CA1294088C - Chemically modified protein - Google Patents
Chemically modified proteinInfo
- Publication number
- CA1294088C CA1294088C CA000540507A CA540507A CA1294088C CA 1294088 C CA1294088 C CA 1294088C CA 000540507 A CA000540507 A CA 000540507A CA 540507 A CA540507 A CA 540507A CA 1294088 C CA1294088 C CA 1294088C
- Authority
- CA
- Canada
- Prior art keywords
- protein
- chemically modified
- polyethylene glycol
- islet
- modified protein
- 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 - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
ABSTRACT OF THE DISCLOSURE
An islet-activating protein produced by bacteria belonging to the genus Bordetella is chemically modified with p~lyethylene glycol moiety of the formula:
-O-(-CH2CH2O-)?R
(wherein R is a protective group for hydroxyl and ? is an integer of about 7 to 700), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein.
The chemically modified protein has a strong islet-activating activity and has lower side effects than non-modified IAP, so that it may be employed as a preventive and therapeutic drug for diabetes.
An islet-activating protein produced by bacteria belonging to the genus Bordetella is chemically modified with p~lyethylene glycol moiety of the formula:
-O-(-CH2CH2O-)?R
(wherein R is a protective group for hydroxyl and ? is an integer of about 7 to 700), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein.
The chemically modified protein has a strong islet-activating activity and has lower side effects than non-modified IAP, so that it may be employed as a preventive and therapeutic drug for diabetes.
Description
This application is closely rela ted to Canadian Applications Serial No. 9 475, 743 and 531, 453 both commonly assigned to the applicant.
The present invention relates to a chemically modified protein.
Pertussis toxin, a bacterial toxin, is produced specifically by bacteria belonging to the genus Bor,detel,la, microorganisms causative of pertussis, a disease specific to humans.
It is a well-known fact that pertussis toxin exhibits various biological actions; for example, it acts as a histamine-sensitizing factor (ESF), a leukocytosis (lymphocytosis)-promoting factor (LPF), a hemagglutinin (HA), a mouse protective antigen (MPA) and an islet-activating protein (IAP).
Recent biological and protein-chemical researches have shed light on the - 10 nature of the above-mentioned factors; I,PF has been found to be identical with IAP [Pittman, M.; Review of Infectious Disease, 1, 401- 412 (1979)].
The above-mentioned islet-activating protein (hereinafter also referred toasIAP)hasa pharmacologialaetivity which promotes insulin secretion in mammals while maintaining normal blood glucose levels for a lcllg period; it is thought useful as a therapeutic and prevel;ltive drug for diabetes mellitus.
s a result of investigation, its production method and physico-chemical properties are already known [Yajima, M.; Journal of Biochemistry, 83, 295 -303 (1978)].
:
.
The present invention relates to a chemically modified protein.
Pertussis toxin, a bacterial toxin, is produced specifically by bacteria belonging to the genus Bor,detel,la, microorganisms causative of pertussis, a disease specific to humans.
It is a well-known fact that pertussis toxin exhibits various biological actions; for example, it acts as a histamine-sensitizing factor (ESF), a leukocytosis (lymphocytosis)-promoting factor (LPF), a hemagglutinin (HA), a mouse protective antigen (MPA) and an islet-activating protein (IAP).
Recent biological and protein-chemical researches have shed light on the - 10 nature of the above-mentioned factors; I,PF has been found to be identical with IAP [Pittman, M.; Review of Infectious Disease, 1, 401- 412 (1979)].
The above-mentioned islet-activating protein (hereinafter also referred toasIAP)hasa pharmacologialaetivity which promotes insulin secretion in mammals while maintaining normal blood glucose levels for a lcllg period; it is thought useful as a therapeutic and prevel;ltive drug for diabetes mellitus.
s a result of investigation, its production method and physico-chemical properties are already known [Yajima, M.; Journal of Biochemistry, 83, 295 -303 (1978)].
:
.
On the other hand, IAP has side effects such as leukocytosis promotion, histamine sensitization and hemagglutin-ation; as a heteroprotein of microbial origin, it also has immunogenicity. It is therefore desired that a substance having islet-activaking activity alone with slight or no side effects is developed.
For the above-mentioned purpose, attempts have been made to chemically modify IAP[Nogimori,K.et al.;Biochemische Biophysis-che Acta,801,~20-231(198~) and 801,232-243(1984)]; however, the 10 attenuation of side effects, specifically the reduction of antigenicity or immunogenicity, cannot be expected.
The purpose of the present invention is to produce IAP
derivatives having islet-activating activity with attenuated side effects.
The present invention provides a chemically modified protein, which comprises an islet-activating protein produced by ;~ bacteria belonging to the genus Bordetella and a polyethylene glycol moiety, the polyethylene glycol moiety being bound to a primary amino group of the isle-t-activating protein.
Any IAP produced by the bacteria belonging to the Bordetella, whether purified or semi-purified, can be used as long as it has insulin secretion promoting activity. Examples of such IAP products are those described in the above-mentioned references on IAP production and those produced as directed in the reference example below.
;:;
For the above-mentioned purpose, attempts have been made to chemically modify IAP[Nogimori,K.et al.;Biochemische Biophysis-che Acta,801,~20-231(198~) and 801,232-243(1984)]; however, the 10 attenuation of side effects, specifically the reduction of antigenicity or immunogenicity, cannot be expected.
The purpose of the present invention is to produce IAP
derivatives having islet-activating activity with attenuated side effects.
The present invention provides a chemically modified protein, which comprises an islet-activating protein produced by ;~ bacteria belonging to the genus Bordetella and a polyethylene glycol moiety, the polyethylene glycol moiety being bound to a primary amino group of the isle-t-activating protein.
Any IAP produced by the bacteria belonging to the Bordetella, whether purified or semi-purified, can be used as long as it has insulin secretion promoting activity. Examples of such IAP products are those described in the above-mentioned references on IAP production and those produced as directed in the reference example below.
;:;
Primary amino groups of the islet-activating proteins which can be used include ~-amino gxoups of lysine and primary amino groups at the amino terminal of the protein.
The above-mentioned binding polyethylene glycol residue can be represented by the formula - -~CH2cH2 ~ R wherein R is a protective group for hydro~yl and Q is an integer of about 7 to 700.
As for the chemically modified protein of the present invention, it is preferable that the above-mentioned primary amino group binds to the above-mentioned polyethylene glycol via a spacer.
Any spacer can be used, as long as it binds the above-. mentioned primary amino groups to the polyethylene glycol moiety.
As examples of such spacers, mention may be made of i) groups (triazines), ii) groups of the formula ~CH2 )mN ~
wherein m is an integer from 1 to 3 (alkylenes) and iii) groups of the formula - C -~-CH2-)n wherein n is an integer from 1 NH
to 3 (imidates).
When the spacer is a triazine, the chemically modified portein may have 1 or 2 polyethylene glycol moieties per protein molecule, though the latter number is preferred. When the spacer is an alkylene or imidate, it is preferable that _ or n is the integer 2.
:`
The above-mentioned binding polyethylene glycol residue can be represented by the formula - -~CH2cH2 ~ R wherein R is a protective group for hydro~yl and Q is an integer of about 7 to 700.
As for the chemically modified protein of the present invention, it is preferable that the above-mentioned primary amino group binds to the above-mentioned polyethylene glycol via a spacer.
Any spacer can be used, as long as it binds the above-. mentioned primary amino groups to the polyethylene glycol moiety.
As examples of such spacers, mention may be made of i) groups (triazines), ii) groups of the formula ~CH2 )mN ~
wherein m is an integer from 1 to 3 (alkylenes) and iii) groups of the formula - C -~-CH2-)n wherein n is an integer from 1 NH
to 3 (imidates).
When the spacer is a triazine, the chemically modified portein may have 1 or 2 polyethylene glycol moieties per protein molecule, though the latter number is preferred. When the spacer is an alkylene or imidate, it is preferable that _ or n is the integer 2.
:`
It is recommended that the chemically modified protein of the present invention has IAP molecules to which 1 to 30, preferably 1 to 10 polyethylene glycol molecules or spacers having such polyethylene glycols are bound. It is also recommend-ed that about 3 to 80%, preferably about 3 to 40~, of the primary amino groups in the IAP molecule are bound to the above-mentioned polyethylene glycol moieties, or spacers having the polyethylene glycol moieties.
