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 stable conjugated peptide complexes, wherein the peptide is coupled to a polymer containing lipophilic and hydrophilic groups, in particular to oral or parenteral insulin compositions, wherein insulin is combined with The polymer of the linear polyglycol group and the lipophilic group is covalently combined, and the mutual arrangement of the insulin, the linear polyglycol group and the lipophilic group in conformation enhances the resistance of insulin to enzymatic hydrolysis in vivo. The conjugates of the invention are useful for both therapeutic and non-therapeutic (eg, diagnostic) uses. Peptides and polymers can be covalently bonded or associated with each other through hydrogen bonding or the like.

Description

Combined stable polypeptide compositions, therapeutic and diagnostic formulations thereof and methods of preparation and use thereof

The present invention relates to binding stabilized polypeptide and protein compositions and formulations, and to methods of making and using them.

The use of peptides and proteins for systemic treatment of certain diseases is now well accepted in medical practice. The role played by polypeptides in replacement therapy is so important that much research activity is devoted to their synthesis in large quantities using recombinant DNA techniques. Many of these polypeptides are endogenous molecules, which are strong and specific in producing physiological effects.

A major factor limiting the usefulness of these substances in desired applications is their susceptibility to metabolism by plasma proteases when administered parenterally. The oral route of administration of these substances is more problematic because the high acidity of the stomach destroys them before they reach the desired target tissue, in addition to protein breakdown in the stomach. Polypeptides and protein fragments produced by the action of gastric and pancreatic enzymes are cleaved by exogenous and endogenous peptidases on the brush border membrane of the intestine to obtain dipeptides and tripeptides. Degraded by brush border peptidases. Ingested peptides preserved at any point after passage through the stomach are further metabolized in the intestinal mucosa, which has a permeable barrier preventing its entry into cells.

Despite these obstacles, there is substantial evidence in the literature suggesting that nutritional and medicinal proteins are absorbed through the intestinal mucosa. On the other hand, nutritive and pharmaceutical (poly)peptides are taken up by specific transport systems on intestinal mucosal cells. These findings suggest that properly formulated (poly)peptides and proteins can be administered orally retaining sufficient biological activity for their intended use. However, if it is possible to modify these peptides so as to retain their physiological activity completely or at least to a significant degree, while stabilizing them against the action of proteolytic enzymes and enhancing their ability to penetrate through the intestinal mucosa, then it can be properly utilized They serve their intended purpose. The products thus obtained will offer the advantage of resulting in more efficient absorption and correspondingly lower dosages can be used to produce an optimal therapeutic effect.

The problems associated with oral or parenteral administration of proteins are well known in the pharmaceutical industry and various strategies are being employed in an attempt to overcome them. These strategies include the co-administration of penetration enhancers, such as salicylates, lipid-bile acid mixed micelles, glycidol, and acylcarnitines, but they have often been found to cause serious local toxicity problems, such as local irritation and toxic , complete abrasion of the epithelial cell layer and tissue inflammation. These problems arise because enhancers are usually administered at the same time as the peptide preparation, and leakage from the dosage form often occurs. Other strategies to improve oral dosing include mixing the peptides with protease inhibitors such as aprotinin, soybean trypsin inhibitor and aprotinin A in an attempt to limit degradation of the given therapeutic agent. Unfortunately, these protease inhibitors are non-selective and endogenous proteins are also inhibited. This effect is undesirable.

Increasing the penetration of peptides through mucous membranes by altering the physicochemical properties of drug candidates has also been tested. The results show that increasing lipophilicity alone is not sufficient to increase transcellular transport. Peptide-water hydrogen bond cleavage has in fact been suggested to be the major energy barrier to overcome in achieving transmembrane diffusion of peptides (Conradi, R.A., Hilgers, A.R., Ho, N.F.H., and Burton, P.S., "The influence of peptide structure on transport across Caco-2 cells”, Pharm. Res., 8, 1453-1460, (1991)). Some authors describe the problem of protein stabilization.

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

A great deal of work has been published on protein stabilization. Abuchowski and Davis disclose various methods of conjugating enzymes to polymers (supra), more specifically dextran, polyvinylpyrrolidone, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides reportedly retain their biological activity and solubility in water required for parenteral applications. The same authors disclose in US Patent No. 4,179,337 that polyethylene glycol confers solubility and non-immunogenicity on proteins after conjugation to these proteins. However, these polymers do not contain segments suitable for binding to the intestinal mucosa, nor do they contain any components that promote or increase membrane penetration. Although these conjugates are water soluble, they are not intended for oral use.

Meisner et al. in US Patent No. 4,585,754 show that proteins can be stabilized by conjugation with chondroitin sulfate. The products of this conjugation are usually polyanionic, highly hydrophilic, and lack cell-permeability. They are usually not intended to be taken by mouth.

Mill et al. in US Patent 4,003,792 indicate that certain acidic polysaccharides (such as pectin, algesic acid, hyaluronic acid, and carrageenan) can be coupled to proteins to produce soluble and insoluble products. These polysaccharides are polyanionic substances from food plants. They lack cell penetration and are generally not considered for oral use.

In Pharmacological Research Communication 14, 11-120 (1982), Boccu et al. showed that polyethylene glycol can be coupled to proteins such as superoxide dismutase ("SOD"). The resulting conjugation product has increased stability against denaturation and enzymatic digestion. Polymers do not contain the components necessary for membrane interaction and therefore suffer from the same problems mentioned above, they are not suitable for oral administration.

The following article reveals other techniques for stabilizing peptide and protein drugs in combination with lower molecular weight compounds such as aminolethicin, fatty acids, vitamin _B12 , and glycosides: R. Igarishi et al., “Proceed . Intern. Symp. Control. Rel. Bioact. Materials, 17, 366, (1990); T. Taniguchi et al. Ibid 19, 104, (1992); GJ Russel-Jones, Ibid, 19, 102, (1992); M .Baudys et al., Ibid, 19, 210(1992).These modified compounds are not polymeric and therefore do not contain the components required to impart solubility and membrane affinity, which are oral and parenteral Necessary for bioavailability after administration. Many formulations of this class lack oral bioavailability.

Another method employed to prolong the time of action of proteinaceous substances in vivo is the technique of encapsulation. M.Saffan et al. proposed that protein drugs be sealed in an azo polymer film for oral administration in Science, 223, 1081, (1986), and reported that the film was not digested in the stomach, but was absorbed by microorganisms in the large intestine. The system degrades, thereby releasing the encapsulated protein there. This technique utilizes physical mixtures and does not facilitate transmembrane absorption of the released protein.

Ecanow proposed in US Patent No. 4,963,367 that physiologically active compounds (including proteins) can be encapsulated by a membrane formed by condensation, and the resulting product can be suitable for transmembrane administration. Other formulations of the same invention can be administered by inhalation, oral, parenteral and transdermal routes. These approaches do not provide the properties required for oral administration - complete stability to gastrointestinal acidity and proteolytic enzymes.

Another approach taken to stabilize protein drugs for oral and parenteral administration is to entrap the therapeutic agent in liposomes. For a review of this technology see Y.W. Chien, "New Drug Delivery Systems", Marcel Dekker, New York, NY, 1992. Liposome-protein complexes are physical mixtures and their administration produces irregular and unpredictable results. Undesirable accumulation of protein components in certain organs has been reported during the use of such liposome-protein complexes. In addition to these factors, there are disadvantages when using liposomes such as high cost, difficult preparation methods requiring complex freeze-drying cycles, and solvent incompatibility. Furthermore, variable biodistribution and antigenic organization are constraints when developing clinically useful liposomal formulations.

The use of "proteinoids" has recently been described (Santiago, N., Milstein, S.J., Rivera, T., Garcia, E., Chang., T.C., Baughman, R.A., and Bucher, D., "Oral Immunization of Rats with Influenza Virus M Protein (M1) Microspheres", Abstract #A221, Proc. Int. Symp. Control. Rel. Bioac. Mater., 19, 116 (1992)). This paper reports the oral administration of several classes of therapeutic agents using this system, which encapsulates the drug of interest within a polymer sheath composed of highly branched amino acids. As in the case of liposomes, the drug is not chemically bound to the proteinoid sphere and leakage of the drug from the dosage form components is possible.

The peptide that has been the focus of much synthetic work aimed at improving its administration and bioabsorption is insulin.

The use of insulin in the treatment of diabetes can be traced back to 1922, when Banting et al. ("Pancreatic Extracts in the Treatment of Diabetes mellitus," Can.Med.Assoc.J., 12,141-146(1922)) proved The resulting active extract has a therapeutic effect on diabetic dogs. That same year, treatment of diabetic patients with pancreatic extracts led to exciting clinical improvements that saved lives. The process of daily insulin injections is necessary for long-term recovery.

The insulin molecule consists of two chains of amino acids connected by disulfide chains, and the molecular weight of insulin is about 6,000. The beta cells of the pancreatic islets secrete a single-chain precursor of insulin, called proinsulin. Proteolysis of proinsulin results in the removal of 4 basic amino acids (at positions No. 31, 32, 64 and 65 of the insulin chain: Arg, Arg, Lys, Arg, respectively) and the connecting ("C") peptide. In the resulting two-chain insulin molecule, the amino terminus of the A chain is glycine, and the amino terminus of the B chain is phenylalanine.

Insulin can exist as monomers, dimers and hexamers formed from three dimers. The hexamer is coordinated to two Zn ⁺⁺ ions. The biological activity is present in the monomer. Although until recently, almost exclusively bovine and porcine insulins have been used to treat diabetes in humans; numerous differences in insulin between strains are known. Porcine insulin is most similar to human insulin, the only difference being an alanine rather than a threonine at the C-terminus of the B chain. Despite these differences, most mammalian insulins have similar specific activities. Until recently, animal extracts provided all the insulin used to treat the disease. The advent of recombinant technology has made possible the commercial-scale production of human insulin (eg, Humulin ^(TM) insulin, available from Eli Lilly, Indianapolis, IN, USA).

Although insulin has been used in the treatment of diabetes for more than 70 years, until two recent publications, there have been few reports on the stability of its preparations. (Brange, J., Langkjaer, L., Havelund, S., and Volund, A., "Chemical sta-bility of insulin. 1. Degradation during storage of pharmaceutical preparations," 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 publications, the authors describe in detail the chemical stability of several insulin preparations under conditions of temperature and pH changes. Earlier reports focused almost entirely on biological potency as a measure of stability of insulin preparations. However, the advent of several new powerful analytical techniques, such as disk electrophoresis, size-exclusion chromatography, and HPLC, has made it possible to characterize the chemical stability of insulin in detail. Early chemical studies of insulin stability were difficult because the recrystallized insulin tested was found to be no higher than 80-90% pure. Recently, high-purity insulin with a single component has become available. The impurities contained in the single-component insulin are lower than the detection level of the current analysis technology.

Formulated insulins are prone to several types of degradation. Non-enzymatic deamidation occurs when the side chain amide group on a glutamine or asparagine residue is hydrolyzed to the free carboxylic acid. There are six possible positions for this deamidation of insulin: Gln ^A5 , Gln ^A15 , Asn ^A18 , Asn ^A21 , Asn ^B3 , and Gln ^B4 . Published reports suggest that three Asn residues are the most sensitive to this response.

