MXPA98000883A - Compositions of therapeutic agent stabilized by conjugation, supply and formulations of diagnost - Google Patents

Abstract

The present invention relates to a complex of stabilized conjugated therapeutic agent comprising a therapeutic agent conjugatively coupled to a polymer that includes lipophilic and hydrophilic portions. In a particular aspect, the invention comprises an insulin composition suitable for parenteral or non-parenteral administration, preferably oral or parenteral administration comprising insulin covalently coupled to a polymer that includes: (i) a linear polyalkylene glycol portion and (ii) a lipophilic portion, wherein the insulin, the linear polyalkylene glycol portion and the lipophilic portion are arranged conformationally in relation to one another so that the insulin in the composition has increased resistance in vivo to enzymatic degradation, relative to the insulin alone. One, two or three polymeric constituents can be covalently bound to the therapeutic agent molecule, with a polymeric constituent being preferred. The conjugates of the invention are usefully used in therapeutic as well as non-therapeutic, eg diagnostic, applications, and the therapeutic agent and polymer may be covalently coupled to one another, or alternatively may be coupled associatively to one another, v. gr. by hydrogen bonding or other association binding

Description

THERAPEUTIC AGENT COMPOSITIONS STABILIZED BY CONJUGATION, SUPPLY AND DIAGNOSTIC FORMULATIONS DESCRIPTION Field of the Invention The present invention relates to conjugate stabilized compositions and formulations of therapeutic agents and methods for forming and using them. The compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anticoagulants, etc., and precursors of prodrugs, derivatives and intermediates thereof. Description of the Related Art In the field of pharmaceutical therapeutic invention and the treatment of physiological disease and condition, a wide variety of therapeutic agents have been used including various proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anticoagulants, etc., and precursors of prodrugs, derivatives and intermediates of the foregoing. For example, the use of polypeptides and proteins for the systemic treatment of specific diseases is now well accepted in medical practice. The role played by peptides in replacement therapy is so important that many research activities have been directed to the synthesis of large quantities by recombinant DNA technology. Many of these polypeptides are endogenous molecules that are very powerful and specific to produce their biological actions. Other non-polypeptide therapeutics are equally important and pharmaceutically effective. A major factor limiting the usefulness of these therapeutic substances for their intended application is that they are easily metabolized by plasma proteases when they are given parenterally. The oral route of administration of these substances is even more problematic because in addition to the proteolysis in the stomach, the acidity of the stomach destroys them before they reach their intended target tissue. For example, polypeptides and protein fragments, produced by the action of gastric and pancreatic enzymes, are separated by exo and endopeptidases in the intestinal hair-restricting membrane to give di- and tripeptides and even if proteolysis by pancreatic enzymes is avoided, the polypeptides are subjected to degradation by the peptides of the hairline. Any of the therapeutic agents that survive the passage through the stomach also undergo metabolism in the intestinal mucosa where a penetration barrier prevents entry into the cells. Despite these obstacles, there is substantial evidence in the literature to suggest that nutritional and pharmaceutical therapeutic agents such as proteins are absorbed through the intestinal mucosa. On the other hand, the nutritional and drug polypeptides are absorbed by the specific peptide transporters in the cells of the intestinal mucosa. These findings indicate that appropriately formulated therapeutic agents such as (poly) peptides and proteins can be administered by the oral route with retention of sufficient biological activity for their intended use. However, if it were possible to modify these therapeutic agents so that their physiological activities were maintained totally, or at least to a significant degree and at the same time stabilize them against the proteolytic enzymes and increase their capacity of penetration through the intestinal mucosa, it may be possible to use them appropriately for their intended purpose. The product thus obtained could offer advantages in that the most efficient absorption could result with the concomitant ability to use lower doses to produce the optimal therapeutic effect. The problems associated with oral or parenteral administration of therapeutic agents such as proteins are well known in the pharmaceutical industry and several strategies have been used in an attempt to solve them. These strategies include the incorporation of penetration enhancers such as salicylates, lipids-mixed bile salts-micelles, glycerides, and acylcarnitines, but these are found to frequently cause serious problems of local toxicity such as local irritation and toxicity, complete abrasion of the epithelial layer, and tissue inflammation. These problems arise because the enhancers are usually co-administered with the therapeutic agent and leakage of the dosage form often occurs. Other strategies for improving oral delivery include mixing the therapeutic agent with protease inhibitors, such as aprotinin, soybean trypsin inhibitor and amastatin, in an attempt to limit the degradation of the therapeutic agent administered. Unfortunately, these protease inhibitors are not selective, and endogenous proteins are also inhibited. This effect is not desirable. It has also been considered that the increased penetration of therapeutic agents through mucosal membranes modifies the physico-chemical properties of the candidate drugs. The results indicate that simply raising lipophilicity is not sufficient to increase cell transport. It is also suggested that the separation of the hydrogen bonds of the peptide-water is the main energy barrier to be overcome in obtaining diffusion of peptide therapeutics through the membranes (Conradi, RA, Hilgers, AR, Ho, NFH, and Burton, PS, "The influence of peptide structure on transport across Caco-2 cells", Pharm. Res., 8, 1453-1460, (1991)). The stabilization of proteins has been described by several authors. Abuchowski and Davis ("Soluble polymers-Enzyme adducts", In: Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, NY, (1981)) described several methods of derivatizing enzymes to provide stable products. in vivo non-immunogenic soluble in water. Many papers dealing with the stabilization of proteins have been published. Abuchowski and Davis describe several ways to conjugate enzymes with polymeric materials (I bid.). More specifically these polymers are dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and poly-amino acids. The resulting conjugated polypeptides are reported to retain their biological activities and water solubility for parenteral applications. The same authors, in the patent of E. U .A. No. 4,179,337, discloses that polyethylene glycol forms soluble and non-immunogenic proteins when coupled to said proteins. These polymeric materials, however, do not contain fragments suitable for the binding of intestinal mucus, nor do they contain portions that could facilitate or increase membrane penetration. While these conjugates were soluble in water, they were not used for oral administration. Meisner et al., Patent of E. U.A. No. 4, 585,754, teaches that proteins can be stabilized by conjugating them with chondroitin sulfates. The products of this combination are usually polyanionic, very hydrophilic and lack cellular penetration capacity. They are usually not used for oral administration.

