CN1080575C - Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same - Google Patents

Abstract

The present invention relates to a stably conjugated peptide compound, wherein peptide is coupled on a polymer containing lipophilic and hydrophilic groups. The present invention particularly relates to an insulin composition for oral administration or non-gastrointestinal tract administration, wherein insulin and a polymer containing linear carbowax groups and lipophilic groups are covalently combined. The tolerance of the insulin to in vivo enzymolysis is enhanced by the mutual arrangement of the insulin, the linear carbowax groups and the lipophilic groups on conformation. The conjugate of the present invention can be used for treatment and non-treatment (such as diagnose) purposes. The peptide and the polymer can be covalently combined, and can also be mutually associated by hydrogen bonds, etc.

Description

In conjunction with stable peptide composition, its treatment, diagnostic preparation and preparation and using method

The present invention relates to also relate to its preparation and using method in conjunction with stable polypeptide and protein compositions and preparation.

The whole body therapeutic that polypeptide and protein are used for some disease now is widely acceptance in medical practice.Polypeptide role in replacement therapy is so important, so that a lot of research activities is dropped in recombinant DNA technology synthetic in a large number.Much be endogenous molecule in these polypeptide, they are very strong producing on the physiological action, and are specific.

Limit these materials the principal element of the serviceability in the desired application be during when parenteral administration they easily by the plasma protein enzymes metabolism.The oral administration route of these materials more is a problem because before their arrive desired target tissue except albuminolysis under one's belt, the high acidity of stomach also can be destroyed it.Polypeptide that is produced by the effect of harmonization of the stomach pancreatin and protein fragments are obtained dipeptides and tripeptides by the exogenous and endogenous peptide enzymatic lysis on the brush border membrane of intestinal, even avoided the albuminolysis of pancreatin, polypeptide also is subjected to the degraded of brush border peptidases.The peptide of taking by any point of preserving behind the stomach stops its intestinal mucosa that enters cell by further metabolism having permeability barrier.

Although these obstacles are arranged, there are material evidence prompting trophism and pharmaceutical protein to be absorbed in the document by intestinal mucosa.On the other hand, trophism and medicine (many) peptide class is absorbed by the special movement system on the intestinal mucosa cells.These discoveries show, prepare appropriate (many) but peptide class and protein per os give, keep the enough biological activitys of desired use.Yet, if can modify these peptide classes, so that fully or at least degree keeps their physiologically active significantly, stablize them simultaneously, be not subjected to the effect of protease, and strengthen their penetrating powers, so just can utilize them to reach its intended purposes rightly by intestinal mucosa.The product that obtains like this will provide following advantage: produce more effective absorption, and correspondingly available than the suitableeest therapeutic effect of low dosage generation.

Proteinic problem oral or that parenteral route gives to be followed is known in pharmaceuticals industry, is just adopting various strategies to try hard to solve them.These strategies comprise and share penetration enhancers; as Salicylate (ester), fat one bile acid mixed micelle, (+)-2,3-Epoxy-1-propanol and fatty acyl carnitine; but they often have been found and have caused serious local toxicity problem, wear and tear fully and tissue inflammation as local excitation and toxicity, epithelium layer.Causing of these problems is that normal the appearance spills from dosage form because reinforcing agent is common and peptides products gives simultaneously.Other strategies that improve oral administration comprise peptide are mixed with protease inhibitor such as aprotinin, soybean trypsin inhibitor and aminopeptidase inhibition A to scheme to limit the degraded of the therapeutic agent of being given.Unfortunately, these protease inhibitor are nonselective, and endogenous protein also is suppressed.This effect is undesirable.

Increasing the peptide class by the physicochemical property that changes drug candidate also tests by the osmosis of mucosa.The result shows, improves lipotropy merely and is not enough to increase transcellular transport.In fact existing people proposes, and peptide-water hydrogen bond rupture is main energy barrier (Conradi, the R.A. that will overcome in the film diffusion that reaches peptide, Hilgers, A.R., Ho, N.F.H., and Bur-ton, P.S., " The influence of peptide structure on transport acrossCaco-2 cells ", Pharm.Res., 8,1453-1460, (1991)).Some authors have described the problem of protein stabilization.

Abuchowski and Davis (" Soluble polymers-Enzyme adducts ", In:Enzymes as Drugs, Eds.Holcenberg and Roberts, J.Wiley andSons, New York, NY, (1981)) disclosed various enzyme derivatization methods, so that water solublity, non-immunogenic, stable product in vivo to be provided.

Delivered at present extensive work about protein stabilization.Abuchowski and Davis disclose the whole bag of tricks (the same) that enzyme combines with polymer, and more specifically, these polymer are glucosan, polyvinylpyrrolidone, glycopeptide, Polyethylene Glycol and polyamino acids.It was reported, gained keep its parenteral route to use required biological activity and the dissolubility in water in conjunction with polypeptide.Same author is in U.S. Patent No. 4,179, disclose in 337 Polyethylene Glycol with the protein coupling after give these protein solubilities and non-immunogenic.But these polymer do not contain and are suitable for the bonded segment of intestinal mucosa, do not contain any composition that can promote or increase membrane permeation yet.Although these are combined into water solublity, they are not intended to oral.

Meisner etc. are in U.S. Patent No. 4,585, point out and can make protein stabilization by combining with chondroitin sulfate in 754.This bonded product is normally polyanionic, and hydrophilic is very strong, lacks the Premeabilisation of cells ability.They are not intended to oral usually.

Mill etc. are at United States Patent (USP) 4,003, point out that some acidic polysaccharose (as pectin, algesic acid, hyaluronic acid and carrageenin) can be coupled on the protein in 792, produce solubility and insoluble product.These polysaccharide are the polyanionic materials from food plant.They lack the Premeabilisation of cells ability, are not considered for oral usually.

At Pharmacological Research Communication 14, on the 11-120 (1982), Boccu etc. have disclosed can be with polyethylene glycol conjugation on protein such as peroxide dismutase (" SOD ").Improving of gained in conjunction with the antitypy of product and the stability of enzymic digestion.Polymer does not contain and carries out the necessary composition of membrane interaction, therefore suffers from same problem above-mentioned, and they are unsuitable for oral.

Below article disclosed the other technologies of stabilized peptide and pharmaceutical grade protein, wherein, pharmaceutical grade protein is that chemical compound with lower molecular weight is (as aminolethicin, fatty acid, vitamin B ₁₂And glucoside) bonded: R.Igarishi et al., " Proceed.Intern.Symp.Control.Rel.Bioact.Materials, 17,366, (1990); T.Taniguchi etal.Ibid 19,104, (1992); G.J.Russel-Jones, Ibid, 19,102, (1992); M.Baudys et al., Ibid, 19,210 (1992).These modified compounds are not polymer, therefore do not contain and give dissolubility and the required composition of film affinity, and dissolubility and film affinity then are oral and the parenteral administration artifact utilizes necessary.Many such preparations lack the oral bioavailability rate.

Being used the other method that prolongs action time in the protein material body is encapsulated technology.M.Saffan etc. are at Science, and 223,1081, (1986) proposed on pharmaceutical grade protein is sealed in and be used for orally in the azobenzene polymer film, reported this film and do not digested under one's belt, but in large intestine, degraded by microbiota, thus, discharge sealed protein at this place.This technology is utilized physical mixture, does not promote to be released proteinicly to absorb through film.

Ecanow is in U.S. Patent No. 4,963, proposes the film phonograph seal that physiologically active compound (comprising protein) can be formed by cohesion in 367, and the product of making can be suitable for through the film administration.Other preparations of same invention can through suck, oral, parenteral route and percutaneous administration.These methods do not provide the character required as oral administration-to the complete stability of gastrointestinal tract acidity and protease.

For the stable protein medicine is that therapeutic agent is wrapped in the liposome to make other method oral and that parenteral administration is taked.The summary of this technology is seen Y.W.Chien, " NewDrug Delivery Systems ", Marcel Dekker, New York, NY, 1992.Liposome-albumen composition is a physical mixture, and their dispensing produces irregular and can not expected result.It is reported, in the use of this liposome-albumen composition, produce undesirable protein component in some organ and pile up.Except these factors, when using liposome, also have such as shortcomings such as expense height, the preparation method of difficulty that needs complicated lyophilization cycle and solvent incompatibilities.In addition, when the clinical useful Liposomal formulation of exploitation, the bio distribution of variation and antigenicity tissue become restraining factors.

The someone has described use (Santiago, N., Milstein, the S.J. of " proteinoid " recently, Rivera, T., Garcia, E., Chang., T.C., Baughman, R.A., and Bucher, D., " Oral Immunization of Rats with InfluenzaVirus M Protein (M1) Microspheres ", Abstract#A221, Proc.Int.Symp.Control.Rel.Bioac.Mater., 19,116 (1992)).This article has been reported a few class therapeutic agents with this system's oral administration, and this system is sealed in the interested medicine of people in the polymeric jacket of being made up of highly branched aminoacid.The same with the situation of liposome, medicine be not chemical bond on the proteinoid ball, it is possible that medicine spills from the dosage form composition.

Become the focus of a lot of synthetic works, the peptide of being devoted to improve its administration and bio-absorbable is an insulin.

Insulin is used for can tracing back to nineteen twenty-two to treatment of diabetes, at that time, Banting etc. (" Pancreatic Extracts in the Treatment of Diabetes mellitus; " Can.Med.Assoc.J., 12,141-146 (1922)) prove that the activity extract that obtains from pancreas has therapeutic effect to the diabetes Canis familiaris L..In the same year, treat the exciting clinical improvement that diabetics causes saving life with pancreatic extract.Every day, the process of insulin injection was necessary for long-term rehabilitation.

Insulin molecule is formed the molecular weight of insulin about 6,000 by two amino acid chains that connect through disulfide bond.The strand precursor of the β emiocytosis insulin of islets of langerhans is called proinsulin.Insulinogenic albuminolysis cause 4 basic amino acids removal (respectively the No.31,32 of insulin chain, 64 with 65: Arg, Arg, Lys, Arg) with (" the C ") peptide that is connected.In the double-stranded insulin molecule of gained, A chain amino terminal is a glycine, and B chain amino terminal is a phenylalanine.

Insulin can monomer, the form of dimer and six aggressiveness that formed by three dimers exists.Six aggressiveness and two Zn ⁺⁺The ion coordination combination.Biological activity is present in the monomer.Though as of late, almost have only cattle and Iletin II (Lilly) to be used to treat people's diabetes; But learnt that islets of langerhans have numerous differences between kind of the system.Iletin II (Lilly) is the most similar to insulin human, and difference is alanine rather than threonine at the C-of B chain end only.Although these difference is arranged, most of mammals islets of langerhans have similar specific activity.As of late, animal extracts provides and has treated the used whole insulins of this disease.The appearance of recombinant technique makes the commercial-scale system of insulin human respectively become possibility, (for example, Humulin ^TMInsulin can be buied from the Eli Lilly company of indiana ,US Minneapolis).

Though insulin was used for treating diabetes existing more than 70 year, two pieces of publications as of late, the research that relevant its preparation stability seldom occurs is reported.(Brange，J.，Langkjaer，L.，Havelund，S.，and Volund，A.，“Chemical sta-bility of insulin.1.Degradation during storage of pharmaceuticalpreparations，”Pharm.Res.，9，715-726，(1992)；and Brange，J.Havelund，S.，and Hougaard，P.，“Chemical stability of insulin.2.Formulation of higher molecular weight transformation prod-ucts during storage of pharmaceutical preparations，”Pharm.Res.，9，727-734(1992))。In these two pieces of publications, the author has described the chemical stability of several insulin preparations under the condition of temperature and pH variation in detail.Report early almost all concentrates on the biological value of measuring as insulin preparation stability.But several new strong analytical technologies as the appearance of disc electrophoresis, volume removing chromatogram and HPLC, make the chemical stability feature of detailed mensuration insulin become possibility.The chemical research of early stage insulin stability is difficult, because the insulin purity of the recrystallization that discovery is tried is not higher than 80-90%.Can obtain the high purity insulin of single component recently.This single component insulin impurities is lower than the level that detects of existing analytical technology.