Brange et al. (supra) report that under acidic conditions insulin is rapidly degraded by extensive deamidation at position ^A21 of Asn. In contrast, in the neutral formulation, deamidation occurs at Asn ^B3 at a much slower rate independent of insulin concentration and the strain of insulin origin. However, temperature and type of formulation play an important role in determining the rate of hydrolysis at the _B3 position. For example, if insulin is crystalline compared to amorphous, hydrolysis at position _B3 is minimal. The flexibility of the crystalline form (ternary structure) decreases, significantly reducing the reaction rate. Incorporation of phenols into neutral formulations to stabilize the ternary structure resulted in a reduced rate of deamidation.

In insulin preparations, in addition to hydrolytic degradation products, high molecular weight conversion products are also formed. Brange et al. used size exclusion chromatography to demonstrate that the main product formed when insulin preparations were stored between 4-45°C was a covalent insulin dimer. In protamine-containing formulations, a covalent insulin protamine product is also formed. The rate of formation of insulin dimers and insulin-protamine products was significantly affected by temperature. For human or porcine insulin, the formation time of 1% high molecular weight product (conventional N1 preparation) was reduced from 154 months to 1.7 months at 37°C compared to 4°C. For the zinc suspension formulation of porcine insulin, the same conversion takes 357 months at 4°C, but only 0.6 months at 37°C.

These types of degradation of insulin may be of great significance to diabetic patients. Although the formation of high molecular weight products is generally slower than the formation of the aforementioned hydrolytic (chemical) degradation products, its effects can be more severe. There is clear evidence that immune responses to insulin may arise from the presence of insulin covalent polymers (Robbins, D.C. Cooper, S.M. Fineberg, S.E., and Mead, P.M., "Antibodies to covalent aggregates 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 circulating aggregate 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)). As many as 30% of insulin-treated diabetic patients display specific antibodies to covalent insulin dimers. Very pronounced lymphocyte stimulation has been reported in only 2% of allergic patients due to the presence of covalent insulin dimers. When the dimer content was in the range of 0.3-0.6%, the reaction was not significant. Therefore, the level of covalent insulin dimer in the preparation should be controlled below 1% to avoid various clinical manifestations.

There are several commercially available insulin formulations; although stability has improved to such an extent that rerefrigeration of all formulations is no longer necessary, there remains a need for insulin formulations with increased stability. Improved insulins that are less likely to form high molecular weight products would be a substantial advance in the fields of pharmacy and medical technology, and improvements that provide this stability (and also the possibility of oral administration of insulin) would make a significant contribution to the treatment of diabetes.

In addition to their in vivo use as therapeutic agents, peptides and proteins are also being used substantially more and more in diagnostic reagent applications. In many of these applications, peptides and proteins are used in solution environments where they are sensitive to temperature and enzymatic degradation of (poly)peptides and proteins such as those used in immunoassays , Antibodies—enzymes for hapten binding, peptide and protein hormones, antibodies, enzyme-protein conjugates, viral proteins used in large quantities in the detection methodologies for diagnosing or screening AIDS, hepatitis and rheumatic diseases, for example tissue Peptide and protein growth factors for cultures, enzymes for clinical chemistry and insoluble enzymes such as those used in the food industry. As a more specific example, alkaline phosphatase is used as a reagent in a kit for colorimetric determination of antibodies or antigens in biological fluids. Although this enzyme is commercially available in various forms, including free enzyme and antibody conjugates, its storage stability and solutions are often limited. As a result, alkaline phosphatase conjugates are often freeze-dried and additives such as bovine serum albumin and Tween 20 are used to increase the stability of the enzyme preparation. Although these methods are beneficial in some cases to enhance the resistance of peptide and protein reagents to degradation, they have various disadvantages that limit their general application.

The present invention generally relates to binding-stabilized (poly)peptide and protein compositions and formulations, and methods of making and using the same.

More specifically, the present invention relates in a broad combinatorial aspect to covalently bound peptide complexes in which the peptide is bound to one or more polymer molecules (such as linear polyglycols) incorporating hydrophilic groups as part of them. , the polymer incorporates a lipophilic group as its constituent.

In another particular aspect, the present invention relates to a physiologically active peptide composition comprising a physiologically active peptide covalently linked to a polymer containing (i) a linear polyglycol group and (ii) a lipophilic group, wherein the peptide, linear The conformational arrangement of the polyglycol group and the lipophilic group makes the physiologically active peptide in the physiologically active peptide composition relatively independent of the physiologically active peptide (that is, the non-conjugated form lacking the polymer coupled thereto) Increased in vivo tolerance to enzymatic degradation.

In another aspect, the present invention relates to a three-dimensional conformation of a physiologically active peptide composition comprising a physiologically active peptide covalently linked to a polysorbate complex comprising (i) a linear polyglycol group and (ii) a lipophilic group. Peptides wherein the physiologically active peptide, the linear polyglycol group and the lipophilic group are arranged conformationally relative to each other such that (a) the lipophilic group is exposed in a three-dimensional conformation and (b) the physiologically active peptide in a physiologically active composition Increased in vivo resistance to enzymatic degradation relative to the physiologically active peptide alone.

Still further, the present invention relates to multivalent ligand-bound peptide complexes comprising triglyceride backbone groups and

a bioactive peptide covalently bound to the triglyceride backbone group via a polyglycol spacer bonded to a carbon atom of the triglyceride backbone group; and

At least one fatty acid group covalently bonded directly to a carbon atom on the triglyceride backbone group or through a polyglycol spacer.

In such multivalent ligand-bound peptide complexes, the α' and β carbon atoms of the triglyceride bioactive group can have direct covalent attachment to it, or indirect covalent attachment through a polyglycol spacer. resin acid groups on it. Alternatively, the fatty acid groups can be covalently attached to the α and α' carbons of the triglyceride backbone group, directly or through a polyglycol spacer, while the bioactive peptide is covalently attached to the β carbon of the triglyceride, directly bound thereto either covalently or indirectly through a polyglycol spacer. It will be appreciated that, within the scope of the above discussion, a wide variety of structures, compositions and dosage forms are possible for multivalent ligand-binding peptide complexes containing triglyceride backbone groups.

It is within the broad scope of the present invention that the biologically active peptides in such polyvalent ligand-bound peptide complexes be linked to the modified backbone of the triglyceride via an alkyl spacer or other acceptable spacer. Groups are advantageously bound covalently. Spacer acceptability as used herein refers to steric, structural and end-use specific acceptability characteristics.

In yet another aspect, the present invention relates to polysorbate complexes comprising a polysorbate component comprising a triglyceride backbone having carbon atoms covalently bonded to its alpha, alpha' and beta carbon atoms functional groups, which include:

(i) fatty acid groups; and

(ii) A polyglycol group having a physiologically active group covalently bonded thereto, eg, a physiologically active group covalently bonded to an appropriate functionality of the polyglycol group.

Such covalent attachment can be direct, for example, to the hydroxyl terminal functional groups of the polyglycol groups, or, covalent attachment can be indirect, for example, by reacting a terminal carboxyl functional spacer onto the polyglycol. , so that the resulting capped polyglycol has terminal carboxyl functionality to which physiologically active groups can be covalently bonded.

The invention still further relates to stable, water-soluble binding peptide complexes comprising a physiologically active peptide covalently bound to a physiologically compatible polyglycol-modified glycolipid moiety. In this complex, the physiologically active peptide can be covalently bound to the physiologically compatible polyglycol-modified glycolipid group through the unstable covalent bond of the free form amino acid group of the polypeptide, wherein the unstable covalent bond Can be cleaved in vivo by biochemical hydrolysis and/or proteolysis. Physiologically compatible polyglycol-modified glycolipid groups to include polysorbate polymers, e.g. containing monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate Polysorbate polymers of fatty acid esters of monooleate, dioleate, trioleate, monostearate, distearate and tristearate are advantageous. In this complex, the physiologically compatible polyglycol-modified glycolipid moiety may suitably comprise a polymer selected from polyglycol ethers and polyglycol esters of fatty acids, wherein the fatty acid is, for example, selected from the group consisting of lauryl Fatty acids of palmitic, oleic and stearic acids.

In the above complex, as an example, the physiologically active peptide may include a peptide selected from the group consisting of: insulin, calcitonin, ACTH, glucagon, somatostatin, growth hormone, growth-promoting factor, parathyroid Glandular hormones, erythropoietin, hypothalamic releasing factor, prolactin, thyroid-stimulating hormone, endorphins, enkephalins, vasopressin, non-naturally occurring orphinoids (opiods), superoxide dismutase , interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, and papain.

In another aspect, the present invention relates to an oral dosage form for mediating insulin deficiency comprising a pharmaceutically acceptable carrier and a stable water-soluble bound insulin complex comprising covalently linked to a physiologically compatible polyglycol Insulin or proinsulin with modified glycolipid groups.

Still further, the present invention relates to a method of treating insulin deficiency in a human or non-human mammalian subject exhibiting insulin deficiency comprising orally administering to the subject an effective amount of a physiologically compatible polyglycol-modified polyglycol containing a covalently conjugated A stable water-soluble conjugated insulin complex of the glycolipid moiety of insulin or proinsulin.

As used herein, the term "peptide" is broadly meant to include polypeptides with a molecular weight ≤ about 10,000 and proteins with a molecular weight > about 10,000, where molecular weight is a number average molecular weight. As used herein, the term "covalently bonded" means that certain groups are either directly covalently bonded to each other, or indirectly covalently bonded to each other through one or several spacer groups, such as bridges, spacers or linking groups. The term "conjugated" means that certain groups are either covalently coupled to each other or non-covalently coupled to each other, such as by hydrogen bonding, ionic bonding, van der Waals attraction, and the like.

Thus, the present invention includes various compositions for therapeutic use (in vivo) wherein the peptide component of the conjugated peptide complex is a physiologically or biologically active peptide. In such peptide-containing compositions, the conjugation of the peptide component to the polymer containing hydrophilic and lipophilic groups can be direct covalent bonding or indirect (via suitable spacer) bonding, hydrophilic The lipophilic groups may be arranged in any suitable manner in the polymer conjugated structure, including direct or indirect covalent bonding with each other. Thus, a wide variety of peptides can be tailored for a wide variety of uses in the present invention as needed or desired for a particular end-use therapeutic application.

In another aspect, covalently bound peptide compositions such as those described above may utilize peptide components intended for use in diagnostic or in vitro applications, where the peptide is such as a diagnostic reagent, an immunoassay or other diagnostic or ex vivo Applied diagnostic conjugated complement. In such non-therapeutic applications, the peptide complexes of the invention are highly useful as stable compositions that can be formulated in compatible solvents or other solution-based formulations to provide robust degradation-resistant Stable composition dosage form.

In the aforementioned therapeutic and non-therapeutic (e.g. diagnostic) applications, the present invention relates in a broad combinatorial aspect to covalently bound peptide complexes, wherein the peptide is covalently bound to one or more polymer molecules, the polymer The molecules incorporate hydrophilic groups such as polyglycol groups and lipophilic groups such as fatty acid groups as part of the polymer. In a preferred aspect, the peptide can be covalently bonded to one or more linear polyglycol polymer molecules into which lipophilic groups (such as fatty acid groups) are incorporated as constituents. part.