Mili et al. Patent of E. U.A. No. 4,003,792 teaches that certain acidic polysaccharides, such as pectins, alginic acid, hyaluronic acid and carrageenan, can be coupled to proteins to produce both soluble and insoluble products. Said polysaccharides are polyanionic, derived from food plants. They lack cellular penetration capacity and are usually not used for oral administration. In Pharmacological Researach Communication 14, 11-120 (1982), Boccu et al. Reported that polyethylene glycol could be bound to a protein such as superoxide dismutase ("SDO"). The resulting conjugate product showed increased stability against denaturation and enzymatic digestion. The polymers did not contain portions that are necessary for membrane interaction and therefore suffer from the same problems as noted above in that they are not suitable for oral administration. Other techniques for stabilizing peptide and protein drugs in which substances of proteinaceous drugs are conjugated to relatively low molecular weight compounds such as aminoleticin, fatty acids, vitamin B12 and glycosides are described in the following articles: R. Igarishi et al. , "Proceed, Intern, Symp. Control Reí. Bioact. Materials, 17, 366, (1990), T. Taniguchi et al., Ibid 1, 9, 104, (1992), GJ Russel-Jones, Ibid, 19., 102, (1992), M. Baudys et al., Ibid 1, 9, 210, (1992) Modification compounds are not polymers and consequently do not contain portions necessary to impart both the solubility and the membrane affinity necessary for bioavailability following the administration oral as well as parenteral Many of these preparations lack oral bioavailability Another approach that has been taken to extend the in vivo duration of action of proteinaceous substances is the encapsulation technique. Saffan et al., In Science, 223, 1081, (1986) teaches the encapsulation of proteinaceous drugs in a film of azopolomers for oral administration. The film always survives digestion in the stomach but is degraded by the icroflora in the large intestine when the encapsulated protein is released. The technique uses a physical mixture and does not facilitate the absorption of protein released through the membrane. Ecanow, Patent of E. U.A. No. 4, 963, 367, teaches that physiologically active compounds, including proteins, can be encapsulated by a coacervative-derived film and the finished product can be suitable for transmucosal administration. Other formulations of the same invention can be administered by inhalation, orally, or parenteral or transdermal routes. These approaches do not provide an intact stability against acidity and proteolytic enzymes of the gastrointestinal tract, the property as desired for oral delivery. Another approach taken to stabilize protein drugs for oral as well as parenteral administration involves trapping the therapeutic agent in liposomes. A review of this technique is found in Y. W. Chien, "New Drug Delivery Systems," Marcel Dekker, New York, NY, 1992. Liposome-protein complexes are physical mixtures. Its administration gives erratic and unpredictable results. The unwanted accumulation of the protein component in certain organs has been reported in the use of said liposome-protein complexes. In addition to these factors, there are additional drawbacks associated with the use of liposomes such as cost, difficult manufacturing processes that require complex Ippoliflcation cycles and solvent incompatibilities. In addition, altered biodistribution and antigenicity aspects have emerged as limiting factors in the development of clinically useful liposomal formulations. The use of "proteinoids" has been recently 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 #A 221, Proc. Int. Sump. Control. I laughed Bioac. Mater., 19, 116 (1992)). Oral supply of various classes of therapeutics has been reported using this system, which encapsulates the drug of interest in a polymeric shell composed of highly branched amino acids. As is the case with liposomes, the drugs are not chemically bound to the sphere of proteinoids and leakage of the drug outside the components of the dosage form is possible. A peptide that has been the focus of much synthetic work and the effort to improve this administration and bioassimilation, is insulin.