Groups which can be used for R as protective groups for hydroxyl in the above-mentioned polyethylene glycol include lower (e.g. 1 to 3 carbon atoms) alkyls and lower (e.g. 1 to 3 carbon atoms) alkanoyls. As examples of such alkyls, mention may be made of methyl, ethyl, propyl and isoprop,,vl; and as examples of such alkanoyls, formyl, acetyl and propionyl. As the protective groups for hydroxyl, methyl is preferred. It is preferable that Q is about from 80 to 300~ and that the average molecular weight of the polyethylene glycol is about from 350 to 30,000, preferably about 1,900 to 15,000.
The chemically modified protein of the present invention can be produced by reacting islet-activating protein (IAP) produced by bacteria belonging to the genus Bordetella with a pol~ethylene glycol-bound compound (activated PEG).
Thus, another aspect of the present invention provides:
[A] reacting the islet-activating protein with an activated polyethylene glycol-bound compound, thereby producing , the chemically modified protein in'which the polyethylene glycol moiety is bound to the protein without spacer, :
Groups which can be used for R as protective groups for hydroxyl in the above-mentioned polyethylene glycol include lower (e.g. 1 to 3 carbon atoms) alkyls and lower (e.g. 1 to 3 carbon atoms) alkanoyls. As examples of such alkyls, mention may be made of methyl, ethyl, propyl and isoprop,,vl; and as examples of such alkanoyls, formyl, acetyl and propionyl. As the protective groups for hydroxyl, methyl is preferred. It is preferable that Q is about from 80 to 300~ and that the average molecular weight of the polyethylene glycol is about from 350 to 30,000, preferably about 1,900 to 15,000.
The chemically modified protein of the present invention can be produced by reacting islet-activating protein (IAP) produced by bacteria belonging to the genus Bordetella with a pol~ethylene glycol-bound compound (activated PEG).
Thus, another aspect of the present invention provides:
[A] reacting the islet-activating protein with an activated polyethylene glycol-bound compound, thereby producing , the chemically modified protein in'which the polyethylene glycol moiety is bound to the protein without spacer, :
[B] reacting the islet-activating protein with a ~: compound of the formula:
N ~ [ ~ CH2CH2O ~ R~
; X -(~ N
N~/
(wherein X is a halogen atom, p is an integer of 1 or 2 r and .~ the other symbols are as defined above), thereby producing the chemically modified protein in which ~he polyethylene glycol moiety is bound to the protein via the triazine spacer, [C] reacting the islet-activating protein with a compound of the formula:
2--t--m-l t CH2CH2o ~ R (II) (wherein the symbols are as defined above) in the presence of a reducing ageni, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the alkylene spacer, or [D] reacting the islet-activating protein with a ~ : compound of the formula:
:'~
., : R - C-~CH2 ~ ~ 2 2 ~ R (III) : NH
(wherein R' is a lower alkoxy, and the other symbols arP as defined above) :::
~: .
:~ :
- 5a 24205-738 or an acid addition salt thereof, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein vla the imidate spacer.
The chemically modified protein of the present invention wherein the spacer is a triazine can be produced by reacting IAP
and a compound of the formula N ~ [O ( CH2CH2O ~ R]p (I) N
wherein R and Q are as defined above, X is a halogen and p is an integer of 1 or 2. It is preferable that X is a chlorine atom.
The reaction itself is known and is usually carried out in an aqueous solution of a buffer such as phosphate or borate at pH of about 8 to 10 and at about 0C to room temperature for about 1 to 24 hours. Compound (I) is used in a molar ratio of 1 - 500 to 1 of IAP, preferably 5 - 200 to 1 of IAP.
The chemidally modified protein of the present invention wherein the spacer is an alkylene can be produced by reacting IAP with a compound of the formula ~~~CH2 ~m-l -t~CH2CH2o ~ R (II) wherein R, _ and Q arè as defined above, in the presence of a ~; 20 reducing agent.
The reaction itself is known and is usually carried out in an a~ueous solution of a buffer such as phosphate or borate at pH about 6.0 to 9.0 and at about 0C to 50C for about 10 to 80 ~ . .
- 5b - 24205-738 hours. Reducing agents which work well for the reaction include borohydride reducing agents such as sodium borohydride and sodium cyanoborohydride. Compound (II) is used in a molar ratio of 1 - 1000 to 1 of IAP, preferably 5 - 200 to 1 of IAP; the reducer is used in a molar ratio of 1 - 100 to 1 of IAP.
The chemically modified protein of the present invention wherein the spacer is an imidate can be produced by reacting IAP
with a compound of the formula R'--Cl ~CH2~ t- 2 2 ~ R (III) NH
wherein R, n and Q are of the same meanings as defined above, and R' is a lower alkoxy. It is preferable that alkoxys having from 1 to 3 carbon atoms (e.g. methoxy, ethoxy and propoxy) are used for R'. The above-mentioned Compound (III) can also be used in the form of an acid addition salt such as hydrochloride, sulfate or acetate.
; The reaction is carried out in an aqueous solution of a buffer such as phosphate or borate under weakly alkaline conditions at pH of about 7.0 to 9.0 and at about 0C to 40C
for about 3 to 30 hours.
The amino group modification ratio can be varied ad libitum according to the amount of the above-mentioned activated PEG used.
Said amount to 1 mole in I~P is preferably about 5 to 1000 moles, more preferably about 50 to 500 moles in the case of which the spacer is a triazine, and preferably about 10 to 2000 - 5c - 24205-738 moles, more preferably about 50 to 1000 moles in the case of which the spacer is an imidate or an alkylene.
If necessary, the desired chemically modified protein can be purified from the reaction liquid by using an ordinary method of protein purification such as dialysis, salting-out, ultrafiltration, ion exchange chromatography, gel filtration, high performance liquid chromatography and electrophoresis.
Ultrafiltration and gel filtration are particularly efficient for the elimination of non-reacted PEG. The degree of amino group modification can be calculated by e.g.amino acid analysis following acid decomposition.
Compounds (I) and (II~, starting materials, are both known substances; their production method and physico-chemical characteristics are described respectively, e.g., in Chemistry Letters, 773(1980) and European Patent Publication No. 154316.
Compounds (III) can be produced by hydrolyzing the known compound of the formula NC t CH2 ~ 0 -t CH2CH2 ~ R (IV) wherein R, n and ~ are of the samei meanings as defined above in the presence of a lower alkanol (methanol, ethanol, propanol etc.) and an acid (hydrogen chloride, hydrochloric acid, sulfuric acid, acetic acid etc.) in accordance with the routine procedure.
f~3~8~3 - 5d - 24205-738 The chemically modified protein of the present invention has a strong islet-activating aativity with notably attenuated leukocytosis promoting, histamine-sensitizing and hemagglutin-ating action, which are side effects of non-modified IAP.
In addition, the protein is notably lower in antigenicity and immunogenicity than any known non-modified IAP or relatives thereof. It is ' ~ `
: ' also low in toxicity.
The chemically modified protein of the present invention therefore functions very well as a preventive and therapeutic drug for diabetes mellitus in mammals (rats, mice, dogs, cats, humans etc.).
For example, when used as a therapeutic drug for diabetes mellitus, the chem;cally modified protein of the present invention is administered to adults in the form of injection at a dose of from 10 ng/kg to 500 L~g/kg daily or in the form of oral drug in a dose of 1 mg/kg to 500 mg/kg daily, calculated on the protein basis.
Figures 1, 2 and 3 respectively show the time-based changes in glucose tolerance (plasma glucose value), body weight and leukocyte count in diabetes mellitus model rats, described in Experimental Example 7. In Figures 1 and 2, - O -, - X ~ and - ~ - respectively indicate control group, normal rat group, IAP group and PEG-IAP group.
Action The action of the chemically modified protein of the present invention is also shown.in the experinents below.
Experiment 1 Islet Activating Activity Non-modif~led IAP and the PEG-IAP obtained in Examples 1 to 4 were each dissolved in a physiological salt solution. One ml (4 ~g, calculated on the protein basis) of each resulting solution was injected to SD rats (5 males in each group) via a tail veinj 3 days after which time-based changes in blood 1ucoselevel and t~lood insulin level were measured following an injection of a glucose solution. The animals were fasted for 24 hours before the experiment.