In another particularly broad aspect, the invention relates to non-covalently bound peptide complexes wherein the peptide is non-covalently associated with one or more polymer molecules incorporating a hydrophilic group (e.g. Polyglycol groups) and lipophilic groups (such as fatty acid groups) as constituents. The polymer may be various structural and arrangement analogs of the polymers in the covalently bound peptide complexes described above, but where the peptide is not covalently bound to the polymer molecule, but rather, for example, through hydrogen bonding, ionic bonding or ionic coordination. Associative bonds such as position, van der Waals bond, special peptide microencapsulation or association are associated with the polymer.

This non-covalent association of a peptide component and a polymeric group may utilize a therapeutic (eg in vivo) peptide component as well as a non-therapeutic peptide component such as a diagnostic or other (in vitro) use.

In such association-bound peptide compositions, it is within the skill of the art to select a method (for example, to give the hydrogen bonding force between the peptide and the polymer) to properly construct, modify the polymer component, or make it properly Functionalized to give it the ability to associate.

Other aspects, features and modifications of the present invention will become more fully apparent from the following disclosure and appended claims.

Figure 1 is the time (hour) function curve of serum glucose (mg/dL) after administration of insulin itself and compound dosage forms of insulin.

Fig. 2 is the time (minute) function curve of serum glucose (mg/dL) after administration of various dosage forms of insulin.

Figure 3 is a graph of the digestion of insulin and OT insulin by chymotrypsin as a function of time.

Figure 4 is a graph of serum calcium in rats as a function of time after oral administration of polymeric calcitonin conjugates to rats.

Figure 5 is a bar graph showing the calcium level in rat serum as a function of time after oral administration of polymer calcitonin OT 1-ct or OT 2-ct polymer calcitonin conjugates to rats.

Figure 6 is a bar graph of the polymer-insulin effect on a diabetic rat model.

Fig. 7 is an effect diagram showing the percentage change of blood glucose over time after oral administration of 001-insulin to monkeys (cynomolgous monkeys).

Fig. 8 is an effect graph showing the dose-dependent effect over time of oral administration of 001-insulin to monkeys (cynomolgous monkeys).

Modification of peptides with non-toxic, non-immunogenic polymers may provide certain benefits. If the modification is carried out so that the product (polymer-peptide conjugate) can retain all or most of its biological activity, the following properties can be produced: it can enhance the permeability of epithelial cells; it can protect the modified peptide from proteolytic digestion and its It can improve the affinity for the endogenous transport system; it can impart chemical stability to gastric acid; it can make the balance between lipophilicity and hydrophilicity of the polymer reach the optimum speed. Proteinaceous substances having the improved properties described above may be effective as replacement therapy after oral or parenteral administration. Other routes of administration, such as nasal and transdermal administration, may also be used with the modified peptide.

In non-therapeutic applications, incorporation of diagnostic and/or reagent peptidoids, including precursors and intermediates of final product peptides or other products, when the peptide component is covalently bound to the polymer in the manner of the present invention provides stable corresponding benefits. The resulting covalently bound peptides are resistant to environmental degradation factors, including solvent or solution-mediated degradation processes. As a result of this enhanced resistance to degradation, the shelf-life of the active peptide component can be significantly extended, with a concomitant increase in the reliability of the peptide-containing composition in its intended use.

Covalent conjugation of peptides and polymers by the methods of the present invention effectively minimizes hydrolytic degradation and achieves in vitro and in vivo stability.

Similar benefits are seen when therapeutic, diagnostic or reagent peptoids are non-covalently, associatively linked to polymers according to the methods of the present invention.

Using insulin covalently bound to a polymer component as an illustrative example of the invention, the nature of the binding including the cleavable covalent chemical bond allows for control over the time course with which the polymer is cleavable from the peptide (insulin). This cleavage can be enzymatic or chemical. Full activity was seen after complete cleavage of the polymer from the volt. Furthermore, chemical modification allows the attached peptide (eg, insulin) to permeate through cell membranes. In a preferred aspect of the invention, the membrane permeation promoting properties of lipophilic fatty acid residues are incorporated into the binding polymer. In this regard, reusing insulin as the peptide of interest, fatty acid polymer derivatives of insulin improve the intestinal absorption of insulin: carbamoylation of Phe ^B1 and Lys ^B29 with long-chain fatty acid polymers affords certain Compounds that lower blood sugar activity. This derivatization improves the stability of insulin in the intestinal mucosa and increases its absorption from the small intestine.

Although the following description mainly and illustratively revolves around the use of insulin as a peptide component in various compositions and formulations of the present invention, it should be understood that the application of the present invention is not limited thereto, but extends to Methods Peptides of any class, covalently or associatively bound, including but not limited to the following classes: calcitonin, ACTH, glucagon, somatostatin, growth hormone, growth-promoting factor, parathyroid Hormones, erythropoietin, hypothalamic releasing factor, prolactin, thyroid stimulating hormone, endorphins, antibodies, hemoglobin, soluble CD-4, coagulation factors, tissue plasminogen activator, enkephalins, vascular Vasin, non-naturally occurring morphinoids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, pancreatic Proteases, chymotrypsin, and papain, alkaline phosphatase, and other suitable enzymes, hormones, proteins, polypeptides, enzyme-protein conjugates, antibody-hapten conjugates, viral epitopes, and the like.

It is an object of the present invention to provide suitable polymers for conjugation with peptides in order to obtain the desirable characteristics enumerated above. Another aim is to utilize such modified peptides for durable transport of peptides in vivo. A further object is to use this technology to administer peptides orally in active form.

A further object is the use of the associated peptides in immunoassays, diagnostics and other non-therapeutic (eg in vitro) applications. Yet another object of the present invention is to provide stable conjugated peptide compositions, including covalently conjugated compositions suitable for both in vivo and non-vivo applications, or to provide various peptides suitable for both in vivo and non-vivo applications. Non-covalent, associatively binding peptide compositions.

Within the broad scope of the invention, a single polymer molecule can be used to bind a wide variety of peptides. In the broad practice of the invention, it may also be beneficial to utilize multiple polymers as binding agents for a certain peptide; A combination of these methods is used. Furthermore, the stably bound peptide compositions have utility in both in vivo and non-vivo applications. In addition, it is recognized that the bonded polymers may utilize any other groups, components, or other bonded species if desired for the end application. As an example, in certain applications covalently bound to polymers, it may be useful to impart functional groups to polymers that are resistant to UV degradation, oxidation, or other properties or characteristics. As a further example, in certain applications where polymers are functionalized to impart certain reactive or cross-linkable properties, it may be beneficial to enhance various properties or characteristics of the overall binding mass. Accordingly, a polymer may contain any functionality, repeating groups, linkages, or other constituent structures that do not interfere with the performance of the binding composition for its intended purpose. Other objects and advantages of the present invention will be more fully apparent from the following description and appended claims.

Illustrative polymers that can be used to achieve these desired characteristics are described below in the enumerated reaction schemes. In covalently bound peptide applications, polymers can be functionalized and then attached to free amino acids of the peptide to form labile linkages that retain activity in the presence of intact labile linkages. This bond is then removed by chemical hydrolysis and proteolysis, increasing the activity of the peptide.

The polymers utilized in the present invention can be suitably incorporated into their molecules such as edible fatty acids (lipophilic end), polyglycols (water-soluble end), acceptable sugar groups (receptor interacting end) and peptide linkages. components such as spacers. Among the polymers selected, polysorbates are particularly preferred and, in the following discussion, are selected to illustrate various embodiments of the invention. The scope of the present invention is of course not limited to polysorbates, and various other polymers containing the groups described above may be usefully employed in the broad practice of the invention. Sometimes it may be desirable to remove one of these groups in the polymer structure, leaving the other without loss of purpose. When it is desired to do so, groups which are preferably removed without losing the object and benefit of the invention are sugars and/or spacer groups.

Preferably used polymers have molecular weights falling between 500-1000 Daltons.

In the practice of the present invention, the polyglycol residue of a _C2 - _C4 alkyl polyglycol, preferably polyethylene glycol (PEG), is advantageously incorporated into the polymer system of interest.

The presence of these PEG residues imparts hydrophilicity to the polymer and the corresponding polymer-peptide conjugate. Certain glycolipids are known to stabilize proteins and peptides. This stabilization mechanism may involve the binding of the fatty acid groups of the glycolipid to the hydrophobic regions of the peptide or protein, thereby preventing aggregation of the protein or peptide. It is also known that condensed peptides are less absorbed in the small intestine than native peptides. Accordingly, the present invention contemplates polymer-peptide products in which a peptide, such as insulin, is bound to either hydrophilic or hydrophobic residues of the polymer. The fatty acid portion of the polymer serves to associate with the hydrophobic regions of the peptide, preventing aggregation in solution. The resulting polymer-peptide conjugates will thus be stable to chemical and enzymatic hydrolysis; water-soluble due to PEG residues; and not prone to aggregation due to fatty acid-hydrophobic domain interactions.

In the practice of the present invention, polyglycol derivatives have many beneficial properties in the formulation of polymer-peptide conjugates, as related to the following properties of polyglycol derivatives: improved water solubility, while not causing antigenic or immunogenic reactivity; high biocompatibility; no in vivo biodegradation of polyglycol derivatives; and easy elimination by living organisms.

Thus, the polymers used in the practice of the present invention contain lipophilic and hydrophilic groups, making the resulting polymer-peptide conjugates highly effective (bioactive) in oral as well as parenteral and other physiological modes of administration , is also highly effective in non-physiological applications.

其中：w+x+y+z＝20；

结合物2：

结合物3：

Listed below as illustrative examples of polymer-peptide conjugates of the present invention are the molecular formulas of the covalently bound conjugates, hereinafter referred to as Conjugate 1, Conjugate 2, and Conjugate 3, wherein "Ins" is insulin , specific values of m, n, w, x, and y will be described in the following discussion. Conjugate 1:

Among them: w+x+y+z=20;

Conjugate 2:

Conjugate 3:

Conjugate 1 is at the center of the polymer system commercially available polysorbate monopalmitate, a sugar derivative used in many pharmaceutical applications. The lipophilic and absorption-enhancing properties are imparted by the oleic acid chains, while polyethylene glycol (PEG) residues provide a hydrophilic (receptive hydrogen bonding) environment. Insulin is attached via urethane linkages adjacent to the PEG region of the polymer.

In conjugate 2 there is no sugar residue, but the insulin is attached to the polymer via a urethane bond adjacent to the PEG region of the polymer. Therefore, the lipophilic fatty acid region of the polymer is some distance from the point of attachment of insulin.

Conjugate 3 is the opposite of the arrangement described above for conjugate 2, still containing no sugar residues, but in this structure the lipophilic fatty acid residues are closest to the point of attachment to insulin, while the hydrophilic PEG region Away from the point of attachment, the attachment is also via a urethane bond.

