monitoring the flow with a magnetic bloodflow meter. Either native UK
or PEG-UK dissolved in saline solution was administered from the stopcock. To maintain the anaesthesia, sodium barbiturate was injected.
For the preparation of artifical thrombus, A.
fermoralis of an anaesthetized dog was exposed by the same procedure. A transfusion catheter was fixed at a branch of A. femoralis, then the distal portion of the A. femoralis was clamped with two b~lldo~ clamps at a distance of 2 cm from each other and was emptied, then washed with saline solution. Dry oxygen was passed therethrough at a rate of 2 l/min for 10 minutes to injure intima. After injection of a fibrinogen solution followed by thrombin solution, the formed thrombus was allowed to age for 10 minutes. Then, the bulldog clamps were removed. Thrombus formation was confirmed angiographically by an injection of 10 ml of 60% Urografin~ from the catheter. UK dissolved in 16~
- ~o-saline was injected from the same catheter and angiographies were taken periodically thereafter.
Example 30 - CHANGES IN Fl, F3 AND FDP AFTER UK
INJECTION
Changes in the haemostatic indices are good indications of UK treatment. Often used are Fl (plasmin inhibi~or), F3 (plasminogen) and FDP (fibrin and/or fibrinogen degradation product). Native UK or PEG-UK was injected into a dog at the superficial vein of the fore leg at dosages shown in FIGURES 13a, b and c. Blood was sampled from the opposite side at certain time intervals and the Fl, F3 and FDP contents of the samples were determined.
A decrease in both Fl and F3 levels is a direct proof of plasminogen activation. The consumption of F
and F3 indicates that plasmin activated by UK from plasminogen (F3) was deactivated by its inhibitor (Fl) and was removed from the circulatory system. Another proof is a prominent increase in FDP. In a dog injected with PEG-UK, the FDP level was kept higher for several hours although its enæymatic activity to activate plasminogen was lower than that of native UK. These findings reveal clearly that chemical modification with PEG amplifies the physiological activity of UK.
7'~
FIGURE 13a illustrates a comparison of the Plasmin Inhibitor in Fl from Citrated Dog Plasma at certain time intervals after injection of native UK or PEG-UK
into a dog.
FIGURE 13b illustrates a comparison of the Plasminogen in F3 from Citrated Dog Plasma at certain time intervals after injection of native UK or PEG-UK
in~o a dog.
FIGURE 13c illustrates a comparison of the FDP in Dog Serum at certain time intervals after i.v.
injection of UK or PEG-UK into a dog.
Example 31 - THROMBOLYTIC EFFECT OF PEG-UK I~
EXPERIMENTAL THROMBOSIS
Thrombolytic activities of native UK and PEG-UK
were compared in dogs with experimental thrombosis. A
shunt having a fine filter was fixed between A.
femoralis and V. femoralis and then blood was run through it. After a complete stop of flow (several minutes) either native UK or PEG-UK was administered through the three-way stopcock at a dose of 10,000 ~/kg. As a result, the flow of blood in the dog with PEG-UK recovered while not in the dog having native UK administered even after repeated flushes of saline ~olution.
1' ;LI'i7.~3 -~2-Example 32 - INHIBITION OF THROMBUS FORMATION WITH
PEG-UK
The inhibitory effect of PEG-UK on the thrombus formation was investigated as compared with native UK
with a saline solution-filled shunt prepared by the same procedures de~cribed above. In this experiment either native UK or P~G-UK was administered prior to the blood circulation. The dose was 20,000 ~/kg in both cases. Results obtained are as follows:
1) In the dog without UK, the blood flow stopped within 5 minutes.
2) In the dog with native UK, the blood flow stopped within 5 minutes.
3) In the dog with PEG-UK, the blood flow continued during the experiment for >1 hour.
At this stage, the three filters were washed with saline. Complete inhibition of thrombus formation was observed in PEG-UK, which is probably due to the thrombin inhibition by FDP.
Example 33 - LYSIS OF ARTIFICIAL THROM~US WITH PEG-UK
USING ANGIOGRAM
The thombolytic effect of PEG-UK was evaluated in dogs bearing artifical thrombus in A. femoralis. After confirmation of thrombus formation, either native UK
(dog "A" ) or PEG-UK (dog "B") was injected from the transfusion catheter fixed on a proximal branch of the ~7~
thrombus at a dose of 10,00~ ~/kg. Angiographies were taken periodically to observe ~he recovery of blood flow. It is concluded from these angiographies that PEG-UK dissolved the artifical ~hrombus formed in A.
femoralis while native UK was unable to do so. FIGURE
14 illustrates a comparison of FDP levels in dogs bearing artifical thrombus in A. femoralis. Dog A had native UK injected into a proximal branch of the thrombus, while dog B had PEG-UK injected into the same.
Example 34 - INHIBITORY EFFECT ON THROMBUS FORMATION
Using a dog, a shunt was established under anesthesia by a blood transfu~ion filter ("Terufusion"
blood transfusion Set Type-l) between A. femoralis and V. femoralis and a blood flowmeter ("MF-26", rectangular wave electromagnetic bl~od f~-owmeter manufactured by Nippon Koden K.K.) was connected at the vein side. The filter had in advance been filled with physiological saline and, after administration of a drug, the blood was caused to flow through the shunt by opening a three-way stopcock. Without administration of the drug, thrombus was formed in the filter within 4-5 minutes after the blood had started flowing through the filter, whereby any further flow of the blood ~hrough the shunt was preven~ed. The time period until 1~ o 7 ~
the stoppage of the blood flow due to the formation of thrombus was not prolonged at all even when 200,000 units of unmodified urokinase were administered. On the contrary, thrombus was not formed nor was the blood flow rate reduced at all even after 40 minutes after the initiation of the blood flow through the shunt when PEG-DCT-UK, which pertains to the present invention, had been administered.
In summary, Examples 30-34 dealt with the thrombolysis by PEG-UK wherein the thrombolytic ability thereof was compared with that of native UK using two thrombosis models. The superiority of PEG-UK to native UK with respect to fibrinolytic activation is due to the PEG chains which appear to protect the UK molecule from deactivating interactions with inhibitors. This protection serves to extend the UK circulating life.
Having fully described the pre5ent-~nvention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.
Claims (5)
PROPERTY OF PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS.
1. A method of extending the circulating life of a plasminogen activator in a mammalian bloodstream which comprises chemically modifying said plasminogen activator by bonding at least one polyalkylene glycol moiety there-to by means of a coupling agent to the amino acid side chains of said plasminogen activator.
2. The method of Claim 1, wherein said plasminogen activator is urokinase.
3. The method of Claim 1, wherein said mammalian bloodstream is a human bloodstream.
4. The method of Claim 1, wherein said polyalkylene glycol moiety is a methoxypolyethylene glycol moiety.
5. The method of Claim 4, wherein said methoxy-polyethylene glycol has a molecular weight of about 5,000.
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56172908A JPS5896026A (en) | 1981-10-30 | 1981-10-30 | Novel urokinase derivative, its preparation and thrombolytic agent containing the same |
BR8206347A BR8206347A (en) | 1981-10-30 | 1982-10-27 | PROCESS TO PRODUCE PLASMINOGEN ACTIVATING DERIVATIVES |
US06/437,009 US4495285A (en) | 1981-10-30 | 1982-10-27 | Plasminogen activator derivatives |
CH6304/82A CH658669A5 (en) | 1981-10-30 | 1982-10-28 | DERIVATIVES OF PLASMINOGEN ACTIVATORS. |
DE19823240174 DE3240174A1 (en) | 1981-10-30 | 1982-10-29 | Stable plasminogen activator derivs. - contg. poly:alkylene glycol linked to side chains |
FR8218223A FR2515684B1 (en) | 1981-10-30 | 1982-10-29 | NEW DERIVATIVE OF A NON-IMMUNOGENIC PLASMINOGEN ACTIVATOR OF HUMAN ORIGIN |
CA000414556A CA1203764A (en) | 1981-10-30 | 1982-10-29 | Plasminogen activator derivatives |
GB08230987A GB2110219B (en) | 1981-10-30 | 1982-10-29 | Plasminogen activator derivatives |
SE8206173A SE457800B (en) | 1981-10-30 | 1982-10-29 | PLASMINOGENAKTIVATORDERIVAT |
US06/546,590 US4640835A (en) | 1981-10-30 | 1983-10-28 | Plasminogen activator derivatives |
CA000443230A CA1217718A (en) | 1981-10-30 | 1983-12-14 | Plasminogen activator derivatives |
Applications Claiming Priority (3)
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JP56172908A JPS5896026A (en) | 1981-10-30 | 1981-10-30 | Novel urokinase derivative, its preparation and thrombolytic agent containing the same |
US06/546,590 US4640835A (en) | 1981-10-30 | 1983-10-28 | Plasminogen activator derivatives |
CA000443230A CA1217718A (en) | 1981-10-30 | 1983-12-14 | Plasminogen activator derivatives |
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CA000443230A Expired CA1217718A (en) | 1981-10-30 | 1983-12-14 | Plasminogen activator derivatives |
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GB8430252D0 (en) * | 1984-11-30 | 1985-01-09 | Beecham Group Plc | Compounds |
US4791192A (en) * | 1986-06-26 | 1988-12-13 | Takeda Chemical Industries, Ltd. | Chemically modified protein with polyethyleneglycol |
US5045190A (en) * | 1988-11-08 | 1991-09-03 | Carbonell Ruben G | Chromatography apparatus |
US5166322A (en) * | 1989-04-21 | 1992-11-24 | Genetics Institute | Cysteine added variants of interleukin-3 and chemical modifications thereof |
JPH05501855A (en) * | 1989-05-17 | 1993-04-08 | リサーチ、コーポレーション、テクノロジーズ、インコーポレーテッド | Methods and compositions for treating thrombosis in mammals |
FR2661920B1 (en) * | 1990-05-10 | 1995-10-06 | Serbio | METHOD FOR DETERMINING A PLASMINOGEN ACTIVATOR AND ITS INHIBITOR. |
US5595732A (en) * | 1991-03-25 | 1997-01-21 | Hoffmann-La Roche Inc. | Polyethylene-protein conjugates |
US5272076A (en) * | 1991-06-13 | 1993-12-21 | Eli Lilly And Company | Compounds and methods for treatment of thromboembolic disorders using an adduct of t-PA |
US5382657A (en) * | 1992-08-26 | 1995-01-17 | Hoffmann-La Roche Inc. | Peg-interferon conjugates |
US5877016A (en) | 1994-03-18 | 1999-03-02 | Genentech, Inc. | Human trk receptors and neurotrophic factor inhibitors |
US6458762B1 (en) | 1994-03-28 | 2002-10-01 | Baxter International, Inc. | Therapeutic use of hemoglobin for preserving tissue viability and reducing restenosis |
US5795569A (en) * | 1994-03-31 | 1998-08-18 | Amgen Inc. | Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation |
HU218893B (en) * | 1994-03-31 | 2000-12-28 | Amgen Inc. | Water soluble compositions and methods for stimulating megakaryocyte growth and differentiation |
US5708142A (en) | 1994-05-27 | 1998-01-13 | Genentech, Inc. | Tumor necrosis factor receptor-associated factors |
DE4423131A1 (en) * | 1994-07-01 | 1996-01-04 | Bayer Ag | New hIL-4 mutant proteins as antagonists or partial agonists of human interleukin 4 |
MX9704137A (en) * | 1994-12-07 | 1997-09-30 | Novo Nordisk As | Polypeptide with reduced allergenicity. |
EA001220B1 (en) | 1995-06-07 | 2000-12-25 | Глаксо Груп Лимитед | Peptides or peptidemimetics bound to thrombopoietin receptor, pharmaceutical composition and method of treatment |
US5869451A (en) | 1995-06-07 | 1999-02-09 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
BR9510676A (en) * | 1995-12-29 | 1999-11-23 | Procter & Gamble | Detergent compositions comprising immobilized enzymes |
US6998116B1 (en) * | 1996-01-09 | 2006-02-14 | Genentech, Inc. | Apo-2 ligand |
US6046048A (en) * | 1996-01-09 | 2000-04-04 | Genetech, Inc. | Apo-2 ligand |
US6030945A (en) * | 1996-01-09 | 2000-02-29 | Genentech, Inc. | Apo-2 ligand |
US20050089958A1 (en) * | 1996-01-09 | 2005-04-28 | Genentech, Inc. | Apo-2 ligand |
US20020165157A1 (en) * | 1996-04-01 | 2002-11-07 | Genentech, Inc. | Apo-2LI and Apo-3 polypeptides |
US6469144B1 (en) | 1996-04-01 | 2002-10-22 | Genentech, Inc. | Apo-2LI and Apo-3 polypeptides |
US7091311B2 (en) * | 1996-06-07 | 2006-08-15 | Smithkline Beecham Corporation | Peptides and compounds that bind to a receptor |
US6159462A (en) * | 1996-08-16 | 2000-12-12 | Genentech, Inc. | Uses of Wnt polypeptides |
US5851984A (en) * | 1996-08-16 | 1998-12-22 | Genentech, Inc. | Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides |
US6462176B1 (en) * | 1996-09-23 | 2002-10-08 | Genentech, Inc. | Apo-3 polypeptide |
AU725133B2 (en) * | 1997-01-15 | 2000-10-05 | Polaris Group | Modified tumor necrosis factor |
ATE356212T1 (en) | 1997-01-31 | 2007-03-15 | Genentech Inc | O-FUKOSYL TRANSFERASE |
US20020102706A1 (en) * | 1997-06-18 | 2002-08-01 | Genentech, Inc. | Apo-2DcR |
ES2236634T3 (en) | 1997-04-07 | 2005-07-16 | Genentech, Inc. | ANTI-VEGF ANTIBODIES. |
KR100794454B1 (en) | 1997-04-07 | 2008-01-16 | 제넨테크, 인크. | Anti-VEGF Antibodies |
US20100152426A1 (en) * | 1997-05-15 | 2010-06-17 | Ashkenazi Avi J | Apo-2 receptor fusion proteins |
US6342369B1 (en) * | 1997-05-15 | 2002-01-29 | Genentech, Inc. | Apo-2-receptor |
JP2001511653A (en) | 1997-05-15 | 2001-08-14 | ジェネンテク,インコーポレイテッド | Apo-2 receptor |
CA2293724C (en) | 1997-06-05 | 2010-02-02 | Xiaodong Wang | Apaf-1, the ced-4 human homolog, an activator of caspase-3 |
EP2083079A1 (en) * | 1997-06-18 | 2009-07-29 | Genentech, Inc. | Apo-2DcR |
AU751880B2 (en) * | 1997-06-25 | 2002-08-29 | Novozymes A/S | A modified polypeptide |
US6342220B1 (en) | 1997-08-25 | 2002-01-29 | Genentech, Inc. | Agonist antibodies |
US20030175856A1 (en) * | 1997-08-26 | 2003-09-18 | Genetech, Inc. | Rtd receptor |