After collecting 0.1 ml of blood via a tail vein, immediately a 20% glucose solution in a dose of 1 ml per 100 g body weight was intraperitoneally administered to each rat. 15, 30 and 60 minutes later, 0.1 ml of blood was collected from each rat. The determination of blood gluc~ elevels was bythe ' glucose oxidase method; that of blood insulin levels, by the double antibody technique. As shown in Tables 1 to 3, blood glucose decreased and blood insulin increased due to the administration of IAP or PEG-L~P; i.e., the PEG-modified IAP was also found to have islet-activating activity.
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Experiment 2 Leukocytosis-promoting Effect (~ LPF activity) 0.4 ,ug (calculated on the protein basis) of either non-modified IAP or the PEG-IAP obtained in Example 2 was administered via intravenous 5 injection to each A/J mouse (male), 5 days after which leukocytes were counted, and leukocyte increase rates were calculated by subtracting the leukocyte count in the control group from that in each administration group.
~LPFactivity = (leukocyte count in each administration group) -(leukocyte count in control group) As shown in Table 4, leukocytosis-promoting activity is lost or attenuated with PEG-IAP.
Table4 Leukocytosis-promotingActivity Molar Ratio l~ LPF Activity ( X 102/mm3) Non-modified IAP 72 ' Experiment 3 Histamine-sensitizing Effect 2 ,ug of either non-modified IAP or the PEGI-IAP obtained in Example 2 ~: was intravenously injected to each A/J mouse (10 males in each group), 4 days ~:~ after which 2.5 mg of histamine was intraperitoneally injected to each ~use, and the intensity of the histamine-sensitizing effect of each treatment was determined on the basis of the number of mice which died within 1 hour. The results are shown in Table 5. Histamine-sensitizing activity is lost with PEG-IAP.
,~ ~2~i Tabl~5 ~Iistamine-sensitizingEffect Molar Ratio Histamine-sensitizing Effect .
5 Non-modif-led IAP 10 E~periment 4 Hemagglutinating Effect Non-modi~led IAP and the PE~-IAP obtained in Example 2 were each serially diluted with 10 mM phosphate buf~er solution (p~I 7) containing 0.16M sodium chloride; 60 lll of each resulting dilution was transferred to a multi-well plate with a U-shaped base and mixed with 50 ~ul of 0.6%
suspension of goose erythrocyte (Nihon Seibutsu Zairyo Center) in the same buffer solution. After being kept standing at room temperature for 2 hours, each mixture was visually checked for hemagglutination. As shown in Table 20 6, PEG-IAP had an attenuated hemagglutinating effect.
: :
Table 6 Hemagglutinating Effect Protein Concentration (~g/ml) MolarRatio 25 12.6 6.3 3.1 1.6 0.78 0.39 0.20 Non-modifîed IAP~ + ~ + + ~ -- --PEG - IAP60 + ~ ~ -- ~
Hemagglutination; +: Noted, --: Not noted Experiment5 Antigenicity Antigenicity was determined by the enzyme imnunoassay (sandwich 35 technique). The antibody to IAP used in the experiment was anti~ IgG
obtained from immunized goats by affinity purification. The alkaline phosphatase-labeled anti-IAP-IgG used in the e~periment was prepared by bindingalkalinephosphatase (Miles, Inc. ~SA) to the above-mentioned anti-IAP-IgG by the glutaraldehyde method. The enzyme immunoassay procedure used in the experiment is described in detail below, 100 ~l of a solution of anti-IAP-IgG (0.1 ~g/ml) in 50mM carbonate buffer solution (pH 9.7) was transferred to a 96-well microplate (Nunc, Inc, Denmark) and kept standing at 4C overnight for antibody adsorption. The plate was then washed with 10mM phosphate buffer solution (pH 7.d~) containing 0.14M sodium chloride, 3mM potassium chloride and 0.05% Tween~
20. After adding 100 ~1 of a dilution o~ either non-IAP or the ~EG-IAP
in the same buffer solution, obtained in Examples 1 to 4 ~0 to 200 ng/ml), the plate was kept standing at room temperature for 2 hours. After washing each plate with the same bu~fer solution, 100 ~l of alkaline phosphatase-labeled anti-IAP.-IgG(about 0025 ~g/ml)in the same buf~er 15 was added, after which the plate was kept standing at room temperature for 2 hours. The plate was then washed with the same buffer solution and assayed for alkaline phosphatase activity. Alkaline phosphatase activity was determined by measuring the absorbance at a wavelength o~
405 nm, the sarnples having previously reacted at room temperature for 1 20 hour with 200 ~l of a solution of p-nitrophenylpho~;phate (1 mg/ml) in a lM
diethanolamine buffer solution (pH 9.8)containing 0.01% magnesium chloride hexahydrate, using a Corona~MTP-12 microplate photometer. The results are shown in Table 7. Antigenicity levels are shown in % absorbance ratio, calculated o~ the basis ot` the absorbance of non-modified IAP, which 25 was taken as 100%.
Table 7 Antigenicity .
Preparation Molecular Modification Antigenicity method weight of Ratio (%) Level (%) PEG
Non-modif~led PEG-IAP Example 1 350 22.5 76 PEG-IAP Example 1 750 30.6 59 PEG-IAP Examplel 1900 1~.8 60 PE~-L9P Example 1 5000 16.7 40 PEG-IAP Example 2 5000 3O0 60 ~r~e-~n~
PEG-IAP Example2 5000 19.7 38 PEG-IAP Example 2 5000 35.8 10 PEG-IAP Example 3 5000 37.0 78 PEG-IAP Example 4 5000 16.4 88 _ _ Experiment 6 Immunogenicity This experiment was conducted in accordance with the method described in the Journal of Immunological Methods, 14, 381 (1977). A~ter 10 emulsification with Freund's complete adjuvant (FCA), non-modified LAP or the PEG-IAP obtained in Example 2 (2 llg, calculated on the protein basis) was intraperitoneally administered to each A/J mouse (8 animals in each group), 14 and 28 days after which additional administration was carried out.
Starting on the 14th day following the first administration, blood samples 15 ~vere obtained from the retro orbital plexus of each mouse at 7-day interva~s;
ær~n anti-body production was evaluated b~ the passive cutaneous anaphylactic (PCA) reaction using rats. Each serum salT~le (O.lml), , previously c~iluted, was intracutaneously injected in ~each rat, 4 hours after uhich~ach ratwas intravenously injected ~ith 2 ml of a mixture of 100 ~g non-modified IAP and 20 20 mg Evans blue, to determine the vascular permeability of the dye, which was used as evaluation criterion for PCA titers. The results are shown in Table 8, where the values are shown in maximum dilution rates of serum samples positive for PCA reaction. Anti-IAP antibody production was noted in the case of non-modified IAP, PEG-IAP 10 and PEG-IAP 50, while not noted in 25 the case of PEG-IAP 200.
. .
` Table 8 Immunogenicity Date of PCA-Titer Evaluation __ .
~ 30 Molar ratio14 21 28 35 .. . . _ . _ Non-modifiedIAP --* -- -- 16 ~` PEG-IAP 50 -- -- -- 16 _ _ _ *: <4 . I) Experiment 7 Improvement of Glucose Tolerance in Diabetes Mellitus Model Rats After subcutaneous injection of 120mg/kg streptozotocin at~he age of 1.5 C~Ly~ felraleWistar-KyotO rats were raised until the 8th week of age to obtain 5 diabetes mellitus model rats. Each model rat received via intravenous in-:~ injection 2 ~g of either non-modified IAP or the PEG-IAP obtained in Example 2, 6 days after which the glucose tolerance of each rat was determined. In addition, body weight and leukocyte count were measured 5 days and 11 days 10 after injection. Glucose tolerance was determined as follows: 0.1 ml of bloodwas collected via a tail vein (each rat being fasted for 24 hours before the initiationof the experi~.ent), imr.ec~iately a:Eter whichglucose(2g/kg)was'ora11Y
administered to each rat. 15, 30, 60 and 120 minutes later, 0.1 ml of blood was collected from each rat. Plasma glucose contents of blood samples were 15 determined by the glucose oxidase method. Figures 1, 2 and 3 respectively show the plasma glucose contents, body weights and leukocyte counts.
As shown in }~igure 1, diabetic rats showed a considerably worsened glucose tolerance in comparison to normal rats. In the groups administered non-modified LAP or PEG-IAP, however, glucose tolerance was improved to a Ievel comparable with that in normal rats. In addition, in the group administered non-modified IAP, body weight decreased and leukocyte count increased to a level 3 times that in the control group. On the other hand, the group administered PEG-~AP was hardly different from control group, in the ' Judging from these results, it is obvious that while maintaining a glucose tolerance improving effect nearly equivalent to that of non-modified IAP, some side effects of IAP, i.e. body weight-reducing effect and leukocytosis-promoting effect, are lost when IAP is modified with PEG.