The use of insulin as a treatment for diabetes comes from 1922, when Banting et al. ("Pancreatic Extracts in the Treatment of Diabetes Mellitus," Can. Med. Assoc. J., 12., 141-146 (1922)) showed that the active extract of the pancreas had therapeutic effects in diabetic dogs. The treatment of a diabetic patient in the same year with pancreatic extracts resulted in a dramatic clinical improvement to save lives. A course of daily insulin injections is required for extended recovery. The insulin molecule consists of two chains of amino acids linked by disulfide ligations; The molecular weight of insulin is approximately 6,000. The ß cells of the pancreatic islets are created a precursor of a single chain of insulin, known as proinsulin. The proteolysis of proinsulin results in the removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and connection peptide ("C"). In the resulting two-chain insulin molecule, the A chain has glycine at the amino terminus, and the B chain has phenylalanine at the amino terminus. Insulin can exist as a monomer, dimer or a hexamer formed of three of the dimers. The hexamer is coordinated with two atoms of Zn2 +. The biological activity resides in the monomer. Although until recently bovine and porcine insulin was used almost exclusively to treat diabetes in humans, numerous variations of insulin between species are known. Porcine insulin is very similar to insulin in humans, which differ only in that they have a residue of alanine instead of trionine in the C-terminus of the B chain. Despite these differences, most mammalian insulin has activity specific comparable. Until recently, animal extracts provided all the insulin used to treat the disease. The advent of recombinant technology allows the commercial scale manufacture of human insulin (e.g., Humulin ™ Insulin commercially available from Eli Lilly and Company, Indianapolis, IN). Although insulin has now been used for more than 70 years as a treatment for diabetes, few studies of this formulation appeared stably until two recent publications (Brange, J., Langkjaer, L., Havelund, S., and Volund, A. , "Chemical Stability of insulin II 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 products during storage of pharmaceutical preparations, "Pharm. Res., 9, 727-734, (1992)). In these publications the authors exhaustively describe the chemical stability of various insulin preparations under varying temperature and pH conditions. Previous supports focused almost entirely on biological potency as a measure of insulin formulation stability. However, the arrival of several new and powerful analytical techniques-disk electrophoresis, size exclusion chromatography, and CLAR-allows a detailed examination of the chemical stability profile of insulin. Previous chemical studies on insulin stability were difficult because it was found that the recrystallized insulin under examination was not more than 80-90% pure. More recently, high-purity one-component insulin was made available. This single component insulin contains impurities at levels not detectable by current analysis techniques. The formulated insulin tends to numerous types of degradation. Non-enzymatic deamidation occurs when a side chain amide group of a glutaminyl or asparaginyl residue is hydrolyzed to a free carboxylic acid. There are six possible sites for such insulin deamidation: GlnAS, GlnA1s, AsbA18, AsnA21, AsnB3, and GlnB4. Published reports suggest that the three Asn residues are more susceptible to such reactions. Brange and others. (Ibid) reported that in acidic conditions insulin is rapidly degraded by extensive deamidation in AsnA21. In contrast, in neutral formulations deamidation takes place in AsnB3, at a much slower rate, independent of the concentration of insulin and species of insulin origin. However, the temperature and type of formulation play a very important role in determining the Hydrolysis regime in B3. For example, hydrolysis in B3 is minimal if the insulin is crystalline which is the opposite of amorphous. Apparently the reduced flexibility (tertiary structure) in the crystalline form decreases the reaction rate. Stabilizing the tertiary structure by incorporating phenol in neutral formulations results in reduced deamidation regimes. In addition, of the hydrolytic degradation products in insulin formulations, the high molecular weight transformation products are also formed. Brange and others showed by exclusion chromatography that the main products formed in storage of insulin formulations between 4 and 45 ° C are covalent insulin dimers. In protamine-containing formulations the covalent insulin protamine products are also formed. The insulin-protamine dimer product formulation regimen of insulin is significantly affected by temperature. For human or porcine insulin, (regular N1 preparation) the time of formation of high molecular weight products at 1% is decreased from 154 months to 1.7 months at 37 ° C purchased at 4 ° C. For porcine insulin zinc suspension preparations, the same transformation would require 357 months at 41 ° C but only 0.6 months at 37 ° C. These types of insulin degradation can be of greater significance for diabetic subjects. Although the formation of high molecular weight products is generally slower than the formation of hydrolytic (chemical) degradation products described above, the implications may be more serious. There is significant evidence that the incidence of immunological responses to insulin can result from the presence of covalent insulin aggregates (Robbins, DC Cooper, SM Fineberg, S. E., and 'Mead, PM, "Antibodies to covalent aggregates of insulin in blood of insulin-using diabetic patíents ", 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 I diabetic patients is therpeutic insulin", J. Clin. Invest., 77, 717-723. (1986); and Ratner R.E., Phillips, T. M., and Steiner, M., "Persistent cutaneous insulin allergy resulting from high molecular weight insulin aggregates", Diabetes, 39, 728-733, (1990)). As much as 30% of diabetic subjects receiving insulin show specific antibodies to covalent insulin dimers. At a level as low as 2% it was reported that the presence of covalent insulin dimers generated a highly significant response in lymphocyte stimulation in allergic patients. The answers were not significant when the content of dimers was in the range of 0.3-0.6%. As a result, it is recommended that the level of covalent insulin dimers present in the formulation be maintained below 1% to avoid clinical manifestations. Several formulations of insulin are commercially available; Although the stability has been improved to such an extent that it is no longer necessary to refrigerate all formulations, there remains a need for insulin