DK1009817T3 (en) * | 1997-08-26 | 2010-01-18 | Genentech Inc | RTD receptor |
CA2382506A1 (en) | 1997-09-17 | 1999-03-25 | Genentech, Inc. | Novel polypeptides and nucleic acids encoding pro293 which are useful for treating disorders of the pancreas |
US20040231011A1 (en) * | 2001-06-28 | 2004-11-18 | Genentech, Inc. | DcR3 polypeptide, a TNFR homolog |
WO1999014330A1 (en) | 1997-09-18 | 1999-03-25 | Genentech, Inc. | DcR3 POLYPEPTIDE, A TNFR HOMOLOG |
IL135051A0 (en) | 1997-10-10 | 2001-05-20 | Genentech Inc | Apo-3 ligand polypeptide |
EP2033970A3 (en) | 1997-10-29 | 2009-06-17 | Genentech, Inc. | Polypeptides and nucleic acids encoding the same |
US6387657B1 (en) | 1997-10-29 | 2002-05-14 | Genentech, Inc. | WISP polypeptides and nucleic acids encoding same |
US7192589B2 (en) | 1998-09-16 | 2007-03-20 | Genentech, Inc. | Treatment of inflammatory disorders with STIgMA immunoadhesins |
EP2014677A1 (en) | 1997-11-21 | 2009-01-14 | Genentech, Inc. | A-33 related antigens and their pharmacological uses |
DK1045906T3 (en) | 1998-01-15 | 2009-02-16 | Genentech Inc | APO-2 ligand |
NZ525914A (en) | 1998-03-10 | 2004-03-26 | Genentech Inc | Novel polypeptides and nucleic acids encoding the same |
ES2389387T3 (en) | 1998-03-17 | 2012-10-25 | Genentech, Inc. | Homologous VEGF and BMP1 polypeptides |
EP1075511A1 (en) * | 1998-05-08 | 2001-02-14 | University Of Southern California | Size enhanced fibrinolytic enzymes |
EP3112468A1 (en) | 1998-05-15 | 2017-01-04 | Genentech, Inc. | Il-17 homologous polypeptides and therapeutic uses thereof |
EP2333069A3 (en) | 1998-05-15 | 2011-09-14 | Genentech, Inc. | Therapeutic uses of IL-17 homologous polypeptides |
EP1865061A3 (en) | 1998-05-15 | 2007-12-19 | Genentech, Inc. | IL-17 homologous polypeptides and therapeutic uses thereof |
ES2395693T3 (en) * | 1998-06-12 | 2013-02-14 | Genentech, Inc. | Monoclonal antibodies, cross-reactive antibodies and process for manufacturing them |
US20020172678A1 (en) | 2000-06-23 | 2002-11-21 | Napoleone Ferrara | EG-VEGF nucleic acids and polypeptides and methods of use |
DE69934425T2 (en) | 1998-10-23 | 2007-09-27 | Amgen Inc., Thousand Oaks | THROMBOPOIETIN SUBSTITUTE |
SI1135498T1 (en) | 1998-11-18 | 2008-06-30 | Genentech Inc | Antibody variants with higher binding affinity compared to parent antibodies |
AUPP785098A0 (en) * | 1998-12-21 | 1999-01-21 | Victor Chang Cardiac Research Institute, The | Treatment of heart disease |
EP2075335A3 (en) | 1998-12-22 | 2009-09-30 | Genentech, Inc. | Methods and compositions for inhibiting neoplastic cell growth |
JP5456222B2 (en) | 1998-12-23 | 2014-03-26 | ジェネンテック, インコーポレイテッド | IL-1 related polypeptides |
EP1792989A1 (en) | 1999-04-12 | 2007-06-06 | Agensys, Inc. | 13 Transmembrane protein expressed in prostate cancer |
CA2369413C (en) | 1999-04-12 | 2013-07-09 | Agensys, Inc. | Transmembrane protein expressed in prostate and other cancers |
US6635249B1 (en) | 1999-04-23 | 2003-10-21 | Cenes Pharmaceuticals, Inc. | Methods for treating congestive heart failure |
AU5152700A (en) | 1999-06-15 | 2001-01-02 | Genentech Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
EP1200590B1 (en) | 1999-08-12 | 2009-01-07 | Agensys, Inc. | C-type lectin transmembrane antigen expressed in human prostate cancer and uses thereof |
US7459540B1 (en) * | 1999-09-07 | 2008-12-02 | Amgen Inc. | Fibroblast growth factor-like polypeptides |
WO2001025434A1 (en) | 1999-10-05 | 2001-04-12 | Agensys, Inc. | G protein-coupled receptor up-regulated in prostate cancer and uses thereof |
US7332275B2 (en) | 1999-10-13 | 2008-02-19 | Sequenom, Inc. | Methods for detecting methylated nucleotides |
US6893818B1 (en) * | 1999-10-28 | 2005-05-17 | Agensys, Inc. | Gene upregulated in cancers of the prostate |
KR20020056923A (en) | 1999-11-18 | 2002-07-10 | 코르바스 인터내셔날, 인코포레이티드 | Nucleic acids encoding endotheliases, endotheliases and uses thereof |
US6703480B1 (en) | 1999-11-24 | 2004-03-09 | Palani Balu | Peptide dimers as agonists of the erythropoientin (EPO) receptor, and associated methods of synthesis and use |
CA2490853A1 (en) | 1999-12-01 | 2001-06-07 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US7109299B1 (en) | 1999-12-16 | 2006-09-19 | Affymax, Inc. | Peptides and compounds that bind to the IL-5 receptor |
DK1897945T3 (en) | 1999-12-23 | 2012-05-07 | Genentech Inc | IL-17 homologous polypeptides and therapeutic uses thereof. |
ATE424457T1 (en) * | 2000-01-13 | 2009-03-15 | Genentech Inc | HUMAN STRA6 POLYPEPTIDES |
US7700341B2 (en) * | 2000-02-03 | 2010-04-20 | Dendreon Corporation | Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon |
DK1255558T3 (en) | 2000-02-16 | 2006-10-23 | Genentech Inc | Anti-April antibodies and hybridoma cells |
US7101974B2 (en) | 2000-03-02 | 2006-09-05 | Xencor | TNF-αvariants |
US6740520B2 (en) | 2000-03-21 | 2004-05-25 | Genentech, Inc. | Cytokine receptor and nucleic acids encoding the same |
US20040086970A1 (en) * | 2000-03-22 | 2004-05-06 | Genentech, Inc. | Novel cytokine receptors and nucleic acids encoding the same |
US6667300B2 (en) | 2000-04-25 | 2003-12-23 | Icos Corporation | Inhibitors of human phosphatidylinositol 3-kinase delta |
DK2042597T3 (en) | 2000-06-23 | 2014-08-11 | Genentech Inc | COMPOSITIONS AND PROCEDURES FOR DIAGNOSIS AND TREATMENT OF DISEASES INVOLVING ANGIOGENESIS |
CA2709771A1 (en) | 2000-06-23 | 2002-01-03 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
FR2811323B1 (en) * | 2000-07-07 | 2006-10-06 | Fuma Tech Gmbh | HYBRID MATERIAL, USE OF SAID HYBRID MATERIAL, AND METHOD OF MANUFACTURING THE SAME |
DE60136281D1 (en) | 2000-08-24 | 2008-12-04 | Genentech Inc | METHOD FOR INHIBITING IL-22-INDUCED PAP1 |
PT1313850E (en) | 2000-08-28 | 2008-11-18 | Agensys Inc | Nucleic acid and corresponding protein entitled 85p1b3 useful in treatment and detection of cancer |
EP1944317A3 (en) | 2000-09-01 | 2008-09-17 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US20030044803A1 (en) * | 2000-09-22 | 2003-03-06 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of JAK1 |
US20020164576A1 (en) * | 2000-09-22 | 2002-11-07 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of Nrf2 |
US20020115058A1 (en) * | 2000-09-22 | 2002-08-22 | Pedersen Finn Skou | Methods for diagnosis and treatment of diseases associated with altered expression of Pik3r1 |
US6576452B1 (en) * | 2000-10-04 | 2003-06-10 | Genencor International, Inc. | 2,5-diketo-L-gluconic acid reductases and methods of use |
US6673580B2 (en) * | 2000-10-27 | 2004-01-06 | Genentech, Inc. | Identification and modification of immunodominant epitopes in polypeptides |
EP1355671A2 (en) * | 2000-11-30 | 2003-10-29 | Nektar Therapeutics Al, Corporation | Water-soluble polymer conjugates of triazine derivatives |
US20030165878A1 (en) * | 2000-12-22 | 2003-09-04 | Morris David W. | Novel compositions and methods in cancer associated with altered expression of MCM3AP |
US7700274B2 (en) * | 2000-12-22 | 2010-04-20 | Sagres Discovery, Inc. | Compositions and methods in cancer associated with altered expression of KCNJ9 |
US20030087252A1 (en) * | 2000-12-22 | 2003-05-08 | Morris David W. | Novel compositions and methods in cancer associated with altered expression of PRDM11 |
US20030099963A1 (en) * | 2000-12-22 | 2003-05-29 | Morris David W. | Novel compositions and methods in cancer associated with altered expression of TBX21 |
US7820447B2 (en) | 2000-12-22 | 2010-10-26 | Sagres Discovery Inc. | Compositions and methods for cancer |
US7892730B2 (en) * | 2000-12-22 | 2011-02-22 | Sagres Discovery, Inc. | Compositions and methods for cancer |
US7645441B2 (en) | 2000-12-22 | 2010-01-12 | Sagres Discovery Inc. | Compositions and methods in cancer associated with altered expression of PRLR |
US20030232334A1 (en) | 2000-12-22 | 2003-12-18 | Morris David W. | Novel compositions and methods for cancer |
US7754208B2 (en) | 2001-01-17 | 2010-07-13 | Trubion Pharmaceuticals, Inc. | Binding domain-immunoglobulin fusion proteins |
US7829084B2 (en) * | 2001-01-17 | 2010-11-09 | Trubion Pharmaceuticals, Inc. | Binding constructs and methods for use thereof |
US20020123068A1 (en) * | 2001-01-31 | 2002-09-05 | Dwyer Brian P. | Water-soluble, fluorescent, & electrophoretically mobile peptidic substrates for enzymatic reactions and methods for their use in high-throughput screening assays |
US7087726B2 (en) | 2001-02-22 | 2006-08-08 | Genentech, Inc. | Anti-interferon-α antibodies |
US6924358B2 (en) | 2001-03-05 | 2005-08-02 | Agensys, Inc. | 121P1F1: a tissue specific protein highly expressed in various cancers |
US7125703B2 (en) | 2001-03-13 | 2006-10-24 | Dendreon Corporation | Nucleic acid molecules encoding a transmembrane serine protease 7, the encoded polypeptides and methods based thereon |
US7271240B2 (en) | 2001-03-14 | 2007-09-18 | Agensys, Inc. | 125P5C8: a tissue specific protein highly expressed in various cancers |
KR20040011480A (en) | 2001-03-22 | 2004-02-05 | 덴드레온 샌 디에고 엘엘씨 | Nucleic acid molecules encoding serine protease CVSP14, the encoded polypeptides and methods based thereon |
NZ527971A (en) | 2001-03-27 | 2006-03-31 | Dendreon Corp | Nucleic acid molecules encoding a transmembrane serine protease 9, the encoded polypeptides and methods based thereon |
WO2002083921A2 (en) | 2001-04-10 | 2002-10-24 | Agensys, Inc. | Nuleic acids and corresponding proteins useful in the detection and treatment of various cancers |
US20030191073A1 (en) | 2001-11-07 | 2003-10-09 | Challita-Eid Pia M. | Nucleic acid and corresponding protein entitled 161P2F10B useful in treatment and detection of cancer |
JP2005506832A (en) | 2001-05-14 | 2005-03-10 | デンドレオン・サンディエゴ・リミテッド・ライアビリティ・カンパニー | Nucleic acid molecule encoding transmembrane serine protease 10, encoded polypeptide and method based thereon |
US20070160576A1 (en) | 2001-06-05 | 2007-07-12 | Genentech, Inc. | IL-17A/F heterologous polypeptides and therapeutic uses thereof |
US20060270003A1 (en) | 2003-07-08 | 2006-11-30 | Genentech, Inc. | IL-17A/F heterologous polypeptides and therapeutic uses thereof |
CA2633171C (en) | 2001-06-20 | 2012-11-20 | Genentech, Inc. | Antibodies against tumor-associated antigenic target (tat) polypeptides |
JP2005510208A (en) * | 2001-08-03 | 2005-04-21 | ジェネンテック・インコーポレーテッド | TACIs and BR3 polypeptides and uses thereof |
HU230373B1 (en) | 2001-08-29 | 2016-03-29 | Genentech Inc | Bv8 nucleic acids and polypeptides with mitogenic activity |
US20040235068A1 (en) * | 2001-09-05 | 2004-11-25 | Levinson Arthur D. | Methods for the identification of polypeptide antigens associated with disorders involving aberrant cell proliferation and compositions useful for the treatment of such disorders |
ES2537074T3 (en) | 2001-09-06 | 2015-06-02 | Agensys, Inc. | Nucleic acid and corresponding protein called STEAP-1 useful in the treatment and detection of cancer |
NZ573831A (en) | 2001-09-18 | 2010-07-30 | Genentech Inc | Compositions and methods for the diagnosis and treatment of tumor, particularly breast tumor - TAT193 |
US20070098728A1 (en) * | 2001-09-24 | 2007-05-03 | Pedersen Finn S | Novel compositions and methods in cancer |
US7320789B2 (en) | 2001-09-26 | 2008-01-22 | Wyeth | Antibody inhibitors of GDF-8 and uses thereof |
US7138370B2 (en) | 2001-10-11 | 2006-11-21 | Amgen Inc. | Specific binding agents of human angiopoietin-2 |
US7521053B2 (en) | 2001-10-11 | 2009-04-21 | Amgen Inc. | Angiopoietin-2 specific binding agents |
US20040126762A1 (en) * | 2002-12-17 | 2004-07-01 | Morris David W. | Novel compositions and methods in cancer |
US20040166490A1 (en) * | 2002-12-17 | 2004-08-26 | Morris David W. | Novel therapeutic targets in cancer |
WO2003044179A2 (en) * | 2001-11-20 | 2003-05-30 | Dendreon San Diego Llc | Nucleic acid molecules encoding serine protease 17, the encoded polypeptides and methods based thereon |
US20040180344A1 (en) * | 2003-03-14 | 2004-09-16 | Morris David W. | Novel therapeutic targets in cancer |
US20060040262A1 (en) * | 2002-12-27 | 2006-02-23 | Morris David W | Novel compositions and methods in cancer |
US20040197778A1 (en) * | 2002-12-26 | 2004-10-07 | Sagres Discovery, Inc. | Novel compositions and methods in cancer |
WO2003050276A1 (en) * | 2001-12-05 | 2003-06-19 | Dow Global Technologies Inc. | Method for immobilizing a biologic in a polyurethane-hydrogel composition, a composition prepared from the method, and biomedical applications |
NZ533933A (en) | 2002-01-02 | 2008-06-30 | Genentech Inc | Compositions and methods for the diagnosis and treatment of glioma tumor |
KR20040096592A (en) | 2002-02-21 | 2004-11-16 | 와이어쓰 | Follistatin domain containing proteins |
EP1575480A4 (en) | 2002-02-22 | 2008-08-06 | Genentech Inc | Compositions and methods for the treatment of immune related diseases |
US7303896B2 (en) | 2002-02-25 | 2007-12-04 | Genentech, Inc. | Nucleic acid encoding novel type-1 cytokine receptor GLM-R |
US20100311954A1 (en) * | 2002-03-01 | 2010-12-09 | Xencor, Inc. | Optimized Proteins that Target Ep-CAM |
US20070122406A1 (en) | 2005-07-08 | 2007-05-31 | Xencor, Inc. | Optimized proteins that target Ep-CAM |
US20040023267A1 (en) * | 2002-03-21 | 2004-02-05 | Morris David W. | Novel compositions and methods in cancer |
CA2481507A1 (en) | 2002-04-16 | 2003-10-30 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
CA2484676A1 (en) * | 2002-05-03 | 2003-11-13 | Sequenom, Inc. | Kinase anchor protein muteins, peptides thereof, and related methods |
US7351542B2 (en) | 2002-05-20 | 2008-04-01 | The Regents Of The University Of California | Methods of modulating tubulin deacetylase activity |
US20040001801A1 (en) * | 2002-05-23 | 2004-01-01 | Corvas International, Inc. | Conjugates activated by cell surface proteases and therapeutic uses thereof |