The present invention is more concretely described by the following Re~erence Examples and Working Examples.
Reference Example 1: Preparation of L9P
A high avian hemagglutination value (HA value) fraction containing a small amount of endotoxin, produced in accordance with the method described in Example 1 of European Patent Publication No. 47802, previously heated at . 1, 100C for 3 minutes, was passed through a hydroxyapatite column equilibrated to pH 8.0 to remove :FHA. The fraction not adsorbed to the column was adjusted to pH 6.0 with hydrochloric acid and passed through anotherhydroxyapatitecolumnequilibratedtoP~ 6Ø Adsorbed crude IAP
t~as eluated ~th a 0.1~ phosphate buffer solution (pH 7.0) containing O.~M
. j sodium chloride, then passed through a column packed with anti- FHA-bound Sepharose,~after which it was purified b~ sucrose density gradient centrifugation to obtain purified IAP, which was used in the examples.
Reference Example 2: Preparation of 2,4-bis (0-I~olyethylene glycol methyl 10 ether)- 6-chloro-S-triazine To the mixture containing 40 g of polyethylene glycol methyl ether (average molecular weight: 5000), 200 ml of benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A (~rakoPure Chemicals, Japan ) was added 730 mg of cyanuric chloride. The resultingmixture was heated at 80C
with stirring for 20 hours. Then 400 ml of petroleum ether was added to the heat-ed mixture to precipitate 2.~-bis (0-polyethylene glycol methyl ether)-6-chloro-S-triazine .
The obtained precipitate was dissolved in benzene to remove non-reactedcyanuricchloride. ~fterthisprocedure having been repeated three times, ~ the precipitate was dried in a desiccator under reduced pressure to obtain 36 g of 2,4-bis (0-polyethylene glycol methyl ether)-6-chloro-S-triazine.
he chlorine content of 2,~bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine measured by the P~aricle's method[Analytical Chemistry, 35,683~63)]was 0032%, which was in accord with 0035%,its theoretical value.
Each polyethylene glycol methyl ether(average molecular weight:350, 750 or l900)was treated in the same manner as described above to obtain its corresponding 2,4-bis(~polyethylene glycol methyl ether)-~chloro-S-triazine .
The yields were 10.0 g, 1302 g and 33.6 g, respectively.
The chlorine contents of the obtained 2,4-bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine were 7.5%, 4.2~o and 1.5%, respectively, which were substantially in accord with 7.6%, 4.1% and 107%, their theoreti-cal values, respectively.
he 2,4-bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine having an average molecular weight 350 or 750 was like syrup at ordinary ; ~ temperature.
Reference Example 3: Preparation of polyethylene glycol mono-nethyl ether aldehyde - ~CI f ~ O/~3~
(i) Polyethylene glycol methyl ether (5 g, average molecular weight:5000) was dissolved in 100 ml of methylene chloride. To the solution was added 330 mg of pyridinium chloro-chromate and the solution was then stirred at room temperature for 12 hours.
The obtained reaction solution was diluted with two times its volume of methylene chloride and then poured into a Florisil column (Serva, West Germany, column size: 6 x 10 cm), washed with methylene chloride then chloroform, and then eluated with methanol-chloroform (1:9). After collecting positive fractions in a 2,4-dinitrophenyl hydrazine test, the solvent was evaporated under reduced pressure to obtain the objective compounds as a crystalline wax. Yleld: 1.5 g (30%), Thin layer chromato-graphy: Rf=0.08 (solvent system; chloroform:methanol:acetic acid =
; 9:1:0.5, carrier;silica gel), The absorption of an aldehyde group was recogni2ed at 96.2 PPM in the hydrating form (-CH(OH)2) in 13C-NMR.
Reference Example 4:Preparation of polyethylene glycol imide ester Two grams of polyethylene glycol methyl ether mono-~-cyanoethyl ether prepared from polyethylene glycol methyl ether ~; 20 (average molecular weight~5000) was dissolved in 15 ml of anhydrous methanol. Through the solution was blown dried hydrogen chloride under -20C to saturate it. After being plugged up, it was allowed to stand in a refrigerator for 3 days. To this was added anhydrous ether and the solution was allowed to stand in a refrigerator again. After 4 hours, the upper ether layer was decanted. To the resulting layer was added anhydrous ether and Trademark - 16a - 24205-738 the solution was vigorously stirred and permitted to stand in a refrigerator for an hour to obtain a solid. The solid was well washed with anhydrous ether after pouring out ether, kept in a refrigerator till a solid precipitated and then the resulting ether layer was poured out. The solid was well washed by repeating this procedure twice. The obtained solid was sucked dry in a desiccator containing phosphorus pentoxide and solid NaOH or an hour to obtain 1.5 g of polyethylene ~lycol imide ester (average molecular weight:5000).
In NMR (solvent;d6-DMSO,90MHz), a triplet according to -CH2-CH2-C=NH was recognized at ~2.3, in IR, the absorption according to -CN was found to disappear.
Example l:Preparation of PEG-IAP
, To 2.5 mg of IAP was added 2~ bis (O-polyethylene glycol methyl ether)-6- chloro-S-triazine (average molecular weight: 350, 750, 1900 or 5000) obtained in Reference Example 2 in a molar ratio of 200 to 1 of IAP. The mixture was reacted in 25 ml of 0.1 M borate buffer (pI-I 9.0) at a,oC for 2 hours 5 and then 25 ml of 0.2 M phosphate buffer (p H 7) was added to stop the reaction. The reacted mixture was subjected to ultraflltration (PM-30 membrane, Amicon, USA) to remove non-reacted PEG and 2.0 ml of the concentrate was purified by gel flltration using a column (1.8 x 77 cm) packed with SephacryI S-200 (Pharmacia, Sweden) to obtain PEG-IAP. Protein 10 contents were determined by Lowry's method. Amino group modi~lcation ratios were calculated on the basis of the amount of free amino groups as measuredbythe fluorescamine method[Archives of Biochrnistry and siophysics, 155, 213- 220 (1973)]. The results are shown in Table 9.
Table 9 Amino Group Modi~lcation ratios of PEG-IAP
Molecular Weight of PE GModification Ratio (%) PEG-IAP 350 22.5 PEG-IAP 750 30.5 PEG-IAP1900 15.8 PEG-IAP5000 16.7 .
Example 2: Preparation of PEG-L~P
To 1 mg of IAP was added 2,4-bis (O-polyethylene glycol methyl ether)-6-25 chloro-S-triazine (average molecular weight: 5000) obtained in Reference Example 2 in a molar ratio of 10, 50 or 200 to 1 of IAP. The mixture was reacted in 4.0 ml of 0.1 M borate buffer ( pH 9) at 4C for 2 hours and then 4.0ml of 0.1 M phosphate buffer (pH 7.0) was added to stop the reaction.
The reacted mixture was subjected to ultrafiltration tPM-30 membrane, 30 Amicon, USA) to remove non-reacted PEG and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8 x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-IAP. Protein contents were determined by Lowry's method. Amino group modifilcation ratios were calculated on the basis of the amount of ~ree amino groups as measured by the 35 f1uorescamine method. The results are shavn in Table 10.
*
~ Trademark .
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Table 10 Amino Group Modification Ratios of PEG-IAP
Molar Ratio Modification Ratio (%) PEG-LAP lO 3. 0 PEG-IAP 50 19.7 PEG-IAP 200 35.8 Example 3: Preparation of PEG-IAP
To 2 mg of IAP was added polyethylene glycol methyl ether aldehyde obtained in Reference Example 3 in a molar ratio of 1000 to 1 of IAP. The mixture was reacted in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 2 M urea at room temperature for 3 0 minutes. After 60 ~l of pyridine borane (50 mg/ml) in ~ethanol was added, the nixture was reacted at room temperature for 2 hours. To the reacted mixture was added 2 ml of 1 M glycine to stop the reaction. The reacted mixture was subjected to ultrafiltration (PM-30 membrane, Arnicon, USA~) to remove non- reacted PE~ and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8 x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-IAP.
The amino group modification ratio measured by the fluorescamine method was 37.0%.