In the broad practice of the invention, there may be variable combinations of the hydrophilic and lipophilic regions associated with the polymer and peptide attachment points, such variations resulting in polymers that protect the lipophilic and hydrophilic regions of insulin. In combinations 1, 2 and 3, the urethane linkage point between the polymers is preferably the amine functional group of Gly ^A1 . As noted above, there may be more than one polymer unit attached to each peptide molecule. For example, if the second polymer is attached to insulin, the point of attachment is preferably through the amine function of Phe ^B1 . A third polymer could be attached to the amine functional group of Lys ^B29 , at least in theory. However, experience has shown that linking two polymer molecules per insulin molecule is the highest practical degree of derivatization reasonably obtainable.

Various methods of coupling polymers to peptides are possible in the general practice of the invention and are discussed more fully below. In working with proteins and polypeptides, it is recognized that particular residues on the peptide are important in their biological integrity. It is important to choose an appropriate coupling reagent that does not interfere too much with this residue. In some cases, it may be difficult to avoid coupling of these important residues and thereby hinder their activity, but some activities may be exchanged for increased stability while maintaining existing beneficial properties. For example, in in vivo applications, the frequency of dosing can thus be reduced, resulting in reduced cost and improved patient compliance.

The polymers used in the protein/peptide conjugates according to the present invention are designed to incorporate good physical properties for their intended purpose. Absorption enhancers render the peptide permeable to cell membranes, but do not improve the stability properties of the peptide, so in vivo applications can dispense with the formulation of such penetration enhancers and utilize the polymer-peptide conjugates of the invention. Accordingly, one aspect of the present invention involves the incorporation of fatty acid derivatives into polymers to mimic penetration enhancers.

In the covalently bound polymer-peptide conjugates of the present invention, the peptide can be covalently bound to the water-soluble polymer through a labile chemical bond. This covalent bond between the peptide and the polymer can be broken by chemical or enzymatic reactions. The polymer-peptide product retains an acceptable amount of activity; the full activity of the peptide component is not revealed until the polymer is completely cleaved from the peptide. At the same time, polyethylene glycol moieties are present in the bound polymer, giving the polymer-peptide high water solubility and the ability to circulate in the blood for a long time. Glycolipids are usually associated with polymers in such a way that their fatty acid groups occupy the hydrophobic regions of the peptide, which prevents aggregation. Aggregation of the peptide results in poor absorption in the small intestine. Peptides that do not aggregate are more easily absorbed by the small intestine. Glycolipids are incorporated into the bound polymer to improve stability and prevent aggregation of the bound peptide. These modifications described above endow the peptide with properties such as improved solubility, stability, and membrane affinity. As a result of these improved properties, active polymer-peptides can be administered parenterally and orally, which are subsequently hydrolytically cleaved in vivo and bioavailable.

The polymers used in the embodiments described below can be classified into polyethylene glycol-modified glycolipids and polyethylene glycol-modified fatty acids. Among the preferred binding polymers there may be mentioned polysorbates including monopalmitate, dipalmitate, tripalmitate, monolaurate, dilaurate, trilaurate, monooleate esters, dioleate, trioleate, monostearate, distearate and tristearate. The number average molecular weight of the resulting polymers from each combination is preferably in the range of about 500 to about 10,000 Daltons. Preferred alternative polymers for this embodiment are polyethylene glycol ethers or esters of fatty acids such as lauric acid, palmitic acid, oleic acid and stearic acid, the polymers having a number average molecular weight of from 500-10,000 Daltons . It is preferred to have derivatizable groups in the polymer, such groups may be at the polyethylene glycol terminated terminus or at the fatty acid terminated terminus. The derivatizable group can also be located inside the polymer, thereby acting as a spacer between the peptide and the polymer.

Some methods of modifying the fatty acid sorbitan to obtain the desired polymers are discussed in more detail with structural illustrations. Polysorbate is an ester of sorbitol and its anhydride, which is formed by copolymerizing 1 mole of sorbitol and sorbitan with about 20 moles of ethylene oxide. Shown below are structures of representative polymers. The total number of W, X, Y, Z is 20, R ₁ , R ₂ and R ₃ are each independently selected from the group consisting of lauric acid, oleic acid, palmitic acid and stearate, or R ₁ and R ₂ are each hydroxyl And _R3 is lauric acid, palmitic acid, oleic acid or stearate. These polymers are commercially available for use in pharmaceutical formulations. When higher molecular weight polymers are desired, glycolipids (sorbitol monolaurate, sorbitan monooleate, sorbitol monopalmitate or sorbitol monostearate) and suitable Synthetic polyethylene glycol. The structures of glycolipids useful as starting reagents are described below.

m=10-16

通过调节试剂的摩尔当量和用适当分子量范围的聚乙二醇，按上面的步骤可得到所需分子量范围的单取代或二取代糖脂。其中n和m均可各自改变，其值为适于被稳定化的特定肽的任何适当值，如1-16。In the synthesis of glycolipid polymers having three positions substituted by polyethylene glycol, one end group of the desired polyethylene glycol with two free hydroxyl groups at the end was tritylized with 1 mole of trityl chloride (in pyridine) protected. The one remaining free hydroxyl group of polyethylene glycol is converted to tosylate or its bromide. The desired glycolipid is dissolved in a suitable inert solvent and treated with sodium hydride. The protected polyethylene glycol tosylate or its bromide is dissolved in an inert solvent and added to the glycolipid solution in excess. The product is treated with a solution of p-toluenesulfonic acid in anhydrous inert solvent at room temperature and purified by column chromatography. The structural formula for this transformation is described below.

By adjusting the molar equivalents of the reagents and using polyethylene glycol in an appropriate molecular weight range, the monosubstituted or disubstituted glycolipids in the desired molecular weight range can be obtained according to the above steps. Wherein n and m can each be varied to any suitable value suitable for the particular peptide to be stabilized, such as 1-16.

The sugar moiety of the above-mentioned glycolipids may be substituted by glycerol or aminoglycerol having the following structural formula.

其中m可取任一适当值，如10-16。In such modifications, primary alcohols are etherified or esterified with fatty acid groups such as lauric acid, oleic acid, palmitic acid, or stearic acid, as shown below; and amino groups are derivatized with fatty acids to amide or secondary amino groups.

Wherein m can take any appropriate value, such as 10-16.

The remaining primary alcohol groups were protected with trityl groups, while the secondary alcohol groups were converted to the desired polymers with polyethylene glycol. Typically, polyethylene glycol has a leaving group at one end and a methoxy group at the other end. Polyethylene glycol is dissolved in an inert solvent and added to the solution containing glycolipids and sodium hydride. Deprotection of the product in p-toluenesulfonic acid at room temperature affords the desired polymer shown below.

Sometimes it is necessary to incorporate fatty acid derivatives at different positions in the polyethylene glycol chain to obtain some of the same physical and chemical properties as polysorbate (such as polysorbate trioleate) substituted by 2 or 3 molecules of fatty acid .

Pr＝肽：蛋白质、蛋白质药物；R＝烷基，C₅-C₁₈；n＝5-120； Pr＝肽：蛋白质、蛋白质药物；R＝烷基，C₅-C₁₈；n＝5-120；m＝2-15；

The structure representing the polymer is shown in the reaction scheme below, which is an open-chain polysorbate.

Pr = peptide: protein, protein drug; R = alkyl, _C5 - _C18 ; n = 5-120; Pr = peptide: protein, protein drug; R = alkyl, C ₅ -C ₁₈ ; n = 5-120; m = 2-15;

Pr = peptide: protein, protein drug;

R = alkyl, C ₅ -C ₁₈ ;

m=5-18;

n=2-15

y=5-120;

Wherein, m, n and y can vary independently of each other within the above range.

(Formula 8)

In the synthesis of polymer A, it is necessary to protect the hydroxyl groups on the 1- and 2-position carbons of glycerol [such as Solketal (trade name, DL-α, β-isopropylidene glycerol)]. The remaining hydroxyl group is converted into sodium salt in an inert solvent, and reacted with halogenated or tosylated polyethylene glycol (one end of polyethylene glycol has been esterified and protected). The protection of glycerol was removed and the resulting two free hydroxyl groups were converted to the corresponding sodium salts. These salts are reacted with polyethylene glycol partially derivatized with fatty acids in an inert solvent. The reaction occurs after the free hydroxyl group is converted to the tosylate or its bromide.

Polymer G was synthesized in the same way except that the protected glycerol was first reacted with fatty acid ester whose terminal carbon was halogenated.

In the synthesis of polymer C it is preferred to start from 1,3-dihalo-2-propanol. The dihalogenated compound is dissolved in an inert solvent and reacted with 2 moles of the sodium salt of polyethylene glycol derivatized with 1 mole of fatty acid groups. The product is purified by chromatography or dialysis. The resulting dry product is treated with sodium hydride in an inert solvent. The sodium salt thus formed is reacted with a partially protected halogenated derivative of polyethylene glycol.

Occasionally, it may be desirable to omit the sugar moiety of the polymer. The resulting polymer still contains polyethylene glycol moieties. Appropriate substitution of fatty acids with lipophilic long-chain alkanes preserves the membrane affinity of the fatty acid groups; thereby maintaining biocompatibility. A variant of this embodiment is the treatment of polyethylene glycol with two terminal free hydroxyl groups with sodium hydride in an inert solvent. One equivalent weight of the primary bromide of the fatty acid group was added to the polyethylene glycol solvent mixture. The desired product is extracted with an inert solvent and, if necessary, purified by column chromatography.

When formation of an ester bond between the fatty acid and polyethylene glycol is desired, the acid chloride is treated with an excess of the desired polyethylene glycol in a suitable inert solvent. The polymer is extracted in an inert solvent and, if necessary, further purified by chromatography.

用稀酸或碱处理除去酯保护。 In some modifications of peptides, it is necessary to attach fatty acids directly to the peptide. In this case, the polymer was synthesized with derivatizable functional groups located on the fatty acid groups. A solution of monooxypolyethylene glycol of appropriate molecular weight in an inert solvent is treated with sodium hydride, followed by the addition of a solution of ethyl ester of fatty acid with a leaving group on the terminal carbon. After solvent extraction, if necessary, The product was purified by column chromatography.

Ester protection is removed by treatment with dilute acid or base.

When carbamate formation with a polypeptide is desired, the carboxyl or ester is converted to a hydroxyl group using chemical reduction methods known in the art.

HO-(CH ₂ ) _m -OC ₂ 2H ₄ ) _n XR (Formula 13)

The functional group for conjugation to the polypeptide is usually at the terminal end of the polymer, but sometimes it is preferable for the functional group to be located within the polymer. In this case, the derivatized group functions as a spacer. In one instance of this embodiment, the fatty acid group can be brominated at the alpha carbon of the carboxyl group, while the acid group is esterified. The experimental procedure for such compounds is the same as listed above to obtain the following products:

When extended spacers are desired, polyethylene glycol monoethers can be converted to amino groups and treated with succinic anhydride derivatized with fatty acid groups. The desired polyethylene glycol bearing primary amine groups was dissolved in pH 8.8 sodium phosphate buffer and treated with substituted succinic anhydride fatty acid groups as shown below. The product is isolated by solvent extraction and, if necessary, purified by column chromatography.

It is to be understood that the reaction schemes provided above are for illustrative purposes only and are not limited to such reactions and structures which may be beneficially utilized in the modification of peptides in the broad practice of the invention, for example, to achieve Solubility, stability and cell membrane affinity for enteral and oral administration.