WO2003099320A1 (en) | 2002-05-24 | 2003-12-04 | Zensun (Shanghai) Sci-Tech.Ltd | Neuregulin based methods and compositions for treating viral myocarditis and dilated cardiomyopathy |
EP2305710A3 (en) | 2002-06-03 | 2013-05-29 | Genentech, Inc. | Synthetic antibody phage libraries |
AU2003243400B2 (en) * | 2002-06-07 | 2009-10-29 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
CA2489588A1 (en) | 2002-07-08 | 2004-01-15 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
WO2004011611A2 (en) * | 2002-07-25 | 2004-02-05 | Genentech, Inc. | Taci antibodies and uses thereof |
MXPA05001364A (en) * | 2002-08-02 | 2005-08-03 | Wyeth Corp | Mk2 interacting proteins. |
EP1546316B1 (en) * | 2002-08-12 | 2010-12-01 | Danisco US Inc. | Mutant e. coli appa phytase enzymes and natural variants thereof, nucleic acids encoding such phytase enzymes, vectors and host cells incorporating same and methods of making and using same |
WO2004016733A2 (en) | 2002-08-16 | 2004-02-26 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 251p5g2 useful in treatment and detection of cancer |
AU2003265361A1 (en) * | 2002-08-28 | 2004-03-19 | Pharmacia Corporation | Stable ph optimized formulation of a modified antibody |
WO2004019860A2 (en) * | 2002-08-28 | 2004-03-11 | Pharmacia Corporation | Formulations of modified antibodies and methods of making the same |
AU2003276874B2 (en) | 2002-09-11 | 2009-09-03 | Genentech, Inc. | Novel compositions and methods for the treatment of immune related diseases |
EP2277532A1 (en) | 2002-09-11 | 2011-01-26 | Genentech, Inc. | Novel composition and methods for the treatment of immune related diseases |
CA2498274A1 (en) | 2002-09-16 | 2004-03-25 | Genentech, Inc. | Compositions and methods for the diagnosis of immune related diseases using pro7 |
BR0314591A (en) | 2002-09-18 | 2005-08-09 | Ortho Mcneil Pharm Inc | Platelet augmentation methods and hematopoietic stem cell production |
EP1585482A4 (en) | 2002-09-25 | 2009-09-09 | Genentech Inc | Nouvelles compositions et methodes de traitement du psoriasis |
US20040149235A1 (en) * | 2002-10-04 | 2004-08-05 | Pogue Albert S. | Apparatus and method for removal of waste from animal production facilities |
WO2004039956A2 (en) | 2002-10-29 | 2004-05-13 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
WO2004110345A2 (en) * | 2002-10-29 | 2004-12-23 | Pharmacia Corporation | Differentially expressed genes involved in cancer, the polypeptides encoded thereby, and methods of using the same |
JP2006516094A (en) | 2002-11-08 | 2006-06-22 | ジェネンテック・インコーポレーテッド | Compositions and methods for treatment of natural killer cell related diseases |
EP2308968A1 (en) | 2002-11-26 | 2011-04-13 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
AU2002352976B2 (en) | 2002-11-27 | 2007-11-08 | Agensys, Inc. | Nucleic acid corresponding protein entitled 24P4C12 useful in treatment and detection of cancer |
US7553930B2 (en) * | 2003-01-06 | 2009-06-30 | Xencor, Inc. | BAFF variants and methods thereof |
US20050130892A1 (en) * | 2003-03-07 | 2005-06-16 | Xencor, Inc. | BAFF variants and methods thereof |
US20060014248A1 (en) * | 2003-01-06 | 2006-01-19 | Xencor, Inc. | TNF super family members with altered immunogenicity |
US20050221443A1 (en) * | 2003-01-06 | 2005-10-06 | Xencor, Inc. | Tumor necrosis factor super family agonists |
EP1581904A2 (en) * | 2003-01-08 | 2005-10-05 | Xencor, Inc. | Novel proteins with altered immunogenicity |
CA2860151A1 (en) | 2003-02-10 | 2004-08-26 | Agensys, Inc. | Nucleic acid and corresponding protein named 158p1d7 useful in the treatment and detection of bladder and other cancers |
EP1592708A2 (en) | 2003-02-14 | 2005-11-09 | Sagres Discovery, Inc. | Therapeutic gpcr targets in cancer |
US20070218071A1 (en) * | 2003-09-15 | 2007-09-20 | Morris David W | Novel therapeutic targets in cancer |
US20040170982A1 (en) | 2003-02-14 | 2004-09-02 | Morris David W. | Novel therapeutic targets in cancer |
US7767387B2 (en) * | 2003-06-13 | 2010-08-03 | Sagres Discovery, Inc. | Therapeutic targets in cancer |
KR101118340B1 (en) | 2003-03-12 | 2012-04-12 | 제넨테크, 인크. | Use of bv8 and/or eg-vegf to promote hematopoiesis |
JP4914209B2 (en) * | 2003-03-14 | 2012-04-11 | ワイス | Antibody against human IL-21 receptor and use of the antibody |
MXPA05010411A (en) * | 2003-03-28 | 2006-05-31 | Biopolymed Inc | Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same. |
US20050025763A1 (en) | 2003-05-08 | 2005-02-03 | Protein Design Laboratories, Inc. | Therapeutic use of anti-CS1 antibodies |
US7709610B2 (en) | 2003-05-08 | 2010-05-04 | Facet Biotech Corporation | Therapeutic use of anti-CS1 antibodies |
PT1629088E (en) | 2003-05-30 | 2012-04-10 | Agensys Inc | Prostate stem cell antigen (psca) variants and subsequences thereof |
UA101945C2 (en) | 2003-05-30 | 2013-05-27 | Дженентек, Инк. | Treatment of cancer using bevacizumab |
PT1631313E (en) * | 2003-06-05 | 2015-07-02 | Genentech Inc | Combination therapy for b cell disorders |
US20050163775A1 (en) * | 2003-06-05 | 2005-07-28 | Genentech, Inc. | Combination therapy for B cell disorders |
US7939058B2 (en) | 2003-07-03 | 2011-05-10 | University Of Southern California | Uses of IL-12 in hematopoiesis |
AR046071A1 (en) | 2003-07-10 | 2005-11-23 | Hoffmann La Roche | ANTIBODIES AGAINST RECEIVER I OF THE INSULINAL TYPE GROWTH FACTOR AND THE USES OF THE SAME |
KR20110129988A (en) | 2003-07-18 | 2011-12-02 | 암젠 인코포레이티드 | Specific binding agents to hepatocyte growth factor |
WO2005019258A2 (en) | 2003-08-11 | 2005-03-03 | Genentech, Inc. | Compositions and methods for the treatment of immune related diseases |
WO2005016349A1 (en) * | 2003-08-14 | 2005-02-24 | Icos Corporation | Methods of inhibiting leukocyte accumulation |
US20050043239A1 (en) * | 2003-08-14 | 2005-02-24 | Jason Douangpanya | Methods of inhibiting immune responses stimulated by an endogenous factor |
AR045614A1 (en) * | 2003-09-10 | 2005-11-02 | Hoffmann La Roche | ANTIBODIES AGAINST THE RECEIVER OF INTERLEUQUINA- 1 AND USES OF THE SAME |
US8883147B2 (en) | 2004-10-21 | 2014-11-11 | Xencor, Inc. | Immunoglobulins insertions, deletions, and substitutions |
US8399618B2 (en) | 2004-10-21 | 2013-03-19 | Xencor, Inc. | Immunoglobulin insertions, deletions, and substitutions |
US20060134105A1 (en) * | 2004-10-21 | 2006-06-22 | Xencor, Inc. | IgG immunoglobulin variants with optimized effector function |
US20070281896A1 (en) * | 2003-09-30 | 2007-12-06 | Morris David W | Novel compositions and methods in cancer |
WO2005035569A2 (en) * | 2003-10-10 | 2005-04-21 | Five Prime Therapeutics, Inc. | Kiaa0779, splice variants thereof, and methods of their use |
WO2005035564A2 (en) | 2003-10-10 | 2005-04-21 | Xencor, Inc. | Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders |
PT2161283E (en) | 2003-11-17 | 2014-08-29 | Genentech Inc | Compositions comprising antibodies against cd79b conjugated to a growth inhibitory agent or cytotoxic agent and methods for the treatment of tumor of hematopoietic origin |
HUE026132T2 (en) | 2004-01-07 | 2016-05-30 | Novartis Vaccines & Diagnostics Inc | M-csf-specific monoclonal antibody and uses thereof |
US20050169970A1 (en) * | 2004-02-02 | 2005-08-04 | Unilever Bestfoods, North America | Food composition with fibers |
AU2005230848B9 (en) * | 2004-03-31 | 2011-06-02 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
US7794713B2 (en) * | 2004-04-07 | 2010-09-14 | Lpath, Inc. | Compositions and methods for the treatment and prevention of hyperproliferative diseases |
EP2067789A1 (en) | 2004-04-13 | 2009-06-10 | F. Hoffmann-La Roche Ag | Anti-P selectin antibodies |
EP3943494A1 (en) * | 2004-05-13 | 2022-01-26 | Icos Corporation | Quinazolinones as inhibitors of human phosphatidylinositol 3-kinase delta |
JP2008500338A (en) * | 2004-05-25 | 2008-01-10 | イコス・コーポレイション | Method for treating and / or preventing abnormal proliferation of hematopoietic cells |
US7361493B1 (en) | 2004-05-26 | 2008-04-22 | The United States Of America As Represented By The Secretary Of The Department Of Veterans Affairs | Production of urokinase in a three-dimensional cell culture |
WO2005118864A2 (en) | 2004-05-28 | 2005-12-15 | Agensys, Inc. | Antibodies and related molecules that bind to psca proteins |
US20060014680A1 (en) * | 2004-07-13 | 2006-01-19 | Caiding Xu | Peptides and compounds that bind to the IL-5 receptor |
US20060024677A1 (en) | 2004-07-20 | 2006-02-02 | Morris David W | Novel therapeutic targets in cancer |
ES2339789T3 (en) | 2004-07-20 | 2010-05-25 | Genentech, Inc. | PROTEIN 4 INHIBITORS OF ANGIOPOYETINE TYPE, COMBINATIONS AND ITS USE. |
TWI309240B (en) | 2004-09-17 | 2009-05-01 | Hoffmann La Roche | Anti-ox40l antibodies |
AU2005286662B2 (en) | 2004-09-23 | 2011-10-06 | Vasgene Therapeutics, Inc. | Polypeptide compounds for inhibiting angiogenesis and tumor growth |
SI1797127T1 (en) * | 2004-09-24 | 2017-09-29 | Amgen Inc. | Modified fc molecules |
DE602005021534D1 (en) | 2004-10-15 | 2010-07-08 | Us Gov Health & Human Serv | AMPHIPATHIC HELIXFUL MULTIDOMADE PEPTIDES AND METHOD FOR THE APPLICATION |
US8802820B2 (en) | 2004-11-12 | 2014-08-12 | Xencor, Inc. | Fc variants with altered binding to FcRn |
WO2006053301A2 (en) | 2004-11-12 | 2006-05-18 | Xencor, Inc. | Fc variants with altered binding to fcrn |
JP2008521411A (en) | 2004-11-30 | 2008-06-26 | キュラジェン コーポレイション | Antibodies against GPNMB and uses thereof |
US20060134698A1 (en) * | 2004-12-20 | 2006-06-22 | Evanston Northwestern Healthcare Research Institute | Methods for treating cardiac disease by modifying an N-terminal domain of troponin I |
AU2006208226A1 (en) * | 2005-01-24 | 2006-08-03 | Amgen Inc. | Humanized anti-amyloid antibody |
CA2598409A1 (en) * | 2005-02-17 | 2006-08-24 | Icos Corporation | Phosphoinositide 3-kinase inhibitors for inhibiting leukocyte accumulation |
AU2006230563B8 (en) | 2005-03-31 | 2010-06-17 | Agensys, Inc. | Antibodies and related molecules that bind to 161P2F10B proteins |
TW200720289A (en) * | 2005-04-01 | 2007-06-01 | Hoffmann La Roche | Antibodies against CCR5 and uses thereof |
EP2062591A1 (en) | 2005-04-07 | 2009-05-27 | Novartis Vaccines and Diagnostics, Inc. | CACNA1E in cancer diagnosis detection and treatment |
EP2083088A3 (en) | 2005-04-07 | 2009-10-14 | Novartis Vaccines and Diagnostics, Inc. | Cancer-related genes |
US7592429B2 (en) | 2005-05-03 | 2009-09-22 | Ucb Sa | Sclerostin-binding antibody |
US8003108B2 (en) | 2005-05-03 | 2011-08-23 | Amgen Inc. | Sclerostin epitopes |
US20060271262A1 (en) * | 2005-05-24 | 2006-11-30 | Mclain Harry P Iii | Wireless agricultural network |
NZ563341A (en) | 2005-06-06 | 2009-10-30 | Genentech Inc | Methods for identifying agents that modulate a gene that encodes for a PRO1568 polypeptide |
US8389469B2 (en) * | 2005-06-06 | 2013-03-05 | The Rockefeller University | Bacteriophage lysins for Bacillus anthracis |
US8252756B2 (en) | 2005-06-14 | 2012-08-28 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US7582291B2 (en) * | 2005-06-30 | 2009-09-01 | The Rockefeller University | Bacteriophage lysins for Enterococcus faecalis, Enterococcus faecium and other bacteria |
CN105012953B (en) | 2005-07-25 | 2018-06-22 | 阿普泰沃研发有限责任公司 | B- cells are reduced with CD37- specificity and CD20- specific binding molecules |
WO2007016240A2 (en) * | 2005-07-28 | 2007-02-08 | Novartis Ag | Use of antibody to m-csf |
EP1913027B1 (en) * | 2005-07-28 | 2015-03-04 | Novartis AG | M-csf specific monoclonal antibody and uses thereof |
US8008453B2 (en) | 2005-08-12 | 2011-08-30 | Amgen Inc. | Modified Fc molecules |
AU2006280321A1 (en) | 2005-08-15 | 2007-02-22 | Genentech, Inc. | Gene disruptions, compositions and methods relating thereto |
CN101297034A (en) | 2005-08-24 | 2008-10-29 | 洛克菲勒大学 | PLY-GBS mutant lysins |
CA2624900A1 (en) | 2005-10-04 | 2007-04-19 | The Research Foundation Of State University Of New York | Fibronectin polypeptides and methods of use |
US20080213274A1 (en) * | 2005-10-28 | 2008-09-04 | Sabbadini Roger A | Compositions and methods for the treatment and prevention of fibrotic, inflammatory, and neovascularization conditions of the eye |
MX2008005405A (en) * | 2005-10-28 | 2008-09-11 | Florida Internat University Bo | Horse: human chimeric antibodies. |
US20090074720A1 (en) * | 2005-10-28 | 2009-03-19 | Sabbadini Roger A | Methods for decreasing immune response and treating immune conditions |
RS54111B1 (en) | 2005-11-18 | 2015-12-31 | Glenmark Pharmaceuticals S.A. | Anti-alpha2 integrin antibodies and their uses |
ZA200804162B (en) | 2005-11-21 | 2009-12-30 | Genentech Inc | Novel gene disruptions, compositions and methods relating thereto |
AU2006318539B2 (en) * | 2005-11-23 | 2012-09-13 | Genentech, Inc. | Methods and compositions related to B cell assays |
US20070213264A1 (en) | 2005-12-02 | 2007-09-13 | Mingdong Zhou | Neuregulin variants and methods of screening and using thereof |
ES2664086T3 (en) | 2005-12-30 | 2018-04-18 | Zensun (Shanghai) Science & Technology, Co., Ltd. | Extended release of neurregulin to improve cardiac function |
WO2007102946A2 (en) | 2006-01-23 | 2007-09-13 | Amgen Inc. | Crystalline polypeptides |
WO2007114979A2 (en) | 2006-02-17 | 2007-10-11 | Genentech, Inc. | Gene disruptons, compositions and methods relating thereto |
TW200745163A (en) * | 2006-02-17 | 2007-12-16 | Syntonix Pharmaceuticals Inc | Peptides that block the binding of IgG to FcRn |
TWI417301B (en) | 2006-02-21 | 2013-12-01 | Wyeth Corp | Antibodies against human il-22 and uses therefor |
TW200744634A (en) | 2006-02-21 | 2007-12-16 | Wyeth Corp | Methods of using antibodies against human IL-22 |
JP2009529915A (en) | 2006-03-20 | 2009-08-27 | ゾーマ テクノロジー リミテッド | Human antibodies and methods specific for gastrin substances |