~; ~ Example 4: Preparation of PEG-IAP
To 2 mg of IAP was added polyethylene glycol imide ester obtained in Reference Example 4 in a molar ratio of 2000 to 1 of IAP. The mixture was ~; ~ 25 reacted in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 2M urea at 4C
for 2 hours. To the reacted mixture was added 40 ml of 1 M ammonium acetate (pH 6.0) to stop the reaction. The reacted mixture was subjected to ultrafiltration (PM-30 membrane, Amicon, USA) to remove non-reacted PEG
and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-~AP. The amino group modification ratio measured by the fluorescamine method was 16.4%.
N ~ [ ~ CH2CH2O ~ R~
; X -(~ N
N~/
(wherein X is a halogen atom, p is an integer of 1 or 2 r and .~ the other symbols are as defined above), thereby producing the chemically modified protein in which ~he polyethylene glycol moiety is bound to the protein via the triazine spacer, [C] reacting the islet-activating protein with a compound of the formula:
2--t--m-l t CH2CH2o ~ R (II) (wherein the symbols are as defined above) in the presence of a reducing ageni, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the alkylene spacer, or [D] reacting the islet-activating protein with a ~ : compound of the formula:
:'~
., : R - C-~CH2 ~ ~ 2 2 ~ R (III) : NH
(wherein R' is a lower alkoxy, and the other symbols arP as defined above) :::
~: .
:~ :
- 5a 24205-738 or an acid addition salt thereof, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein vla the imidate spacer.
The chemically modified protein of the present invention wherein the spacer is a triazine can be produced by reacting IAP
and a compound of the formula N ~ [O ( CH2CH2O ~ R]p (I) N
wherein R and Q are as defined above, X is a halogen and p is an integer of 1 or 2. It is preferable that X is a chlorine atom.
The reaction itself is known and is usually carried out in an aqueous solution of a buffer such as phosphate or borate at pH of about 8 to 10 and at about 0C to room temperature for about 1 to 24 hours. Compound (I) is used in a molar ratio of 1 - 500 to 1 of IAP, preferably 5 - 200 to 1 of IAP.
The chemidally modified protein of the present invention wherein the spacer is an alkylene can be produced by reacting IAP with a compound of the formula ~~~CH2 ~m-l -t~CH2CH2o ~ R (II) wherein R, _ and Q arè as defined above, in the presence of a ~; 20 reducing agent.
The reaction itself is known and is usually carried out in an a~ueous solution of a buffer such as phosphate or borate at pH about 6.0 to 9.0 and at about 0C to 50C for about 10 to 80 ~ . .
- 5b - 24205-738 hours. Reducing agents which work well for the reaction include borohydride reducing agents such as sodium borohydride and sodium cyanoborohydride. Compound (II) is used in a molar ratio of 1 - 1000 to 1 of IAP, preferably 5 - 200 to 1 of IAP; the reducer is used in a molar ratio of 1 - 100 to 1 of IAP.
The chemically modified protein of the present invention wherein the spacer is an imidate can be produced by reacting IAP
with a compound of the formula R'--Cl ~CH2~ t- 2 2 ~ R (III) NH
wherein R, n and Q are of the same meanings as defined above, and R' is a lower alkoxy. It is preferable that alkoxys having from 1 to 3 carbon atoms (e.g. methoxy, ethoxy and propoxy) are used for R'. The above-mentioned Compound (III) can also be used in the form of an acid addition salt such as hydrochloride, sulfate or acetate.
; The reaction is carried out in an aqueous solution of a buffer such as phosphate or borate under weakly alkaline conditions at pH of about 7.0 to 9.0 and at about 0C to 40C
for about 3 to 30 hours.
The amino group modification ratio can be varied ad libitum according to the amount of the above-mentioned activated PEG used.
Said amount to 1 mole in I~P is preferably about 5 to 1000 moles, more preferably about 50 to 500 moles in the case of which the spacer is a triazine, and preferably about 10 to 2000 - 5c - 24205-738 moles, more preferably about 50 to 1000 moles in the case of which the spacer is an imidate or an alkylene.
If necessary, the desired chemically modified protein can be purified from the reaction liquid by using an ordinary method of protein purification such as dialysis, salting-out, ultrafiltration, ion exchange chromatography, gel filtration, high performance liquid chromatography and electrophoresis.
Ultrafiltration and gel filtration are particularly efficient for the elimination of non-reacted PEG. The degree of amino group modification can be calculated by e.g.amino acid analysis following acid decomposition.
Compounds (I) and (II~, starting materials, are both known substances; their production method and physico-chemical characteristics are described respectively, e.g., in Chemistry Letters, 773(1980) and European Patent Publication No. 154316.
Compounds (III) can be produced by hydrolyzing the known compound of the formula NC t CH2 ~ 0 -t CH2CH2 ~ R (IV) wherein R, n and ~ are of the samei meanings as defined above in the presence of a lower alkanol (methanol, ethanol, propanol etc.) and an acid (hydrogen chloride, hydrochloric acid, sulfuric acid, acetic acid etc.) in accordance with the routine procedure.
f~3~8~3 - 5d - 24205-738 The chemically modified protein of the present invention has a strong islet-activating aativity with notably attenuated leukocytosis promoting, histamine-sensitizing and hemagglutin-ating action, which are side effects of non-modified IAP.
In addition, the protein is notably lower in antigenicity and immunogenicity than any known non-modified IAP or relatives thereof. It is ' ~ `
: ' also low in toxicity.
The chemically modified protein of the present invention therefore functions very well as a preventive and therapeutic drug for diabetes mellitus in mammals (rats, mice, dogs, cats, humans etc.).
For example, when used as a therapeutic drug for diabetes mellitus, the chem;cally modified protein of the present invention is administered to adults in the form of injection at a dose of from 10 ng/kg to 500 L~g/kg daily or in the form of oral drug in a dose of 1 mg/kg to 500 mg/kg daily, calculated on the protein basis.
Figures 1, 2 and 3 respectively show the time-based changes in glucose tolerance (plasma glucose value), body weight and leukocyte count in diabetes mellitus model rats, described in Experimental Example 7. In Figures 1 and 2, - O -, - X ~ and - ~ - respectively indicate control group, normal rat group, IAP group and PEG-IAP group.
Action The action of the chemically modified protein of the present invention is also shown.in the experinents below.
Experiment 1 Islet Activating Activity Non-modif~led IAP and the PEG-IAP obtained in Examples 1 to 4 were each dissolved in a physiological salt solution. One ml (4 ~g, calculated on the protein basis) of each resulting solution was injected to SD rats (5 males in each group) via a tail veinj 3 days after which time-based changes in blood 1ucoselevel and t~lood insulin level were measured following an injection of a glucose solution. The animals were fasted for 24 hours before the experiment.
After collecting 0.1 ml of blood via a tail vein, immediately a 20% glucose solution in a dose of 1 ml per 100 g body weight was intraperitoneally administered to each rat. 15, 30 and 60 minutes later, 0.1 ml of blood was collected from each rat. The determination of blood gluc~ elevels was bythe ' glucose oxidase method; that of blood insulin levels, by the double antibody technique. As shown in Tables 1 to 3, blood glucose decreased and blood insulin increased due to the administration of IAP or PEG-L~P; i.e., the PEG-modified IAP was also found to have islet-activating activity.
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Experiment 2 Leukocytosis-promoting Effect (~ LPF activity) 0.4 ,ug (calculated on the protein basis) of either non-modified IAP or the PEG-IAP obtained in Example 2 was administered via intravenous 5 injection to each A/J mouse (male), 5 days after which leukocytes were counted, and leukocyte increase rates were calculated by subtracting the leukocyte count in the control group from that in each administration group.
~LPFactivity = (leukocyte count in each administration group) -(leukocyte count in control group) As shown in Table 4, leukocytosis-promoting activity is lost or attenuated with PEG-IAP.
Table4 Leukocytosis-promotingActivity Molar Ratio l~ LPF Activity ( X 102/mm3) Non-modified IAP 72 ' Experiment 3 Histamine-sensitizing Effect 2 ,ug of either non-modified IAP or the PEGI-IAP obtained in Example 2 ~: was intravenously injected to each A/J mouse (10 males in each group), 4 days ~:~ after which 2.5 mg of histamine was intraperitoneally injected to each ~use, and the intensity of the histamine-sensitizing effect of each treatment was determined on the basis of the number of mice which died within 1 hour. The results are shown in Table 5. Histamine-sensitizing activity is lost with PEG-IAP.