The present invention provides conjugates of biocompatible polymers with bioactive macromolecules, diagnostic reagents, etc. (which may consist of substances such as peptides, proteins, enzymes, growth hormones, growth factors, and the like). Such macromolecular compounds can be prepared by α-amino acids entering amide bonds to form peptide oligomers and polymers. Depending on the function of these substances, the peptide components can be proteins, enzymes, growth hormones, etc. For simplicity, these substances are collectively referred to as peptides here, denoted by Pr. In each case, the biologically active peptide contains free amino or carboxyl groups. The linkage between the polymer and the peptide generally occurs through free amino or carboxyl groups.

The peptides selected for illustrative purposes in this specification are of particular interest in the context of pharmaceutical, agricultural, scientific and domestic and industrial applications. They can be enzymes used in replacement therapy; hormones that promote growth in animals or cells in cell culture; or active protein substances for a variety of uses, such as biotechnology and biological and medical diagnostics. Enzymes that may be mentioned are superoxide dismutase, interferon, asparaginase, glutaminase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, pancreatic Protease, chymotrypsin, and papain. Among the peptide hormones, mention may be made of: insulin, calcitonin, ACTH, glucfurin, somatostatin, somatotropin, somatotropin, parathyroid hormone, erythropoietin, hypothalamus Release factor, prolactin, thyroid stimulating hormone, endorphins, enkephalins, and vasopressin.

The reaction of polymers with peptides to form covalently bound products is readily performed. For simplicity of discussion herein, the polymer is referred to as (P). When the polymer contains hydroxyl groups, it is first converted into activated carbonate derivatives, such as p-nitrophenyl carbonate. This activated derivative is then reacted with the peptide under mild conditions for a short period of time to produce a carbamate derivative which retains biological activity.

The reactions and reagents described above are illustrative only and not exclusive; other activators leading to the formation of urethane or other bonds may also be used. A hydroxy group can be converted to an amino group using reagents known in the art. Sequential coupling of peptides via their carboxyl groups leads to amide formation.

When the polymer contains a carboxyl group, it can be converted into a mixed anhydride and react with the amino group of the peptide to form a conjugate containing an amide bond. In another approach, a water-soluble carbodiimide can be treated and reacted with the peptide to produce a conjugate containing an amide bond.

The activity and stability of peptide conjugates can be altered in several ways, such as changing the molecular ratio of polymer to peptide, and using polymers of different molecular sizes. By varying the proportion and size of the polyethylene glycol moieties incorporated into the polymer composition, the solubility of the conjugate can be varied. Hydrophilic and hydrophobic properties are balanced by careful incorporation of fatty acid and polyethylene glycol groups.

Listed below are some illustrative modifications of the polymer-peptide conjugates of the invention.

In the above reaction scheme comprising I, J and K, avenues to improve the hydrophilic/lipophilic balance of bound polymers are indicated. The ester groups in conjugated polymers are susceptible to hydrolysis by esterases; therefore, conjugated polymers containing ester groups can be modified such that the ester groups are converted to ether groups, which are more resistant to hydrolysis. The reaction scheme with L and M shows the conversion of the hydroxyl group to the carboxylic acid group. In this regard, the carboxyl group will provide the carboxylate anion, which is a more stable functional group (forming ionic coordination complexes) than the hydroxyl group, which does not form such a complex. Other suitable anion-derived functional groups for the formation of coordinating ion complexes with the various polymers of the invention include sulfate and phosphate groups.

In summary, various techniques can be beneficially applied to improve the stability of the polymer-peptide conjugates of the invention, these techniques include: functionalization of polymers with groups that are highly resistant to hydrolysis, such as the conversion of the aforementioned ester groups to ether groups; improving the lipophilicity/hydrophilicity balance of the bound polymer to be suitable for the peptide stabilized by the polymer; and tailoring the molecular weight of the polymer to a level suitable for the molecular weight of the peptide stabilized by the polymer.

A unique property of polyglycol-derived polymers of value for therapeutic applications of the present invention is general biocompatibility. The polymers have varying water solubility and are non-toxic. They are non-antigenic, non-immunogenic and do not interfere with the biological activity of the enzyme. They circulate in the blood for a long time and are easily eliminated from living organisms.

The products of the invention are found to be useful in maintaining the biological activity of the peptides and can be prepared for therapeutic administration, for example by dissolving in water or an acceptable liquid medium. Administration can be by parenteral or oral routes. Microcolloidal suspensions can be prepared for parenteral administration to produce a depot effect, or for administration via the oral route.

In the dry lyophilized state, the peptide-polymer conjugates of the invention have good storage stability, and the solution preparations of the conjugates of the invention are likewise characterized by good storage stability.

Therapeutic polymer-peptide conjugates of the invention can be used to prevent or treat disease states in any condition for which the peptide moiety is effective.

Furthermore, the polymer-peptide conjugates of the invention can be used in the diagnosis of components, conditions or disease states of biological systems or specimens, as well as in non-physiological systems for diagnostic purposes.

Furthermore, the polymer-peptide conjugates of the present invention may have application in the prevention or treatment of plant systemic conditions or disease states. As an example, the peptide component of the conjugate may have amenable to use insecticidal, herbicidal, fungicidal and/or pesticide efficacy on various plant systems.

Still further, the conjugates of the invention may be appropriate antibodies or antigens for diagnostic, immunological and/or testing purposes.

In therapeutic use, the present invention contemplates the treatment of animal subjects suffering from or potentially susceptible to these conditions or disease states and in need of such treatment, comprising administering to such animals an effective amount of or disease state therapeutically effective polymer-peptide conjugates of the invention.

Subjects to be treated with the polymer-peptide conjugates of the present invention include human and non-human animal (eg, bird, dog, cat, cow, horse) subjects, preferably mammalian subjects, most preferably human subjects.

The polymer-peptide conjugates of the present invention can be administered to animal subjects in any suitable therapeutically effective and safe dose (which can be readily determined in the art without undue experimentation) according to the specific situation or disease state faced.

Generally speaking, the appropriate dose of the compound of formula (1) to achieve therapeutic efficacy is in the range of 1 μg-100 mg per kilogram of body weight of the recipient per day, preferably in the range of 10 μg-50 mg per kilogram of body weight per day, and most preferably in the range of 1 μg-50 mg per kilogram of body weight per day. In the range of 10μg-50mg. The desired dose may conveniently be administered in 2, 3, 4, 5, 6 or more divided doses at appropriate intervals throughout the day. These sub-doses can be administered in the form of unit doses, for example, each unit dose contains 10 μg-1000 mg of the active ingredient, preferably 50 μg-500 mg, most preferably 50 μg-250 mg. Alternatively, these doses may be given by continuous infusion if the condition of the recipient so requires.

The mode of administration and dosage form will of course affect the therapeutic amount of the compound which is desirable and effective for a particular therapeutic application.

For example, for the same active ingredient, oral administration doses are typically at least twice, eg, 2-10 times, the dosage levels used for parenteral administration.

The polymer-peptide conjugates of the present invention can be administered as they are, or in the form of their pharmaceutically acceptable esters, salts and other physiologically functional derivatives.

The invention also includes pharmaceutical formulations for veterinary and human medicine which comprise as active agent one or more polymer-peptide conjugates of the invention.

In such pharmaceutical formulations, the active agent is preferably used together with one or more pharmaceutically acceptable carriers, and optionally with any other therapeutic ingredients. The carrier must be pharmaceutically acceptable in the sense of being compatible with the ingredients of its formulation and must not be unduly deleterious to the recipient thereof. As noted above, the active agent is provided in an amount effective to achieve the desired pharmacological effect and in an amount suitable to achieve the desired daily dosage.

Preparations include those suitable for parenteral use (injection) and parenteral use (non-injection), specific administration methods include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal , intrathecal, intraarticular, intraarterial, subarachnoid, bronchial, lymphatic, vaginal and intrauterine administration. Preparations suitable for oral and parenteral administration are preferred.

When the active agent is used in a formulation comprising a liquid solution, the formulation may advantageously be administered orally or parenterally. When the active agent is used in the form of a liquid suspension formulation or as a powder formulation in a biocompatible carrier, the formulation is advantageous for oral, rectal or bronchial administration.

Oral administration is advantageous when the active agent is used directly in the form of a powdered solid. Alternatively, administration can be bronchial by spraying the powder (in a gaseous carrier) to form an aerosol of the powder, which is inhaled by the patient from a respirator containing a suitable nebulizer.

The formulations containing the active agents of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. Typically, the active compound is uniformly and intimately combined with liquid carriers, finely divided solid carriers or both, and then, if necessary, the product is shaped into the desired formulation dosage forms.

Formulations of the present invention suitable for oral administration may be prepared as discrete units, such as capsules, cachets, tablets or dragees, each containing a predetermined amount of powdered or granular active ingredient; or in an aqueous or non-aqueous liquid Suspensions in liquids such as syrups, spirits, emulsions or drenches.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be formed by compressing in a suitable machine, the active compound in a free-flowing form such as powder or granules, which may optionally be mixed with binders, disintegrants, lubricants, inert diluents, surface-active agents. or expellants are mixed together to make tablets. Molded tablets may be made by molding in a suitable machine, containing the powdered active compound with a suitable carrier.

A syrup may be prepared by adding the active compound to a concentrated solution of a sugar, such as sucrose, with any optional ingredients. These additional ingredients may include flavoring agents, suitable preservatives, agents to delay the crystallization of sugars and agents to increase the solubility of any other ingredients, for example polyhydric alcohols (eg, glycerol or sorbitol).

Formulations suitable for parenteral administration suitably comprise a sterile aqueous formulation of the active compound, which is preferably isotonic with the blood of the recipient (eg, physiological saline). Such formulations may contain suspending agents and thickening agents or other particulate systems for carrying the compound to the blood component or to one or more organs. These formulations may be presented in unit dose or multiple unit dose form.

Nasal spray formulations contain a purified aqueous solution of the active compound along with preservatives and isotonic agents. Such formulations are preferably adjusted to a pH and isotonicity compatible with the nasal mucosa.

Formulations for rectal administration may be presented as suppositories with suitable carriers, such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared in a manner similar to nasal sprays, except that pH and isotonic factors are preferably adjusted for ophthalmic use.

Topical formulations contain the active compound dissolved or suspended in one or several vehicles such as mineral oil, petroleum, polyhydric alcohols, or other bases used in topical pharmaceutical formulations.

In addition to the above-mentioned ingredients, the preparation of the present invention may also include one selected from diluents, buffers, flavoring agents, disintegrants, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. or one or more additional ingredients.

In non-therapeutic applications of the invention, polymer-peptide conjugates may utilize either a covalent or non-covalent association between the peptide and polymer components. In addition, the associated peptide and polymer components can be exploited in the use of therapeutic peptide agents, i.e., by appropriate methods of use, such as those described above in the illustrative discussion of the covalently bound polymer-peptide conjugates of the invention method.

In these non-therapeutic association peptide-polymer compositions, the peptide and polymer components may first be formulated together to enhance stability and resistance to degradation, or the components may be separate parts such as multi-part compositions, The composition is mixed at the point of use, and in the absence of associative bonds between the polymer and the peptide in the resulting mixture, the composition will be susceptible to rapid decay or other modes of degradation. Regardless of the form of the associative peptide and polymer composition, the present invention focuses on associations that enhance certain properties or aspects of the peptide or increase its utility relative to the peptide component in the absence of these associative polymers.