TWI397535B (en) | 2006-03-21 | 2013-06-01 | Genentech Inc | Combinatorial therapy involving alpha5beta1 antagonists |
US7973142B2 (en) | 2006-04-07 | 2011-07-05 | Warner Chilcott Company | Antibodies that bind human protein tyrosine phosphatase beta (HPTPβ) |
US20090288176A1 (en) | 2006-04-19 | 2009-11-19 | Genentech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
TWI395754B (en) | 2006-04-24 | 2013-05-11 | Amgen Inc | Humanized c-kit antibody |
US7862812B2 (en) * | 2006-05-31 | 2011-01-04 | Lpath, Inc. | Methods for decreasing immune response and treating immune conditions |
CA2654317A1 (en) | 2006-06-12 | 2007-12-21 | Trubion Pharmaceuticals, Inc. | Single-chain multivalent binding proteins with effector function |
US20080227686A1 (en) * | 2006-06-16 | 2008-09-18 | Lipid Sciences, Inc. | Novel Peptides that Promote Lipid Efflux |
US20080206142A1 (en) * | 2006-06-16 | 2008-08-28 | Lipid Sciences, Inc. | Novel Peptides That Promote Lipid Efflux |
US20080199398A1 (en) * | 2006-06-16 | 2008-08-21 | Brewer H Bryan | Novel Peptides That Promote Lipid Efflux |
US7981425B2 (en) | 2006-06-19 | 2011-07-19 | Amgen Inc. | Thrombopoietic compounds |
CA2787343C (en) | 2006-06-26 | 2016-08-02 | Amgen Inc. | Compositions comprising modified lcat and uses thereof |
EP2057193B1 (en) | 2006-08-04 | 2013-12-18 | Novartis AG | Ephb3-specific antibody and uses thereof |
TWI454480B (en) | 2006-08-18 | 2014-10-01 | Novartis Ag | Prlr-specific antibody and uses thereof |
CL2007002567A1 (en) | 2006-09-08 | 2008-02-01 | Amgen Inc | ISOLATED PROTEINS FROM LINK TO ACTIVINE TO HUMAN. |
WO2008039843A2 (en) * | 2006-09-26 | 2008-04-03 | Lipid Sciences, Inc. | Novel peptides that promote lipid efflux |
BRPI0717512A2 (en) | 2006-09-29 | 2013-11-19 | Hoffmann La Roche | CCR5 ANTIBODIES AND USES OF THE SAME |
US7767206B2 (en) | 2006-10-02 | 2010-08-03 | Amgen Inc. | Neutralizing determinants of IL-17 Receptor A and antibodies that bind thereto |
EP1914303A1 (en) * | 2006-10-09 | 2008-04-23 | Qiagen GmbH | Thermus eggertssonii DNA polymerases |
WO2008055072A2 (en) | 2006-10-27 | 2008-05-08 | Lpath, Inc. | Compositions and methods for treating ocular diseases and conditions |
MX2009004532A (en) | 2006-10-27 | 2009-09-04 | Lpath Inc | Compositions and methods for binding sphingosine-1-phosphate. |
EP3284825B1 (en) | 2006-11-02 | 2021-04-07 | Biomolecular Holdings LLC | Methods of producing hybrid polypeptides with moving parts |
EP2423226A3 (en) | 2006-11-10 | 2012-05-30 | Amgen Inc. | Antibody-based diagnostics and therapeutics |
JP5313909B2 (en) | 2006-11-13 | 2013-10-09 | アイコス コーポレイション | Thienopyrimidinone for the treatment of inflammatory diseases and cancer |
WO2008061019A2 (en) | 2006-11-14 | 2008-05-22 | Genentech, Inc. | Modulators of neuronal regeneration |
EP3156415A1 (en) | 2006-11-22 | 2017-04-19 | Bristol-Myers Squibb Company | Targeted therapeutics based on engineered proteins for tyrosine kinases receptors, including igf-ir |
WO2008070780A1 (en) | 2006-12-07 | 2008-06-12 | Novartis Ag | Antagonist antibodies against ephb3 |
US8183201B2 (en) * | 2006-12-26 | 2012-05-22 | National Cheng Kung University | Methods of treating αvβ3 integrin-associated diseases by administering polypeptides selective for αvβ3 integrin |
US7943728B2 (en) * | 2006-12-26 | 2011-05-17 | National Cheng Kung University | Disintegrin variants and their use in treating osteoporosis-induced bone loss and angiogenesis-related diseases |
JP2010517944A (en) * | 2007-01-26 | 2010-05-27 | バイオインヴェント インターナショナル アーベー | DLL4 signaling inhibitor and use thereof |
JP2010517529A (en) * | 2007-02-02 | 2010-05-27 | アムジエン・インコーポレーテツド | Hepcidin and hepcidin antibody |
EP2111228B1 (en) | 2007-02-02 | 2011-07-20 | Bristol-Myers Squibb Company | 10Fn3 domain for use in treating diseases associated with inappropriate angiogenesis |
CN103966345A (en) | 2007-02-09 | 2014-08-06 | 西北大学 | Particles for detecting intracellular targets |
US8088887B2 (en) * | 2007-02-13 | 2012-01-03 | Academia Sinica | Peptide-conjugates that bind to VEGF-stimulated or tumor vasculature and methods of treatment |
US8415453B2 (en) * | 2007-02-13 | 2013-04-09 | Academia Sinica | Lung cancer-targeted peptides and applications thereof |
WO2008103962A2 (en) | 2007-02-22 | 2008-08-28 | Genentech, Inc. | Methods for detecting inflammatory bowel disease |
US8808747B2 (en) * | 2007-04-17 | 2014-08-19 | Baxter International Inc. | Nucleic acid microparticles for pulmonary delivery |
CA2689923A1 (en) | 2007-05-30 | 2008-12-11 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
DK2164992T3 (en) * | 2007-05-30 | 2016-08-15 | Lpath Inc | COMPOSITIONS AND METHODS FOR BONDING OF LYTHOPHOSPHATIC ACID |
US9163091B2 (en) * | 2007-05-30 | 2015-10-20 | Lpath, Inc. | Compositions and methods for binding lysophosphatidic acid |
US8759300B2 (en) * | 2007-06-14 | 2014-06-24 | The Research Foundation For The State University Of New York | Polypeptides and methods of use |
US7625555B2 (en) | 2007-06-18 | 2009-12-01 | Novagen Holding Corporation | Recombinant human interferon-like proteins |
EP2167130A2 (en) * | 2007-07-06 | 2010-03-31 | Trubion Pharmaceuticals, Inc. | Binding peptides having a c-terminally disposed specific binding domain |
CN101802013B (en) | 2007-07-16 | 2014-07-02 | 健泰科生物技术公司 | Humanized anti-CD79b antibodies and immunoconjugates and methods of use |
NZ583367A (en) | 2007-07-16 | 2012-10-26 | Genentech Inc | Anti-cd79b antibodies and immunoconjugates and methods of use |
PT2181190E (en) | 2007-07-26 | 2014-02-24 | Amgen Inc | Modified lecithin-cholesterol acyltransferase enzymes |
CN101361968B (en) | 2007-08-06 | 2011-08-03 | 健能隆医药技术(上海)有限公司 | Use of interleukin-22 in treating fatty liver |
BRPI0814763A2 (en) * | 2007-08-09 | 2015-03-03 | Syntonix Pharmaceuticals Inc | IMMUNOMODULATING PEPTIDES |
EP2615113A3 (en) | 2007-08-23 | 2013-11-13 | Amgen Inc. | Antigen binding proteins to proprotein convertase subtilisin kexin type 9 (PCSK9) |
JOP20080381B1 (en) | 2007-08-23 | 2023-03-28 | Amgen Inc | Antigen Binding Proteins to Proprotein Convertase subtillisin Kexin type 9 (pcsk9) |
NZ583605A (en) | 2007-08-29 | 2012-10-26 | Sanofi Aventis | Humanized anti-cxcr5 antibodies, derivatives thereof and their uses |
US7982016B2 (en) | 2007-09-10 | 2011-07-19 | Amgen Inc. | Antigen binding proteins capable of binding thymic stromal lymphopoietin |
US20090156488A1 (en) | 2007-09-12 | 2009-06-18 | Zensun (Shanghai) Science & Technology Limited | Use of neuregulin for organ preservation |
EP2050764A1 (en) | 2007-10-15 | 2009-04-22 | sanofi-aventis | Novel polyvalent bispecific antibody format and uses thereof |
US8361465B2 (en) * | 2007-10-26 | 2013-01-29 | Lpath, Inc. | Use of anti-sphingosine-1-phosphate antibodies in combination with chemotherapeutic agents |
WO2009058564A2 (en) | 2007-11-01 | 2009-05-07 | Maxygen, Inc. | Immunosuppressive polypeptides and nucleic acids |
US8541543B2 (en) * | 2007-11-20 | 2013-09-24 | Academia Sinica | Peptides specific for hepatocellular carcinoma cells and applications thereof |
EP2231181B1 (en) | 2007-12-17 | 2016-02-17 | Marfl AB | New vaccine for the treatment of mycobacterium related disorders |
WO2009085200A2 (en) | 2007-12-21 | 2009-07-09 | Amgen Inc. | Anti-amyloid antibodies and uses thereof |
SI2808343T1 (en) | 2007-12-26 | 2019-10-30 | Xencor Inc | Fc variants with altered binding to FcRn |
WO2009094551A1 (en) | 2008-01-25 | 2009-07-30 | Amgen Inc. | Ferroportin antibodies and methods of use |
MX2010008437A (en) | 2008-01-31 | 2010-11-25 | Genentech Inc | Anti-cd79b antibodies and immunoconjugates and methods of use. |
JP6018361B2 (en) | 2008-01-31 | 2016-11-02 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Antibodies against human CD39 and their use for inhibiting regulatory T cell activity |
JO2913B1 (en) | 2008-02-20 | 2015-09-15 | امجين إنك, | Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof |
US8697081B2 (en) * | 2008-04-09 | 2014-04-15 | The Regents Of The University Of Michigan | Method of modulating neovascularization |
EP2279412B1 (en) | 2008-04-09 | 2017-07-26 | Genentech, Inc. | Novel compositions and methods for the treatment of immune related diseases |
EP2365003A1 (en) * | 2008-04-11 | 2011-09-14 | Emergent Product Development Seattle, LLC | CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof |
US8921315B1 (en) | 2008-04-24 | 2014-12-30 | Neumedicines, Inc. | Method of increasing survival of a human subject having exposure to an acute exposure to non-therapeutic whole body ionization by administering a therapeutically effective dose of IL-12 |
EP2816059A1 (en) | 2008-05-01 | 2014-12-24 | Amgen, Inc | Anti-hepcidin antibodies and methods of use |
EP3388527A1 (en) | 2008-05-15 | 2018-10-17 | Tetherex Pharmaceuticals Corporation | Anti-psgl-1 antibodies and methods of identification and use |
US8093018B2 (en) | 2008-05-20 | 2012-01-10 | Otsuka Pharmaceutical Co., Ltd. | Antibody identifying an antigen-bound antibody and an antigen-unbound antibody, and method for preparing the same |
JP2011520961A (en) | 2008-05-22 | 2011-07-21 | ブリストル−マイヤーズ スクイブ カンパニー | Scaffold domain protein based on multivalent fibronectin |
JOP20190083A1 (en) | 2008-06-04 | 2017-06-16 | Amgen Inc | Fgf21 mutant fusion polypeptides and uses thereof |
EP2318036B1 (en) | 2008-06-30 | 2015-06-03 | The Regents of the University of Michigan | Lysosomal phospholipase a2 (lpla2) activity as a diagnostic and therapeutic target for identifying and treating systemic lupus erythematosis |
US20100048488A1 (en) * | 2008-08-01 | 2010-02-25 | Syntonix Pharmaceuticals, Inc. | Immunomodulatory peptides |
US8163497B2 (en) | 2008-09-07 | 2012-04-24 | Glyconex Inc. | Anti-extended type I glycosphingolipid antibody, derivatives thereof and use |
SI2344540T1 (en) | 2008-10-02 | 2018-04-30 | Aptevo Research And Development Llc | Cd86 antagonist multi-target binding proteins |
JP2012504946A (en) | 2008-10-07 | 2012-03-01 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Neutralizing antibody against platelet factor 4 variant 1 (PF4V1) and fragments thereof |
EA032727B1 (en) | 2008-10-10 | 2019-07-31 | Амген Инк. | Fgf21 mutant proteolysis-resistant polypeptide and use thereof |
AU2009308293B2 (en) | 2008-10-22 | 2015-02-05 | Genentech, Inc. | Modulation of axon degeneration |
US8871202B2 (en) | 2008-10-24 | 2014-10-28 | Lpath, Inc. | Prevention and treatment of pain using antibodies to sphingosine-1-phosphate |
JP5775458B2 (en) * | 2008-11-06 | 2015-09-09 | グレンマーク ファーマシューティカルズ, エセ.アー. | Treatment using anti-α2 integrin antibody |
US20100202963A1 (en) | 2008-11-13 | 2010-08-12 | Gallatin W Michael | Therapies for hematologic malignancies |
US9492449B2 (en) | 2008-11-13 | 2016-11-15 | Gilead Calistoga Llc | Therapies for hematologic malignancies |
KR101692880B1 (en) | 2008-11-24 | 2017-01-04 | 노오쓰웨스턴 유니버시티 | Polyvalent rna-nanoparticle compositions |
EP2379096B1 (en) | 2008-12-19 | 2019-10-30 | Baxalta GmbH | Tfpi inhibitors and methods of use |
US20100184844A1 (en) * | 2009-01-08 | 2010-07-22 | Northwestern University | Inhibition of Bacterial Protein Production by Polyvalent Oligonucleotide Modified Nanoparticle Conjugates |
US20100233270A1 (en) | 2009-01-08 | 2010-09-16 | Northwestern University | Delivery of Oligonucleotide-Functionalized Nanoparticles |
US20100294952A1 (en) * | 2009-01-15 | 2010-11-25 | Northwestern University | Controlled agent release and sequestration |
EP3002296B1 (en) | 2009-03-17 | 2020-04-29 | Université d'Aix-Marseille | Btla antibodies and uses thereof |
US20120121591A1 (en) | 2009-03-20 | 2012-05-17 | Amgen Inc. | SELECTIVE AND POTENT PEPTIDE INHIBITORS OF Kv1.3 |
KR20120002995A (en) | 2009-03-24 | 2012-01-09 | 길리아드 칼리스토가 엘엘씨 | Atropisomers of 2-purinyl-3-tolyl-quinazolinone derivatives and methods of use |
UA108199C2 (en) | 2009-03-25 | 2015-04-10 | ANTIBODY AGAINST α5β1 AND ITS APPLICATION | |
JP5795306B2 (en) | 2009-04-01 | 2015-10-14 | ジェネンテック, インコーポレイテッド | Treatment of insulin resistance disease |
WO2010120561A1 (en) | 2009-04-01 | 2010-10-21 | Genentech, Inc. | Anti-fcrh5 antibodies and immunoconjugates and methods of use |
WO2010112034A2 (en) | 2009-04-02 | 2010-10-07 | Aarhus Universitet | Compositions and methods for treatment and diagnosis of synucleinopathies |
US8067201B2 (en) * | 2009-04-17 | 2011-11-29 | Bristol-Myers Squibb Company | Methods for protein refolding |
CN102458410A (en) * | 2009-04-20 | 2012-05-16 | 吉联亚·卡利斯托加有限责任公司 | Methods of treatment for solid tumors |
EP2248903A1 (en) | 2009-04-29 | 2010-11-10 | Universitat Autònoma De Barcelona | Methods and reagents for efficient and targeted gene transfer to monocytes and macrophages |
JP2012525847A (en) | 2009-05-05 | 2012-10-25 | アムジエン・インコーポレーテツド | FGF21 variants and uses thereof |
PE20120358A1 (en) | 2009-05-05 | 2012-04-26 | Amgen Inc | FGF21 MUTANTS AND USES OF THEM |
TW201102086A (en) | 2009-06-04 | 2011-01-16 | Hoffmann La Roche | Antibodies against human CCN1 and uses thereof |
CA2797480A1 (en) | 2009-06-15 | 2010-12-23 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotubular myopathy using chimeric polypeptides comprising myotubularin 1 (mtm1) polypeptides |
CA2764835A1 (en) | 2009-06-17 | 2010-12-23 | Amgen Inc. | Chimeric fgf19 polypeptides and uses thereof |
CN102470156A (en) * | 2009-07-20 | 2012-05-23 | 成功大学 | Polypeptides selective for av ss3 integrin conjugated with a variant of human serum albumin (HSA) and pharmaceutical uses thereof |
CA2768843A1 (en) | 2009-07-21 | 2011-01-27 | Gilead Calistoga Llc | Treatment of liver disorders with pi3k inhibitors |
WO2011014750A1 (en) | 2009-07-31 | 2011-02-03 | Genentech, Inc. | Inhibition of tumor metastasis using bv8- or g-csf-antagonists |