,~ ~2~i Tabl~5 ~Iistamine-sensitizingEffect Molar Ratio Histamine-sensitizing Effect .
5 Non-modif-led IAP 10 E~periment 4 Hemagglutinating Effect Non-modi~led IAP and the PE~-IAP obtained in Example 2 were each serially diluted with 10 mM phosphate buf~er solution (p~I 7) containing 0.16M sodium chloride; 60 lll of each resulting dilution was transferred to a multi-well plate with a U-shaped base and mixed with 50 ~ul of 0.6%
suspension of goose erythrocyte (Nihon Seibutsu Zairyo Center) in the same buffer solution. After being kept standing at room temperature for 2 hours, each mixture was visually checked for hemagglutination. As shown in Table 20 6, PEG-IAP had an attenuated hemagglutinating effect.
: :
Table 6 Hemagglutinating Effect Protein Concentration (~g/ml) MolarRatio 25 12.6 6.3 3.1 1.6 0.78 0.39 0.20 Non-modifîed IAP~ + ~ + + ~ -- --PEG - IAP60 + ~ ~ -- ~
Hemagglutination; +: Noted, --: Not noted Experiment5 Antigenicity Antigenicity was determined by the enzyme imnunoassay (sandwich 35 technique). The antibody to IAP used in the experiment was anti~ IgG
obtained from immunized goats by affinity purification. The alkaline phosphatase-labeled anti-IAP-IgG used in the e~periment was prepared by bindingalkalinephosphatase (Miles, Inc. ~SA) to the above-mentioned anti-IAP-IgG by the glutaraldehyde method. The enzyme immunoassay procedure used in the experiment is described in detail below, 100 ~l of a solution of anti-IAP-IgG (0.1 ~g/ml) in 50mM carbonate buffer solution (pH 9.7) was transferred to a 96-well microplate (Nunc, Inc, Denmark) and kept standing at 4C overnight for antibody adsorption. The plate was then washed with 10mM phosphate buffer solution (pH 7.d~) containing 0.14M sodium chloride, 3mM potassium chloride and 0.05% Tween~
20. After adding 100 ~1 of a dilution o~ either non-IAP or the ~EG-IAP
in the same buffer solution, obtained in Examples 1 to 4 ~0 to 200 ng/ml), the plate was kept standing at room temperature for 2 hours. After washing each plate with the same bu~fer solution, 100 ~l of alkaline phosphatase-labeled anti-IAP.-IgG(about 0025 ~g/ml)in the same buf~er 15 was added, after which the plate was kept standing at room temperature for 2 hours. The plate was then washed with the same buffer solution and assayed for alkaline phosphatase activity. Alkaline phosphatase activity was determined by measuring the absorbance at a wavelength o~
405 nm, the sarnples having previously reacted at room temperature for 1 20 hour with 200 ~l of a solution of p-nitrophenylpho~;phate (1 mg/ml) in a lM
diethanolamine buffer solution (pH 9.8)containing 0.01% magnesium chloride hexahydrate, using a Corona~MTP-12 microplate photometer. The results are shown in Table 7. Antigenicity levels are shown in % absorbance ratio, calculated o~ the basis ot` the absorbance of non-modified IAP, which 25 was taken as 100%.
Table 7 Antigenicity .
Preparation Molecular Modification Antigenicity method weight of Ratio (%) Level (%) PEG
Non-modif~led PEG-IAP Example 1 350 22.5 76 PEG-IAP Example 1 750 30.6 59 PEG-IAP Examplel 1900 1~.8 60 PE~-L9P Example 1 5000 16.7 40 PEG-IAP Example 2 5000 3O0 60 ~r~e-~n~
PEG-IAP Example2 5000 19.7 38 PEG-IAP Example 2 5000 35.8 10 PEG-IAP Example 3 5000 37.0 78 PEG-IAP Example 4 5000 16.4 88 _ _ Experiment 6 Immunogenicity This experiment was conducted in accordance with the method described in the Journal of Immunological Methods, 14, 381 (1977). A~ter 10 emulsification with Freund's complete adjuvant (FCA), non-modified LAP or the PEG-IAP obtained in Example 2 (2 llg, calculated on the protein basis) was intraperitoneally administered to each A/J mouse (8 animals in each group), 14 and 28 days after which additional administration was carried out.
Starting on the 14th day following the first administration, blood samples 15 ~vere obtained from the retro orbital plexus of each mouse at 7-day interva~s;
ær~n anti-body production was evaluated b~ the passive cutaneous anaphylactic (PCA) reaction using rats. Each serum salT~le (O.lml), , previously c~iluted, was intracutaneously injected in ~each rat, 4 hours after uhich~ach ratwas intravenously injected ~ith 2 ml of a mixture of 100 ~g non-modified IAP and 20 20 mg Evans blue, to determine the vascular permeability of the dye, which was used as evaluation criterion for PCA titers. The results are shown in Table 8, where the values are shown in maximum dilution rates of serum samples positive for PCA reaction. Anti-IAP antibody production was noted in the case of non-modified IAP, PEG-IAP 10 and PEG-IAP 50, while not noted in 25 the case of PEG-IAP 200.
. .
` Table 8 Immunogenicity Date of PCA-Titer Evaluation __ .
~ 30 Molar ratio14 21 28 35 .. . . _ . _ Non-modifiedIAP --* -- -- 16 ~` PEG-IAP 50 -- -- -- 16 _ _ _ *: <4 . I) Experiment 7 Improvement of Glucose Tolerance in Diabetes Mellitus Model Rats After subcutaneous injection of 120mg/kg streptozotocin at~he age of 1.5 C~Ly~ felraleWistar-KyotO rats were raised until the 8th week of age to obtain 5 diabetes mellitus model rats. Each model rat received via intravenous in-:~ injection 2 ~g of either non-modified IAP or the PEG-IAP obtained in Example 2, 6 days after which the glucose tolerance of each rat was determined. In addition, body weight and leukocyte count were measured 5 days and 11 days 10 after injection. Glucose tolerance was determined as follows: 0.1 ml of bloodwas collected via a tail vein (each rat being fasted for 24 hours before the initiationof the experi~.ent), imr.ec~iately a:Eter whichglucose(2g/kg)was'ora11Y
administered to each rat. 15, 30, 60 and 120 minutes later, 0.1 ml of blood was collected from each rat. Plasma glucose contents of blood samples were 15 determined by the glucose oxidase method. Figures 1, 2 and 3 respectively show the plasma glucose contents, body weights and leukocyte counts.
As shown in }~igure 1, diabetic rats showed a considerably worsened glucose tolerance in comparison to normal rats. In the groups administered non-modified LAP or PEG-IAP, however, glucose tolerance was improved to a Ievel comparable with that in normal rats. In addition, in the group administered non-modified IAP, body weight decreased and leukocyte count increased to a level 3 times that in the control group. On the other hand, the group administered PEG-~AP was hardly different from control group, in the ' Judging from these results, it is obvious that while maintaining a glucose tolerance improving effect nearly equivalent to that of non-modified IAP, some side effects of IAP, i.e. body weight-reducing effect and leukocytosis-promoting effect, are lost when IAP is modified with PEG.
The present invention is more concretely described by the following Re~erence Examples and Working Examples.
Reference Example 1: Preparation of L9P
A high avian hemagglutination value (HA value) fraction containing a small amount of endotoxin, produced in accordance with the method described in Example 1 of European Patent Publication No. 47802, previously heated at . 1, 100C for 3 minutes, was passed through a hydroxyapatite column equilibrated to pH 8.0 to remove :FHA. The fraction not adsorbed to the column was adjusted to pH 6.0 with hydrochloric acid and passed through anotherhydroxyapatitecolumnequilibratedtoP~ 6Ø Adsorbed crude IAP
t~as eluated ~th a 0.1~ phosphate buffer solution (pH 7.0) containing O.~M
. j sodium chloride, then passed through a column packed with anti- FHA-bound Sepharose,~after which it was purified b~ sucrose density gradient centrifugation to obtain purified IAP, which was used in the examples.
Reference Example 2: Preparation of 2,4-bis (0-I~olyethylene glycol methyl 10 ether)- 6-chloro-S-triazine To the mixture containing 40 g of polyethylene glycol methyl ether (average molecular weight: 5000), 200 ml of benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A (~rakoPure Chemicals, Japan ) was added 730 mg of cyanuric chloride. The resultingmixture was heated at 80C
with stirring for 20 hours. Then 400 ml of petroleum ether was added to the heat-ed mixture to precipitate 2.~-bis (0-polyethylene glycol methyl ether)-6-chloro-S-triazine .