The islets of langerhans of preparation have the polytype propensity for degradation.When the amide side chain base on glutamine or the asparagine residue was hydrolyzed into the free carboxy acid, non-enzymatic deamidating appearred.This deamidating of insulin has 6 possible position: Gln ^A5, Gln ^A15, Asn ^A18, Asn ^A21, Asn ^B3, and Gln ^B4The report prompting of having delivered, three Asn residues are the most responsive to this reaction.

Brange etc. (the same) report, under acid condition, insulin passes through Asn ^A21The position extensive deamidating and degraded rapidly.On the contrary, in the neutrality prescription, at Asn ^B3Deamidating takes place in the position, its speed slowly many, do not rely on the kind system in insulin concentration and insulin source.Yet temperature and preparation type are at decision B ₃Play an important role on the hydrolysis rate of position.For example, compare, if insulin is crystallization shape, then B with unbodied ₃The position is hydrolyzed to MIN.The pliability of crystallization shape (ternary structural) reduces, and reduces response speed significantly.Phenol is mixed neutral preparation causes demidizate speed to reduce to stablize ternary structural.

In insulin preparation, except that the hydrolysis catabolite, also form the high molecular converted product.Brange etc. prove that with size exclusion chromatography the primary product that insulin preparation forms is the insulin dimers of covalency under storing between 4-45 ℃.In containing the preparation of protamine, also form the insulin protamine product of covalency.The tangible temperature influence of formation speed of insulin dimers and insulin-protamine product.For people or Iletin II (Lilly), the formation time of 1% high molecular weight product (conventional N1 preparation) is compared with 4 ℃ in the time of 37 ℃, reduces to 1.7 months from 154 months.For the zinc mixed suspension preparation of Iletin II (Lilly), same conversion needs 357 months in the time of 4 ℃, and only needs 0.6 month in the time of 37 ℃.

These degraded types of insulin may be far reachings to diabetics.Though the formation of high molecular weight product is slower than the formation of aforementioned hydrolysis (chemistry) catabolite usually, its influence may be more serious.There is clear evidence to show, immunoreactive generation to insulin may be from (Robbins, D.C.Cooper, the S.M.Fineberg of existing of insulin covalent polymer, S.E., and Mead, P.M., " Antibodies to covalentaggregates of insulin in blood of insulin-using diabetic patients ", Diabetes, 36,838-841, (1987); Maislos, M., Mead, P.M., Gaynor, D.H., and Robbins, D.C., " The source of the circulatingaggregate of insulin in type 1 diabetic patients is therapeutic in-sulin ", J.Clin.Invest., 77.717-723. (1986); And Ratner R.E., Phlilips, T.M., and Steiner, M., " Persistent cutaneous insulinallergy resulting from high molecular weight insulin aggregates " .Diabetes, 39,728-733, (1990)).Having nearly, 30% diabetics of accepting insulinize shows specific antibody to the covalency insulin dimers.According to reports, because the existence of covalency insulin dimers, the ratio that allergia patient produces the LS reaction of highly significant only accounts for 2%.When dimer content is in the 0.3-0.6% scope, react not remarkable.Therefore, answer the level of the covalency insulin dimers in the preparation to be controlled at below 1%, to avoid various clinical manifestations.

Insulin preparation has several commercially available products; Though stability has been improved to the degree of all preparations of cold preservation again that need not, but still maintenance is to the demand of the insulin preparation of stability increase.The improvement insulin that is difficult for the formation high molecular weight product will be substantial progress in pharmacy and medicine technology field, provide these improvement of this stability (probability of oral insulin also is provided) will make significant contribution to treatment of diabetes.

Polypeptide and protein are except using in vivo as therapeutic agent, and on diagnostic reagent was used, polypeptide and protein also obtained the substantial use that increases day by day.In a lot of such application, polypeptide and protein use in solution environmental, in solution environmental, they are to temperature and (many) peptides and proteinic enzymatic degradation sensitivity, these (many) peptides and protein are such as being used for immunoassay, antibody---hapten is in conjunction with the enzyme of usefulness, peptide and proteohormone, antibody, the enzyme-protein conjugate, at diagnosis or screening AIDS, hepatitis and wind such as examine at a large amount of virus proteins that use on detection side's science of law of disease, be used for for example peptide and the protein growth factor of tissue culture, be used for the enzyme of clinical chemistry and such as the used insoluble enzyme of food industries.Example is more specifically, and in the test kit of antibody that is used for biological fluid or antigenic colorimetric determination, alkali phosphatase is used as a kind of reagent.Though this kind of enzyme is commercially available with various forms, comprise resolvase and antibody conjugates, its bin stability and solution are often restricted.As a result, the alkaline phosphatase enzyme conjugates often is cryodesiccated, and uses bovine serum albumin and polysorbas20 and so on additive to increase the stability of enzyme preparation.Though these methods help strengthening polypeptide and the tolerance of protein reagent to degrading in some cases, and various shortcomings are arranged, and limit its widespread usage.

The present invention relates at large in conjunction with stable (many) peptides and protein compositions and preparation, and preparation and using method.

More specifically, the present invention relates to covalently bound peptide complexes a wide combined aspects, wherein peptide is incorporated into one or more polymer molecules (as linear polyglycols) that mix hydrophilic group as its ingredient, and this polymer has mixed a lipophilic group as its ingredient.

In another special aspects, the present invention relates to physiologically active peptide compositions, comprise and contain (i) the linear polyglycols group and the (ii) banded physiologically active peptide of polymer covalency of lipophilic group, wherein peptide, linear polyglycols group and lipophilic group each other the arrangement on conformation make in the physiologically active peptide compositions physiologically active peptide with respect to independent physiologically active peptide (the non-binding shape that promptly lacks coupling polymer thereon) to tolerance enhancing in the body of enzymatic degradation.

On the other hand, the present invention relates to the physiologically active peptide compositions of three-dimensional conformation, comprise and contain (i) the linear polyglycols group and the (ii) banded physiologically active peptide of polysorbate complex covalency of lipophilic group, wherein physiologically active peptide, linear polyglycols group and lipophilic group each other the arrangement on conformation (a) lipophilic group in three-dimensional conformation is exposed and (b) physiologically active peptide in the physiologically active composition with respect to independent physiologically active peptide to tolerance enhancing in the body of enzymatic degradation.

Further, the present invention relates to the bonded peptide complexes of multivalent ligand, this peptide complexes comprise triglyceride main chain group and

By being incorporated into the basic at interval and covalently bound biologically active peptide of triglyceride main chain group of polyglycols on the triglyceride main chain group carbon atom; And

Directly with triglyceride main chain group on carbon atom covalently bound or by at interval basic covalently bound at least one fatty acid group of polyglycols.

In the bonded peptide complexes of this multivalent ligand, the α ' of triglyceride bio-active group and β carbon atom can have direct covalent bond on it, or by the polyglycols base resinic acid group of covalent bond on it indirectly at interval.Perhaps, fatty acid group can be directly or by polyglycols at interval base be covalently bonded in the α and the α ' carbon of triglyceride main chain group, and the β carbon covalency of biologically active peptide and triglyceride connects, directly covalent bond thereon or by polyglycols at interval base indirectly in conjunction with thereon.People will recognize that, discuss in the above in the scope, for the multivalent ligand binding peptide complex that contains triglyceride main chain group, a large amount of different structure, compositions and dosage forms can be arranged.

In very big scope of the present invention, the biologically active peptide in the bonded peptide complexes of this multivalent ligand is so that the basic modified main chain group covalent bond with triglyceride of base or other acceptable intervals is more favourable at interval by alkyl.The acceptability of the interval base that this moment is used is meant the special acceptable feature in spatial, the structural and terminal use.

More on the one hand, the present invention relates to contain the polysorbate complex of the polysorbate composition that comprises the triglyceride main chain, on the triglyceride main chain, have the functional group that is covalently bonded in its α, α ' and β carbon atom, it comprises:

(i) fatty acid group; With

The polyglycols group that (ii) has covalent bond physiologically active group thereon, for example, the physiologically active group is covalently bond to the suitable degree of functionality of polyglycols group.

Such covalent bond can be direct, for example, be attached to the C-terminal functional group of polyglycols group, perhaps, covalent bond can be indirect, for example, at interval base is topped to the C-terminal of polyglycols by reaction with the terminal carboxyl group official, but so that resultingly has physiologically active group covalent bond terminal carboxyl group degree of functionality thereon through topped polyglycols.

The present invention also further relates to stable, water miscible binding peptide complex, and it comprises the physiologically active peptide on the glycolipid part that is covalently bonded in the upward compatible polyglycols modification of physiology.In this complex, physiologically active peptide can be by polypeptide the unsettled covalent bond of free shape amino acid group be covalently bonded in the physiology and go up the glycolipid base that compatible polyglycols is modified, wherein unsettled covalent bond can be cut off by biochemical hydrolysis and/or albuminolysis in vivo.The glycolipid group that the last compatible polyglycols of physiology is modified is to comprise the polysorbate polymer, and the polysorbate polymer that for example contains the fatty acid ester that is selected from monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurin, monoleate, dioleate, trioleate, monostearate, distearate and tristearate is for more favourable.In this complex, the glycolipid part that the last compatible polyglycols of physiology is modified can suitably comprise the polymer of the polyglycols ester of the polyglycol ether that is selected from fatty acid and fatty acid, and wherein fatty acid is selected from lauric acid, Palmic acid, oleic acid and stearic fatty acid for for example comprising.

In above-mentioned complex, separate as example, physiologically active peptide can comprise the peptide that is selected from following material: insulin, calcitonin, ACTH, glucagon, growth hormone release inhibiting hormone, growth hormone, somatomedin, parathyroid hormone, erythropoietin, hypothalamic releasing factor, prolactin antagonist, thyrotropin, endorphins, enkephalin, vasopressin, the class deltorphin delta (opiods) that non-natural exists, the peroxide dismutase, interferon, asparagine amide enzyme, arginase, the arginine deaminase, the adenosine deaminase, ribonuclease, trypsin, chymase and papain.

On the other hand, the present invention relates to regulate (mediation) insulinopenic peroral dosage form, comprise pharmaceutically acceptable carrier and stable water solublity bound insulin complex, the latter comprises the insulin or the proinsulin of the glycolipid group that is covalently attached to the upward compatible polyglycols modification of physiology.

Further, the present invention relates to showing that insulinopenic people or non-human mammal experimenter treat insulinopenic method on one's body, comprise insulin or the insulinogenic stable water solublity bound insulin complex that the covalent conjunct agent physiology goes up the glycolipid group of compatible polyglycols modification that contain to the oral effective dose of experimenter.

The polypeptide of molecular weight≤about 10,000 and the protein of molecular weight＞about 10,000 broadly represented to comprise in term used herein " peptide ", and wherein molecular weight is the number mean molecule quantity.As used herein, term " covalently bound " refer to special groups both can be mutually direct covalent bond, also can pass through one or several spacer groups, as bridge, base or the mutual indirectly covalent bond of chain base at interval.Term " conjugated " refers to both covalent couplings mutually of special groups, and also non-covalent ground combination mutually is for example by hydrogen bond, ionic bond, Van der Waals force etc.