WO2011028952A1 (en) | 2009-09-02 | 2011-03-10 | Xencor, Inc. | Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens |
US8926976B2 (en) | 2009-09-25 | 2015-01-06 | Xoma Technology Ltd. | Modulators |
WO2011038301A2 (en) | 2009-09-25 | 2011-03-31 | Xoma Technology Ltd. | Screening methods |
AU2010308030B2 (en) | 2009-10-12 | 2014-05-29 | Pfizer Inc. | Cancer treatment |
TW201117824A (en) | 2009-10-12 | 2011-06-01 | Amgen Inc | Use of IL-17 receptor a antigen binding proteins |
EP2488643A4 (en) * | 2009-10-15 | 2013-07-03 | Hoffmann La Roche | Chimeric fibroblast growth factors with altered receptor specificity |
WO2011049625A1 (en) | 2009-10-20 | 2011-04-28 | Mansour Samadpour | Method for aflatoxin screening of products |
KR20120105446A (en) | 2009-10-22 | 2012-09-25 | 제넨테크, 인크. | Methods and compositions for modulating hepsin activation of macrophage-stimulating protein |
CN104043126A (en) | 2009-10-22 | 2014-09-17 | 霍夫曼-拉罗奇有限公司 | Modulation of axon degeneration |
WO2011056502A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Bone morphogenetic protein receptor type ii compositions and methods of use |
WO2011056494A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor-like kinase-1 antagonist and vegfr3 antagonist combinations |
WO2011056497A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor type iib compositions and methods of use |
KR20120136345A (en) | 2009-10-30 | 2012-12-18 | 노오쓰웨스턴 유니버시티 | Templated nanoconjugates |
CA2779574C (en) | 2009-11-05 | 2018-12-18 | Rhizen Pharmaceuticals S.A. | Novel kinase modulators |
RU2012124093A (en) | 2009-11-12 | 2013-12-20 | Дженентек, Инк. | METHOD FOR INCREASING DENSITY OF DENDRITIC SPIKES |
US9260517B2 (en) | 2009-11-17 | 2016-02-16 | Musc Foundation For Research Development | Human monoclonal antibodies to human nucleolin |
TW201129379A (en) | 2009-11-20 | 2011-09-01 | Amgen Inc | Anti-Orai1 antigen binding proteins and uses thereof |
AU2010324686B2 (en) | 2009-11-30 | 2016-05-19 | Genentech, Inc. | Antibodies for treating and diagnosing tumors expressing SLC34A2 (TAT211 = SEQID2 ) |
WO2011066511A1 (en) | 2009-11-30 | 2011-06-03 | The U.S.A., As Represented By The Secretary Department Of Health And Human Services | Synthetic apoa-1 mimetic amphipathic peptides and methods of use thereof |
EP2506861A1 (en) * | 2009-12-02 | 2012-10-10 | Amgen Inc. | Binding proteins that bind to human fgfr1c, human b-klotho and both human fgfr1c and human b-klotho |
UA109888C2 (en) | 2009-12-07 | 2015-10-26 | ANTIBODY OR ANTIBODILITY ANTIBODY OR ITS BINDING TO THE β-CLOTE, FGF RECEPTORS AND THEIR COMPLEXES | |
AU2010330794A1 (en) | 2009-12-18 | 2012-06-21 | Amgen Inc. | Wise binding agents and epitopes |
HUE029026T2 (en) | 2009-12-22 | 2017-01-30 | Roche Glycart Ag | ANTI-HER3 Antibodies and uses thereof |
MX2012007416A (en) | 2009-12-23 | 2012-07-23 | Univ Nat Cheng Kung | Compositions and methods for the treatment of angiogenesis-related eye diseases. |
WO2011079308A2 (en) | 2009-12-23 | 2011-06-30 | Emergent Product Development Seattle, Llc | Compositions comprising tnf-alpha and il-6 antagonists and methods of use thereof |
JP5856073B2 (en) | 2009-12-29 | 2016-02-09 | エマージェント プロダクト デベロップメント シアトル, エルエルシー | RON binding construct and method of use thereof |
WO2011097527A2 (en) | 2010-02-04 | 2011-08-11 | Xencor, Inc. | Immunoprotection of therapeutic moieties using enhanced fc regions |
US20110189178A1 (en) * | 2010-02-04 | 2011-08-04 | Xencor, Inc. | Immunoprotection of Therapeutic Moieties Using Enhanced Fc Regions |
CN102781960B (en) | 2010-02-16 | 2014-12-10 | 米迪缪尼有限公司 | HSA-related compositions and methods of use |
US8877897B2 (en) | 2010-02-23 | 2014-11-04 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
US8975376B2 (en) | 2010-02-23 | 2015-03-10 | Sanofi | Anti-alpha2 integrin antibodies and their uses |
US20110212088A1 (en) * | 2010-02-26 | 2011-09-01 | Sabbadini Roger A | Anti-paf antibodies |
US8642557B2 (en) | 2010-03-12 | 2014-02-04 | Abbvie Biotherapeutics Inc. | CTLA4 proteins and their uses |
CN103025345B (en) | 2010-03-19 | 2016-01-20 | 巴克斯特国际公司 | TFPI inhibitor and using method |
MY187990A (en) | 2010-03-31 | 2021-11-07 | Boehringer Ingelheim Int | Anti-cd40 antibodies |
JP2013523726A (en) | 2010-04-01 | 2013-06-17 | オンコレナ エービー | Improved treatment of renal cell carcinoma |
US9517264B2 (en) | 2010-04-15 | 2016-12-13 | Amgen Inc. | Human FGF receptor and β-Klotho binding proteins |
BR112012028010A2 (en) | 2010-05-03 | 2017-09-26 | Genentech Inc | isolated antibody, cell, isolated nucleic acid, method of identifying a first antibody that binds to a tat425 antigenic epitope attached to an antibody, methods of inhibiting cell growth, therapeutic treatment of determining the presence of a tat425 protein and diagnosing the presence of a tumor in a mammal |
EP2571878B1 (en) | 2010-05-17 | 2018-10-17 | Incozen Therapeutics Pvt. Ltd. | Novel 3,5-disubstitued-3h-imidazo[4,5-b]pyridine and 3,5- disubstitued -3h-[1,2,3]triazolo[4,5-b]pyridine compounds as modulators of protein kinases |
DK2571516T3 (en) | 2010-05-18 | 2018-02-05 | Neumedicines Inc | IL-12 FORMULATIONS FOR STIMULATING HEMOPOIES |
EP2576615B1 (en) | 2010-05-26 | 2016-03-30 | Bristol-Myers Squibb Company | Fibronectin based scaffold proteins having improved stability |
WO2011160062A2 (en) | 2010-06-17 | 2011-12-22 | The Usa As Represented By The Secretary, National Institutes Of Health | Compositions and methods for treating inflammatory conditions |
AR082163A1 (en) | 2010-07-15 | 2012-11-14 | Hoffmann La Roche | SPECIFICALLY BINDING ANTIBODIES OF THE HUMAN TSLPR AND METHODS OF USING THEMSELVES |
WO2012010696A1 (en) | 2010-07-23 | 2012-01-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for cancer management targeting co-029 |
CA2806252C (en) | 2010-07-29 | 2019-05-14 | Xencor, Inc. | Antibodies with modified isoelectric points |
US20130177555A1 (en) | 2010-08-13 | 2013-07-11 | Medimmune Limited | Monomeric Polypeptides Comprising Variant FC Regions And Methods Of Use |
US9688735B2 (en) | 2010-08-20 | 2017-06-27 | Wyeth Llc | Designer osteogenic proteins |
KR101630501B1 (en) | 2010-08-20 | 2016-06-15 | 와이어쓰 엘엘씨 | Designer osteogenic proteins |
CN102380091A (en) | 2010-08-31 | 2012-03-21 | 健能隆医药技术(上海)有限公司 | Application of interleukin-22 in curing virus hepatitis |
JP6159660B2 (en) | 2010-09-22 | 2017-07-05 | アムジエン・インコーポレーテツド | Immunoglobulins as carriers and uses thereof |
EP2621954A1 (en) | 2010-10-01 | 2013-08-07 | Oxford Biotherapeutics Ltd. | Anti-rori antibodies |
US9445990B2 (en) | 2010-10-06 | 2016-09-20 | Medtronic, Inc. | TNF inhibitor formulation for use in implantable infusion devices |
WO2012061129A1 (en) | 2010-10-25 | 2012-05-10 | Genentech, Inc | Treatment of gastrointestinal inflammation and psoriasis a |
EA201890548A1 (en) | 2010-11-04 | 2018-07-31 | Бёрингер Ингельхайм Интернациональ Гмбх | ANTIBODIES TO IL-23 |
US9023791B2 (en) | 2010-11-19 | 2015-05-05 | Novartis Ag | Fibroblast growth factor 21 mutations |
WO2012080769A1 (en) | 2010-12-15 | 2012-06-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Anti-cd277 antibodies and uses thereof |
CN108939067A (en) | 2010-12-21 | 2018-12-07 | 瑟莱克斯制药公司 | Anti- palatelet-selectin antibody and its use and identification method |
WO2012085132A1 (en) | 2010-12-22 | 2012-06-28 | Orega Biotech | Antibodies against human cd39 and use thereof |
JOP20210044A1 (en) | 2010-12-30 | 2017-06-16 | Takeda Pharmaceuticals Co | Anti-cd38 antibodies |
WO2012101125A1 (en) | 2011-01-24 | 2012-08-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Specific antibodies against human cxcl4 and uses thereof |
WO2012102679A1 (en) | 2011-01-24 | 2012-08-02 | National University Of Singapore | Pathogenic mycobacteria-derived mannose-capped lipoarabinomannan antigen binding proteins |
TW201238976A (en) | 2011-02-23 | 2012-10-01 | Hoffmann La Roche | Antibodies against human IL33R and uses thereof |
AR085911A1 (en) | 2011-03-16 | 2013-11-06 | Sanofi Sa | SAFE THERAPEUTIC DOSE OF A SIMILAR PROTEIN TO AN ANTIBODY WITH VUAL REGION |
EA035351B1 (en) | 2011-03-31 | 2020-06-01 | Инсэрм (Инститют Насиональ Де Ля Сантэ Э Де Ля Решерш Медикаль) | Antibodies directed against icos and uses thereof |
KR102001686B1 (en) | 2011-04-07 | 2019-07-18 | 암젠 인크 | Novel egfr binding proteins |
TR201905909T4 (en) | 2011-04-19 | 2019-05-21 | Pfizer | Combinations of anti-4-1bb antibodies and adcc inducing antibodies for cancer therapy. |
MX365160B (en) | 2011-05-04 | 2019-05-24 | Rhizen Pharmaceuticals Sa | Novel compounds as modulators of protein kinases. |
JOP20200043A1 (en) | 2011-05-10 | 2017-06-16 | Amgen Inc | Methods of treating or preventing cholesterol related disorders |
MX344219B (en) | 2011-05-18 | 2016-12-07 | Mederis Diabetes Llc | Improved peptide pharmaceuticals for insulin resistance. |
EP4286400A2 (en) | 2011-05-18 | 2023-12-06 | Eumederis Pharmaceuticals, Inc. | Improved peptide pharmaceuticals |
US9127065B2 (en) | 2011-05-19 | 2015-09-08 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Anti-human HER3 antibodies and uses thereof |
ES2894398T3 (en) | 2011-06-03 | 2022-02-14 | Xoma Technology Ltd | Specific antibodies to TGF-beta |
WO2012171031A1 (en) | 2011-06-10 | 2012-12-13 | Baxter International Inc. | Treatment of coagulation disease by administration of recombinant vwf |
EP2718456B1 (en) | 2011-06-13 | 2019-04-24 | Neumedicines, Inc. | Mitigation of cutaneous injury with il-12 |
US9045526B2 (en) | 2011-06-23 | 2015-06-02 | The Regents Of The University Of Michigan | Compound and method for modulating opioid receptor activity |
EP2543678A1 (en) | 2011-07-08 | 2013-01-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antibodies for the treatment and prevention of thrombosis |
EP2543677A1 (en) | 2011-07-08 | 2013-01-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antibodies for the treatment and prevention of thrombosis |
EP2543679A1 (en) | 2011-07-08 | 2013-01-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antibodies for the treatment and prevention of thrombosis |
WO2013012855A1 (en) | 2011-07-18 | 2013-01-24 | Amgen Inc. | Apelin antigen-binding proteins and uses thereof |
US20140234330A1 (en) | 2011-07-22 | 2014-08-21 | Amgen Inc. | Il-17 receptor a is required for il-17c biology |
WO2013022855A1 (en) | 2011-08-05 | 2013-02-14 | Xencor, Inc. | Antibodies with modified isoelectric points and immunofiltering |
US9605083B2 (en) | 2011-08-16 | 2017-03-28 | Emory University | JAML specific binding agents, antibodies, and uses related thereto |
KR20140068877A (en) | 2011-08-17 | 2014-06-09 | 제넨테크, 인크. | Inhibition of angiogenesis in refractory tumors |
AU2012301769B2 (en) | 2011-08-31 | 2016-05-19 | Amgen Inc. | FGF21 for use in treating type 1 diabetes |
AU2012308302A1 (en) | 2011-09-14 | 2014-03-20 | Northwestern University | Nanoconjugates able to cross the blood-brain barrier |
EA027900B1 (en) | 2011-09-22 | 2017-09-29 | Эмджен Инк. | Cd27l antigen binding proteins |
US9458214B2 (en) | 2011-09-26 | 2016-10-04 | Novartis Ag | Dual function fibroblast growth factor 21 proteins |
US10851178B2 (en) | 2011-10-10 | 2020-12-01 | Xencor, Inc. | Heterodimeric human IgG1 polypeptides with isoelectric point modifications |
WO2013053076A1 (en) | 2011-10-10 | 2013-04-18 | Zensun (Shanghai)Science & Technology Limited | Compositions and methods for treating heart failure |
AU2012323287B2 (en) | 2011-10-10 | 2018-02-01 | Xencor, Inc. | A method for purifying antibodies |
US8999325B2 (en) | 2011-10-13 | 2015-04-07 | Aerpio Therapeutics, Inc | Treatment of ocular disease |
CN104039351A (en) | 2011-10-13 | 2014-09-10 | 阿尔皮奥治疗学股份有限公司 | Methods for treating vascular leak syndrome and cancer |
CN104053670A (en) | 2011-10-31 | 2014-09-17 | 百时美施贵宝公司 | Fibronectin binding domains with reduced immunogenicity |
SG11201402283PA (en) | 2011-11-16 | 2014-06-27 | Boehringer Ingelheim Int | Anti il-36r antibodies |
MX2014006272A (en) | 2011-11-23 | 2014-10-24 | Igenica Biotherapeutics Inc | Anti-cd98 antibodies and methods of use thereof. |
JP2015502368A (en) | 2011-12-16 | 2015-01-22 | カロス セラピューティクス,インコーポレーテッド | Methods and uses of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide) -related peptides and their derivatives for the treatment of retinal disorders and diseases |
US9988439B2 (en) | 2011-12-23 | 2018-06-05 | Nicholas B. Lydon | Immunoglobulins and variants directed against pathogenic microbes |
EP2793944A4 (en) | 2011-12-23 | 2015-09-02 | Nicholas B Lydon | Immunoglobulins and variants directed against pathogenic microbes |
US9636381B2 (en) | 2012-01-18 | 2017-05-02 | Neumedicines, Inc. | Methods for radiation protection by administering IL-12 |
EP2807187B1 (en) | 2012-01-26 | 2017-07-26 | Christopher J. Soares | Peptide antagonists of the calcitonin cgrp family of peptide hormones and their use |
SI2834241T1 (en) | 2012-03-05 | 2021-06-30 | Gilead Calistoga Llc | Polymorphic forms of (s)-2-(1-(9h-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3h)-one |
KR102143887B1 (en) | 2012-03-16 | 2020-08-12 | 유니버시티 헬스 네트워크 | Methods and compositions for modulating toso activity |
CN104302306B (en) | 2012-03-21 | 2019-03-22 | 百深有限责任公司 | TFPI inhibitor and its application method |
US9592289B2 (en) | 2012-03-26 | 2017-03-14 | Sanofi | Stable IgG4 based binding agent formulations |
CA2868883C (en) | 2012-03-30 | 2022-10-04 | Sorrento Therapeutics Inc. | Fully human antibodies that bind to vegfr2 |
KR20140144726A (en) | 2012-03-30 | 2014-12-19 | 리젠 파마슈티컬스 소시에떼 아노님 | Novel 3,5-disubstitued-3h-imidazo[4,5-b]pyridine and 3,5-disubstitued -3h-[1,2,3]triazolo[4,5-b] pyridine compounds as modulators of c-met protein kinases |
WO2013151649A1 (en) | 2012-04-04 | 2013-10-10 | Sialix Inc | Glycan-interacting compounds |