The obtained precipitate was dissolved in benzene to remove non-reactedcyanuricchloride. ~fterthisprocedure having been repeated three times, ~ the precipitate was dried in a desiccator under reduced pressure to obtain 36 g of 2,4-bis (0-polyethylene glycol methyl ether)-6-chloro-S-triazine.
he chlorine content of 2,~bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine measured by the P~aricle's method[Analytical Chemistry, 35,683~63)]was 0032%, which was in accord with 0035%,its theoretical value.
Each polyethylene glycol methyl ether(average molecular weight:350, 750 or l900)was treated in the same manner as described above to obtain its corresponding 2,4-bis(~polyethylene glycol methyl ether)-~chloro-S-triazine .
The yields were 10.0 g, 1302 g and 33.6 g, respectively.
The chlorine contents of the obtained 2,4-bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine were 7.5%, 4.2~o and 1.5%, respectively, which were substantially in accord with 7.6%, 4.1% and 107%, their theoreti-cal values, respectively.
he 2,4-bis(~polyethylene glycol methyl ether)-6-chloro-S-triazine having an average molecular weight 350 or 750 was like syrup at ordinary ; ~ temperature.
Reference Example 3: Preparation of polyethylene glycol mono-nethyl ether aldehyde - ~CI f ~ O/~3~
(i) Polyethylene glycol methyl ether (5 g, average molecular weight:5000) was dissolved in 100 ml of methylene chloride. To the solution was added 330 mg of pyridinium chloro-chromate and the solution was then stirred at room temperature for 12 hours.
The obtained reaction solution was diluted with two times its volume of methylene chloride and then poured into a Florisil column (Serva, West Germany, column size: 6 x 10 cm), washed with methylene chloride then chloroform, and then eluated with methanol-chloroform (1:9). After collecting positive fractions in a 2,4-dinitrophenyl hydrazine test, the solvent was evaporated under reduced pressure to obtain the objective compounds as a crystalline wax. Yleld: 1.5 g (30%), Thin layer chromato-graphy: Rf=0.08 (solvent system; chloroform:methanol:acetic acid =
; 9:1:0.5, carrier;silica gel), The absorption of an aldehyde group was recogni2ed at 96.2 PPM in the hydrating form (-CH(OH)2) in 13C-NMR.
Reference Example 4:Preparation of polyethylene glycol imide ester Two grams of polyethylene glycol methyl ether mono-~-cyanoethyl ether prepared from polyethylene glycol methyl ether ~; 20 (average molecular weight~5000) was dissolved in 15 ml of anhydrous methanol. Through the solution was blown dried hydrogen chloride under -20C to saturate it. After being plugged up, it was allowed to stand in a refrigerator for 3 days. To this was added anhydrous ether and the solution was allowed to stand in a refrigerator again. After 4 hours, the upper ether layer was decanted. To the resulting layer was added anhydrous ether and Trademark - 16a - 24205-738 the solution was vigorously stirred and permitted to stand in a refrigerator for an hour to obtain a solid. The solid was well washed with anhydrous ether after pouring out ether, kept in a refrigerator till a solid precipitated and then the resulting ether layer was poured out. The solid was well washed by repeating this procedure twice. The obtained solid was sucked dry in a desiccator containing phosphorus pentoxide and solid NaOH or an hour to obtain 1.5 g of polyethylene ~lycol imide ester (average molecular weight:5000).
In NMR (solvent;d6-DMSO,90MHz), a triplet according to -CH2-CH2-C=NH was recognized at ~2.3, in IR, the absorption according to -CN was found to disappear.
Example l:Preparation of PEG-IAP
, To 2.5 mg of IAP was added 2~ bis (O-polyethylene glycol methyl ether)-6- chloro-S-triazine (average molecular weight: 350, 750, 1900 or 5000) obtained in Reference Example 2 in a molar ratio of 200 to 1 of IAP. The mixture was reacted in 25 ml of 0.1 M borate buffer (pI-I 9.0) at a,oC for 2 hours 5 and then 25 ml of 0.2 M phosphate buffer (p H 7) was added to stop the reaction. The reacted mixture was subjected to ultraflltration (PM-30 membrane, Amicon, USA) to remove non-reacted PEG and 2.0 ml of the concentrate was purified by gel flltration using a column (1.8 x 77 cm) packed with SephacryI S-200 (Pharmacia, Sweden) to obtain PEG-IAP. Protein 10 contents were determined by Lowry's method. Amino group modi~lcation ratios were calculated on the basis of the amount of free amino groups as measuredbythe fluorescamine method[Archives of Biochrnistry and siophysics, 155, 213- 220 (1973)]. The results are shown in Table 9.
Table 9 Amino Group Modi~lcation ratios of PEG-IAP
Molecular Weight of PE GModification Ratio (%) PEG-IAP 350 22.5 PEG-IAP 750 30.5 PEG-IAP1900 15.8 PEG-IAP5000 16.7 .
Example 2: Preparation of PEG-L~P
To 1 mg of IAP was added 2,4-bis (O-polyethylene glycol methyl ether)-6-25 chloro-S-triazine (average molecular weight: 5000) obtained in Reference Example 2 in a molar ratio of 10, 50 or 200 to 1 of IAP. The mixture was reacted in 4.0 ml of 0.1 M borate buffer ( pH 9) at 4C for 2 hours and then 4.0ml of 0.1 M phosphate buffer (pH 7.0) was added to stop the reaction.
The reacted mixture was subjected to ultrafiltration tPM-30 membrane, 30 Amicon, USA) to remove non-reacted PEG and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8 x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-IAP. Protein contents were determined by Lowry's method. Amino group modifilcation ratios were calculated on the basis of the amount of ~ree amino groups as measured by the 35 f1uorescamine method. The results are shavn in Table 10.
*
~ Trademark .
, ~: . , ., . .. ~ ~ . .
Table 10 Amino Group Modification Ratios of PEG-IAP
Molar Ratio Modification Ratio (%) PEG-LAP lO 3. 0 PEG-IAP 50 19.7 PEG-IAP 200 35.8 Example 3: Preparation of PEG-IAP
To 2 mg of IAP was added polyethylene glycol methyl ether aldehyde obtained in Reference Example 3 in a molar ratio of 1000 to 1 of IAP. The mixture was reacted in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 2 M urea at room temperature for 3 0 minutes. After 60 ~l of pyridine borane (50 mg/ml) in ~ethanol was added, the nixture was reacted at room temperature for 2 hours. To the reacted mixture was added 2 ml of 1 M glycine to stop the reaction. The reacted mixture was subjected to ultrafiltration (PM-30 membrane, Arnicon, USA~) to remove non- reacted PE~ and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8 x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-IAP.
The amino group modification ratio measured by the fluorescamine method was 37.0%.
~; ~ Example 4: Preparation of PEG-IAP
To 2 mg of IAP was added polyethylene glycol imide ester obtained in Reference Example 4 in a molar ratio of 2000 to 1 of IAP. The mixture was ~; ~ 25 reacted in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 2M urea at 4C
for 2 hours. To the reacted mixture was added 40 ml of 1 M ammonium acetate (pH 6.0) to stop the reaction. The reacted mixture was subjected to ultrafiltration (PM-30 membrane, Amicon, USA) to remove non-reacted PEG
and 2.0 ml of the concentrate was purified by gel filtration using a column (1.8x 77 cm) packed with Sephacryl S-200 (Pharmacia, Sweden) to obtain PEG-~AP. The amino group modification ratio measured by the fluorescamine method was 16.4%.
Claims (23)
1. A chemically modified protein which comprises an islet-activating protein produced by bacteria belonging to the genus Bordetella and polyethylene glycol moiety of the formula -O-(CH2CH2O)?R
wherein R is a protective group for hydroxyl and ? is an integer of about 7 to 700, the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein.
wherein R is a protective group for hydroxyl and ? is an integer of about 7 to 700, the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein.
2. A chemically modified protein as claimed iniClaim 1, wherein the islet-activating protein is bound to the polyethylene glycol through a spacer.
3. A chemically modified protein as claimed in Claim 2, wherein the spacer is a group of the formula
4. A chemically modified protein as claimed in Claim 2, wherein the spacer is a group of the formula -(CH2?m wherein m is an integer from 1 to 3.