Thereby, the present invention includes the various compositionss that (in the body) used in treatment, wherein the peptide components of the peptide complexes that closes of yoke is physiologically active or biologically active peptide.Contain in the peptide combinations this, it can be combining of direct covalent bond or indirect (by the proper spacing base) that peptide composition closes with the yoke of the polymer that contains hydrophilic and lipophilic group, hydrophilic and lipophilic group can be arranged in the polymer yoke in any appropriate manner and close in the structure, comprise direct or indirect each other covalent bond.Therefore, miscellaneous peptide can be regulated to be used for use widely of the present invention by the needs or the hope of specific final treatment application.

On the other hand, those covalently bound peptide combinations can utilize and intend being used to diagnosing or the peptide composition of external application as described above, wherein peptide is such as diagnostic reagent, a kind of diagnosis that is used for using in immunoassay or other diagnosis or non-body complement of conjugates.In such non-therapeutic was used, usefully as stable compositions, they can be formulated in the preparation of compatible solvent or other solution substrate peptide complexes height of the present invention, can effectively degradation-resistant stable compositions dosage form to provide.

In aforementioned therapies and non-therapeutic (as diagnostic) application, the present invention relates to covalently bound peptide complexes a wide combined aspects, wherein, one or more polymer molecules of peptide covalent conjunct agent, this polymer molecule mix hydrophilic radical (as the polyglycols group) and lipophilic group (as fatty acid group) as described in the ingredient of polymer.Aspect preferable, peptide can mix lipophilic group (as fatty acid group) as ingredient by covalent bond and one or more linear polyglycols polymer molecule covalent bond in this polyglycols polymer.

Another wide especially aspect, the present invention relates to non-covalent bonded peptide complexes, wherein peptide and one or more polymer molecules associate non-covalently, and fusion hydrophilic radical in the polymer molecule (as the polyglycols group) and lipophilic group (as fatty acid group) are as ingredient.This polymer can be the various structures and the homotaxis thing of polymer in the above-mentioned covalent bond peptide complexes, but wherein peptide is not to be incorporated into polymer molecule with covalent manner, and for example by such as association key and polymer associate such as the microcapsule parcel of hydrogen bond, ionic bond or ion coordination, Van der Waals key, special peptide or associations.

The non-covalent association of this peptide composition and polymeric groups can utilize treatment (in body) to use the non-therapeutic peptide composition with peptide composition and as diagnosis or other (external).

In the bonded peptide combinations of this association, can select in the technical scope of this area that a kind of mode (for example giving the hydrogen bond force of peptide and polymer) makes up rightly, modified polymer component or make it suitably functionalized, to give its bonded ability of associating.

In the open and additional claim below, other aspects of the present invention, feature and modification will be clear more fully.

Fig. 1 be time of giving serum glucose (mg/dL) behind insulin itself and the compound dosage form insulin (hour) function curve.

Fig. 2 is time (branch) function curve that gives serum glucose (mg/dL) behind the various dosage form insulins.

Fig. 3 is that insulin and OT insulin are by the time function curve of pancreas milk reducing protease digesting.

Fig. 4 is the time function curve that rat oral gives the serum calcium of rat behind the polymer calcitonin conjugates.

Fig. 5 is that the expression rat oral gives behind polymer calcitonin OT 1-ct or the OT 2-ct polymer calcitonin conjugates time function bar chart of calcium level in the rat blood serum.

Fig. 6 is the bar chart of polymer on the diabetes rat model-insulin effect.

Fig. 7 be expression monkey (cynomolgous monkey) per os give glucose in the 001-insulin bleeding from anus through the time percentage effect figure that changes.

Fig. 8 be expression monkey (cynomolgous monkey) per os give 001-insulin post dose through the time effect effect figure.

Can provide some benefit with polymer-modified peptide nontoxic, non-immunogenicity. If the product (polymer-peptide conjugates) that makes of modifying can keep its whole or most of biologically actives, then can produce following character: can strengthen the epithelial cell penetrating power; Can protect modified peptide to exempt from protein breakdown digestion and subsequent loss of activity; Can improve the compatibility to the endogenous movement system; Can give the chemical stability to hydrochloric acid in gastric juice; Can make the balance between polymer lipophilicity and the hydrophily reach optimum velocity. It can be effective that protein material with above-mentioned improved properties is treated as an alternative behind oral or parenteral administration. Peptide with improvement also can adopt other administration route, such as intranasal and percutaneous dosing.

In non-therapeutic was used, when peptide composition was covalently bonded in polymer in mode of the present invention, comprising that the combination of the diagnosis of the precursor of the peptide of final product or other products and intermediate and/or reagent class peptide is stable provided corresponding benefit. The covalent bond peptide of gained comprises that to the environment degradable factor degradation process of solvent or solution mediation has tolerance side. This result that the degraded tolerance is strengthened, the shelf-life of active peptide component can obviously prolong, and incident is the increase that contains the reliability of peptide composition in being applied to specific use.

The peptide that carries out with the inventive method and the covalent bond of polymer make hydrolytic degradation minimize effectively, obtain external and the body internal stability.

Work as therapeutic, diagnostic or reagent class peptide presses method of the present invention and polymer is non-covalent, when associating binding, also see similar benefit.

Utilization is covalently bonded in the insulin of polymers compositions as illustrative embodiment of the present invention, comprises that the essence of the combination of cleavable covalent chemical bond is considered with polymer and can be controlled from the time-histories of peptide (insulin) cracking. This cracking can exist enzyme or chemical mechanism. Visible all active after lie prostrate complete cracking at polymer. And chemical modification can make peptide (such as the insulin) infiltration that links pass through cell membrane. In a preferred aspect of the present invention, the promotion membrane permeability matter of lipophilicity fatty acid residue participates in conjugated polymer. In this respect, recycling is as the insulin of concerned peptide, and the fatty acid polymer derivative of insulin has improved the intestinal absorption of insulin: use the long-chain fat acid polymer with Phe^B1And Lys^B29Carbamoylation obtains providing the compound that reduces to a certain degree the blood sugar activity. This derivatization improves insulin in the stability of intestinal mucosa, increases it from the absorption of small intestine.

Although following explanation mainly, illustratively in various compositions of the present invention and the preparation insulin as the application of peptide composition, yet should understand like this, application of the present invention is not so limited, and expand to any kind peptide with the inventive method covalency or the ground combination of associating, include but not limited to the peptide of following kind: calcitonin, ACTH, hyperglycemic factor, growth hormone-release inhibiting factor, growth hormone, somatomedin, parathyroid hormone, hematopoietin, hypothalamic releasing factor, prolactin, thyroid-stimulating hormone (TSH), endorphin, antibody, hemoglobin, solubility CD-4, clotting factor, tissue plasminogen activator, enkephalins, vasopressing, the class deltorphin delta that non-natural exists, the peroxide dismutase, interferon, asparagine acid amides enzyme, arginase, the arginine deaminase, the adenosine deaminase, ribalgilase, trypsase, chymotrypsin, and papain, alkaline phosphatase, and other suitable enzymes, hormone, protein, polypeptide, the enzyme-protein bond, antibody-hapten conjugates, virus epitopes etc.

An object of the present invention is to provide for the suitable polymer of peptide combination, in order to obtain above-named gratifying feature. Another purpose is to utilize this modified peptide with transit peptides enduringly in vivo. Another purpose is with the oral peptide that gives of activity form with this technology.

Further purpose is that peptide with the association combination is applied to immunoassays, diagnosis and other non-therapeutics (as external) application facet. The peptide composition that also has a purpose to provide stable bond of the present invention, comprise all covalently bound compositions that not only had been suitable in the body but also had been suitable for using in the non-body, or all non-covalent, association binding peptide compositions that not only is suitable in the body but also is suitable for using in the non-body are provided.

In broad scope of the present invention, can with the single polymers molecular application in the combination of numerous kinds peptide, in broad practice of the present invention, utilizing multiple polymers also may be useful as the bond of a certain peptide; Also can adopt the combination of these methods. And the peptide composition of stable bond all has practicality in using in vivo with in the non-body. In addition, can recognize, if conform with final application, in conjunction with polymer can utilize any other group, composition or other are in conjunction with kind. As an example, use in some that is covalently bonded in polymer, to polymer give anti-UV degraded, functional group anti-oxidant or other character or characteristic may be useful. As a further example, polymers function is used with some of giving some activity or crosslinkable characteristic, it may be useful strengthening total various character or characteristic in conjunction with material. Therefore, polymer can contain and not hinder binding compositions to any degree of functionality, the recurring group of usefulness of expection purpose, key, or other form structures. Other purposes of the present invention and advantage will be more fully clear from following explanation and appended claims.

The below describes the illustrative polymer of the feature that can be used for obtaining these expectations in the reaction process of enumerating. In the application of covalently bound peptide, polymer can be functionalized, and then is attached on the free amino acid of peptide, forms labile bond, under the state that complete labile bond exists, is still keeping activity. Then, remove this key by chemical hydrolysis and protein breakdown, increase the activity of peptide.

The polymer that the present invention utilizes can suitably mix the compositions such as interval base that are connected usefulness such as edible fatty acid (lipophilicity is terminal), polyglycols (water-soluble end), acceptable glycosyl (acceptor interaction is terminal) with peptide in its molecule. In selected polymer, polysorbate is desirable especially, in following discussion, is chosen to illustrate various embodiments of the present invention. Scope of the present invention is not limited to polysorbate certainly, can useful application in broad practice of the present invention contains various other polymer of above-mentioned group. Sometimes it may be desirable removing in these groups one in polymer architecture, keep other group and do not lose purpose. When hope is done like this, do not lose purpose of the present invention and interests and the group that should remove is sugar and/or interval group.

The polymer molecular weight of better employing drops between the 500-1000 dalton.

In practice of the present invention, C₂-C ₄The polyglycols residue of alkyl polyglycols (being preferably polyethylene glycol (PEG)) is advantageously mixed in the interested polymer system.

The existence of these PEG residues is given hydrophily to polymer and corresponding polymer-peptide conjugates. Known some glycolipid can stable protein and peptide. This stable mechanism may comprise the combination of the water repellent region of the fatty acid group of glycolipid and peptide or protein, thereby has prevented the cohesion of protein or peptide. Known also has, and the peptide of cohesion is difficult to be absorbed than native peptides at small intestine. Therefore, the present invention expects such polymer-peptide prod, and wherein peptide (such as insulin) is combined with hydrophily or the hydrophobic residue of polymer. The fatty acid part of polymer associates for the hydrophobic region with peptide, thereby prevents the cohesion in solution. Thereby resulting polymer-peptide bond will be, and will be stable to chemistry and enzyme hydrolysis; Because of PEG residue tool water-soluble; And be difficult for aggegation because of aliphatic acid-hydrophobic region reciprocation.

In practice of the present invention, the polyglycols derivative has a lot of useful character in the polymer-peptide conjugates preparation, and as relevant with following polyglycols derivative character: water solubility improves, and does not cause the reaction of antigen or immunogene simultaneously; High degree of biocompatibility; Without biodegradation in the body of polyglycols derivative; And the body that is easy to be lived is got rid of.

Therefore, the polymer of using in the present invention's practice comprises lipophilicity and hydrophilic radical, the polymer-peptide conjugates that makes gained highly effectively (biologically active) in oral and parenteral route and other physiological administering modes is also highly effective in non-physiologic is used.

The below classify as polymer-peptide conjugates of the present invention illustrative embodiment be after be called for short the molecular formula of the represented covalently bound bond of bond 1, bond 2 and bond 3, wherein " Ins " is insulin, and the occurrence of m, n, w, x and y will be described in the following discussion. Bond 1:

Wherein: w+x+y+z=20;

Bond 2:

Bond 3:

Bond 1 is for being positioned at the commercially available polysorbate monoleate (polysorbate monopalmitate) at polymer system center, and this is a kind of sugar derivatives for a lot of medicinal applications. The character that lipophilicity and absorption increase is given by the oleic acid chain, and polyethylene glycol (PEG) residue provides the environment of hydrophily (accepting hydrogen bond). Insulin links up by the amino-formate bond in the PEG district of polymer by the neighbour.