Thus, the present invention includes providing suitable polymers for the in vitro stabilization of peptides in solution, as a better illustrative application for non-therapeutic applications. For example, polymers can be used to increase the temperature stability and resistance of the peptides to enzymatic degradation. The improved temperature stability properties of peptides bound by the methods of the invention provide a means of improving the shelf life, room temperature stability and robustness of diagnostic and research reagents and kits (eg, immunoassay kits). As a specific example, alkaline phosphatase can be covalently or associatively linked to a suitable polymer according to the method of the present invention, so that this phosphatase can be used as a reagent in a colorimetric assay kit for antibodies or antigens in biological fluids. when stable.

The following examples serve to illustrate the invention and should not be considered as limiting it.

Example 1

Conjugate 1

Polysorbate trioleate (polysorbate trioleate) p-nitrophenyl carbonate

To a solution of p-nitrophenyl chloroformate (0.8 g, 4 mmole) in 50 ml of anhydrous acetone was added dry polysorbate trioleate (7 g, 4 mmole), followed by dimethylaminopyridine (0.5 g , 4 mmole). The reaction mixture was stirred at room temperature for 24 hours, the solvent was removed under reduced pressure, and the resulting precipitate was diluted with dry benzene and filtered through celite. The residue was refrigerated overnight in dry benzene, and the excess precipitate was removed by filtration. The solvent was removed under reduced pressure, and the remaining benzene was volatilized and removed under reduced pressure to obtain 6.4 g of polysorbate trioleate p-nitrophenyl carbonate.

Linkage of insulin to the activated polymer

To a solution of activated polysorbate trioleate p-nitrophenyl chloroformate (1 g) in distilled water was added a solution of bovine insulin (50 mg) in 0.1M pH 8.8 phosphate buffer. 1N NaOH was added to maintain the pH if necessary. The reaction mixture was stirred at room temperature for 2.5 hours, then, the mixture was subjected to gel filtration chromatography on Sephadex G-75. It was eluted with 0.1M pH7.0 phosphate buffer, and the fractions were collected by an automatic fractional collector, and the conjugate 1 was purified. The polymer content was determined by the trinitrobenzenesulfonic acid (TNBS) test, and the protein concentration was determined by the Biuret method. The molar ratio of polymer to insulin was determined to be 1:1.

Example 2

Conjugate 2

The terminal hydroxyl groups of polyethylene glycol monostearate were activated by reaction with p-nitrophenyl chloroformate as described above. To a solution of the activated polymer (1 g) in distilled water was added bovine insulin (80 mg) dissolved in 0.1 M phosphate buffer (pH 8.8). The pH was maintained by carefully adjusting with 1 N NaOH. After stirring for 3 hours, the reaction was quenched with excess glycine and subjected to gel filtration chromatography using Sephadex G-75. The insulin/polymer conjugate was collected and lyophilized. The protein content was determined using the Biuret test to obtain quantitative yields.

Example 3

Conjugate 3

Tetrahydro-2-(12-bromododecyloxy)-2Hpyran

To a solution of 12-bromo-1-dodecanol (1 mol) in dichloromethane containing pyridinium p-toluenesulfonate (P-TSA) was added dihydropyran (2 mol). The reaction mixture was stirred for 24 h, then washed twice with water and dried over anhydrous _MgSO4 . Dichloromethane was removed under reduced pressure. The resulting product is purified, if desired, by chromatography on silica gel.

Polyethylene Glycol and Tetrahydrofuran Derivatives Conjugated

The above tetrahydropyran derivative dissolved in dry benzene was added to a solution of polyethylene glycol (1 mole) in dry benzene containing NaH (1 mole). The reaction mixture was stirred at room temperature for 24 hours. Then, the mixture was passed through a silica gel column, eluting with benzene. Purification by column chromatography is carried out if necessary. Treatment with P-TSA at room temperature removes the protecting tetrahydropyranyl group. The final product is purified by column chromatography if necessary. The hydroxyl groups of the polymer were activated by reaction with p-nitrophenyl chloroformate as described above. Conjugation with insulin was performed as described for conjugate 1.

Example 4

Comparative studies of polymer-insulin conjugates and native insulin were performed using bovine insulin to determine their relative stability and activity in animal models. In animal studies, the effectiveness of polymer-insulin in lowering blood sugar levels was compared to that of natural insulin. Female and male mice weighing an average of 25 g were fasted overnight in groups of 5 for the treatment in fractional periods over 2 days.

At time zero, each animal received a single dose of native insulin (group 1, 100 μg/kg, subcutaneously); native insulin (group 2, 1.5 mg/kg, intragastrically); conjugate 1 (group 3 group, 100 μg/kg, intragastric administration); or conjugate 1 (group 4, 100 μg/kg, subcutaneous injection). Another group (Group 5) was not given any kind of insulin, but was given glucose 30 minutes before the prescribed sampling time. Animals were fasted overnight prior to treatment and between studies. All test substances were formulated in phosphate-buffered saline (pH 7.4). Thirty minutes before the scheduled sampling time at 0.5, 1, 2, 4, 8 and 24 hours after insulin treatment, animals were given a large dose of glucose (5 g/kg, 50% solution, gavage), so that each animal received only one Insulin or Conjugate 1 and a glucose burst. At the specified sampling time, blood was taken from the tail vein, and the glucose content was analyzed immediately with a one-touch digital glucose tester (Life Scan). The test results are shown in Figure 1, groups 1-5.

At 30 minutes, the blood glucose levels of animals in group 1 (native insulin, injected subcutaneously) were approximately 30% of those in animals in the control group (group 5, untreated). The hypoglycemic effect of animals in group 1 lasted only 3.5 hours. Oral administration of native insulin (Group 2) lowered blood glucose levels by up to 60% of that of the control group, with the maximum response occurring 30 minutes after insulin treatment. In contrast, group 3 animals (conjugate 1, 100 μg/kg, p.o.) had decreased glucose levels and a significantly delayed onset of hypoglycemic activity. The hypoglycemic activity of animals in group 3 was greater than that of animals in group 2, even though the insulin dose given to group 3 was only 1/15 of that given to group 2. Glucose levels in Group 3 animals were lower than in any of the other treatment groups at various times after 3 hours, with the greatest difference between the 4-8 hour sampling times. During the first 4 hours of the study, the glucose levels of group 4 animals (conjugate 1, 100 μg/kg, s.c.) followed the same course as group 1 animals. After 4 hours, the glucose level in group 2 remained above the control group (untreated, group 5), while at 8 hours, the glucose level in group 4 dropped to 62% of the level of group 5 and remained at the level of group 5 under.

Example 5

Insulin efficacy studies were carried out in male and female mice with unconjugated insulin and conjugate 1 as test substances. An aim of this study was to determine whether insulin in the conjugate 1 form, when administered subcutaneously, would affect blood glucose levels in the same manner. The second objective was to determine whether the insulin complex of Conjugate 1, unlike free insulin, acts to lower blood glucose levels when administered orally. The results are shown in Figure 2, where "insulin complex" refers to conjugate 1.

Basic blood samples were obtained from 10 untreated fasted mice (5 males and 5 females) for serum glucose analysis; the basic value is represented by the symbol "O" in Fig. 2 . The other three groups (5 males and 5 females in each group) were fasted overnight and given glucose load (5 g/kg body weight) by intragastric administration only. Ten animals were sacrificed at 30, 60 and 120 minutes after dosing, and blood samples were obtained for glucose analysis. Fasted mice were given oral (p.o.) and parenteral (s.c.) commercially available insulin and conjugated Object 1 (5 males and 5 females were sacrificed at three times for blood analysis) to provide different treatment methods. The treatment, route of administration and symbols used to represent the results in Figure 2 include: (i) glucose (5g/kg, p.o.), symbol "·"; (ii) insulin (100 μg/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) conjugate 1 (100μg/kg. s.c.) and glucose (5g/kg, p.o.), symbol: "□"; (v) conjugate 1 (250μg/kg, s.c.) and glucose (5k/kg, p.o.), symbol: "□"; (vi ) conjugate 1 (1.5 mg/kg, s.c.) and glucose (5 g/kg, p.o.), symbol: "△", in these tests of conjugate 1, the concentration of protein in the administration solution was 0.1 mg protein /ml solution; for the purpose of comparison, the modified covalently bonded insulin-polymer conjugate with a protein concentration of 0.78 mg protein/ml solution in the dosing solution is also included, (vii) modified conjugate 1 (100 μg/kg, s.c.) and glucose (5 g/kg, p.o.), symbol: "△".

Insulin was given 15 minutes before the glucose load.

Except for the animals in the basic group, glucose was given to all animals by intragastric administration at a dose of 5 g/kg (50% w/v solution prepared with physiological saline, 10 ml/kg). When insulin is injected subcutaneously, the dose is 100 μg/kg (0.004% (w/v) solution in physiological saline, 2.5 ml/kg). When the conjugate 1 polymer-insulin complex was administered by intragastric administration, the dose was 1.56 mg/kg (2.0 ml/kg undiluted test substance). When the conjugate 1 polymer-insulin complex is administered by subcutaneous injection, the dose is 100 μg/kg (1.28ml/kg, a 1:10 dilution of the solution received at 0.78 mg/ml) or 250 μg/kg (3.20 ml/kg of a 1:10 diluent of the receiving solution). The modified conjugate 1 contains 0.1 mg insulin/ml, and the dose is 1.0 ml/kg, ie 100 μg/kg.

Glucose was measured with Gemini centrifugal Analyzer and commercial grape kit. The test method is a combined enzymatic reaction, that is, the reaction of glucose and ATP catalyzed by hexokinase is combined with the reaction of glucose-6-phosphate dehydrogenase to obtain NADH. Duplicate samples were analyzed and average values reported. Some serum samples had to be diluted (1:2 or 1:4) in order to measure the very high glucose concentrations present in some samples.

Mean serum glucose rose to high levels 30 minutes after the glucose load, decreased by 60 minutes, and was below baseline by 120 minutes. If commercially available insulin (100 μg/kg body weight) is injected subcutaneously, it is highly effective in preventing blood glucose rise. However, oral administration of insulin (a high dose of 1.5 mg/kg) has no effect on the increase in blood sugar. This is expected, since insulin is a protein that is easily hydrolyzed in the digestive tract and not fully absorbed into the bloodstream.

When injected subcutaneously at 100 or 250 [mu]g/kg Conjugate 1 was highly effective in inhibiting blood glucose rise after a glucose load. After subcutaneous injection of 1 100 μg/kg conjugate, mean serum glucose values were significantly lower at 30 and 60 minutes than those obtained with subcutaneous injection of 100 μg/kg free insulin. Following subcutaneous injection of 1 250 μg/kg of the conjugate, mean serum glucose decreased (though not significantly) at 30 minutes, significantly decreased at 60 minutes, and returned to baseline by 120 minutes. Glucose levels were below baseline at 120 min with either free insulin 100 μg/kg or conjugated compound 1 100 μg/kg subcutaneously.

1100 μg/kg of the conjugate to be modified produced a significant decrease in blood glucose at 30 minutes after administration.