WO2013155346A1 (en) | 2012-04-11 | 2013-10-17 | The Regents Of The University Of California | Diagnostic tools for response to 6-thiopurine therapy |
EA039663B1 (en) | 2012-05-03 | 2022-02-24 | Амген Инк. | Use of an anti-pcsk9 antibody for lowering serum cholesterol ldl and treating cholesterol related disorders |
CN109206516A (en) | 2012-05-03 | 2019-01-15 | 勃林格殷格翰国际有限公司 | Anti-il-23 p 19 antibodies |
EP2847219A1 (en) | 2012-05-07 | 2015-03-18 | Amgen Inc. | Anti-erythropoietin antibodies |
EP2850095B1 (en) | 2012-05-17 | 2019-10-09 | RA Pharmaceuticals, Inc. | Peptide and peptidomimetic inhibitors |
UY34813A (en) | 2012-05-18 | 2013-11-29 | Amgen Inc | ANTIGEN UNION PROTEINS DIRECTED AGAINST ST2 RECEIVER |
EP3553086A1 (en) | 2012-05-31 | 2019-10-16 | Sorrento Therapeutics Inc. | Antigen binding proteins that bind pd-l1 |
KR20150030706A (en) | 2012-06-11 | 2015-03-20 | 암젠 인코퍼레이티드 | Dual receptor antagonistic antigen-binding proteins and uses thereof |
CA2877573A1 (en) | 2012-06-21 | 2013-12-27 | Sorrento Therapeutics, Inc. | Antigen binding proteins that bind c-met |
US9315579B2 (en) | 2012-06-22 | 2016-04-19 | Sorrento Therapeutics, Inc. | Antigen binding proteins that bind CCR2 |
EP2864355B1 (en) | 2012-06-25 | 2016-10-12 | Orega Biotech | Il-17 antagonist antibodies |
WO2014004549A2 (en) | 2012-06-27 | 2014-01-03 | Amgen Inc. | Anti-mesothelin binding proteins |
EP3431497B1 (en) | 2012-06-27 | 2022-07-27 | The Trustees of Princeton University | Split inteins, conjugates and uses thereof |
WO2014039189A1 (en) | 2012-08-01 | 2014-03-13 | Mcnally Elizabeth | Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (ltbp4) |
WO2014022759A1 (en) | 2012-08-03 | 2014-02-06 | Dana-Farber Cancer Institute, Inc. | Agents that modulate immune cell activation and methods of use thereof |
EP2892928B1 (en) | 2012-09-03 | 2018-05-30 | INSERM - Institut National de la Santé et de la Recherche Médicale | Antibodies directed against icos for treating graft-versus-host disease |
TWI595007B (en) | 2012-09-10 | 2017-08-11 | Neotope Biosciences Ltd | Anti-mcam antibodies and associated methods of use |
EP3738605A1 (en) | 2012-09-10 | 2020-11-18 | Xencor, Inc. | Methods of treating neurological diseases |
EP2906598A1 (en) | 2012-10-09 | 2015-08-19 | Igenica Biotherapeutics, Inc. | Anti-c16orf54 antibodies and methods of use thereof |
MX360816B (en) | 2012-11-20 | 2018-11-15 | Mederis Diabetes Llc | Improved peptide pharmaceuticals for insulin resistance. |
EP2922877B1 (en) | 2012-11-20 | 2018-09-05 | Eumederis Pharmaceuticals, Inc. | Improved peptide pharmaceuticals |
JP6324399B2 (en) | 2012-11-20 | 2018-05-16 | サノフイ | Anti-CEACAM5 antibody and use thereof |
TW201425336A (en) | 2012-12-07 | 2014-07-01 | Amgen Inc | BCMA antigen binding proteins |
EA201591219A1 (en) | 2012-12-27 | 2015-12-30 | Санофи | ANTIBODIES AGAINST LAMP1 AND CONJUGATES ANTIBODIES AND MEDICINES AND THEIR APPLICATION |
US9701759B2 (en) | 2013-01-14 | 2017-07-11 | Xencor, Inc. | Heterodimeric proteins |
US11053316B2 (en) | 2013-01-14 | 2021-07-06 | Xencor, Inc. | Optimized antibody variable regions |
US10131710B2 (en) | 2013-01-14 | 2018-11-20 | Xencor, Inc. | Optimized antibody variable regions |
US10487155B2 (en) | 2013-01-14 | 2019-11-26 | Xencor, Inc. | Heterodimeric proteins |
US10968276B2 (en) | 2013-03-12 | 2021-04-06 | Xencor, Inc. | Optimized anti-CD3 variable regions |
US9605084B2 (en) | 2013-03-15 | 2017-03-28 | Xencor, Inc. | Heterodimeric proteins |
AU2014205086B2 (en) | 2013-01-14 | 2019-04-18 | Xencor, Inc. | Novel heterodimeric proteins |
CA2897987A1 (en) | 2013-01-15 | 2014-07-24 | Xencor, Inc. | Rapid clearance of antigen complexes using novel antibodies |
JO3519B1 (en) | 2013-01-25 | 2020-07-05 | Amgen Inc | Antibody constructs for CDH19 and CD3 |
ES2728936T3 (en) | 2013-01-25 | 2019-10-29 | Amgen Inc | Antibodies directed against CDH19 for melanoma |
CA2899889A1 (en) | 2013-02-01 | 2014-08-07 | Santa Maria Biotherapeutics, Inc. | Administration of an anti-activin-a compound to a subject |
US20150361159A1 (en) | 2013-02-01 | 2015-12-17 | Bristol-Myers Squibb Company | Fibronectin based scaffold proteins |
US9580486B2 (en) | 2013-03-14 | 2017-02-28 | Amgen Inc. | Interleukin-2 muteins for the expansion of T-regulatory cells |
ES2759061T3 (en) | 2013-03-15 | 2020-05-07 | Biomolecular Holdings Llc | Hybrid immunoglobulin containing non-peptidyl binding |
SI2970449T1 (en) | 2013-03-15 | 2019-11-29 | Amgen Res Munich Gmbh | Single chain binding molecules comprising n-terminal abp |
UA118843C2 (en) | 2013-03-15 | 2019-03-25 | Дженентек, Інк. | Il-22 polypeptides and il-22 fc fusion proteins and methods of use |
EP2970486B1 (en) | 2013-03-15 | 2018-05-16 | Xencor, Inc. | Modulation of t cells with bispecific antibodies and fc fusions |
US10519242B2 (en) | 2013-03-15 | 2019-12-31 | Xencor, Inc. | Targeting regulatory T cells with heterodimeric proteins |
WO2014152006A2 (en) | 2013-03-15 | 2014-09-25 | Intrinsic Lifesciences, Llc | Anti-hepcidin antibodies and uses thereof |
US10106624B2 (en) | 2013-03-15 | 2018-10-23 | Xencor, Inc. | Heterodimeric proteins |
EP2970446A1 (en) | 2013-03-15 | 2016-01-20 | Amgen Research (Munich) GmbH | Antibody constructs for influenza m2 and cd3 |
PL3587448T3 (en) | 2013-03-15 | 2021-11-29 | Xencor, Inc. | Heterodimeric proteins |
US9260527B2 (en) | 2013-03-15 | 2016-02-16 | Sdix, Llc | Anti-human CXCR4 antibodies and methods of making same |
US10858417B2 (en) | 2013-03-15 | 2020-12-08 | Xencor, Inc. | Heterodimeric proteins |
SG10201800800YA (en) | 2013-05-06 | 2018-03-28 | Scholar Rock Inc | Compositions and methods for growth factor modulation |
US10005839B2 (en) | 2013-05-17 | 2018-06-26 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Antagonist of the BTLA/HVEM interaction for use in therapy |
PL3004167T3 (en) | 2013-05-30 | 2019-01-31 | Kiniksa Pharmaceuticals, Ltd. | Oncostatin m receptor antigen binding proteins |
CN111423511B (en) | 2013-05-31 | 2024-02-23 | 索伦托药业有限公司 | Antigen binding proteins that bind to PD-1 |
JP6267792B2 (en) | 2013-06-28 | 2018-01-24 | アムジエン・インコーポレーテツド | Methods for treating homozygous familial hypercholesterolemia |
AR097648A1 (en) | 2013-09-13 | 2016-04-06 | Amgen Inc | COMBINATION OF EPIGENETIC FACTORS AND BIESPECTIVE COMPOUNDS THAT HAVE LIKE DIANA CD33 AND CD3 IN THE TREATMENT OF MYELOID LEUKEMIA |
EP3757130A1 (en) | 2013-09-26 | 2020-12-30 | Costim Pharmaceuticals Inc. | Methods for treating hematologic cancers |
WO2015049355A1 (en) | 2013-10-04 | 2015-04-09 | Roche Diagnostics Gmbh | Antibodies specifically binding to her3 |
DK3055331T3 (en) | 2013-10-11 | 2021-03-22 | Oxford Bio Therapeutics Ltd | CONJUGATED ANTIBODIES TO LY75 FOR CANCER TREATMENT |
WO2015057583A1 (en) | 2013-10-14 | 2015-04-23 | The United States Of America, As Represented By The Secretary | Treatment of chronic kidney disease with sahps |
WO2015057908A1 (en) | 2013-10-18 | 2015-04-23 | Novartis Ag | Methods of treating diabetes and related disorders |
EP3063317B1 (en) | 2013-10-28 | 2020-06-03 | DOTS Technology Corp. | Allergen detection |
WO2015066550A1 (en) | 2013-10-31 | 2015-05-07 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
CN104623637A (en) | 2013-11-07 | 2015-05-20 | 健能隆医药技术(上海)有限公司 | Application of IL-22 dimer in preparation of intravenous injection drugs |
EP3083623A1 (en) | 2013-12-20 | 2016-10-26 | Gilead Calistoga LLC | Polymorphic forms of a hydrochloride salt of (s) -2-(9h-purin-6-ylamino) propyl) -5-fluoro-3-phenylquinazolin-4 (3h) -one |
NZ720867A (en) | 2013-12-20 | 2018-01-26 | Gilead Calistoga Llc | Process methods for phosphatidylinositol 3-kinase inhibitors |
AU2015204446A1 (en) | 2014-01-13 | 2016-07-14 | Valerion Therapeutics, Llc | Internalizing moieties |
RU2701434C2 (en) | 2014-01-24 | 2019-09-26 | Нгм Биофармасьютикалс, Инк. | Binding proteins and methods for use thereof |
CN105873949A (en) | 2014-01-31 | 2016-08-17 | 勃林格殷格翰国际有限公司 | Novel anti-BAFF antibodies |
EP4008726A1 (en) | 2014-02-20 | 2022-06-08 | Allergan, Inc. | Complement component c5 antibodies |
RU2018135371A (en) | 2014-02-27 | 2018-12-10 | Аллерган, Инк. | ANTIBODIES TO THE COMPLEX FACTOR Bb |
KR20160127817A (en) | 2014-03-07 | 2016-11-04 | 유니버시티 헬스 네트워크 | Methods and compositions for modifying the immune response |
AU2015229186B2 (en) | 2014-03-14 | 2021-01-28 | Biomolecular Holdings Llc | Hybrid immunoglobulin containing non-peptidyl linkage |
RS59907B1 (en) | 2014-03-28 | 2020-03-31 | Xencor Inc | Bispecific antibodies that bind to cd38 and cd3 |
US10544231B2 (en) | 2014-04-16 | 2020-01-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antibodies for the prevention or the treatment of bleeding episodes |
FR3020063A1 (en) | 2014-04-16 | 2015-10-23 | Gamamabs Pharma | ANTI-HER4 HUMAN ANTIBODY |
US9856306B2 (en) | 2014-05-28 | 2018-01-02 | Spitfire Pharma, Inc. | Peptide pharmaceuticals for insulin resistance |
CN106714830B (en) | 2014-05-30 | 2020-08-25 | 上海复宏汉霖生物技术股份有限公司 | anti-Epidermal Growth Factor Receptor (EGFR) antibodies |
US10106579B2 (en) | 2014-06-12 | 2018-10-23 | Ra Pharmaceuticals, Inc. | Modulation of complement activity |
US11021467B2 (en) | 2014-06-13 | 2021-06-01 | Gilead Sciences, Inc. | Phosphatidylinositol 3-kinase inhibitors |
JP6655074B2 (en) | 2014-06-20 | 2020-02-26 | ジェネンテック, インコーポレイテッド | Scugacin-based scaffold compositions, methods and uses |
AR101669A1 (en) | 2014-07-31 | 2017-01-04 | Amgen Res (Munich) Gmbh | ANTIBODY CONSTRUCTS FOR CDH19 AND CD3 |
TW201609811A (en) | 2014-07-31 | 2016-03-16 | 安美基研究(慕尼黑)公司 | Bispecific single chain antibody construct with enhanced tissue distribution |
MX2017001401A (en) | 2014-07-31 | 2017-08-07 | Amgen Res (Munich) Gmbh | Optimized cross-species specific bispecific single chain antibody constructs. |
AU2015305894A1 (en) | 2014-08-22 | 2017-04-06 | Sorrento Therapeutics, Inc. | Antigen binding proteins that bind CXCR3 |
AU2015306608B2 (en) | 2014-08-27 | 2020-03-05 | Amgen Inc. | Variants of tissue inhibitor of metalloproteinase type three (TIMP-3), compositions and methods |
US10323088B2 (en) | 2014-09-22 | 2019-06-18 | Intrinsic Lifesciences Llc | Humanized anti-hepcidin antibodies and uses thereof |
US20190194654A1 (en) | 2014-10-24 | 2019-06-27 | Astrazeneca Ab | Combination |
CA2967595A1 (en) | 2014-11-12 | 2016-05-19 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
US9879087B2 (en) | 2014-11-12 | 2018-01-30 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
AU2015349680A1 (en) | 2014-11-21 | 2017-06-08 | Northwestern University | The sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates |
CN116333153A (en) | 2014-11-26 | 2023-06-27 | 森科股份有限公司 | Heterodimeric antibodies that bind CD3 and tumor antigens |
US10259887B2 (en) | 2014-11-26 | 2019-04-16 | Xencor, Inc. | Heterodimeric antibodies that bind CD3 and tumor antigens |
KR20170084327A (en) | 2014-11-26 | 2017-07-19 | 젠코어 인코포레이티드 | Heterodimeric antibodies that bind cd3 and cd38 |
CN105669863B (en) | 2014-12-05 | 2019-09-13 | 鸿运华宁(杭州)生物医药有限公司 | It is a kind of can with human endothelin receptor specifically bind antibody and its application |
US11220545B2 (en) | 2014-12-08 | 2022-01-11 | Dana-Farber Cancer Institute, Inc. | Methods for upregulating immune responses using combinations of anti-RGMb and anti-PD-1 agents |
US11697825B2 (en) | 2014-12-12 | 2023-07-11 | Voyager Therapeutics, Inc. | Compositions and methods for the production of scAAV |
US10428155B2 (en) | 2014-12-22 | 2019-10-01 | Xencor, Inc. | Trispecific antibodies |
EP3916017A1 (en) | 2014-12-22 | 2021-12-01 | PD-1 Acquisition Group, LLC | Anti-pd-1 antibodies |
EP3240801B1 (en) | 2014-12-31 | 2021-01-20 | Checkmate Pharmaceuticals, Inc. | Combination tumor immunotherapy |
PL3247725T3 (en) | 2015-01-23 | 2021-01-11 | Sanofi | Anti-cd3 antibodies, anti-cd123 antibodies and bispecific antibodies specifically binding to cd3 and/or cd123 |
US9937222B2 (en) | 2015-01-28 | 2018-04-10 | Ra Pharmaceuticals, Inc. | Modulators of complement activity |
WO2016128349A1 (en) | 2015-02-09 | 2016-08-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antibodies specific to glycoprotein (gp) of ebolavirus and uses for the treatment and diagnosis of ebola virus infection |
AR103675A1 (en) | 2015-02-13 | 2017-05-24 | Sorrento Therapeutics Inc | ANTI-CTLA4 THERAPEUTIC ANTIBODIES |
WO2016138160A1 (en) | 2015-02-24 | 2016-09-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Middle east respiratory syndrome coronavirus immunogens, antibodies, and their use |
WO2016141387A1 (en) | 2015-03-05 | 2016-09-09 | Xencor, Inc. | Modulation of t cells with bispecific antibodies and fc fusions |
JP7068825B2 (en) | 2015-04-08 | 2022-05-17 | ソレント・セラピューティクス・インコーポレイテッド | Antibodies therapeutic agents that bind to CD38 |
CR20170510A (en) | 2015-04-10 | 2018-02-26 | Amgen Inc | INTERUQUINE MUTEINS 2 FOR THE EXPANSION OF REGULATORY T-CELLS |
WO2016164937A2 (en) | 2015-04-10 | 2016-10-13 | Amgen Inc. | Interleukin-2 muteins for the expansion of t-regulatory cells |
DK3283524T3 (en) | 2015-04-17 | 2023-05-30 | Amgen Res Munich Gmbh | BISPECIFIC ANTIBODY CONSTRUCTIONS AGAINST CDH3 and CD3 |
US20160347848A1 (en) | 2015-05-28 | 2016-12-01 | Medimmune Limited | Therapeutic combinations and methods for treating neoplasia |
CA2988588A1 (en) | 2015-06-12 | 2016-12-15 | Georgia State University Research Foundation, Inc. | Compositions and methods for treating opioid tolerance |
ES2828694T3 (en) | 2015-07-29 | 2021-05-27 | Allergan Inc | Heavy chain-only antibodies to ANG-2 |
TWI796283B (en) | 2015-07-31 | 2023-03-21 | 德商安美基研究(慕尼黑)公司 | Antibody constructs for msln and cd3 |
TW202346349A (en) | 2015-07-31 | 2023-12-01 | 德商安美基研究(慕尼黑)公司 | Antibody constructs for dll3 and cd3 |