In bond 2 without saccharide residue, but insulin again the amino-formate bond in the PEG district by contiguous polymer be linked to polymer. Therefore, the tie point of the lipophilicity aliphatic acid district of polymer and insulin has some distances.

The situation of bond 3 is with top opposite to bond 2 described arrangements, still do not contain saccharide residue, but in this structure, the most approaching tie point with insulin of lipophilicity fatty acid residue, and hydrophily PEG district is away from tie point, and this connection is also undertaken by amino-formate bond.

In broad practice of the present invention, may there be changeable combination in hydrophily and the lipophilicity zone relevant with the peptide tie point with polymer, and such variation can produce the lipophilicity of protection insulin and the polymer of hydrophilic region. In bond 1,2 and 3, the amino-formate bond tie point between the polymer is with Gly^A1Amine functional group be advisable. As mentioned above, may there be an above polymer unit to be connected on each peptide molecule. For example, if second polymer is connected on the insulin, point of contact preferably passes through Phe^B1Amine functional group. At least in theory, the 3rd polymer can be connected to Lys^B29Amine functional group on. But experience shows, connects two polymer molecules on each insulin molecule and be reasonably the highest available actual derivative degree.

In general practice of the present invention, the whole bag of tricks that polymer is coupled on the peptide all is feasible, and does hereinafter to beg for more completely. In the work of carrying out proteins and peptides, it should be understood that the specific residue on the peptide is important in its biology integrality. Importantly select suitable coupling agent, it is this residue of interference within reason also. In some cases, may be difficult to avoid the coupling of these important residues thereby hinder its activity, but some activity may bring the stability raising and keep existing beneficial property. For example, in using in vivo, administration frequency can thereby reduce, and expense is reduced, and patient compliance improves.

Physical characteristic for the polymer by proteins/peptides bond of the present invention is designed to mix accomplishes the end in view it. Sorbefacient makes Toplink permeation cell film, but does not improve the stability characteristic of peptide, so application can be exempted from dosage form with this penetration enhancer in the body, and utilizes polymer-peptide conjugates of the present invention. Therefore, one aspect of the present invention relate in polymer, mix derivative of fatty acid with the simulation penetration enhancer.

In covalently bound polymer of the present invention-peptide conjugates, peptide can be covalently bonded in water-soluble polymer by unstable chemical key. This covalent bond between peptide and the polymer can be through chemistry or enzyme reaction and is ruptured. But polymer-peptide prod keeps the activity of receiving amount; When polymer fully and after the peptide cracking, whole activity of peptide composition just display. Simultaneously, polyalkylene glycol moiety is present in the polymer of combination, the ability that makes polymer-peptide have highly-water-soluble and circulate in blood for a long time. Glycolipid occupies mode and the polymer associate of the hydrophobic region of peptide usually with its fatty acid group, this has just prevented cohesion. The cohesion of peptide causes it poor at intestinal absorption. UA peptide is more easily by intestinal absorption. Glycolipid mixes the polymer of combination, thereby reaches the purpose of improving stability and preventing from producing in conjunction with rear peptide cohesion. The character such as dissolubility, stability and film compatibility that peptide improves are given in above-mentioned these modifications. The result that these characteristics are improved, available living polymer-peptide carries out parenteral route and oral administration, and peptide is hydrolytic rupture in vivo subsequently, by biological utilisation.

Used polymer can be categorized as polyethyleneglycol modified glycolipid and polyethyleneglycol modified aliphatic acid in the described embodiment below. In better conjugated polymer, polysorbate be can mention, monopalmitate, dipalmitate, tripalmitate, monolaurate, dilaurate, trilaurin, monoleate, dioleate, trioleate, monostearate, distearate and tristearate comprised. Several mean molecule quantities of the polymer that obtains from every kind of combination should be about 500 to about 10,000 dalton's scopes. Polyglycol ether or ester that the preferential alternative polymer of this embodiment is aliphatic acid, such aliphatic acid is laurate, palmitic acid, oleic acid and stearic acid, polymer is for counting mean molecule quantity from 500-10,000 dalton. But deriveding group is preferably arranged in polymer, and this type of group can be at the end that stops with polyethylene glycol or at the end that stops with aliphatic acid. But deriveding group also can be positioned at polymer inside, thereby can be used as the sept between peptide and the polymer.

The modified fatty acid sorbitan will be described in a more detailed discussion with the structure diagram with the certain methods that obtains required polymer.Polysorbate is the ester of Sorbitol and acid anhydride thereof, is formed by 1 mole of Sorbitol and sorbitan and about 20 moles of ethylene oxide combined polymerizations.Below shown in be the structure of representative polymer. W, X, Y, Z add up to 20, R ₁, R ₂And R ₃Be selected from the group that lauric acid, oleic acid, Palmic acid and stearate radical are formed independently of one another, or R ₁And R ₂Respectively be hydroxyl R ₃Be lauric acid, Palmic acid, oleic acid or stearate radical.These polymer have commercially available product, are used for pharmaceutical preparation.When the needs higher molecular weight polymer, can be from glycolipid (sorbityl monododecanoate, sorbitol monooleate, Sorbitol monopalmitate or Sorbitol monostearate) and suitable synthetic the obtaining of Polyethylene Glycol.The structrual description of glycolipid that can be used as initial reagent is as follows.

m＝10-16

In the glycolipid polymer that has three positions to be replaced by Polyethylene Glycol synthetic, the end group that required end has a Polyethylene Glycol of two free hydroxyl groups carries out tritylation (in pyridine) with 1 mole of trityl chloride and is protected.A remaining free hydroxyl group of Polyethylene Glycol is transferred to tosylate or its bromide.Required glycolipid is dissolved in suitable atent solvent, and handles with sodium hydride.Tosylate or its bromide of protected Polyethylene Glycol are dissolved in atent solvent, and with in the excessive adding glycolipid solution.At room temperature, use this product of the solution-treated of p-methyl benzenesulfonic acid in anhydrous inert solvent, and purify with column chromatography.The structural formula of this conversion is described below.

Molar equivalent by regulating reagent and with the Polyethylene Glycol of suitable molecular weight ranges can obtain the single of desired molecule weight range by top step and replace or two replacement glycolipids. Wherein n and m all can change separately, and its value is for being suitable for any appropriate value of stabilized particular peptide, as 1-16.

The sugar moieties of above-mentioned glycolipid can be replaced by structural formula following glycerol or amino glycerol.

In this modification, as follows, with fatty acid group (as lauric acid, oleic acid, Palmic acid or stearic acid) etherificate or esterification primary alconol; And make amino be derivatized to amide or secondary amine with fatty acid.

The desirable arbitrary appropriate value of m wherein is as 10-16.

Remaining primary alconol base trityl as protecting group, and secondary alcohol groups is converted into required polymer with Polyethylene Glycol.Usually, but Polyethylene Glycol at one end has a leaving group, and has methoxyl group at the other end.Polyethylene Glycol is dissolved in atent solvent, and is added in the solution that contains glycolipid and sodium hydride.Product in slough protection under the room temperature in p-methyl benzenesulfonic acid, is obtained required polymer as follows.

Sometimes need to mix derivative of fatty acid, obtaining and some the identical physicochemical properties of polysorbate (as the polysorbate trioleate) that replaced by 2 or 3 molecules of fatty acids at the different parts of polyglycol chain.

Show the structure of representation polymer in the following reaction process, be the open chain polysorbate.

Pr=peptide: protein, pharmaceutical grade protein; The R=alkyl, C ₅-C ₁₈N=5-120; Pr=peptide: protein, pharmaceutical grade protein; The R=alkyl, C ₅-C ₁₈N=5-120; M=2-15;

Pr=peptide: protein, pharmaceutical grade protein;

The R=alkyl, C ₅-C ₁₈

m＝5-18；

n＝2-15

y＝5-120；

Wherein, m, n and y can change in above-mentioned scope independently of each other.

(formula 8)

In polymer A synthetic, need the hydroxyl on 1 and 2 carbon of protection glycerol (for example Solketal (trade name, DL-α, β-isopropylidene glycerol)).Remaining hydroxyl transfers sodium salt in atent solvent, and with the Polyethylene Glycol of halogenation or tosylation (wherein an end of Polyethylene Glycol become ester and protected) reaction.The protection of deglycerizin is converted into corresponding sodium salts with two free hydroxyl groups of gained.These salt are reacted with the Polyethylene Glycol of using the fatty acid part derivatization in atent solvent.Reaction occurs in free hydroxyl group and is converted into after tosylate or its bromide.

Except protected glycerol is reacted by halogenated fatty acid ester with end carbon earlier, with same method synthetic polymer G.

In polymer C synthetic, with from 1,3-dihalo-2-propanol begins to good.The dihalo chemical compound is dissolved in atent solvent, and reacts with the sodium salt with 2 moles of Polyethylene Glycol of 1 moles of fatty acids group derivatization.Product is purified with chromatography or dialysis.The dry labor thing of gained is handled with sodium hydride in atent solvent.Halide derivative reaction with sodium salt that generates like this and the Polyethylene Glycol of partly being protected.

Sometimes, may need to omit the sugar moieties of polymer.Resulting polymers still contains the Polyethylene Glycol segment.With the suitable substituted fatty acid of lipotropy long chain alkane, can protect the film affinity of fatty acid group; Thereby maintenance biocompatibility.A kind of situation of the present embodiment is with the Polyethylene Glycol of sodium hydride processing with two free end hydroxyls in atent solvent.The one-level bromide of the fatty acid group of 1 equivalent weight is added in the Polyethylene Glycol solvent mixture.Extract required product with atent solvent, if need, available column chromatography is purified.

When needing to form ester bond between fatty acid and the Polyethylene Glycol, in suitable atent solvent, handle this sour chloride thing with excessive required Polyethylene Glycol.In atent solvent, extract polymer,, do further to purify with chromatography if need.

In some modification of peptide, need fatty acid directly is attached on the peptide.In the case, but with being positioned at derived functionalized groups synthetic polymer on the fatty acid group.Single oxygen base polyglycol solution of suitable molecular weight that will be in atent solvent is handled with sodium hydride, but then is added in the solution of the ethyl ester of the fatty acid that has leaving group on the end carbon, after the solvent extraction, if need, uses the column chromatography purified product.

Remove the ester protection with diluted acid or alkali treatment.

When needs form carbamate with polypeptide, carboxyl or ester are changed into hydroxyl with chemical reduction method known in the art.

HO-(CH ₂) _m-OC ₂2H ₄) _nXR (formula 13)

Be used for the bonded functional group of polypeptide usually at the end of polymer, but be positioned at polymer for good with functional group sometimes.At this moment, deutero-group plays base at interval.Under a kind of situation of this embodiment, fatty acid group can be by bromination on the α of carboxyl carbon, and acid groups is esterified.The experimental procedure of this compounds obtains following product with top listed identical:

When the interval that need prolong is basic, the Polyethylene Glycol monoether can be converted into amino, and handles in order to the succinic anhydrides of fatty acid group derivatization.The required Polyethylene Glycol that will have primary amine group is dissolved in the sodium phosphate buffer of pH8.8, and the succinic anhydrides fatty acid group that replaces with being shown below is handled.Product separates with solvent extraction method, if need, the reuse column chromatography is purified.

Should be understood that, the reaction equation that provides above is just to illustrative purposes, be not limited to such reaction and structure, can be utilized valuably in the modification of their peptides in broad practice of the present invention, for example, reach dissolubility, stability and the cell membrane affinity that is suitable for parenteral route and oral administration.