5. A chemically modified protein as claimed in Claim 4, wherein m is 2.
6. A chemically modified protein as claimed in Claim 2, wherein the spacer is a group of the formula wherein n is an integer from 1 to 3.
7. A chemically modified protein as claimed in Claim 6, wherein n is 2.
8. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the average molecular weight of the polyethylene glycol is 350 to 30000,
9. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the average molecular weight of the polyethylene glycol is 1900 to 15000.
10. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the average molecular weight of the polyethylene glycol is 5000.
11. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the islet-activating protein is bound with 1 to 30 spacers.
12. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the islet-activating protein is bound with 1 to 10 spacers.
13. 9 chemically modified protein as claimed in Claim 2, 3 or 5, wherein the modification ratio of the primary amino group is 3 to 80%.
14. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein the modification ratio of the primary amino group is 3 to 40%.
15. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein R is C1-3 alkyl or C1-3 alkanoyl.
16. A chemically modified protein as claimed in Claim 2, 3 or 5, wherein R is methyl.
17. A process for producing a chemically modified protein, the protein being an islet-activating protein produced by bacteria belonging to the genus Bordetella and being modified with a polyethylene glycol moiety of the formula:
(wherein R is a protective group for hydroxyl, and ? is an integer of about 7 to 700), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein optionally via a spacer selected from the group consisting of triazine of the formula:
, alkylene of the formula:
(wherein m is an integer of 1 to 3), and imidate of the formula:
(wherein n is an integer of 1 to 3), which process comprises:
[A] reacting the islet-activating protein with an activated polyethylene glycol-bound compound, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein without spacer, [B] reacting the islet-activating protein with a compound of the formula:
(I) (wherein X is a halogen atom, p is an integer of 1 or 2, and the other symbols are as defined above), thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the triazine spacer, [C] reacting the islet-activating protein with a compound of the formula:
(II) (wherein the symbols are as defined above) in the presence of a reducing agent, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the alkylene spacer, or [D] reacting the islet-activating protein with a compound of the formula:
(III) (wherein R' is a lower alkoxy, and the other symbols are as defined above) or an acid addition salt thereof, thereby producing the chemical modified protein in which the polyethylene glycol moiety is bound to the protein via the imidate spacer.
(wherein R is a protective group for hydroxyl, and ? is an integer of about 7 to 700), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein optionally via a spacer selected from the group consisting of triazine of the formula:
, alkylene of the formula:
(wherein m is an integer of 1 to 3), and imidate of the formula:
(wherein n is an integer of 1 to 3), which process comprises:
[A] reacting the islet-activating protein with an activated polyethylene glycol-bound compound, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein without spacer, [B] reacting the islet-activating protein with a compound of the formula:
(I) (wherein X is a halogen atom, p is an integer of 1 or 2, and the other symbols are as defined above), thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the triazine spacer, [C] reacting the islet-activating protein with a compound of the formula:
(II) (wherein the symbols are as defined above) in the presence of a reducing agent, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via the alkylene spacer, or [D] reacting the islet-activating protein with a compound of the formula:
(III) (wherein R' is a lower alkoxy, and the other symbols are as defined above) or an acid addition salt thereof, thereby producing the chemical modified protein in which the polyethylene glycol moiety is bound to the protein via the imidate spacer.
18. A process as claimed in claim 17, wherein process variant [B] is carried out in an aqueous buffer solution having a pH of about 8 to 10 at about 0°C to room temperature for about 1 to 24 hours employing about 1 to 1,000 moles of the compound (I) in which X is chlorine per mole of the islet-activating protein;
process variant [C] is carried out in an aqueous buffer solution having a pH of about 6 to 9 at about 0°C to 50°C for about 10 to 80 hours using 1 to 2,000 moles of the compound (II) and 1 to 100 moles of a borohydride reducing agent each per mole of the islet-activating protein; or process variant [D] is carried out in a aqueous buffer solution having a pH of about 7 to 9 at about 0°C to 40°C
for about 3 to 30 hours using 1 to 2,000 moles of the compound (III) per mole of the islet-activating protein, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via one of the spacers.
process variant [C] is carried out in an aqueous buffer solution having a pH of about 6 to 9 at about 0°C to 50°C for about 10 to 80 hours using 1 to 2,000 moles of the compound (II) and 1 to 100 moles of a borohydride reducing agent each per mole of the islet-activating protein; or process variant [D] is carried out in a aqueous buffer solution having a pH of about 7 to 9 at about 0°C to 40°C
for about 3 to 30 hours using 1 to 2,000 moles of the compound (III) per mole of the islet-activating protein, thereby producing the chemically modified protein in which the polyethylene glycol moiety is bound to the protein via one of the spacers.
19. A chemically modified protein, the protein being an islet-activating protein produced by bacteria belonging to the genus Bordetella and being chemically modified with a polyethylene glycol moiety including a spacer of the formula:
(I-a), ?CH2-??O ?CH2CH2O??R (II-a), or (III-a) (wherein R is Cl_3 alkyl or Cl 3 alkanoyl, ? is an integer of 7 to 700, m is an integer of 1 to 3, n is an integer of 1 to 3, and p is an integer of 1 or 2), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein to such an extent that 3 to 80% of the primary amino groups are bound to the moiety.
(I-a), ?CH2-??O ?CH2CH2O??R (II-a), or (III-a) (wherein R is Cl_3 alkyl or Cl 3 alkanoyl, ? is an integer of 7 to 700, m is an integer of 1 to 3, n is an integer of 1 to 3, and p is an integer of 1 or 2), the polyethylene glycol moiety being bound to a primary amino group of the islet-activating protein to such an extent that 3 to 80% of the primary amino groups are bound to the moiety.
20. A chemically modified protein as claimed in claim 19, wherein 3 to 40% of the primary amino groups are bound to the polyethylene glycol moiety which has an average molecular weight of about 1,900 to 15,000.
21. A chemically modified protein as claimed in claim 19 or 20, wherein the spacer-polyethylene glycol moiety has formula (I-a) in which p is 2.
22. A chemically modified protein as claimed in claim 19 or 20, wherein the spacer-polyethylene glycol moiety has formula (II-a) in which m is 2.
23. A pharmaceutical composition comprising an insulin secretion promotion effective amount of the chemically modified protein as defined in claim 1 in admixture with a pharmaceutically acceptable diluent or carrier.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15109886 | 1986-06-26 | ||
| JP151098/1986 | 1986-06-26 | ||
| JP97990/1987 | 1987-04-21 | ||
| JP62097990A JPS63126900A (en) | 1986-06-26 | 1987-04-21 | Chemically modified protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1294088C true CA1294088C (en) | 1992-01-07 |
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ID=26439106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000540507A Expired - Lifetime CA1294088C (en) | 1986-06-26 | 1987-06-25 | Chemically modified protein |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US4791192A (en) |
| EP (1) | EP0251717A3 (en) |
| CA (1) | CA1294088C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
| JPS6023084B2 (en) * | 1979-07-11 | 1985-06-05 | 味の素株式会社 | blood substitute |
| US4640835A (en) * | 1981-10-30 | 1987-02-03 | Nippon Chemiphar Company, Ltd. | Plasminogen activator derivatives |
| JPS5896026A (en) * | 1981-10-30 | 1983-06-07 | Nippon Chemiphar Co Ltd | Novel urokinase derivative, its preparation and thrombolytic agent containing the same |
| JPS59206316A (en) * | 1983-05-11 | 1984-11-22 | Kaken Pharmaceut Co Ltd | Active substance derivative for enhancing secretion of insulin and production thereof |
| JPS6098988A (en) * | 1983-11-01 | 1985-06-01 | Chemo Sero Therapeut Res Inst | Purification of lpf-ha |
| US4496689A (en) * | 1983-12-27 | 1985-01-29 | Miles Laboratories, Inc. | Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer |
| EP0154316B1 (en) * | 1984-03-06 | 1989-09-13 | Takeda Chemical Industries, Ltd. | Chemically modified lymphokine and production thereof |
-
1987
- 1987-06-18 US US07/063,400 patent/US4791192A/en not_active Expired - Fee Related
- 1987-06-25 EP EP87305685A patent/EP0251717A3/en not_active Withdrawn
- 1987-06-25 CA CA000540507A patent/CA1294088C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| US4791192A (en) | 1988-12-13 |
| EP0251717A3 (en) | 1990-05-16 |
| EP0251717A2 (en) | 1988-01-07 |
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