formulations with increased stability. A modified insulin that is not prone to the formation of high molecular weight products could be a substantial advance in the pharmaceutical and medical techniques and the modification that provide this stability (and in addition that provide the possibility of oral insulin availability) could form a significant contribution to the management of diabetes. In addition to the in vivo use of therapeutic agents, polypeptides, nucleoside proteins, and other molecules that are bioactive in vivo, it also finds substantial and growing use in diagnostic reagent applications. Many of these applications, these agents are used in solution environments where they are susceptible to thermal and enzymatic degradation. Examples of such diagnostic agents include enzymes, peptide hormones and proteins, antibodies, enzyme-protein conjugates used for immunoassays, hapten-antibody conjugates, viral proteins such as those used in a large number of diagnostic methodologies for diagnosis or screening. of diseases such as AIDS, hepatitis, and rubella, the growth factors of peptides and proteins used for example in tissue culture, enzymes used in clinical chemistry and insoluble enzymes such as those used in the food industry. As a further specific example, alkaline phosphatase is widely used as a reagent in equipment used for chlorimetric detection of antibody or antigen in biological fluids. Although said enzyme is commercially available in various forms including enzyme and antibody conjugates, its stability to storage and solution is often limited. As a result the alkaline phosphatase conjugates are frequently freeze-dried and the additives such as bovine serum albumin, and Tween 20 are used to extend the stability of the enzyme preparations. While such approaches are advantageous in some cases to improve resistance to degradation of therapeutic and / or diagnostic agents, they have several drawbacks that limit their general applicability. SUMMARY OF THE INVENTION The present invention relates generally to compositions and formulations of therapeutic and / or diagnostic agents stabilized by conjugation and methods for making use thereof. More particularly, the present invention relates in a broad compositional aspect to covalently conjugated therapeutic and / or diagnostic complexes wherein the peptide of the therapeutic and / or diagnostic agent is cavalently linked to one or more molecules of a polymer that it incorporates as a an integral part thereof a hydrophilic portion, e.g., a linear polyethylene glycol, and wherein said polymer incorporates a lipophilic portion as an integral part thereof. In a particular aspect, the present invention relates to a physiologically active therapeutic agent composition comprising a physiologically active therapeutic agent covalently coupled to a polymer comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the therapeutic agent, linear polyalkylene glycol portion, and the lipophilic portion are disposed conformationally in relation to one another so that the physiologically active therapeutic agent in the physiologically active therapeutic agent composition has increased resistance in vivo to degradation enzymatic, in relation to the physiologically active therapeutic agent alone (ie in a non-conjugated form without the polymer coupled thereto). In another aspect, the invention relates to a three-dimensional conformationally physiologically active therapeutic agent composition comprising a physiologically active therapeutic agent coupled with a polysorbate complex comprising (i) linear polyalkylene glycol portion and (ii) a lipophilic portion wherein the physiologically active therapeutic agent, the linear polyalkylene glycol portion, and the lipophilic portion are disposed conformationally in relation to one another so that (a) the lipophilic portion is available externally in the three-dimensional conformation, and (b) the The physiologically active therapeutic agent in the physiologically active therapeutic agent composition has an increased resistance in vivo to enzymatic degradation relative to the physiologically active therapeutic agent alone.

In a further aspect, the invention relates to a multiligand conjugated therapeutic agent complex comprising a portion of the triglyceride base structure, which has: a bioactive therapeutic agent covalently coupled to the structure portion of the triglyceride base through a glycol and polyalkylene spacer group attached to a carbon atom of the triglyceride base structure portion; and at least a portion of fatty acids covalently linked either directly to a carbon atom of the portion of the triglyceride base structure or covalently bound through a polyalkylene glycol separator portion. In said multiligand conjugated therapeutic agent complex the carbon atoms a 'and β of the bioactive portion of triglycerides can have portions of fatty acids linked by the covalent bond either directly thereto or indirectly covalently linked thereto. through separate portions of polyalkylene glycol. Alternatively, a portion of fatty acids may be covalently linked either directly or through a polyalkylene glycol separation portion to the carbons a and 'of the portion of the triglyceride base structure, with the bioactive therapeutic agent being covalently coupled. with the β-carbon with the portion of the triglyceride base structure, either covalently linked directly to it or indirectly bound thereto through a polyalkylene separation moiety. It should be recognized that a wide variety of structural, compositional and conformational forms are possible for the multiligand conjugated therapeutic agent complex comprising the portion of the triglyceride base structure, within the scope of the above discussion. In such a multiligand conjugated therapeutic agent complex, the bioactive therapeutic agent may be advantageously covalently coupled to the modified triglyceride base structure portion through alkyl spacer groups or alternatively other acceptable spacer groups, within the broad scope of the invention. Used in such a context, the acceptability of the spacer group refers to steric, compositional characteristics and the specific acceptability of end-use application. In yet another aspect, the invention relates to a polysorbate complex comprising a polysorbate portion that includes a triglyceride base structure that is covalently coupled to α, α 'and β carbon atoms thereof and using groups that include : (i) A fatty acid group; and (ii) A polyethylene glycol group having a physiologically active portion covalently attached thereto eg, a physiologically active portion is covalently linked to an appropriate functionality of the polyethylene glycol group.