The invention provides the conjugate of biocompatible polymer and bioactive macromolecule, diagnostic reagent etc. (they can by forming) such as peptide, protein, enzyme, growth hormone, somatomedin and similar substance.Such macromolecular compound can enter amido link by a-amino acid, forms peptide oligomer and polymer and produces.According to the function of these materials, peptide components can be protein, enzyme, growth hormone etc.For for simplicity, these materials are referred to as peptide here, represent with Pr.In all cases, biologically active peptide contains free amine group or carboxyl.Bonding between polymer and the peptide generally produces by free amine group or carboxyl.

This description is that the selected peptide of purposes of illustration is gaze at especially in the scope of medicine, agricultural, science and family and commercial Application.They can be the enzymes that is used for replacement therapy; Promote the hormone of cell growth in growth of animal or the cell culture; Or be used for the activated protein metallic substance of various uses, biological example technology and biology and medical diagnosis.The enzyme that can mention is peroxide dismutase, interferon, asparagine amide enzyme, glutamic acid enzyme, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymase, and papain.In peptide hormone, what can mention is: insulin, calcitonin, ACTH, the high blood bran of pancreas element, somatostatin, growth hormone, somatomedin, parathyroid hormone, erythropoietin, hypothalamic releasing factor, prolactin antagonist, thyrotropin, endorphins, enkephalin and vasopressin.

The reaction that polymer and peptide generate the covalent bond product is easy to carry out.This paper is called polymer (P) for discussing for simplicity.During the polymer hydroxyl, change it into active carbonic acid ester derivant earlier, as p-nitrophenyl carbonate ester.Then under the condition of gentleness, should activatory derivant and the reaction of peptide short time, produce carbamate derivatives, this derivant keeps biological activity.

Above-mentioned reaction and reagent only play illustrative effect, and nonexcludability; Cause other activator of the formation of urethanes or other keys also can use.Can transfer hydroxyl to amino with reagent known in the art.By their carboxyl and peptide coupling in succession, cause the formation of amide again.

When polymer contains carboxyl, can be translated into mixed anhydride, and with the reaction of the amino of peptide, form the conjugate of amide containing key.In other method, available water dissolubility carbodiimide treatment, and and reactive polypeptide, the conjugate of generation amide containing key.

The activity of peptide conjugate and stability can several mode changes, as changing the molecular proportion of polymer and peptide, and the polymer of use different molecular size.Mix pulsating ratio of Polyethylene Glycol and size in the polymer composition by change, can change the dissolubility of conjugate.But by with fatty acid and and the careful hydrophilic and hydrophobic property of combination balance of polyethylene group.

Below listed be some illustrative modification reaction of polymer one peptide conjugate of the present invention.

Comprise in the above in the reaction process of I, J and K, shown and improved the equilibrated approach of conjugated polymer hydrophilic/lipophilic.Ester group in the conjugated polymer is to the esterase hydrolyzed sensitivity; Therefore, the conjugated polymer that contains ester group can be modified, and ester group changes ether into, and the latter has more anti-hydrolysis properties.The reaction process that contains L and M shows that hydroxyl changes the carboxylic acid group into.In this respect, carboxyl will provide carboxylate anion, and it is a better functional group (forming the ion coordination complex) of stability than hydroxyl, and hydroxyl does not form such complex.Other the suitable anion source functional group of formation that is used to contain the coordination ion complex of various polymer of the present invention comprises sulfate and phosphate group.

In a word, can use various technology valuably to improve the stability of polymer-peptide conjugates of the present invention, these technology comprise: have the functionalization of the polymer of high resistance hydrolysising group, ester group transfers ether to as described above; Improve the lipotropy/hydrophilic balance of conjugated polymer, make it to be suitable for being aggregated the stable peptide of thing; And the molecular weight of reducing polymer is to the level of the molecular weight that is suitable for being aggregated the stable peptide of thing.

Using valuable peculiar property by the polyglycols derived polymers for treatment of the present invention is general biocompatibility.Polymer has different water solublity, and nontoxic.Their no antigens, non-immunogenicity, the not biological activity of interferases.Their circulation times in blood are long, and are easy to get rid of from the body of living.

It is useful that product of the present invention is found on the biological activity of keeping peptide, and can be used for the treatment of administration by for example being dissolved in water or acceptable liquid medium prepares.Can pass through parenteral route or oral administration.Can prepare fine suspension colloid and be used for parenteral administration with generation depots effect, or the oral route administration.

At exsiccant lyophilised state, peptide-polymer conjugate of the present invention has stability for storage, and the pharmaceutical solutions of conjugate of the present invention is feature equally with the stability for storage.

Treatment of the present invention can be used to prevent with polymer-peptide conjugates or treat peptide components to the morbid state of effective any situation.

In addition, polymer-peptide conjugates of the present invention can be used for the diagnosis of composition, situation or the morbid state of biosystem or specimen, also can be used for the diagnostic purpose of non-physiological system.

And polymer-peptide conjugates of the present invention can have application in the prevention of botanical system situation or morbid state or treatment.As an example, the peptide components of conjugate can have parasite killing, weeding, antifungal and/or the efficacy of agricultural chemical of the service test stood on various botanical systems.

Further, conjugate of the present invention can be antibody or the antigen that matches for diagnosis, immunology and/or test purpose.

On treatment is used, the present invention has considered to suffer from these situations or morbid state or meticulously to it potential sensitivity and need make the Therapeutic Method of the animal examination body of this treatment, comprise to this animal throw give effective dose described situation or morbid state are treated effective polymer-peptide conjugates of the present invention.

Examination body with polymer-peptide conjugates treatment of the present invention comprises people and non-human animal (for example bird, Canis familiaris L., cat, cattle, horse) examination body, is preferably mammal examination body, preferably tries body for the people.

By concrete condition that is faced or morbid state, can any suitable treatment effectively and safe dose (in the present technique field, can easily determine, need not undue experimentation) to animal examination body throwing give polymer-peptide conjugates of the present invention.

In general, formula (1) chemical compound reaches the suitable dose of therapeutic efficiency in the scope of receiver's per kilogram heavy every day of 1 μ g-100mg, more fortunately in the scope of per kilogram of body weight 10 μ g-50mg every day, be preferably in the scope of per kilogram of body weight 10 μ g-50mg every day.Required dosage whole day proper spacing with 2,3,4,5,6 or more divided doses throw to give and be advisable.These divided doses can give by unit dosage form, and for example the per unit dosage form contains effective composition 10 μ g-1000mg, is preferably 50 μ g-500mg, is preferably 50 μ g-250mg.Perhaps, if receiver's the state of an illness needs, but these dosage continuous infusions.

Administering mode and dosage form can influence the treatment total amount of chemical compound certainly, and they are used for specific treatment is desirable and effective.

For example, for same active component, oral administration dosage typically is at least 2 times of the used dosage level of parenteral administration method, and for example 2-10 doubly.

Itself can offer medicine polymer-peptide conjugates of the present invention, also can its materia medica on the form dispensing of acceptable ester, salt and other physiological function derivatives.

The present invention comprises that also the veterinary uses and people's medical preparation, and they comprise that one or more polymer-peptide conjugates of the present invention are as activating agent.

In such pharmaceutical preparation, activating agent should with one or or more than one materia medicas on acceptable carrier, and can randomly use with any other therapeutic component.On the meaning compatible with its preparation composition, carrier must be acceptable on the materia medica, and must be within reason harmful to its receiver.As mentioned above, with the effective dose that obtains required pharmacodynamics effect and the amount that is suitable for reaching required every day of dosage activating agent is provided.

Preparation comprises and is suitable for (the non-injection) that (injection) that non-gastrointestinal uses and non-parenteral route use, that concrete administering mode comprises is oral, rectum, oral cavity, part, nasal cavity, eye, subcutaneous, muscle, vein, percutaneous, sheath are interior, under the intraarticular, intra-arterial, arachnoidea, bronchus, lymphatic vessel, vagina and intrauterine administration.To be suitable for oral and preparation parenteral administration is good.

When activating agent was used to contain the preparation of liquid solution, preparation was with oral or parenteral administration is favourable.When activating agent used with the physiologically acceptable carrier dosage form with the liquid suspension dosage form or as powder, preparation was favourable with oral, rectum or bronchus administration.

When activating agent directly uses with the powdery solid form, favourable with oral administration.Perhaps, can to form the gas suspended matter of powder, be sucked by the patient through the administration of powder spray (in carrier gas) bronchus from the respiratory organ that comprises suitable sprayer unit.

The preparation that contains activating agent of the present invention can provide with unit dosage form easily, any known method preparation of available pharmaceutical field.These methods generally include the step that reactive compound and the carrier that constitutes one or more supplementary elements are merged.Be typically, equably, closely with the solid carrier of reactive compound and liquid-carrier, segmentation or with these two merging, then, if need again product is configured as required galenic dosage form.

The preparation of the present invention that is suitable for oral administration can be made into isolating unit, splits, respectively closes the powder or the graininess active component of scheduled volume as capsule, cachet, tablet or sugar; Or the suspension in water liquid or on-aqueous liquid, as syrup, spirit, Emulsion or draught.

Randomly with one or more supplementary elements, by compacting or the molded tablet that can be made into.The tablet of compacting can be suppressed in suitable machine and form, and reactive compound is a free-flow shape, and as powder or granule, they can randomly be mixed together film-making with binding agent, disintegrating agent, lubricant, inert diluent, surfactant or eliminant.The mold pressing tablet that contains powdered activated chemical compound and suitable carrier can be made in suitable machine in mold pressing.

Reactive compound is added in the fortified aqueous of sugar (as sucrose), wherein also can adds any supplementary element, can be made into syrup.These supplementary elements can comprise flavoring agent, suitable antiseptic, delay sugared crystalline reagent and increase the reagent of any other composition dissolubility, for example polyhydroxy-alcohol (as glycerol or Sorbitol).

The preparation that is suitable for parenteral administration with the aquesterilisa preparation that comprises reactive compound for suitable, it preferably and receiver's blood etc. ooze (as normal saline).Such preparation can comprise suspensoid and thickening agent or be used for chemical compound is taken to other microparticulate systems of blood component or one or more organs.These preparations can unit dose or the multiple-units dosage form provide.

The nose spray agent comprises purification of aqueous solutions and the antiseptic and the isotonic agent of reactive compound.Such preparation preferably is adjusted to the pH compatible with nasal mucosa and waits and oozes state.

The preparation of rectally can be used suitable carriers, as cocoa butter, hydrogenated fat or hydrogenated fat carboxylic acid, makes suppository.

Except pH and wait the factor of oozing preferably to be adjusted to be fit to eye with, use with the similar method of nasal spray to prepare ophthalmic preparation.

Topical preparation comprises and is dissolved in or is suspended in one or more media reactive compound of (being used for the alkali of topical pharmaceutical formulation as Dormant oils, oil, polyhydroxy-alcohol or other).

Outside mentioned component, preparation of the present invention also can comprise one or more supplementary elements that are selected from diluent, buffer, flavoring agent, disintegrating agent, surfactant, thickening agent, lubricant, antiseptic (comprising antioxidant) etc.

In non-therapeutic of the present invention was used, polymer-peptide conjugates can be utilized covalent bond or the non-covalent marriage relation between peptide and component of polymer.In addition, associating peptide and component of polymer can be used in therapeutic peptide reagent uses, and promptly by suitable using method, described those methods are discussed in the explanation of the relevant covalently bound polymer-peptide conjugates of the present invention as mentioned.

In these non-therapeutic association peptide-polymer compositionss, peptide and component of polymer can be earlier formulated together with enhanced stability and anti-degradation capability, perhaps, these compositions can be the divided portion such as many parts compositions, said composition is mixed in use, and when not associating key between polymer and the peptide in resulting mixture, said composition will be to quick decay or other degraded mode sensitivities.No matter for the form of association peptide and polymer composition, the peptide components during with respect to these association polymers of shortage, the association that the present invention pays attention to strengthen some characteristic or the aspect of peptide or increases its practicality.