TWI829617B (en) | 2015-07-31 | 2024-01-21 | 德商安美基研究(慕尼黑)公司 | Antibody constructs for flt3 and cd3 |
TWI717375B (en) | 2015-07-31 | 2021-02-01 | 德商安美基研究(慕尼黑)公司 | Antibody constructs for cd70 and cd3 |
TWI744242B (en) | 2015-07-31 | 2021-11-01 | 德商安美基研究(慕尼黑)公司 | Antibody constructs for egfrviii and cd3 |
US10308711B2 (en) | 2015-08-14 | 2019-06-04 | Allergan, Inc. | Heavy chain only antibodies to PDGF |
WO2017040566A1 (en) | 2015-09-01 | 2017-03-09 | Boehringer Ingelheim International Gmbh | Use of anti-cd40 antibodies for treatment of lupus nephritis |
TWI799366B (en) | 2015-09-15 | 2023-04-21 | 美商建南德克公司 | Cystine knot scaffold platform |
EA201890613A1 (en) | 2015-09-21 | 2018-10-31 | Аптево Рисёрч Энд Девелопмент Ллс | POLYPEPTIDES CONNECTING CD3 |
JP2018535655A (en) | 2015-09-29 | 2018-12-06 | アムジエン・インコーポレーテツド | ASGR inhibitor |
AU2016332725A1 (en) | 2015-09-29 | 2018-03-22 | Celgene Corporation | PD-1 binding proteins and methods of use thereof |
EP3355908A1 (en) | 2015-10-01 | 2018-08-08 | Amgen Inc. | Treatment of bile acid disorders |
EP3359575B1 (en) | 2015-10-09 | 2020-07-22 | Florida State University Research Foundation, Inc. | Antibodies specific for 4,6-diamino-5-(formylamino) pyrimidine and uses thereof |
EP3362074B1 (en) | 2015-10-16 | 2023-08-09 | President and Fellows of Harvard College | Regulatory t cell pd-1 modulation for regulating t cell effector immune responses |
KR20180088381A (en) | 2015-11-12 | 2018-08-03 | 시아맙 쎄라퓨틱스, 인코포레이티드 | Glycan-interacting compounds and methods of use |
KR20180093010A (en) | 2015-12-04 | 2018-08-20 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | New Cancer Therapeutic Antibodies |
US11623957B2 (en) | 2015-12-07 | 2023-04-11 | Xencor, Inc. | Heterodimeric antibodies that bind CD3 and PSMA |
TWI745320B (en) | 2015-12-16 | 2021-11-11 | 美商Ra製藥公司 | Modulators of complement activity |
CN116333124A (en) | 2016-01-29 | 2023-06-27 | 索伦托药业有限公司 | Antigen binding proteins that bind to PD-L1 |
MX2018009386A (en) | 2016-02-03 | 2018-11-09 | Amgen Res Munich Gmbh | Psma and cd3 bispecific t cell engaging antibody constructs. |
KR20180103084A (en) | 2016-02-03 | 2018-09-18 | 암젠 리서치 (뮌헨) 게엠베하 | BCMA and CD3 bispecific T cell engrafting antibody constructs |
EA039859B1 (en) | 2016-02-03 | 2022-03-21 | Эмджен Рисерч (Мюник) Гмбх | Bispecific antibody constructs binding egfrviii and cd3 |
JP7157981B2 (en) | 2016-03-07 | 2022-10-21 | チャールストンファーマ, エルエルシー | anti-nucleolin antibody |
CN109328069B (en) | 2016-04-15 | 2023-09-01 | 亿一生物医药开发(上海)有限公司 | Use of IL-22 in the treatment of necrotizing enterocolitis |
JOP20170091B1 (en) | 2016-04-19 | 2021-08-17 | Amgen Res Munich Gmbh | Administration of a bispecific construct binding to CD33 and CD3 for use in a method for the treatment of myeloid leukemia |
TWI826351B (en) | 2016-05-31 | 2023-12-21 | 大陸商鴻運華寧(杭州)生物醫藥有限公司 | R antibodies, their pharmaceutical compositions and uses |
RU2767357C2 (en) | 2016-06-14 | 2022-03-17 | Ксенкор, Инк. | Bispecific checkpoint inhibitors antibodies |
EP3475304B1 (en) | 2016-06-28 | 2022-03-23 | Xencor, Inc. | Heterodimeric antibodies that bind somatostatin receptor 2 |
MA45674A (en) | 2016-07-15 | 2019-05-22 | Takeda Pharmaceuticals Co | METHODS AND MATERIALS FOR EVALUATING A RESPONSE TO PLASMOBLAST AND PLASMOCYTE DEPLÉTION TREATMENTS |
TWI790206B (en) | 2016-07-18 | 2023-01-21 | 法商賽諾菲公司 | Bispecific antibody-like binding proteins specifically binding to cd3 and cd123 |
US10793632B2 (en) | 2016-08-30 | 2020-10-06 | Xencor, Inc. | Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors |
ES2965087T3 (en) | 2016-09-02 | 2024-04-10 | Soares Christopher J | Use of CGRP receptor antagonists in the treatment of glaucoma |
JP2019534858A (en) | 2016-09-09 | 2019-12-05 | ジェネンテック, インコーポレイテッド | Selective peptide inhibitor of FRIZZLED |
AU2017327828B2 (en) | 2016-09-16 | 2023-11-16 | Shanghai Henlius Biotech, Inc. | Anti-PD-1 antibodies |
WO2018053405A1 (en) | 2016-09-19 | 2018-03-22 | Celgene Corporation | Methods of treating immune disorders using pd-1 binding proteins |
JP2019534859A (en) | 2016-09-19 | 2019-12-05 | セルジーン コーポレイション | Method for treating vitiligo using PD-1 binding protein |
JP2019537621A (en) | 2016-10-04 | 2019-12-26 | フェアバンクス ファーマシューティカルズ,インコーポレイテッド | Anti-FSTL3 antibodies and uses thereof |
AU2017343752B2 (en) | 2016-10-14 | 2021-12-16 | Neomatrix Therapeutics, Inc | Peptides derived from fibronectin with improved bioactivity and reduced susceptibility to neutrophil elastase degradation |
US10550185B2 (en) | 2016-10-14 | 2020-02-04 | Xencor, Inc. | Bispecific heterodimeric fusion proteins containing IL-15-IL-15Rα Fc-fusion proteins and PD-1 antibody fragments |
TW202300515A (en) | 2016-10-20 | 2023-01-01 | 法商賽諾菲公司 | Anti-chikv antibodies and uses thereof |
WO2018081318A1 (en) | 2016-10-25 | 2018-05-03 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Prefusion coronavirus spike proteins and their use |
US11414482B2 (en) | 2016-11-08 | 2022-08-16 | University Of Miami | Anti-secretogranin III (SCG3) antibodies and uses thereof |
EP3541847A4 (en) | 2016-11-17 | 2020-07-08 | Seattle Genetics, Inc. | Glycan-interacting compounds and methods of use |
KR102580647B1 (en) | 2016-12-07 | 2023-09-20 | 몰레큘러 템플레이츠, 인코퍼레이션. | Shiga toxin A subunit effector polypeptides, Shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation |
CN110087668A (en) | 2016-12-07 | 2019-08-02 | Ra制药公司 | The regulator of complement activity |
AU2018210404B2 (en) | 2017-01-19 | 2022-01-27 | Novo Nordisk A/S | ApoC-II mimetic peptides |
JOP20190189A1 (en) | 2017-02-02 | 2019-08-01 | Amgen Res Munich Gmbh | Low ph pharmaceutical composition comprising t cell engaging antibody constructs |
WO2018152496A1 (en) | 2017-02-17 | 2018-08-23 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Compositions and methods for the diagnosis and treatment of zika virus infection |
MA47812A (en) | 2017-03-03 | 2021-04-14 | Seagen Inc | COMPOUNDS INTERACTING WITH GLYCAN AND METHODS OF USE |
GB201703876D0 (en) | 2017-03-10 | 2017-04-26 | Berlin-Chemie Ag | Pharmaceutical combinations |
CN110382544B (en) | 2017-03-16 | 2023-12-22 | 先天制药公司 | Compositions and methods for treating cancer |
WO2018183173A1 (en) | 2017-03-27 | 2018-10-04 | Boehringer Ingelheim International Gmbh | Anti il-36r antibodies combination therapy |
US10729741B2 (en) | 2017-03-27 | 2020-08-04 | Neomatrix Therapeutics Inc. | Methods of treating burns with i.v. cP12 in a window from 2 to 6 hours after injury |
WO2018200742A1 (en) | 2017-04-25 | 2018-11-01 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Antibodies and methods for the diagnosis and treatment of epstein barr virus infection |
UY37726A (en) | 2017-05-05 | 2018-11-30 | Amgen Inc | PHARMACEUTICAL COMPOSITION THAT INCLUDES BISPECTIFIC ANTIBODY CONSTRUCTIONS FOR IMPROVED STORAGE AND ADMINISTRATION |
US10793634B2 (en) | 2017-06-09 | 2020-10-06 | Boehringer Ingelheim International Gmbh | Anti-TrkB antibodies |
WO2019006472A1 (en) | 2017-06-30 | 2019-01-03 | Xencor, Inc. | Targeted heterodimeric fc fusion proteins containing il-15/il-15ra and antigen binding domains |
EP3648788A1 (en) | 2017-07-07 | 2020-05-13 | Baxalta Incorporated | Treatment of gastrointestinal bleeding in patients with severe von willebrand disease by administration of recombinant vwf |
BR112020000322A2 (en) | 2017-07-07 | 2020-07-14 | Baxalta Incorporated | use of recombinant von willebrand factor (rvwf) |
EP3655430A1 (en) | 2017-07-19 | 2020-05-27 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection |
CA3073537A1 (en) | 2017-08-22 | 2019-02-28 | Sanabio, Llc | Soluble interferon receptors and uses thereof |
EP3704154A1 (en) | 2017-11-02 | 2020-09-09 | Oxford BioTherapeutics Ltd | Antibodies and methods of use |
US10981992B2 (en) | 2017-11-08 | 2021-04-20 | Xencor, Inc. | Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors |
CA3082383A1 (en) | 2017-11-08 | 2019-05-16 | Xencor, Inc. | Bispecific and monospecific antibodies using novel anti-pd-1 sequences |
MX2020004933A (en) | 2017-11-14 | 2021-01-08 | Arcellx Inc | D-domain containing polypeptides and uses thereof. |
EP3724229A1 (en) | 2017-12-11 | 2020-10-21 | Amgen Inc. | Continuous manufacturing process for bispecific antibody products |
CN111655718A (en) | 2017-12-19 | 2020-09-11 | Xencor股份有限公司 | Engineered IL-2 FC fusion proteins |
UY38041A (en) | 2017-12-29 | 2019-06-28 | Amgen Inc | CONSTRUCTION OF BIESPECFIC ANTIBODY DIRECTED TO MUC17 AND CD3 |
EP3732193A1 (en) | 2017-12-29 | 2020-11-04 | Alector LLC | Anti-tmem106b antibodies and methods of use thereof |
WO2019136158A1 (en) | 2018-01-03 | 2019-07-11 | Spitfire Pharma, Inc. | Improved peptide pharmaceuticals for treatment of nash and other disorders |
KR20200115546A (en) | 2018-01-26 | 2020-10-07 | 제넨테크, 인크. | IL-22 Fc fusion protein and method of use |
BR112020015016A2 (en) | 2018-01-26 | 2020-12-29 | Genentech, Inc. | PHARMACEUTICAL COMPOSITIONS, METHODS OF TREATMENT OF INFLAMMATORY INTESTINAL DISEASE, INHIBITION OF MICROBIAL INFECTION IN THE INTESTINE AND ACCELERATING OR IMPROVING WOUND HEALING |
EP3746476A1 (en) | 2018-01-31 | 2020-12-09 | Alector LLC | Anti-ms4a4a antibodies and methods of use thereof |
BR112020016400A2 (en) | 2018-02-14 | 2020-12-15 | Viela Bio, Inc. | ANTIBODIES FOR THYROSINE KINASE 3 RECEPTOR BINDER SIMILAR TO MCDONOUGH FELINE SARCOMA (FMS) (FLT3L) AND THEIR USES FOR THE TREATMENT OF AUTOIMMUNE AND INFLAMMATORY DISEASES |
AU2019226085A1 (en) | 2018-02-21 | 2020-09-17 | Genentech, Inc. | Dosing for treatment with IL-22 Fc fusion proteins |
US11485782B2 (en) | 2018-03-14 | 2022-11-01 | Beijing Xuanyi Pharmasciences Co., Ltd. | Anti-claudin 18.2 antibodies |
CN117126279A (en) | 2018-03-20 | 2023-11-28 | 鸿运华宁(杭州)生物医药有限公司 | GIPR antibody and fusion protein of GIPR antibody and GLP-1, and pharmaceutical composition and application thereof |
SG11202009213TA (en) | 2018-03-21 | 2020-10-29 | Baxalta Inc | Separation of vwf and vwf propeptide by chromatographic methods |
US10982006B2 (en) | 2018-04-04 | 2021-04-20 | Xencor, Inc. | Heterodimeric antibodies that bind fibroblast activation protein |
WO2019195561A2 (en) | 2018-04-06 | 2019-10-10 | BioLegend, Inc. | Anti-tetraspanin 33 agents and compositions and methods for making and using the same |
CN110357959B (en) | 2018-04-10 | 2023-02-28 | 鸿运华宁(杭州)生物医药有限公司 | GCGR antibody, fusion protein of GCGR antibody and GLP-1, and pharmaceutical composition and application of GCGR antibody and fusion protein |
WO2019204655A1 (en) | 2018-04-18 | 2019-10-24 | Xencor, Inc. | Tim-3 targeted heterodimeric fusion proteins containing il-15/il-15ra fc-fusion proteins and tim-3 antigen binding domains |
JP2021521784A (en) | 2018-04-18 | 2021-08-30 | ゼンコア インコーポレイテッド | PD-1 targeted heterodimer fusion proteins containing IL-15 / IL-15RaFc fusion proteins and PD-1 antigen binding domains and their use |
JP2021522286A (en) | 2018-04-30 | 2021-08-30 | メディミューン リミテッド | Conjugate to target and clear aggregates |
PE20210419A1 (en) | 2018-04-30 | 2021-03-08 | Takeda Pharmaceuticals Co | CANNABINOID TYPE 1 (CB1) RECEPTOR BINDING PROTEINS AND USES OF THEM |
EP3788071A1 (en) | 2018-05-02 | 2021-03-10 | The United States Of America, As Represented By The Secretary, Department of Health and Human Services | Antibodies and methods for the diagnosis, prevention, and treatment of epstein barr virus infection |
UA128113C2 (en) | 2018-05-25 | 2024-04-10 | ЕЛЕКТОР ЕлЕлСі | Anti-sirpa antibodies and methods of use thereof |
CN110655577A (en) | 2018-06-13 | 2020-01-07 | 鸿运华宁(杭州)生物医药有限公司 | APJ antibody and fusion protein thereof with Elabela, and pharmaceutical composition and application thereof |
GB201809746D0 (en) | 2018-06-14 | 2018-08-01 | Berlin Chemie Ag | Pharmaceutical combinations |
BR112020024412A8 (en) | 2018-06-18 | 2023-03-21 | Innate Pharma | ANTIBODIES, PHARMACEUTICAL COMPOSITION, KIT, NUCLEIC ACID, HOST CELL, METHODS OF TREATMENT OR PREVENTION OF CANCER, OF REDUCING ACTIVITY, OF INCREASE OF ACTIVITY AND OF INCREASE OF ACTIVATION |
AU2019291890A1 (en) | 2018-06-29 | 2020-12-17 | Boehringer Ingelheim International Gmbh | Anti-CD40 antibodies for use in treating autoimmune disease |
CA3099176A1 (en) | 2018-06-29 | 2020-01-02 | Alector Llc | Anti-sirp-beta1 antibodies and methods of use thereof |
JOP20200343A1 (en) | 2018-07-02 | 2020-12-31 | Xencor Inc | Anti-steap1 antigen-binding protein |
LT3618928T (en) | 2018-07-13 | 2023-04-11 | Alector Llc | Anti-sortilin antibodies and methods of use thereof |
WO2020021061A1 (en) | 2018-07-26 | 2020-01-30 | Pieris Pharmaceuticals Gmbh | Humanized anti-pd-1 antibodies and uses thereof |
WO2020025532A1 (en) | 2018-07-30 | 2020-02-06 | Amgen Research (Munich) Gmbh | Prolonged administration of a bispecific antibody construct binding to cd33 and cd3 |
CA3107192A1 (en) | 2018-08-03 | 2020-02-06 | Amgen Research (Munich) Gmbh | Antibody constructs for cldn18.2 and cd3 |
JP2021534769A (en) | 2018-08-31 | 2021-12-16 | エーエルエックス オンコロジー インコーポレイテッド | Decoy polypeptide |
CN113164597A (en) | 2018-09-24 | 2021-07-23 | 爱尔皮奥制药公司 | Multispecific antibodies targeting HPTP-beta (VE-PTP) and VEGF |
CN113365697A (en) | 2018-09-25 | 2021-09-07 | 百进生物科技公司 | anti-TLR9 agents and compositions and methods of making and using the same |
CA3114295A1 (en) | 2018-09-28 | 2020-04-02 | Kyowa Kirin Co., Ltd. | Il-36 antibodies and uses thereof |
SG11202103192RA (en) | 2018-10-03 | 2021-04-29 | Xencor Inc | Il-12 heterodimeric fc-fusion proteins |
CA3114802A1 (en) | 2018-10-11 | 2020-04-16 | Amgen Inc. | Downstream processing of bispecific antibody constructs |
KR20210113261A (en) | 2019-01-04 | 2021-09-15 | 리졸브 테라퓨틱스, 엘엘씨 | Treatment of Sjogren's Disease Using Nuclease Fusion Proteins |