Example 6

Preparation of p-nitrophenyl carbonate of polysorbate monopalmitate (Polysorbate Monopalmitate)

Dry polysorbate monopalmitate by azeotropic method with dry benzene.

To a solution of dry polymer (2 g, 2 mmole) in 10 ml dry pyridine was added p-nitrophenyl chloroformate (0.6 g, 3 mmole). The mixture was stirred at room temperature for 24 hours. The reaction mixture was cooled in ice for dilution with benzene and filtered through a filter. Repeat this step and finally remove the solvent on a rotary evaporator. Traces of solvent were removed in vacuo. The yield of product was 1.8 g.

Example 7

Preparation of Polysorbate Monopalmitate and Insulin Conjugate

According to the conjugation reaction method of the aforementioned embodiment 1, but the consumption of polysorbate monopalmitate is 1 g, the consumption of insulin is 80 mg, and the reaction product is separated by HPLC to obtain insulin-polysorbate monopalmitate Conjugates in which acid esters are covalently bonded.

Example 8

Preparation of enzyme-polymer conjugates

Conjugation of alkaline phosphatase (AP) to the polymer was performed in the same manner as described in Example 1 for Conjugate 1. Also, to determine whether a high or low polymer/protein ratio is more beneficial, conjugates were prepared with 140 mol polymer/mol enzyme and 14 mol polymer/mol enzyme. For conjugated AP per molecule, the number of polymer groups in the high and low ratios was 30 and 5, respectively.

The following procedure was performed to obtain approximately 5 groups/mole of alkaline phosphatase: 4.1 mg (salt-free) was dissolved in 0.05M sodium bicarbonate. To this solution was added the activated polymer (0.75 mg) in water/dimethylsulfoxide and the solution was stirred at room temperature for 3-12 hours. The resulting reaction mixture was dialyzed against saline solution (0.3N NaCl) (MW cut-off 12,000-14,000) in dialysis tubing for 12 hours with 4-6 dialysate changes. The same method is used for high ratio conjugates. The total protein concentration of the dialyzed material was determined by the Biuret method.

Activity Assessment and Stability Studies

Phosphatase assay was carried out according to the method of A. Voller et al. Bulletin WHO, 53, 55 (1976). Add an equal amount of enzyme solution (50 μl) to the microwell, mix with 200 μl substrate solution (10 g/l, 4-nitrophenyl phosphate solution in 20% ethanolamine buffer, pH 9.3), and incubate at room temperature 45 minutes. The reaction was stopped with 50 μl 3M NaCl. Absorbance at 405 nm was measured on a microplate reader.

Comparing the activity of phosphatases and native enzymes under various conditions.

Dilute solutions containing the same concentrations of alkaline phosphatase and alkaline phosphatase-polymer conjugate were stored at various temperatures. Test enzyme activity regularly. The two polymers tested at 5°C, 15°C, 35°C and 55°C were compared with a control alkaline phosphatase stored at 5°C.

As shown in Table A, the initial enzyme activity of both polymers was approximately three-fold higher than that of the control. Both polymer-enzyme conjugates had improved thermostability over the native enzyme, especially for the conjugate characterized by a higher polymer/enzyme ratio. DAYTEMP℃ 0 2 3 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

                                                                                       

15 89 74 43 36 2835 53 48 21 20 2055 10 2 1 2

Example 9

Conjugate 1A

To a solution of insulin (50 mg) in 0.05 M sodium bicarbonate buffer at pH 9.2 was added a solution of activated polymer (1 g) in water/dimethylsulfoxide, and stirred at room temperature for 3 hours. Carefully adjust with 1N NaOH to maintain the pH of the mixture. The reaction mixture was then dialyzed against 0.1M phosphate buffer, pH 7.0. The purified product was lyophilized. The protein content (48 mg) was determined by the Biuret method. The number of polymer chains attached to insulin is determined by the TNBB assay and is measured as a ratio of moles of polymer to 1 mole of insulin.

Example 10

Calcitonin- _OT50 was synthesized as follows. To a solution of salmon calcitonin (5 mg, Becham) in deionized water (1.5 ml) and borate buffer was added a stirred solution of activated OT (160 mg) in 2.0 ml of water at 5°C. The resulting solution was stirred at 20°C for 1.5 hours, and the pH of the solution was adjusted to 8.8. The reaction was stirred for an additional 0.5 hour, then the pH was brought to 3.8 with 1M dilute hydrochloric acid. The reaction mixture was stored overnight at 5°C. Firstly, the solution was dialyzed against PBS buffer (pH7.5, 2 L) with permeable membrane (MWCO 3500), and then dialyzed against PBS buffer (adjusted to pH3.6, 4×1 L). The dialysate was filtered through a 0.22[mu] filter and stored at 5[deg.]C. The protein concentration was measured by Biuret method and HPLC, with natural calcitonin as the standard. Purity analysis was carried out by HPLC on a size exclusion chromatography column and eluting with 0.05M phosphate buffer (adjusted to pH 3.8).

Example 11

Calcitonin-001 was synthesized as follows. At 5°C, add activated 001 (130mg ) in dimethylsulfoxide (1ml). The solution was stirred at 20°C for 1.5 hours, and the pH was adjusted to 8.8 with 1N hydrochloric acid. The reaction was stirred for an additional hour, then the pH was adjusted to 3.8, and the reaction mixture was stored at 5°C overnight. 4ml of deionized water was added to the reaction mixture, and the supernatant was dialyzed against phosphate buffered saline (PBS) (pH7.4, 2L) in a dialysis membrane (MWCO3500), and then against PBS buffer (adjusted to pH3. 6) Dialysis 4 times. The dialysate was filtered through a 0.22[mu] filter and stored at 5[deg.]C. The protein concentration was measured by Biuret method and HPLC method, with natural calcitonin as the standard. Purity was analyzed by HPLC using a C-8 reverse phase column eluting with a gradient of acetonitrile/0.1% TFA (30-55% over 25 minutes).

Example 12

Insulin-OT was prepared as follows. Weigh 2.0 g of activated OT polymer into a 250 ml round bottom flask. Completely dissolve OT polymer in 10ml of anhydrous DMSO, add 20ml of deionized water, and stir for 5 minutes. The resulting OT solution was cooled to 10 °C. 100mg insulin (Sigma/bovine pancreas/Zn) dissolved in 40ml 0.1M sodium borate buffer (pH9.3) was added to the OT polymer solution all at once. The reaction mixture turned pale yellow. After 30 minutes; the pH of the solution was adjusted to 8.8 with 2N HCl. The reaction solution was stirred for another 1.5 hours, and the pH was adjusted to 8.4. The reaction mixture was filtered through a 0.8 μ filter and dialyzed against PBS buffer (pH 7.4). The dialyzed mixture was filtered through a 0.22 μ filter and concentrated. The concentrated sample was chromatographed with Sephadex G-75 (0.05M sodium phosphate buffer as eluent). Column: 2.5cm (diameter) x 32cm (height). Analysis showed that the yield of insulin conjugate was quantitative at 2 moles of polymer per mole of insulin.

Example 13

001-insulin was orally administered to cynomolgus monkeys in doses of 0.5 mg, 2 mg and 5 mg, respectively, to the treatment group monkeys (cynomolgus monkeys) who had been given pulse doses of glucose. The area under the curve (AUC) was calculated for each treatment group. Using this approach, lower AUC values indicate maximal blood glucose lowering efficacy. The AUC results are shown in Figure 18. These results indicate that a single oral dose of 001-insulin is effective in preventing blood glucose rise in cynomlgus monkeys after a bolus dose of glucose. The AUC after oral administration of 001-insulin was 1/2 less than that of untreated animals, demonstrating the significant hypoglycemic activity of 001-insulin in monkeys.

Example 14

Calcium-lowering activity of orally administered polymer-calcitonin conjugates was evaluated in a rat model of hypocalcemia. Two polymer-calcitonin derivatives (OT-calcitonin and 001-calcitonin) were evaluated as follows. Male Sprague-Dawley rats (average weight 54 g) were fasted overnight. The animals were randomly assigned to each treatment group (5 animals in each group), and the basal serum calcium level of each group was measured. The treatment groups then received single doses of modified calcitonin (50 mg/kg), OT-calcitonin (25 ng/kg) and 001-calcitonin, (250 ng/kg, 2.5 μg/kg and 25 μg/kg ). These doses are given orally. Serum calcium was measured 2 and 4 hours after dosing. Results are presented in Figure 4 as a percentage of naive serum calcium levels versus time. □ stands for calcitonin; ○ stands for 001, 250 ng; ■ stands for 001, 2.5 μg; △ stands for 001, 25 μg; ● stands for OT, 2.5 μg. The results showed a significant dose-dependent decrease in serum calcium, indicating that the conjugate was active over 4 hours from the start of the assessment. The prolonged duration of action may be consistent with the significant bioavailability after oral administration.

Example 15

The calcium-lowering activity of the polymer-calcitonin after oral administration in a rat model was demonstrated as follows. Male Sprague-Dawley rats, weighing an average of 54 g, were fasted overnight. Animals were randomly assigned to treatment groups (4 animals per group), and basal serum calcium levels were determined for each group. Control groups received a single dose of unmodified calcitonin (ct) by subcutaneous (s.c.) route (50 ng/kg) or orally (2.5 μg/kg). The treatment group received OT1-ct or OT2-ct (25 μg/kg) by oral route. Serum calcium was measured 2 and 4 hours after dosing. Results are presented in Figure 5 as a percentage of naive serum calcium levels versus time. OT1-ct and OT2-ct were duplicate formulations of the same calcitonin-polymer conjugate, which were evaluated one by one to demonstrate consistency of response from one batch to the other. The results showed a significant reduction in serum calcium, indicating that the conjugate was active over 4 hours from the initial assessment.

Example 16

Polymer-insulin was evaluated on the diabetic (BB) rat model as follows. The BB rat is a reliable model of human insulin-dependent diabetes. Without daily subcutaneous (sc) insulin injections, BB rats died within 2 days. During the 14-day study, subcutaneous insulin was discontinued and animals were switched to oral low (100 μg/kg/day), medium (3 mg/kg/gd) and high (6 mg/kg/day) doses of the polymer - Insulin for treatment. The results (mean survival time) are shown in FIG. 6 . The sudden switch from subcutaneous injection to oral constitutes a particularly rigorous test for polymer insulins. The results showed that animals administered the low dose did not receive sufficient amounts of insulin to survive; the mid-dose group showed increased survival time; and some animals in the high-dose group survived the entire study period with only oral polymer insulin (14 days). Polymer insulins are administered in non-optimized formulations. The study also showed a statistically significant reduction in blood sugar levels in the afternoon compared to the morning, and blood sugar levels would be better controlled if the animals received multiple daily doses instead of a single bolus. Daily oral administration of polymer-insulin to normal (non-diabetic) animals caused a marked decrease in blood bran levels in the morning.