Therefore, the present invention includes the external stable suitable polymers that provides, as the illustrative preferably application of non-therapeutic application for peptide in solution.For example, polymer can be used to improve the temperature stability and the antienzyme degradability of peptide.Through the bonded peptide of the inventive method, the raising of its temperature stabilization characteristic provides the shelf-life of improving diagnosis and research reagent and test kit (as immunoassay kit), the method for room temperature stability and fastness.As object lesson, alkali phosphatase covalently or can be linked in suitable polymers by the inventive method with associating, make this phosphatase as the reagent in antibody in the biofluid or the antigen colorimetric determination test kit time, have stability.

The following examples are used to set forth the present invention, and should not regard limitation ot it as.

Embodiment 1

Conjugates 1

Polysorbate trioleate (polysorbate trioleate) p-nitrophenyl carbonate ester

To the p-nitrophenyl chloroformate ester in the 50ml anhydrous propanone (0.8g, 4mmole) add in the solution exsiccant polysorbate trioleate (7g, 4mmole), add again dimethyl aminopyridine (0.5g, 4mmole).Reactant mixture was stirred under room temperature 24 hours, removal of solvent under reduced pressure, the gained precipitation is passed through diatomite filtration with exsiccant benzene dilution.Residue cold preservation in dried benzene is spent the night, and removes by filter unnecessary precipitation.Removal of solvent under reduced pressure, remaining benzene are under low pressure volatilized and are removed, and obtain 6.4g polysorbate trioleate p-nitrophenyl carbonate ester.

The connection of insulin and activated polymer

In the solution of activated polysorbate trioleate p-nitrophenyl chloroformate ester (1g), add the solution of bovine insulin (50mg) at 0.1M pH8.8 phosphate buffer at distilled water.If need, add 1N NaOH to keep pH.Under the room temperature reactant mixture was stirred 2.5 hours, then, mixture is made gel filtration chromatography with Sephadex G-75.With 0.1MpH7.0 phosphate buffer eluting, collect flow point with automatic fraction collector, purify conjugate 1.With trinitro-benzene-sulfonic acid (TNBS) test determination polymer content, survey protein concentration with the Biuret method.Polymer is measured as 1: 1 to the mol ratio of insulin.

Embodiment 2

Conjugates 2

As mentioned above, by the terminal hydroxyl of polyethylene glycol mono stearate being activated with the p-nitrophenyl chloroformate ester reaction.In the solution of activated polymer (1g), add the bovine insulin (80mg) that is dissolved in 0.1M phosphate buffer (pH 8.8) at distilled water.Regulate with 1NNaOH carefully and keep this pH.Stir after 3 hours,, and make gel filtration chromatography with Sephadex G-75 with excessive glycine cessation reaction.Collect and lyophilizing insulin/polymer conjugate.With Biuret test determination protein content, obtain quantitative yield.

Embodiment 3

Conjugates 3

Tetrahydrochysene-2-(12-bromododecane oxygen base)-2H pyrans

Containing to 12-bromo-1-lauryl alcohol (1 mole) in the dichloromethane solution of p-methyl benzenesulfonic acid pyridiniujm (P-TSA) and adding dihydropyran (2 moles).Reactant mixture was stirred 24 hours, wash secondary then with water, use anhydrous MgSO ₄Dry.Dichloromethane is removed in decompression.If desired, products therefrom is purified with silica gel chromatography.

Polyethylene Glycol and tetrahydrofuran derivatives connect

The above-mentioned tetrahydropyran derivatives that will be dissolved in the dried benzene is added to Polyethylene Glycol (1 mole) in the dried benzole soln that contains NaH (1 mole).Reactant mixture stirred under room temperature 24 hours.Then, mixture by silicagel column, is used the benzene eluting.If need, carry out column chromatography again and purify.Handle with P-TSA under the room temperature, remove the THP trtrahydropyranyl of protectiveness.If need, with the column chromatography end product of purifying.As mentioned above, by with the hydroxyl of p-nitrophenyl chloroformate ester reacting activation polymer.As described, carry out closing with the yoke of insulin for conjugates 1.

Embodiment 4

Carry out polymer-insulin conjugate compound and natural insulin comparative study with bovine insulin, on animal model, measure their relative stability and activity.In zooscopy, the polymer-usefulness of insulin blood sugar lowering level and the blood sugar lowering usefulness of natural insulin compare.With the average female and male white mouse overnight fasting of heavy 25g, use 5 one group, the processing that is used to last 2 days the mark phase carries out.

In the zero-time, every animal subject is accepted single agent natural insulin (the 1st group, 100 μ g/kg, subcutaneous injection); Natural insulin (the 2nd group, 1.5mg/kg irritates stomach); Conjugates 1 (the 3rd group, 100 μ g/kg irritate stomach); Or conjugates 1 (the 4th group, 100 μ g/kg, subcutaneous injection).Also have one group (the 5th group) not give the insulin of any kind of, but give glucose in preceding 30 minutes of the sample time of regulation.Animal is overnight fasting before processing, and in research interval fasting.All substances are all used phosphate buffered saline (PBS) (pH7.4) preparation.Handled 0.5,1,2,4,8 and 24 hour the scheduled sampling time in back preceding 30 minutes at insulin, give animal High dose glucose (5g/kg, 50% solution are irritated stomach), impact so that every animal is only accepted potion insulin or conjugates 1 and a glucose.Stipulating sample time, get blood from the tail vein, analyze glucose content with once touching digital glucose tester (Life Scan) immediately.Result of the test is seen Fig. 1, the 1-5 group.

In the time of 30 minutes, the blood sugar level of the 1st treated animal (natural insulin, subcutaneous injection) is about 30% of matched group (the 5th group an is untreated) animal.The 1st treated animal hypoglycemic effect only continues 3.5 hours.The natural insulin per os gives 60% of the as many as matched group of (the 2nd group) blood sugar lowering level, and this maximum reflects present insulin and handled back 30 minutes.On the contrary, and the 3rd treated animal (conjugates 1,100 μ g/kg, p.o.) glucose level reduces, and hypoglycemic activity obviously postpones to take place.The hypoglycemic activity of the 3rd treated animal is greater than the 2nd treated animal, though give the 3rd group insulin dose only for give the 2nd group 1/15.Each time after 3 hours, the 3rd treated animal glucose level is lower than other any processed group, and maximum difference was 4-8 hour sample time.In preceding 4 hours of research, (conjugates 1,100 μ g/kg, glucose level s.c.) is by the course variation same with the 1st treated animal for the 4th treated animal.After 4 hours, the 2nd group of glucose level remains on the matched group (being untreated the 5th group), and the 4th group of glucose level drops to 62% of the 5th group of level in the time of 8 hours, maintains under the 5th group of level.

Embodiment 5

On male and female white mice, carry out insulin efficiency research, with the insulin of non-binding form and conjugates 1 as substances.Whether a purpose of this research is can work to blood sugar level with the same manner when determining the insulin subcutaneous administration of conjugates 1 form.Second purpose is to determine whether the insulin complex substance of conjugates 1 is different with free insulin, can play the blood sugar lowering level during oral administration.The results are shown in Figure 2, wherein " insulin complex substance " refers to conjugates 1.

Untreatedly obtained basic blood sample from 10, be used for the serum glucose analysis by fasting white mice (5 male 5 female); Basic value is represented with symbol " O " among Fig. 2.With other three groups (every group 5 male 5 female) overnight fastings, only irritate stomach and give glucose load (5g/kg body weight).After administration, respectively put to death 10 animals in 30,60 and 120 minutes, obtain blood sample and be used for glucose analysis, give commercially available insulin and conjugates 1 (respectively putting to death the 5 male 5 female haemanalysises of doing) to fasting its mouse oral (p.o.) and parenteral route (s.c.) respectively, so that different processing modes to be provided three moment.The processing, route of administration and the symbol that are used for presentation graphs 2 results comprise: (i) glucose (5g/kg, p.o.), symbol " "; (ii) insulin (100 μ g/kg, s.c.) and glucose (5g/kg, p.o.), symbol: " "; (iii) insulin (1.5mg/kg, p.o.) and glucose (5g/kg, p.o.), symbol: " "; (iv) conjugates 1 (100 μ g/kg.s.c.) and glucose (5g/kg, p.o.), symbol: " "; (v) conjugates 1 (250 μ g/kg, s.c.) and glucose (5k/kg, p.o.), symbol: " "; (vi) conjugates 1 (1.5mg/kg, s.c.) and glucose (5g/kg, p.o.), symbol: " △ ", in these tests of conjugates 1, give that proteinic concentration is 0.1mg albumen/ml solution in the drug solns; Be correlated purpose, also being included in to protein concentration in the drug solns is the modified covalently bound insulin-polymer conjugate of 0.78mg albumen/ml solution, (vii) modified conjugates 1 (100 μ g/kg, s.c.) and glucose (5g/kg, p.o.), symbol: " △ ".

Gave insulin in preceding 15 minutes at glucose load.

Except the base set animal, all animals are irritated stomach give glucose, dosage 5g/kg is (with the 50%w/v solution of normal saline preparation, 10ml/kg).When the subcutaneous injection insulin, dosage is that 100 μ g/kg are (with 0.004% (w/v) solution of normal saline preparation, 2.5ml/kg).When conjugates 1 polymer-insulin complex substance gave to irritate stomach, dosage was 1.56mg/kg (the undiluted thing that tried of 2.0ml/kg).When conjugates 1 polymer-insulin complex substance during with the subcutaneous injection administration, dosage is 100 μ g/kg (1.28ml/kg, 1: 10 diluent of the solution that 0.78mg/ml accepted) or 250 μ g/kg (1: 10 diluent of solution that 3.20ml/kg accepts).Modified conjugates 1 contains 0.1mg insulin/ml, and dosage is 1.0ml/kg, i.e. 100 μ g/kg.

With Gemini centrifugal Analyzer and commercially available Fructus Vitis viniferae kit measurement glucose.Method of testing is the associating enzymatic reaction, that is, reaction and glucose-6-phosphate dehydrogenase (G6PD) reaction coupling with catalytic glucose of hexokinase and ATP obtain NADH.Analyze the double sample, the report meansigma methods.In order to measure the very high concentration of glucose that is present in some sample, must dilution (1: 2 or 1: 4) some blood serum sample.

Behind the glucose load 30 minutes, the average serum glucose rose to high level, reduced in the time of 60 minutes, was lower than baseline in the time of 120 minutes.If the commercially available insulin of subcutaneous injection (100 μ g/kg body weight), prevent on the blood sugar increasing highly effective.Yet,, invalid to the rising of blood glucose if per os gives insulin (high dose of 1.5mg/kg).This is in accordance with expectation, because insulin is a kind of protein, at the digestive tract facile hydrolysis, can not intactly absorb into blood flow.

When with 100 or 250 μ g/kg subcutaneous injection conjugatess 1, suppressing highly effective on the blood sugar increasing behind the glucose load.Behind the subcutaneous injection conjugates 1 100 μ g/kg, in the time of 30 and 60 minutes, the situation when the average serum dextrose equivalent all is lower than the free shape insulin of subcutaneous injection 100 μ g/kg significantly.Behind the subcutaneous injection conjugates 1 250 μ g/kg, the average serum glucose reduces (though not remarkable) in the time of 30 minutes, significantly reduces in the time of 60 minutes, gets back to baseline in the time of 120 minutes.No matter be free shape insulin 100 μ g/kg of subcutaneous injection or compound conjugate 1 100 μ g/kg, G/W is on average below baseline in the time of 120 minutes.

The conjugates 1100 μ g/kg that modify produce blood glucose 30 minutes the time after administration obviously reduces.

Embodiment 6

The preparation of the p-nitrophenyl carbonate ester of polysorbate monopalmitate (Polysorbate Monopalmitate)

First with polysorbate monopalmitate drying with dried benzene through azeotropic method.