CN116063520A (en) | 2019-01-30 | 2023-05-05 | 真和制药有限公司 | anti-GAL 3 antibodies and uses thereof |
MX2021009114A (en) | 2019-02-01 | 2021-10-13 | Takeda Pharmaceuticals Co | Methods of prophylactic treatment using recombinant vwf (rvwf). |
EP3693023A1 (en) | 2019-02-11 | 2020-08-12 | Sanofi | Use of anti-ceacam5 immunoconjugates for treating lung cancer |
MX2021009514A (en) | 2019-02-07 | 2021-11-04 | Sanofi Sa | Use of anti-ceacam5 immunoconjugates for treating lung cancer. |
KR20210134725A (en) | 2019-03-01 | 2021-11-10 | 젠코어 인코포레이티드 | Heterodimeric Antibodies that Bind to ENPP3 and CD3 |
AU2020248645A1 (en) | 2019-03-27 | 2021-10-28 | Tigatx, Inc. | Engineered IgA antibodies and methods of use |
CN114040800A (en) | 2019-04-09 | 2022-02-11 | 阿波科有限责任公司 | Killer cell lectin-like receptor subfamily G member 1(KLRG1) depleting antibodies |
CA3130449A1 (en) | 2019-04-30 | 2020-11-05 | Gigagen, Inc. | Recombinant polyclonal proteins and methods of use thereof |
CA3137377A1 (en) | 2019-05-09 | 2020-11-12 | Boehringer Ingelheim International Gmbh | Anti-sema3a antibodies and their uses for treating eye or ocular diseases |
TW202045711A (en) | 2019-06-13 | 2020-12-16 | 美商安進公司 | Automated biomass-based perfusion control in the manufacturing of biologics |
TW202115112A (en) | 2019-06-27 | 2021-04-16 | 德商百靈佳殷格翰國際股份有限公司 | Anti-angpt2 antibodies |
BR112021024997A2 (en) | 2019-07-03 | 2022-01-25 | Oxford Biotherapeutics Ltd | Antibodies, polynucleotide, expression vector, host cell, pharmaceutical composition, methods and use |
CN112239507A (en) | 2019-07-17 | 2021-01-19 | 鸿运华宁(杭州)生物医药有限公司 | Fusion protein of ETA antibody and TGF-beta Trap, and pharmaceutical composition and application thereof |
CN112300279A (en) | 2019-07-26 | 2021-02-02 | 上海复宏汉霖生物技术股份有限公司 | Methods and compositions directed to anti-CD 73 antibodies and variants |
KR20220058540A (en) | 2019-07-31 | 2022-05-09 | 알렉터 엘엘씨 | Anti-MS4A4A antibodies and methods of use thereof |
US20210032370A1 (en) | 2019-08-02 | 2021-02-04 | Immatics Biotechnologies Gmbh | Recruiting agent further binding an mhc molecule |
DE102019121007A1 (en) | 2019-08-02 | 2021-02-04 | Immatics Biotechnologies Gmbh | Antigen binding proteins that specifically bind to MAGE-A |
US20220281967A1 (en) | 2019-08-02 | 2022-09-08 | Orega Biotech | Novel il-17b antibodies |
US20220289807A1 (en) | 2019-08-13 | 2022-09-15 | Amgen Inc. | Interleukin-2 muteins for the expansion of t-regulatory cells |
MX2022002981A (en) | 2019-09-10 | 2022-04-06 | Amgen Inc | Purification method for bispecific antigen-binding polypeptides with enhanced protein l capture dynamic binding capacity. |
JP2022547556A (en) | 2019-09-11 | 2022-11-14 | 武田薬品工業株式会社 | Therapies involving the complex of von Willebrand factor and complement C1Q |
CN112521501A (en) | 2019-09-18 | 2021-03-19 | 鸿运华宁(杭州)生物医药有限公司 | GIPR antibody and fusion protein thereof with GLP-1, and pharmaceutical composition and application thereof |
TW202126685A (en) | 2019-09-24 | 2021-07-16 | 德商百靈佳殷格翰國際股份有限公司 | Anti-nrp1a antibodies and their uses for treating eye or ocular diseases |
CN114667298A (en) | 2019-11-04 | 2022-06-24 | 艾利妥 | SIGLEC-9ECD fusion molecules and methods of use thereof |
AU2020381536A1 (en) | 2019-11-13 | 2022-04-21 | Amgen Inc. | Method for reduced aggregate formation in downstream processing of bispecific antigen-binding molecules |
AU2020397416A1 (en) | 2019-12-03 | 2022-07-14 | Evotec International Gmbh | Interferon-associated antigen binding proteins for use in treating hepatitis B infection |
CN115052625A (en) | 2019-12-03 | 2022-09-13 | 埃沃特克国际有限责任公司 | Interferon-associated antigen binding proteins and uses thereof |
JP2023505256A (en) | 2019-12-05 | 2023-02-08 | ソレント・セラピューティクス・インコーポレイテッド | Compositions and methods comprising anti-CD47 antibodies in combination with tumor-targeting antibodies |
CR20220329A (en) | 2019-12-13 | 2022-11-23 | Alector Llc | Anti-mertk antibodies and methods of use thereof |
IL293471A (en) | 2019-12-17 | 2022-08-01 | Amgen Inc | Dual interleukin-2 /tnf receptor agonist for use in therapy |
EP4090365A1 (en) | 2020-01-15 | 2022-11-23 | Immatics Biotechnologies GmbH | Antigen binding proteins specifically binding prame |
CN115427447A (en) | 2020-01-17 | 2022-12-02 | 百进生物科技公司 | anti-TLR 7 agents and compositions and methods of making and using the same |
US20230093169A1 (en) | 2020-01-22 | 2023-03-23 | Amgen Research (Munch) Gmbh | Combinations of antibody constructs and inhibitors of cytokine release syndrome and uses thereof |
AU2021216945A1 (en) | 2020-02-04 | 2022-09-01 | Takeda Pharmaceutical Company Limited | Treatment of menorrhagia in patients with severe von Willebrand Disease by administration of recombinant VWF |
US20230183377A1 (en) | 2020-02-26 | 2023-06-15 | Sorrento Therapeutics, Inc. | Activatable antigen binding proteins with universal masking moieties |
EP4118113A1 (en) | 2020-03-12 | 2023-01-18 | Amgen Inc. | Method for treatment and prophylaxis of crs in patients comprising a combination of bispecifc antibodies binding to cds x cancer cell and tnfalpha or il-6 inhibitor |
WO2021195089A1 (en) | 2020-03-23 | 2021-09-30 | Sorrento Therapeutics, Inc. | Fc-coronavirus antigen fusion proteins, and nucleic acids, vectors, compositions and methods of use thereof |
CN116075525A (en) | 2020-03-31 | 2023-05-05 | 艾莱克特有限责任公司 | anti-MERTK antibodies and methods of use thereof |
WO2021207662A1 (en) | 2020-04-10 | 2021-10-14 | Genentech, Inc. | Use of il-22fc for the treatment or prevention of pneumonia, acute respiratory distress syndrome, or cytokine release syndrome |
CA3180222A1 (en) | 2020-04-15 | 2021-10-21 | Voyager Therapeutics, Inc. | Tau binding compounds |
EP4138884A1 (en) | 2020-04-20 | 2023-03-01 | Sorrento Therapeutics, Inc. | Pulmonary administration of ace2 polypeptides |
WO2021231976A1 (en) | 2020-05-14 | 2021-11-18 | Xencor, Inc. | Heterodimeric antibodies that bind prostate specific membrane antigen (psma) and cd3 |
AU2021275049A1 (en) | 2020-05-19 | 2022-12-22 | Amgen Inc. | MAGEB2 binding constructs |
CA3181776A1 (en) | 2020-05-26 | 2021-12-02 | Boehringer Ingelheim International Gmbh | Anti-pd-1 antibodies |
JP2023527972A (en) | 2020-05-29 | 2023-07-03 | アムジエン・インコーポレーテツド | Reduced Adverse Effect Administration of Bispecific Constructs that Bind CD33 and CD3 |
CA3177152A1 (en) | 2020-06-12 | 2021-12-16 | David Scott Johnson | Recombinant polyclonal proteins targeting covid-19 and methods of use thereof |
WO2021259227A1 (en) | 2020-06-23 | 2021-12-30 | 江苏康缘药业股份有限公司 | Anti-cd38 antibody and use thereof |
EP4172207A1 (en) | 2020-06-26 | 2023-05-03 | Sorrento Therapeutics, Inc. | Anti-pd1 antibodies and uses thereof |
CA3165342A1 (en) | 2020-06-29 | 2022-01-06 | James Arthur Posada | Treatment of sjogren's syndrome with nuclease fusion proteins |
WO2022031834A1 (en) | 2020-08-05 | 2022-02-10 | Gigagen, Inc. | Recombinant polyclonal proteins targeting zika and methods of use thereof |
KR20230062600A (en) | 2020-09-04 | 2023-05-09 | 메르크 파텐트 게엠베하 | Anti-CEACAM5 Antibodies and Conjugates and Uses Thereof |
US20220089759A1 (en) | 2020-09-21 | 2022-03-24 | Boehringer Ingelheim International Gmbh | Use of anti-cd40 antibodies for treatment of inflammatory conditions |
WO2022081870A1 (en) | 2020-10-14 | 2022-04-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Stabilized norovirus virus-like particles as vaccine immunogens |
US20220127344A1 (en) | 2020-10-23 | 2022-04-28 | Boehringer Ingelheim International Gmbh | Anti-sema3a antibodies and their uses for treating a thrombotic disease of the retina |
WO2022093641A1 (en) | 2020-10-30 | 2022-05-05 | BioLegend, Inc. | Anti-nkg2a agents and compositions and methods for making and using the same |
WO2022093640A1 (en) | 2020-10-30 | 2022-05-05 | BioLegend, Inc. | Anti-nkg2c agents and compositions and methods for making and using the same |
CN116635421A (en) | 2020-11-06 | 2023-08-22 | 安进公司 | Polypeptide constructs that bind CD3 |
KR20230098335A (en) | 2020-11-06 | 2023-07-03 | 암젠 인크 | Antigen binding domains with reduced clipping ratio |
UY39508A (en) | 2020-11-06 | 2022-05-31 | Amgen Res Munich Gmbh | BSPECIFIC ANTIGEN-BINDING MOLECULES WITH MULTIPLE TARGETS OF ENHANCED SELECTIVITY |
BR112023008629A2 (en) | 2020-11-06 | 2023-10-03 | Amgen Inc | POLYPEPTIDE CONSTRUCTS THAT SELECTIVELY BIND CLDN6 AND CD3 |
WO2022132923A1 (en) | 2020-12-16 | 2022-06-23 | Voyager Therapeutics, Inc. | Tau binding compounds |
WO2022147463A2 (en) | 2020-12-31 | 2022-07-07 | Alamar Biosciences, Inc. | Binder molecules with high affinity and/ or specificity and methods of making and use thereof |
JP2024504696A (en) | 2021-01-20 | 2024-02-01 | バイオアントレ エルエルシー | CTLA4-binding proteins and methods of treating cancer |
EP4288457A2 (en) | 2021-02-05 | 2023-12-13 | Boehringer Ingelheim International GmbH | Anti-il1rap antibodies |
IL305736A (en) | 2021-03-09 | 2023-11-01 | Xencor Inc | Heterodimeric antibodies that bind cd3 and cldn6 |
EP4305065A1 (en) | 2021-03-10 | 2024-01-17 | Xencor, Inc. | Heterodimeric antibodies that bind cd3 and gpc3 |
JP2024510435A (en) | 2021-03-18 | 2024-03-07 | シージェン インコーポレイテッド | Selective drug release from internalization complexes of bioactive compounds |
JP2024512002A (en) | 2021-03-18 | 2024-03-18 | アレクトル エルエルシー | Anti-TMEM106B antibody and method of use thereof |
WO2022204274A1 (en) | 2021-03-23 | 2022-09-29 | Alector Llc | Anti-tmem106b antibodies for treating and preventing coronavirus infections |
WO2022204529A1 (en) | 2021-03-26 | 2022-09-29 | Abcuro, Inc. | Anti-klrg1 antibodies |
BR112023019512A2 (en) | 2021-03-26 | 2023-10-31 | Abcuro Inc | ANTI-KLRG1 ANTIBODIES |
CN115141276A (en) | 2021-03-31 | 2022-10-04 | 鸿运华宁(杭州)生物医药有限公司 | Antibody capable of being specifically combined with human endothelin receptor and application thereof in treatment of diabetic nephropathy and chronic nephropathy |
WO2022212836A1 (en) | 2021-04-01 | 2022-10-06 | Pyxis Oncology, Inc. | Gpnmb antibodies and methods of use |
CA3215594A1 (en) | 2021-04-02 | 2022-10-06 | Agnieszka KIELCZEWSKA | Mageb2 binding constructs |
CN117279947A (en) | 2021-05-06 | 2023-12-22 | 安进研发(慕尼黑)股份有限公司 | Antigen binding molecules targeting CD20 and CD22 for use in proliferative diseases |
JP7436711B2 (en) | 2021-06-04 | 2024-02-22 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | Anti-SIRP-alpha antibody |
WO2022261183A2 (en) | 2021-06-08 | 2022-12-15 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating and/or identifying an agent for treating intestinal cancers |
EP4351732A1 (en) | 2021-06-09 | 2024-04-17 | Evotec International GmbH | Interferon-associated antigen binding proteins for use for the treatment or prevention of coronavirus infection |
EP4355783A1 (en) | 2021-06-16 | 2024-04-24 | Alector LLC | Monovalent anti-mertk antibodies and methods of use thereof |
CN117642426A (en) | 2021-06-16 | 2024-03-01 | 艾莱克特有限责任公司 | Bispecific anti-MerTK and anti-PDL 1 antibodies and methods of use thereof |
AU2022320627A1 (en) | 2021-07-26 | 2024-02-08 | Abcuro, Inc. | Killer cell lectin-like receptor subfamily g member 1 (klrg1) depleting antibodies |
WO2023069919A1 (en) | 2021-10-19 | 2023-04-27 | Alector Llc | Anti-cd300lb antibodies and methods of use thereof |
WO2023097119A2 (en) | 2021-11-29 | 2023-06-01 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to modulate riok2 |
WO2023099682A1 (en) | 2021-12-02 | 2023-06-08 | Sanofi | Ceacam5 adc–anti-pd1/pd-l1 combination therapy |
TW202339804A (en) | 2021-12-02 | 2023-10-16 | 法商賽諾菲公司 | Cea assay for patient selection in cancer therapy |
AR128065A1 (en) | 2021-12-22 | 2024-03-20 | Cdr Life Ag | ANTI-C3 ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS THEREOF AND THEIR USES TO TREAT OPHTHALMIC OR EYE DISEASES |
WO2023131901A1 (en) | 2022-01-07 | 2023-07-13 | Johnson & Johnson Enterprise Innovation Inc. | Materials and methods of il-1beta binding proteins |
WO2023172968A1 (en) | 2022-03-09 | 2023-09-14 | Merck Patent Gmbh | Anti-gd2 antibodies, immunoconjugates and therapeutic uses thereof |
WO2023170239A1 (en) | 2022-03-09 | 2023-09-14 | Merck Patent Gmbh | Methods and tools for conjugation to antibodies |
TW202346368A (en) | 2022-05-12 | 2023-12-01 | 德商安美基研究(慕尼黑)公司 | Multichain multitargeting bispecific antigen-binding molecules of increased selectivity |
WO2023240287A1 (en) | 2022-06-10 | 2023-12-14 | Bioentre Llc | Combinations of ctla4 binding proteins and methods of treating cancer |
WO2023250388A1 (en) | 2022-06-22 | 2023-12-28 | Voyager Therapeutics, Inc. | Tau binding compounds |
WO2024013727A1 (en) | 2022-07-15 | 2024-01-18 | Janssen Biotech, Inc. | Material and methods for improved bioengineered pairing of antigen-binding variable regions |
WO2024020051A1 (en) | 2022-07-19 | 2024-01-25 | BioLegend, Inc. | Anti-cd157 antibodies, antigen-binding fragments thereof and compositions and methods for making and using the same |
WO2024026447A1 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Anti-gpnmb antibodies and methods of use thereof |
WO2024040114A2 (en) | 2022-08-18 | 2024-02-22 | BioLegend, Inc. | Anti-axl antibodies, antigen-binding fragments thereof and methods for making and using the same |
WO2024059675A2 (en) | 2022-09-14 | 2024-03-21 | Amgen Inc. | Bispecific molecule stabilizing composition |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
SU1022988A1 (en) * | 1979-09-28 | 1983-06-15 | Всесоюзный кардиологический научный центр АМН СССР | Stabilized urokinase having trombolite activity and method of producing same |
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1983
- 1983-10-28 US US06/546,590 patent/US4640835A/en not_active Expired - Fee Related
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