Said covalent linkage can be either direct, eg, to a terminal hydroxy functionality of the polyethylene glycol group, or alternatively, the covalent linkage can be indirect, e.g., reactively crowning the hydroxy termination of the glycol group of polyethylene with a terminal carboxy-functional spacer group so that the resulting polyethylene glycol glycol group has a terminal carboxy functionality to which the physiologically active portion can be covalently linked. The invention relates to a further aspect for an aqueous soluble, stable therapeutic agent or conjugate complex comprising a physiologically active therapeutic agent covalently coupled to a modified glycol lipid portion of physiologically compatible polyethylene glycol. In said complex, the physiologically active therapeutic agent may be covalently coupled to the modified glycolipid portion of physiologically compatible polyethylene glycol by a labile covalent bond in a group of free amino acids of the therapeutic agent, wherein the labile covalent linkage can be separated live by hydrolysis and / or proteolysis. The modified glycolipid portion of physiologically compatible polyethylene glycol may advantageously comprise a polysorbate polymer, e.g., a polysorbate polymer comprising fatty acid ester groups selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate, dioleate, trioleate, monostearate, distearate, and tristearate. In said complex, the modified glycolipid portion of physiologically compatible polyethylene glycol may suitably comprise a polymer selected from a group consisting of polyethylene glycol ethers or fatty acids and polyethylene glycol fatty acid ethers wherein the fatty acids for example they comprise a fatty acid selected from the group consisting of lauric, palmitic, oleic, and stearic acids. In the above complex, the physiologically active therapeutic agent by way of illustration may comprise a peptide, protein, nucleoside, nucleotide, antineoplastic agent, antibiotic, anti-aging agent, antiarrhythmic agent, antiviral agent, or prodrugs, precursors as intermediates, or derivatives thereof. . For example, the therapeutic agent may comprise peptides selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, and hypothalamic release factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, opioids not present in nature, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chymotrypsin, and papain. As other examples, the therapeutic agent may comprise: An antiviral such as: Ara-A (Arabinofuranosyladenine). Acylguanosine, nordeoxyguanosine, Azidothymidine, Dideoxydenosine, or Dideoxycytidine; an anti-cancer agent such as Dideoxyinosine Fluxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, or L-Darubicin; and antibiotic such as Erythromycin, Vancomycin, oleandomycin, or Ampicillin; an anti-arrhythmic such as Quinidine, or an anti-aging agent such as Heparin. In another aspect, the present invention relates to a dosage form of oral administration for the mediation of insulin deficiency, comprising a pharmaceutically acceptable carrier and an aqueous, soluble, stable conjugated insulin complex comprising insulin or proinsulin covalently coupled to a portion of physiologically compatible modified polyethylene glycol glycolipids. A further aspect, the present invention relates to a method for treating insulin deficiency in a human or non-human mammalian subject exhibiting said deficiency, comprising orally administering to the subject an effective amount of a conjugated insulin composition comprising an insulin complex. aqueous, soluble, conjugated conjugate comprising insulin or proinsulin covalently coupled to a portion of physiologically compatible polyethylene glycol modified glycolipids. The term "peptide" as used herein is intended to be broadly constructed even as polypeptides by themselves having molecular weights of up to about 10,000, as well as proteins having molecular weights of greater than about 10,000, wherein the molecular weights are molecular weights average. As used herein, the term "covalently coupled" means that the specified portions are attached either covalently linked directly to one another or covalently linked indirectly to one another through an intermediate portion or portions such as a bridge, separator or portion or portions of ligature. The term "conjugatively coupled" means that the specific portions are covalently coupled to one another or are non-covalently coupled with ones, eg, by hydrogen bonding, ionic bonding, Van der Waals forces, etc. The term "therapeutic agent" means an agent that is therapeutically useful e.g., an agent for the treatment, remission or attenuation of a disease state, physiological condition, symptoms or etiological factors, or for the evaluation or diagnosis thereof. . The invention therefore comprises various compositions for therapeutic application (in vivo), wherein the therapeutic agent component of the conjugated therapeutic agent complex is a physiologically active, or bioactive, therapeutic agent. In such compositions containing therapeutic agent, conjugation of the therapeutic agent to the polymer comprising hydrophilic and lipophilic portions can be direct covalent binding or indirect binding (by appropriate spacer groups) and the hydrophilic and lipophilic portions can also be structurally arranged in the structure of the therapeutic agent. polymer conjugation in any suitable manner involving direct or indirect covalent binding in relation to one another. Therefore, a wide variety of species of therapeutic agents can be adapted in the broad practice of the present invention, as necessary or convenient in a given end-use therapeutic application. In another aspect, the covalently coupled therapeutic agent compositions such as those described above can utilize therapeutic agent components, intended for diagnosis or applications, wherein the therapeutic agent for example is a diagnostic reagent, or a complement of a diagnostic conjugate. for immunoassay or other diagnostic applications or not in vivo. In such non-therapeutic applications, the complexes of the invention are used with great utility as stabilizing