To dry polymeric (2g, 2mmole) in the solution of the dried pyridine of 10ml, add p-nitrophenyl chloroformate ester (0.6g, 3mmole).Mixture was at room temperature stirred 24 hours.Reactant mixture is cooled off in ice, be used for the benzene dilution, filter through filter.Repeat this step, on rotary evaporator, remove at last and desolvate.Vacuum is removed trace solvent.The output of product is 1.8g.

Embodiment 7

The preparation of polysorbate monopalmitate and insulin conjugate compound

Yoke according to foregoing embodiment 1 closes reaction method, but the consumption of polysorbate monopalmitate is 1g, and the consumption of insulin element is 80mg, uses the HPLC reaction product isolated, obtains insulin-covalently bound conjugates of polysorbate monopalmitate.

Embodiment 8

The preparation of enzyme-polymer conjugate

With with embodiment 1 in about the described coupling of carrying out alkali phosphatase (AP) and polymer with quadrat method of conjugates 1.And, for determining that polymer/protein is at high proportion or low ratio is more favourable, prepare conjugates with 140 moles of polymer/mole enzyme and 14 moles of polymer/mole enzyme.For the AP that the per molecule yoke closes, high and low ratio of polymer group number is respectively 30 and 5.

Following operation is in order to obtain the alkali phosphatase of about 5 group/moles: 4.1mg (salt-free) is dissolved in the 0.05M sodium bicarbonate.Water/the dimethyl sulfoxide solution that in this solution, adds activated polymer (0.75mg), under the room temperature with solution stirring 3-12 hour.With the gained reactant mixture in Dialysis tubing to saline solution (0.3N NaCl) dialysis (MW cutoff 12,000-14,000) 12 hours, change dialysis solution 4-6 time.Be used for conjugates at high proportion with quadrat method.Measure the total protein concentration of dialysis back material with the Biuret method.

Active assessment and stability study

Press Bulletin WHO such as A.Voller, 53,55 (1976) method is carried out phosphatase test.(50 μ l) is added in the micropore with equivalent amounts of enzyme liquid, mixes with 200 μ l substrate solutions (pH 9.3 for 10g/l, the solution of phosphatase 24-nitro phenyl ester in 20% ethanolamine buffer), cultivates 45 minutes under room temperature.With 50 μ l 3M NaCl cessation reactions.On microplate reader, measure 405nm place absorption value.

The activity that compares phosphatase and native enzyme under various conditions.

The weak solution that will contain same concentration alkali phosphatase and alkali phosphatase-polymer conjugate is stored at various temperatures.The routine test enzymatic activity.Two kinds of polymer that tried under 5 ℃, 15 ℃, 35 ℃ and 55 ℃ are compared with alkali phosphatase with the contrast of storing in 5 ℃.

Shown in Table A, the initial enzymatic activity of two kinds of polymer is all than high about three times of matched group.Two kinds of polymer-enzyme conjugates all improve heat stability than native enzyme, are that the conjugates of feature is particularly evident with polymer/enzyme higher proportion.DAYTEMP℃ 0 2 3 4 5 6AP/HIGH 5 399 360 321 371 343 337

15 158 115 126 24 184

35 132 112 135 138 123

55 36 25 10 14AP/LOW

5 324 252 210 220 162 159

15 83 47 40 38 51

35 61 36 35 33 32 55 17 6 2 2 AP/CONTROL 5 100 100 100 100 100 100

15 89 74 43 36 28 35 53 48 21 20 20 55 10 2 1 2

Embodiment 9

Conjugate 1A

In insulin (50mg) solution, add the solution of the activated polymer (1g) that water/dimethyl sulfoxine is made into, under room temperature, stirred 3 hours with the preparation of the 0.05M sodium bicarbonate buffer liquid of pH9.2.Regulate with 1N NaOH carefully, keep the pH of mixture.Then with the phosphate buffer dialysis of reactant mixture to 0.1M pH7.0.With the product lyophilizing of purifying.Measure protein content (48mg) with the Biuret method.Be linked in the number of the polymer chain of insulin with the TNBB test determination, the ratio of recording is that moles of polymer is to 1 mole of insulin.

Embodiment 10

Synthesis of calcitonin-OT as follows ₅₀Under 5 ℃, (5mg Becham) adds the solution of activation OT (160mg) in 2.0ml water while stirring in the solution at the salmon calcitonin with deionized water (1.5ml) and borate buffer solution preparation.Gained solution stirred 1.5 hours down in 20 ℃, and the pH of solution is transferred to 8.8.With reactant liquor restir 0.5 hour, make pH reach 3.8 with the 1M dilute hydrochloric acid then.Reactant mixture is stored in 5 ℃ and is spent the night.(pH7.5,2L) dialysis (transfer to pH3.6,4 * 1L) dialysis to the PBS buffer again to the PBS buffer with solution with saturating plate film (MWCO 3500) earlier.Dialysis solution is filtered by 0.22 μ filter, and store in 5 ℃.Surveying protein concentration with Biuret method and HPLC, is standard with the natural calcitonin.Use the HPLC method, on the volume removing chromatogram post,, carry out purity analysis with 0.05M phosphate buffer (transferring to pH3.8) eluting.

Embodiment 11

Synthesis of calcitonin-001 as follows.Under 5 ℃, (1.5ml, 0.1M pH8.8) in Pei Zhi salmon calcitonin (5mg) solution, add dimethyl sulfoxine (1ml) solution of activation 001 (130mg) while stirring to remove to use deionized water (2ml) and borate buffer solution.This solution was stirred 1.5 hours down in 20 ℃, regulate pH to 8.8 with 1N hydrochloric acid.With reactant liquor restir 1 hour, then pH is transferred to 3.8, reactant mixture is stored in 5 ℃ and is spent the night.In reactant mixture, add the 4ml deionized water, and (pH7.4,2L) dialysis is again to PBS buffer (being adjusted to pH3.6) dialysis 4 times to phosphate buffer (PBS) earlier in dialyzer (MWCO3500) with supernatant.Dialysis solution filters by 0.22 μ filter, and stores in 5 ℃.Surveying protein concentration with Biuret method and HPLC method, is standard with the natural calcitonin.Use the HPLC purity assay, use the C-8 reversed-phase column, make gradient elution (30-55% was through 25 minutes) with acetonitrile/0.1%TFA.

Embodiment 12

Prepare insulin-OT as follows.Take by weighing 2.0g activation OT polymer in the 250ml round-bottomed flask.The OT polymer is dissolved in the anhydrous DMSO of 10ml fully, adds the 20ml deionized water, stirred 5 minutes.The OT solution of gained is chilled to 10 ℃.To be dissolved in 100mg insulin (the Sigma/ ox pancreas/Zn) once all be added in the OT polymer solution of 40ml 0.1M sodium borate buffer liquid (pH9.3).Reactant mixture transfers to light yellow.After 30 minutes; With 2N HCl the pH of solution is transferred to 8.8.Again reactant liquor was stirred 1.5 hours, regulate pH to 8.4.Reactant mixture is by 0.8 μ membrane filtration, and dialysis in PBS buffering (pH7.4).Mixture through dialysis filters and concentrates by 0.22 μ filter.With Sephadex G-75 spissated sample is made chromatograph chromatography (eluent is the 0.05M sodium phosphate buffer).Post: 2.5cm (diameter) * 32cm (height).The analysis showed that the yield of insulin conjugate compound is quantitative, be every mole of insulin 2 moles of polymer.

Embodiment 13

The 001-insulin is given to give the treatment group monkey (cynomolgus monkey) of buckles glucose respectively with 0.5mg, 2mg and 5mg dosage per os.Calculate the area under a curve (AUC) of each treatment group.Use this method, the lower hypoglycemic maximum efficiency of AUC value representation.AUC the results are shown in Figure 18.These results show that it is effective for preventing on the blood sugar increasing of cynomlgus monkey after giving the buckles glucose that single agent per os gives the 001-insulin.The AUC that per os gives behind the 001-insulin lacks 1/2 than the AUC of untreated animal, proves the remarkable hypoglycemic activity of 001-insulin on monkey.

Embodiment 14

On rat hypocalcemia model, the blood droping calcium activity of polymer-calcitonin conjugates that the assessment per os gives.With following method assessment two polymer-calcitonin derivants (OT-calcitonin and 001-calcitonin).Male Sprague-Dawley rat (on average heavy 54g) is by overnight fasting.Animal is assigned randomly to each treatment group (5 every group), surveys the basic serum calcium level of each group.Treatment group is then accepted calcitonin (50mg/kg), OT-calcitonin (25 ng/kg) and the 001-calcitonin that single agent is modified, (250ng/kg, 2.5 μ g/kg and 25 μ g/kg) respectively.These dosage per os give.2 hours and 4 hours mensuration serum calciums after the administration.The result in Fig. 4 with the percent of former serum calcium level to time representation.represents calcitonin; Zero represents 001,250 ng; ■ represents 001,2.5 μ g; △ represents 001,25 μ g, ● represent OT, 2.5 μ g.The dependent serum calcium of show dose significantly reduces as a result, shows that conjugates has activity in more than 4 hour of beginning to assess.May be consistent the action time that prolongs with the remarkable bioavailability after oral.

Embodiment 15

Prove the blood droping calcium activity after oral administration polymer-calcitonin on the rat model as follows.Male Sprague-Dawley rat on average weighs 54 grams, overnight fasting.Animal is assigned randomly to each treatment group (4 every group), measures every group basic serum calcium level.Matched group is accepted the calcitonin (ct) of single agent unmodified by subcutaneous injection (s.c.) approach (50ng/kg) or oral (2.5 μ g/kg).Treatment group by oral route is accepted OT1-ct or OT2-ct (25 μ g/kg).Administration is measured serum calcium after 2 hours and 4 hours.The result in Fig. 5 with the percent of former serum calcium level to time representation.OT1-ct is the repetition preparation of identical calcitonin-polymer conjugate with OT2-ct, and they are assessed in turn to prove a batch and another reaction concordance in batches.The result shows the remarkable reduction of serum calcium, shows that conjugates has activity in more than 4 hour of beginning to assess.

Embodiment 16

On diabetes (BB) rat model, assess polymer-insulin as follows.BioBreeding rat is the reliable model of insulin human dependent diabetes mellitus.Subcutaneous injection every day (sc) insulin not, BioBreeding rat are dead in 2 days.In 14 days research, the subcutaneous injection insulin of stopping using, animal changes into oral low dosage (100 μ g/kg/ days), and the polymer-insulin of middle dosage (3mg/kg/gd) and high dose (6mg/kg/ days) is treated.Result's (mean survival time) sees Fig. 6.Change oral strict especially the test that constituted from subcutaneous injection to the polymer insulin suddenly.The result shows that the animal of low dosage administration does not absorb the insulin of the q.s that can survive; Middle dosed administration group shows that the time-to-live increases; Some animal oral administration polymer insulin whole study period (14 days) of promptly having survived only in the high dose administration group.The polymer insulin is with the administration of non-optimization dosage form.Research also show afternoon than the morning blood sugar level reduction of statistical significance is arranged, if animal is accepted multiple dosing and replace the single heavy dose every day, then blood sugar level will be subjected to better control.Give polymer-insulin to normal (non-glycosuria sugar) animal per os every day, cause that the morning, blood bran level obviously reduced.