Example 17

The effects of oral administration of 001-insulin to monkeys (cynomolgous monkeys) are shown below. Six monkeys (3 males and 3 females, with an average weight of 2.5 kg) were fasted overnight. The basal blood glucose level was measured, and then 50% glucose solution was administered by gavage so that the animals received a shock dose of 3 mg/kg glucose. Blood glucose levels were reported at various times (0-4 hours). Two days later, the animals were fasted again, and 3 g/kg glucose was injected. At this time the animals received 001-insulin (equivalent to insulin 5 mg/kg) orally. Blood glucose levels were recorded at various times. The effect of 001-insulin at doses of 2 mg/kg and 0.5 mg/kg was again determined using the same animals and protocol, with a two-day exclusion period between treatments. The results of each treatment (control, 0.5, 2 or 5 mg/kg) were calculated as the percentage change of blood glucose versus time, and the results are shown in Figure 7, where □ represents glucose, △ represents 5 mg, · represents 2 mg, and ○ represents 0.5 mg. After 2 hours of administration of a bolus of glucose, the average increase in blood glucose levels in the treatment group was 1/3 of that in the non-treatment group.

Example 18

Chymotrypsin digestion of insulin and OT insulin was determined as follows. Chymotrypsin (purified Sigam type 1S) was incubated with insulin (bovine) or OT-insulin conjugate dissolved in phosphate-buffered saline (pH 8) at 37°C. A certain amount was taken out periodically, and 1/10 volume of 2% trifluoroacetic acid (TFA) was added to stop the reaction. The samples were analyzed by HPLC, and the results are shown in Figure 3, where OT-insulin is represented by ■ and insulin by □.

Best Mode for Carrying Out the Invention

Optimal binding and stable polypeptide polymer compositions and formulations provided by the present invention involve covalently conjugated peptide complexes in which peptides are covalently bound to incorporated hydrophilic components such as linear polyglycols as part of their One or more polymer molecules, and wherein said polymer incorporates a lipophilic component as a constituent thereof.

A particularly preferred aspect of the present invention relates to a physiologically active peptide composition comprising a physiologically active peptide covalently linked to a polymer containing (i) a linear polyglycol group and (ii) a lipophilic group, wherein the peptide, linear polydiol The conformational arrangement of the alcohol group and the lipophilic group makes the physiologically active peptide in the physiologically active peptide composition relatively independent of the physiologically active peptide (i.e., lacking the unbound form of the polymer coupled thereto) to the enzyme Enhanced in vivo tolerance to degradation.

In another aspect, the present invention relates to three-dimensionally structured physiologically active peptide compositions comprising physiologically active peptides covalently linked to polysorbate complexes containing (i) linear polyglycol groups and (ii) lipophilic groups. Active peptide, wherein the conformational arrangement of the physiologically active peptide, the linear polyglycol group and the lipophilic group with each other results in (a) exposure of the lipophilic group in a three-dimensional conformation and (b) physiological activity in a physiologically active composition The in vivo resistance of the peptide to enzymatic degradation is enhanced relative to the physiologically active peptide alone.

In another more preferred aspect, the present invention relates to a multivalent ligand-bound peptide complex comprising a triglyceride backbone group and a polydiglycol bound to a carbon atom of the triglyceride backbone group a bioactive peptide having an alcohol spacer covalently bonded to a triglyceride backbone group; and at least one fatty acid covalently bonded directly to a carbon atom on the triglyceride backbone group or covalently bonded through a polyglycol spacer group.

In such polyvalent ligand-bound peptide complexes, the α and β carbon atoms of the triglyceride bioactive group can be covalently bound directly to it, or indirectly through a polyglycol spacer. fatty acid groups on the Alternatively, the fatty acid groups can be covalently attached to the α and α′ carbons of the triglyceride backbone group, either directly or through a polyglycol spacer, while the bioactive peptide is covalently attached to the β carbon of the triglyceride, directly bound thereto either covalently or indirectly through a polyglycol spacer.

The present invention includes the use of the stabilized polypeptide conjugates discussed herein in orally administered doses for the amelioration and treatment of disease states involving peptide deficiency.

Claims (31)

CLAIMS 1. A peptide composition characterized by comprising a peptide stably and covalently linked to one or more non-naturally occurring polymer molecules containing lipophilic and hydrophilic groups. 2. The peptide composition according to claim 1, wherein the peptide is a physiologically active therapeutic peptide. 3. The peptide composition according to claim 1, wherein the peptide is a diagnostic peptide for reactive determination of in vitro or in vivo conditions or the presence or absence of components. 4. The peptide composition according to claim 1, wherein the peptide is a preventive peptide for preventing diseases of plants or components thereof. 5. The peptide composition of claim 1, wherein the non-naturally occurring polymer comprises (i) a linear polyglycol group and (ii) a lipophilic group. 6. The peptide composition of claim 1, wherein Non-naturally occurring polymers include polysorbates; peptides include physiologically active peptides. 7. The peptide composition of claim 1, wherein the polymer has a molecular weight of 500-10,000 Daltons. 8. The peptide composition of claim 1, wherein the peptide is selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, growth hormone, growth-stimulating factor, parathyroid hormone, Erythropoietin, hypothalamic releasing factor, prolactin, thyroid stimulating hormone, endorphins, enkephalins, vasopressin, non-naturally occurring morphines, superoxide dismutase, interferon, aspartate Amidase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, and papain. 9. The peptide composition of claim 1, wherein the peptide comprises insulin. 10. A physiologically active peptide composition characterized by comprising a physiologically active peptide covalently linked to one or more polymer molecules containing polysorbate. 11. A physiologically active peptide composition in a three-dimensional conformation, characterized in that it comprises a physiologically active peptide covalently linked to a polysorbate complex containing (i) a linear polyglycol group and (ii) a lipophilic group, The conformational arrangement among the physiologically active peptide, the linear polyglycol group and the lipophilic group exposes the lipophilic group in the three-dimensional conformation. 12. A peptide complex combined with multivalent ligands, characterized in that: it comprises a triglyceride main chain group and a polyglycol spacer bonded to a triglyceride main chain group carbon atom and a triglyceride main chain and at least one fatty acid group covalently bonded directly to a carbon atom on a triglyceride backbone group or through a polyglycol spacer. 13. The multivalent ligand-binding peptide complex according to claim 12, wherein the α' and β carbon atoms of the triglyceride bioactive group are directly covalently bonded thereto, or via polyglycol A spacer is indirectly covalently bound to a fatty acid group thereon. 14. The multivalent ligand-bound peptide complex of claim 12, wherein the fatty acid group is covalently bound to the alpha and alpha' carbons of the triglyceride backbone group either directly or via a polyglycol spacer , while the bioactive peptide is covalently bound to the beta carbon of the triglyceride, either directly covalently bound thereto or indirectly through a polyglycol spacer. 15. A conjugated peptide complex, characterized in that it comprises a biologically active peptide covalently bound to a polysorbate moiety comprising a triglyceride backbone having covalently bound a fatty acid group at one of its α, α' and β carbon atoms, and has a polyethylene glycol group covalently bonded to one of its α, α' and β carbon atoms. 16. The conjugated peptide complex of claim 15, wherein the biologically active peptide is covalently bound to a polyethylene glycol group. 17. A stable, water-soluble, conjugated peptide complex characterized by comprising a peptide stably covalently linked to a polyethylene glycol-modified glycolipid moiety. 18. The peptide complex according to claim 17, wherein the peptide is covalently linked to the glycolipid component modified by polyethylene glycol through an unstable covalent bond on the free amino acid in the polypeptide, wherein the unstable covalent bond can be recovered in vivo Cleavage by biochemical hydrolysis and/or proteolysis. 19. The peptide complex of claim 17, wherein the polyethylene glycol-modified glycolipid component comprises a polysorbate polymer. 20. The peptide complex according to claim 19, wherein the polysorbate polymer comprises a compound selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monooleate Fatty acid ester groups of ester, dioleate, trioleate, monostearate, distearate and tristearate. 21. The peptide complex according to claim 17, wherein the glycolipid moiety modified by polyglycol comprises a polymer selected from polyglycol ethers of fatty acids and polyglycol esters of fatty acids, wherein the fatty acids comprise polyglycol esters selected from lauric acid, Fatty acids of palmitic, oleic and stearic. 22. The peptide complex according to claim 17, wherein the peptide is selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, growth hormone, growth-stimulating factor, parathyroid hormone, erythrocyte-stimulating hormone Propoietin, hypothalamic releasing factor, prolactin, thyroid stimulating hormone, endorphins, enkephalins, vasopressin, non-naturally occurring morphines, superoxide dismutase, interferon, asparagine enzyme, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, and papain. 23. The peptide complex according to claim 18, further characterized in that it comprises a polysorbate component comprising a triglyceride backbone on which α, α' and A fatty acid radical at one of the beta carbons and having a polyethylene glycol group covalently bonded to one of its alpha, alpha' and beta carbon atoms. 24. The peptide complex of claim 23, wherein the peptide is covalently bound to the beta carbon atom of the triglyceride backbone. 25. The peptide complex of claim 17, wherein the peptide is a protein. 26. The peptide complex of claim 23, wherein the triglyceride backbone comprises a biocompatible divalent spacer between its beta carbon atom and the alpha or alpha' carbon atom. 27. A conjugated peptide complex, characterized in that it comprises a biologically active peptide covalently bound to a polysorbate component comprising a triglyceride backbone having a respective covalent Functional groups bound to its α, α' and β carbon atoms: (i) fatty acid groups; and (ii) polyglycol groups with covalently bound bioactive peptides contained thereon. 28. The conjugated peptide complex of claim 27, wherein the biologically active peptide is covalently bound to the terminal functional group of the polyethylene glycol group. 29. The polymer-peptide conjugate, characterized in that it comprises a conjugated polymer selected from polymers represented by the following molecular formula Wherein the total number of W, X, Y, Z is 4-100, R ₁ , R ₂ and R ₃ are each selected from lauric acid, oleic acid, palmitic acid and stearic acid, or each of R ₁ -R ₂ is a hydroxyl group and R ₃ is lauric acid, palmitic acid, oleic acid or stearic acid;

m=10-16 Total number of x, y, z = 8-240 Wherein m is 10-16, n+m=8-240, or when there is more than one n, the total number of all n+m is 8-240, and wherein the sugar moiety in the glycolipid can be arbitrarily replaced by glycerol or aminoglycerol replace;

Z=OTs or Br wherein m and n are as defined above;

Wherein m=2-15, n=5-120;

Wherein m=2-15, n=5-120; Wherein m=5-18, n=2-15;

Wherein m=2-15, n=5-120;

Wherein m=2-15, n=5-120;

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

Wherein m=2-15, n=5-120; Wherein m=2-15, n=5-120; Wherein R ₁ =(CH ₂ ) _m , m=2-15, n=5-120; wherein R ₁ =(CH ₂ ) _m , m=2-15, n=5-120; and wherein said polymer is covalently linked to the peptide. 30. An oral dosage form for alleviating insulin deficiency, characterized in that it comprises a pharmaceutically acceptable carrier and a stable water-soluble conjugated insulin complex comprising stably and covalently linked to a physiologically compatible Insulin with polyglycol-modified glycolipid groups. 31. A conjugated peptide polymer composition for the prophylactic or modulatory treatment of an underlying or developed condition or disease with an effective peptide agent, characterized in that said composition comprises a stable, water-soluble, conjugated A peptide complex comprising a peptide agent covalently bound to a physiologically compatible polyethylene glycol-modified glycolipid component.

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