compositions which for example, can be formulated in compatible solvents or other formulations based on solutions to provide stable composition forms having increased resistance to degradation. In the previous therapeutic and non-therapeutic applications (v.gr., diagnostic), the present invention relates in a broad compositional aspect to complexes of covalently conjugated therapeutic agents wherein the therapeutic agent is covalently bound to one or more molecules of a polymer that incorporates as an integral part of said polymer a hydrophilic moiety , e.g., a portion of polyalkylene glycol and a lipophilic portion, e.g., a portion of fatty acid. In a preferred aspect, the therapeutic agent may be covalently conjugated by covalent attachment to one or more molecules of a linear polyalkylene glycol polymer incorporated in which, as an integral part thereof, there is a lipophilic moiety, e.g., a fatty acid portion. In another particular broad aspect, the present invention relates to complexes of non-covalently conjugated therapeutic agents wherein the therapeutic agent is non-covalently associated with one or more molecules of a polymer that incorporates as an integral part thereof a hydrophilic moiety, v. g., a portion of polyalkylene glycol and a lipophilic portion, e.g., a portion of fatty acid. The polymer may be variously structured and arranged analogous to the description of the polymer in the complexes of therapeutic agents, conjugates covalently described above, but wherein the therapeutic agent is not bound to the polymer molecule (s) in a covalently, but nevertheless it is associated with the polymer, as for example by associative binding, such as hydrogen bonding, ionic binding or complexation, Van der Waals binding, encapsulation or micellular association (of the specific therapeutic agent), etc. Such non-covalent associations of a therapeutic agent component and polymeric portion (s) for example may use a therapeutic agent component for therapeutic applications (e.g., in vivo) as well as components of non-therapeutic therapeutic agents, v .gr., for diagnosis or other use (in vitro).

In such associatively conjugated therapeutic agent compositions, the polymer component may be suitably constructed, modified, or appropriately functionalized to impart the associative conjugating ability in a selective manner (eg, to impart the hydrogen binding capacity to the polymer and vice versa) , to the therapeutic agent), within the technique of matter. Other aspects, features, and modifications of the invention will be more fully apparent from the description and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of glucose, in mg / dL, as a function of time, in minutes, for the administration of insulin by itself and in complexed forms. Figure 2 is a graph of serum glucose, in mg / dL, as a function of time, in hours, for the administration of insulin in various forms. DETAILED DESCRIPTION OF THE INVENTION. AND PREFERRED MODES TO CARRY OUT THE SAME The modification of therapeutic agents with non-toxic, non-immunogenic polymers could offer certain advantages. If the modifications are made in such a way that the products (conjugates of polymeric-therapeutic agents) retain all or most of their biological activities, the following properties may result: the epithelial penetration capacity may be increased; the modified therapeutic agent can be protected from proteolytic digestion and subsequent abolition of activity; the affinity for endogenous transport systems can be improved; the chemical stability against stomach acidity can be imparted; the balance between lipophilicity and hydrophobicity of the polymers could be optimized. The Protein substances considered with the improved properties described above may be effective as replacement therapy following both oral and parenteral administration. Other routes of administration, such as nasal and transdermal, may also be possible using the modified therapeutic agent. In non-therapeutic applications (eg, diagnostic), diagnostic conjugation-stabilization and / or reagent species of peptides, nucleosides, or other therapeutic agents, including precursors and intermediates of end-use nucleosides, peptides or other products , provides corresponding advantages, when the conjugation component is covalently bound to a polymer in the manner of the present invention. The resulting covalently conjugated agent is resistant to environmental degradation factors, including degradation processes mediated by solvent or solution. As a result of said increased resistance to degradation, the shelf life of the active ingredient is capable of being significantly increased, with concomitant reliability of the composition containing the therapeutic agent in the specific end use for which it is used. The covalent conjugation of therapeutic agents with polymers in the manner of the present invention effectively minimizes hydrolytic degradation and achieves stabilization in vitro and in vivo. The analogous benefits are achieved when the therapeutic, diagnostic or reagent species are not associatively non-covalently conjugated to the polymer molecule (s) in the manner of the present invention. Using insulin bound covalently to the polymer component as an illustrative embodiment of the invention, the nature of the conjugation, involving separable covalent chemical bonds, allows control in terms of the passage of time over which the polymer can be separated from the peptide (insulin) . This separation can occur through enzymatic or chemical mechanisms. The conjugated polymer-peptide complex will be intrinsically active. The total activity will be achieved by following the enzymatic cleavage of the peptide polymer. In addition, chemical modification will allow penetration to the bound peptide, of eg, insulin, to the cell membranes. In a preferred aspect of the present invention, the properties that increase membrane penetration of the lipophilic fatty acid residues are incorporated into the conjugation polymer body. In this regard, using again insulin as the peptide of interest, the polymers derived from fatty acids improve insulin improve intestinal