Embodiment 17

Effect is as follows behind the oral 001-insulin of monkey (cynomolgous monkey).6 monkeys (3 male 3 is female, on average heavy 2.5kg) overnight fasting.Measure basic blood sugar level, irritate the buckles glucose that stomach makes animals received 3mg/kg with 50% glucose solution then.In different time (0-4 hour) report blood sugar level.After two days,, impact with the 3g/kg glucose again with the animal fasting.At this moment the animal per os is accepted 001-insulin (being equivalent to insulin 5mg/kg).In each of blood sugar level time sheet.Animal that reuse is same and scheme are measured the effect of 2mg/kg and 0.5mg/kg dosage 001-insulin, give two days eliminating time between treatment.The result of each treatment (contrast, 0.5,2 or 5mg/kg) the results are shown in Figure 7 with the percentage change calculations of blood glucose to the time, and wherein represents glucose, and △ represents 5mg, represents 2mg, and zero represents 0.5mg.The glucose that gives buckles is after 2 hours, and the average rising of treatment group blood sugar level is 1/3 of a non-treatment group.

Embodiment 18

Measure the pancreas milk reducing protease digesting of insulin and OT insulin as follows.Chymase (the Sigam 1S type of purification) is cultivated down at 37 ℃ with insulin that is dissolved in phosphate buffered saline (PBS) (pH8) (cattle) or OT-insulin conjugate compound.Taking-up regularly is a certain amount of, and 2% trifluoroacetic acid (TFA) that adds 1/10 volume is with stopped reaction.Use the HPLC analytical sample, the results are shown in Figure 3, wherein the OT-insulin is represented with ■, and insulin is represented with.

Implement best mode of the present invention

Polypeptide polymer compositions and preparation that best combination provided by the present invention is stable relate to the peptide complexes that the covalency yoke closes, wherein, peptide is covalently bonded in and mixes hydrophilic component (as linear polyglycols) one or more polymer molecules as its ingredient, and wherein said polymer mixes a low-polarity component as its ingredient.

The good aspect of spy of the present invention relates to physiologically active peptide compositions, comprise and contain (i) the linear polyglycols group and the (ii) banded physiologically active peptide of polymer covalency of lipophilic group, wherein peptide, linear polyglycols group and lipophilic group each other the arrangement on conformation make physiologically active peptide in the physiologically active peptide compositions with respect to independent physiologically active peptide (the non-binding shape that promptly lacks coupling polymer thereon) to tolerance enhancing in the body of enzymatic degradation.

On the other hand, the present invention relates to the physiologically active peptide compositions of three-dimensional structure rubber, comprise and contain (i) the linear polyglycols group and the (ii) banded physiologically active peptide of polysorbate complex covalency of lipophilic group, wherein physiologically active peptide, linear polyglycols group and lipophilic group each other the arrangement on conformation (a) lipophilic group in three-dimensional conformation is exposed and (b) physiologically active peptide in the physiologically active composition with respect to independent physiologically active peptide to tolerance enhancing in the body of enzymatic degradation.

Another better aspect, the present invention relates to the bonded peptide complexes of multivalent ligand, this peptide complexes comprises triglyceride main chain group and by being incorporated into the polyglycols at interval base and the covalently bound biologically active peptide of triglyceride main chain group on the triglyceride main chain group carbon atom; And directly with triglyceride main chain group on carbon atom covalently bound or by at interval basic covalently bound at least one fatty acid group of polyglycols.

In the bonded peptide complexes of this multivalent ligand, the α of triglyceride bio-active group and β carbon atom can have direct covalent bond on it, or by the polyglycols base fatty acid group of covalent bond on it indirectly at interval.Perhaps, fatty acid group can be directly or by polyglycols at interval base be covalently bonded in the α and the α ' carbon of triglyceride main chain group, and the β carbon covalency of biologically active peptide and triglyceride connects, directly covalent bond thereon or by polyglycols at interval base indirectly in conjunction with thereon.

The present invention includes this paper discussion in conjunction with stable polypeptide conjugates for alleviating and treatment relates to the application of the oral administration dosage of the morbid state that peptide lacks.

Claims (31)

1. peptide combinations, the peptide that the polymer molecule that it is characterized in that comprising stably, covalently exists with one or more non-naturals that contain lipophilic group and hydrophilic radical connects.

2. by the described peptide combinations of claim 1, wherein peptide is physiology active treatment peptide.

3. by the described peptide combinations of claim 1, wherein peptide is to be used for the reactive diagnosis peptide whether situation in external or the body or composition exist of measuring.

4. by the described peptide combinations of claim 1, wherein peptide is the prevention peptide that is used to prevent the disease of plant or its composition.

5. by the described peptide combinations of claim 1, wherein the polymer that exists of non-natural comprises (i) linear polyglycols group and (ii) lipophilic group.

6. by the described peptide combinations of claim 1, wherein

The polymer that non-natural exists comprises polysorbate; Peptide comprises physiologically active peptide.

7. by the described peptide combinations of claim 1, wherein polymer molecular weight is 500-10,000 dalton.

8. by the described peptide combinations of claim 1, wherein peptide is selected from: insulin, calcitonin, ACTH, glucagon, somatostatin, growth hormone, somatomedin, parathyroid hormone, erythropoietin, hypothalamic releasing factor, prolactin antagonist, thyrotropin, endorphins, enkephalin, vasopressin, the class deltorphin delta that non-natural exists, the peroxide dismutase, interferon, asparagine amide enzyme, arginase, the arginine deaminase, the adenosine deaminase, ribonuclease, trypsin, chymase, and papain.

9. by the described peptide combinations of claim 1, wherein peptide comprises insulin.

10. physiologically active peptide compositions is characterized in that: comprise and the banded physiologically active peptide of one or more polymer molecule covalency that contains polysorbate.

11. the physiologically active peptide compositions of three-dimensional conformation, it is characterized in that: comprise and contain (i) the linear polyglycols group and the (ii) banded physiologically active peptide of polysorbate complex covalency of lipophilic group, wherein physiologically active peptide, linear polyglycols group and lipophilic group each other the arrangement on conformation make that lipophilic group exposes in the three-dimensional conformation.

12. the bonded peptide complexes of multivalent ligand is characterized in that: comprise triglyceride main chain group and by being incorporated into the polyglycols at interval base and the covalently bound biologically active peptide of triglyceride main chain group on the triglyceride main chain group carbon atom; And directly with triglyceride main chain group on carbon atom covalently bound or by at interval basic covalently bound at least one fatty acid group of polyglycols.

13. by the bonded peptide complexes of the described multivalent ligand of claim 12, wherein, the α ' of triglyceride bio-active group and β position carbon atom have direct covalent bond on it, or by the polyglycols base fatty acid group of covalent bond on it indirectly at interval.

14. by the bonded peptide complexes of the described multivalent ligand of claim 12, wherein, fatty acid group directly or by polyglycols at interval base be covalently bonded in the α and the α ' carbon of triglyceride main chain group, and the β carbon covalency of biologically active peptide and triglyceride connects, directly covalent bond thereon or by polyglycols at interval base indirectly in conjunction with thereon.

15. the peptide complexes that yoke closes, it is characterized in that: comprise the biologically active peptide that is covalently bonded in the polysorbate part, polysorbate partly comprises the triglyceride main chain, have on the triglyceride main chain and be covalently bonded in its α, the fatty acid group of one of α ' and β carbon atom, and have the polyethylene group that is covalently bonded in one of its α, α ' and β carbon atom.

16. by the peptide complexes that the described yoke of claim 15 closes, wherein biologically active peptide is covalently bonded in polyethylene group.

17. the peptide complexes stable, water miscible, that yoke closes is characterized in that comprising covalency stably and is linked in peptide on the polyethyleneglycol modified glycolipid composition.

18. by the described peptide complexes of claim 17, wherein peptide is by unstable covalent bond on the free amino acid in the polypeptide and polyethyleneglycol modified glycolipid composition covalency connection, and wherein unstable covalent bond can be cut off by biochemical hydrolysis and/or albuminolysis in vivo.

19. by the described peptide complexes of claim 17, wherein polyethyleneglycol modified glycolipid composition comprises the polysorbate polymer.

20. by the described peptide complexes of claim 19, wherein the polysorbate polymer comprises the fatty acid ester group that is selected from monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurin, monoleate, dioleate, trioleate, monostearate, distearate and tristearate.

21. by the described peptide complexes of claim 17, wherein the glycolipid modified of polyglycols partly comprises the polymer of the polyglycols ester of the polyglycol ether that is selected from fatty acid and fatty acid, and wherein fatty acid comprises and is selected from lauric acid, Palmic acid, oleic acid and stearic fatty acid.

22. by the described peptide complexes of claim 17, wherein peptide is selected from insulin, calcitonin, ACTH, glucagon, growth hormone release inhibiting hormone, growth hormone, somatomedin, parathyroid hormone, erythropoietin, hypothalamic releasing factor, prolactin antagonist, thyrotropin, endorphins, enkephalin, vasopressin, the class deltorphin delta that non-natural exists, the peroxide dismutase, interferon, asparagine amide enzyme, arginase, the arginine deaminase, the adenosine deaminase, ribonuclease, trypsin, chymase and papain.

23. by the described peptide complexes of claim 18, its feature also is: contain the polysorbate composition that comprises the triglyceride main chain, on the triglyceride main chain, have and be covalently bonded in one of its α, α ' and β carbon atom fatty acid group, and have the polyethylene group that is covalently bonded in one of its α, α ' and β carbon atom.

24. by the described peptide complexes of claim 23, wherein peptide is covalently bonded in the β carbon atom of triglyceride main chain.

25. by the described peptide complexes of claim 17, wherein peptide is a protein.

26. by the described peptide complexes of claim 23, wherein the triglyceride main chain comprises biocompatible bivalence base at interval between its β carbon atom and α or α ' carbon atom.

27. the peptide complexes that yoke closes, it is characterized in that: comprise the biologically active peptide that is covalently bonded in the polysorbate composition, the polysorbate composition comprises the triglyceride main chain, and this triglyceride main chain has the functional group that is covalently bonded in respectively on its α, α ' and the β carbon atom: (i) fatty acid group; (ii) have covalent bond and contain the polyglycols group of biologically active peptide thereon.

28. by the peptide complexes that the described yoke of claim 27 closes, wherein biologically active peptide is covalently bonded in the functional end-group of polyethylene group.

29. polymer-peptide conjugates is characterized in that: comprise the yoke that is selected from the polymer that following molecular formula represents and close polymer Wherein W, X, Y, Z's adds up to 4-100, R ₁, R ₂And R ₃Be selected from lauric acid, oleic acid, Palmic acid and stearic acid-base separately, or R ₁-R ₂Respectively be hydroxyl R ₃Be lauric acid, Palmic acid, oleic acid or stearic acid-base;

m＝10-16

X, y, sum=8-240 of z Wherein m is 10-16, n+m=8-240, or when an above n, all n+m add up to 8-240, and wherein the sugar moieties in the glycolipid can at random be replaced by glycerol or amino glycerol;

Wherein m and n definition is as above for Z=OTs or Br;

M=2-15 wherein, n=5-120;

M=2-15 wherein, n=5-120; M=5-18 wherein, n=2-15;

M=2-15 wherein, n=5-120;

M=2-15 wherein, n=5-120;

Wherein X=O or OCH ₂CONH-, m=2-15, n=5-120;

M=2-15 wherein, n=5-120; M=2-15 wherein, n=5-120; R wherein ₁=(CH ₂) _m, m=2-15, n=5-120; R wherein ₁=(CH ₂) _m, m=2-15, n=5-120; And

Wherein said polymer covalency is linked in peptide.

30. alleviate the oral administered dosage form that insulin deficit is used, it is characterized in that: comprise the insulin complex substance that pharmaceutically acceptable carrier and stable water solublity yoke close, the latter comprises stably, covalently is connected in the insulin that the physiology goes up the glycolipid group of compatible polyglycols modification.

31. be used for the peptide polymer compositions that yoke potential with the treatment of effective peptide agent prevention or modulability or the state of an illness that developed or disease closes, it is characterized in that, the peptide complexes that described compositions comprises is stable, water miscible, yoke closes, the latter contains the peptide agent that is covalently bonded in the polyethyleneglycol modified glycolipid composition of physiological compatibility.

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