insulin absorption: carbamylation of the amino groups of PheB1 and LysB29 with polymers of long chain fatty acids produced compounds that provide some degree of hypoglycemia activity. This derivatization increases the stability of insulin in intestinal mucosa and its absorption from the small intestine. While the covered description is principally and illustratively directed to the use of insulin as a peptide component in various compositions and formulations of the invention, it will be appreciated that the utility of the invention is thus not limited, but extends to any species that is conjugatable covalently or associatively in the manner of the invention, including, but not limited to: the following species of peptides: calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic release factors, prolactin, hormone stimulation of thyroid, endorphins, antibodies, hemoglobin, soluble CD-4, quagulating factors, tissue plasminogen activator, lecthalin, vasopressin, opioids that are not present in nature, superoxide dismutase, interferon, arginase, arginine, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chymotrypsin, and papain, phosph alkaline tartar, and other suitable enzymes, hormones, proteins, polypeptides, enzyme-protein conjugates, antibody-hapten conjugates, viral epitopes, etc.; Antivirals such as: Ara-A (Arabinofuranosyladenine) Acylguanosine, Nordeoxyguanosine, Acidothymidine, Dideoxydenosine, and Dideoxyzitidine, anti-cancer agents such as Dideoxinosine Floxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, and L-Darubicin; and antibiotics such as Erythromycin, Vancomycin, oleandomycin, and Ampicillin; antiarrhythmics such as Quinidine, and anticoagulants such as Heparins. An object of the present invention is to provide polymers suitable for conjugation with therapeutic agents in a manner that obtains the desirable characteristics listed above. Another objective is to use said modified therapeutic agents for the sustained in vivo delivery of the therapeutic agent. Still another objective is to use technology to deliver therapeutic agents orally in their active form. Still another object of the present invention is to provide amphiphilic prodrugs that are therapeutically effective by oral or parenteral administration. A further objective is to employ associatively conjugated agents for use in immunoassay, diagnostics, and other non-therapeutic applications (e.g., in vitro). Yet another object of the present invention is to provide stabilized conjugated peptide and nucleoside compositions, including covalently variable covalently suitable compositions for in vivo as well as non-vivo applications and to alternatively provide non-covalent, associatively conjugated peptide and nucleoside compositions, varyingly suitable for applications in vivo as well as not in vivo. Within the broad scope of the present invention, a single polymer molecule can be employed for conjugation with a plurality of therapeutic agent species, and it may also be advantageous in the broad practice of the invention to use a variety of polymers as conjugation agents for a given therapeutic agent; combinations of such approaches can also be employed. In addition, compositions of conjugated therapeutic agents in a stabilizing manner may find utility in both live and non-living applications. Additionally, it can be recognized that the conjugation polymer (s) may use any other groups, portions or other species conjugates as appropriate for the end use application As an example, it may be useful in some applications to covalently bind the polymer to a functional portion that imparts resistance to UV degradation or antioxidation or other properties or characteristics of the polymer As an additional example , it may be advantageous in some applications to functionalize the polymer to make the same reagent of non-interlacing character, to increase the different properties or characteristics of the overall conjugate material Consequently, the polymer may contain any functionality to repeating groups, ligatures or other structures with constituent s that do not affect the effectiveness of the conjugate composition for this intended purpose. Other objects and advantages of the present invention will be fully apparent from the disclosed description and appended claims. Exemplary polymers that can be usefully employed achieve these characteristics conveniently as described below in illustrative reaction scheme. In the applications of covalently linked peptides, the polymers can be functionalized and then coupled to the free amino acid (s) of the peptide (s) to form labile linkages that allow retention of activity with the labile linkages. intact The removal of the binding by chemical hydrolysis and proteolysis then increases the peptide activity. The polymers used in the invention can incorporate in their molecules, constituents such as edible fatty acids (lipophilic end), polyethylene glycols (water soluble end), acceptable sugar portions (receptor interaction end), and spacers for binding of therapeutic agents. Among the polymers of choice, polysorbates are particularly preferred and are chosen to illustrate various embodiments of the invention in the discussion encompassed herein. The scope of this invention is of course not limited to polysorbates and other polymers incorporating the portions described above may be usefully employed in the broad practice of this invention. Sometimes it may be convenient to remove one of said portions and thereby retain others in the polymeric structure. without losing the objectives. When it is convenient to do so, the preferred portions to be eliminated without losing the objectives and benefits of the invention are the sugar portions and / or the separator. It is preferred to operate with polymers whose molecular weights fall between 500 and 10,000 daltons. In the practice of the present invention, the polyalkylene glycol residues of C2-C4 alkyl polyalkylene glycols, preferably polyethylene glycol (PEG), are advantageously incorporated into the polymeric systems of interest. The presence of the PEG residues will impart hydrophilic properties to the polymer and to the conjugates of corresponding polymeric-therapeutic agents. Certain glycolipids are known to stabilize therapeutic agents such as proteins and peptides. The mechanism of this stabilization probably involves the association of the fatty acid portions of glycolipids with the hydrophobic domain of the peptide or protein, therefore the aggregation of the protein or peptide is prevented. It is also known that the aggregated peptides are poorly absorbed in the small intestine compared to the native peptides. The invention therefore contemplates the polymer-peptide products in which the v.g. peptide, insulin, is conjugated with the hydrophilic or hydrophobic polymer residue. The fatty acid portion of the polymer is provided to associate it with the hydrophobic domain of the peptide and thus avoid aggregation in solution. The resultant polymer-peptide conjugates will therefore be: stabilized (to chemical and enzymatic hydrolysis); soluble in water, due to the PEG residue; and, by virtue of the fatty acid-hydrophobic domain interactions, they are not prone to aggregation. The derivatization of polyalkylene glycol has a number of advantageous properties in the formulation of polymer-therapeutic agent conjugates in the practice of the present invention, as it is associated with the following properties of polyalkylene glycol derivatives: it improves the aqueous solubility, while that at the same time produces a non-antigenic or immunogenic response; high grades biocampatibility; absence of in vivo biodegradation of the polyalkylene glycol derivatives; and ease of excretion by living organisms. The polymers employed in the practice of the present invention therefore comprise lipophilic and hydrophilic portions, rendering the resulting polymer-drug conjugate highly effective (bioactive) in oral as well as parenteral and other physiological modes. As used below, the terms "drug" and "therapeutic agent" are used interchangeably. Hereinafter as illustrative examples of the polymer-nucleoside conjugates of the present invention are the formulas of conjugates covalently linked denoted for ease of subsequent reference as Conjugate 1, Conjugate 2, and Conjugate 3, wherein "drug" is insulin or another therapeutic agent , and the specific values of m, n, w, x, yy will be described in the present discussion. Conjugate 1: R = C4-C20; n = 1 to 125. drug where: w + x + y + z = 20; Y OR II CH3 (CH2) 7CH = CH (CH2) 7C-R = oleic acid: or other C4-C20 fatty acid radicals. Conjugate 2: OR II drug NH-C-CCK (C (HgJrnCHa wherein: m and n are each independently from 1 to 125 Conjugate 3: OR II drug - NH- O wherein: m and n are each independently from 1 to 125 The conjugate 1 characterizes polysorbate mono-oleate commercially available in the center of the polymer system, a sugar derivative used in many pharmaceutical applications. The properties that increase the lipophilicity of absorption are imparted by the fatty acid chain, while polyethylene glycol (PEG) residues provide a hydrophilic environment (acceptance of hydrogen bonding). The drug is linked by a carbamate link adjacent to the PEG region of the polymer. In conjugate 2 the sugar residue is excluded, but the drug is again bound to the polymer by a carbamate link adjacent to the hydrophilic PEG region of the polymer. The lipophilic fatty acid region of the polymer is therefore at some distance from the point of drug binding, e.g., insulin.

The arrangement described above for Conjugate 2 is reversed in the case of Conjugate 3. Once again the sugar residue is excluded, but in this structure the lipid fatty acid residue HHCQ is closer to the drug binding site and the region of Hydrophilic PEG is far from the point of attachment, which again is through a carbamate linkage. Varied alignments of the hydrophilic and lipophilic regions in relation to the point of attachment of the polymer to the drug are possible in the broad practice of the invention, and such variation will result in polymers that provide lipophilic and hydrophilic domains to the drug. Conjugates 1, 2 and 3 the point of attachment of the carbamate link between the polymers is preferably the amine function. In the general practice of the invention, various methods of coupling the polymers of the therapeutic agent, e.g., peptide, nucleoside, etc., are available and are more fully discussed hereinafter. The polymers used in the conjugation of therapeutic agent according to the invention are designed to incorporate good physical characteristics that allow them to achieve the desired objectives. The absorption enhancers, while allowing penetration of the drug through the cell membrane, do not improve the stability characteristics of the drug and in vivo applications can therefore use the polymer-drug conjugates of the invention in formulations that do not they have said penetration enhancers. One aspect of the present invention therefore relates to the incorporation of fatty portion within the polymer, to mimic penetration enhancers.

In the covalently conjugated polymer-agent conjugates of the present invention, the drug can be covalently linked to the water-soluble polymer by labile chemical bonding. This covalent binding between the drug and the polymer can be separated by chemical or enzymatic reaction. The polymer-drug product retains an acceptable amount of activity; the complete activity of the component drug is carried out when the polymer is completely separated from the drug. Concurrently, the polyethylene glycol moieties are present in the conjugation polymer for rubbing the polymer-drug conjugate with high aqueous solubility and prolonged blood circulation capacity. The modifications described above confer improved properties of solubility, stability and affinity of membranes on the drug. As a result of these improved characteristics, the invention contemplates the parenteral and oral delivery of both active polymer-drug species and, following the hydrolytic separation, the bioavailability of the drug itself in in vivo applications. The polymers used in the embodiment described above can be classified as polyethylene glycol modified lipids and modified polyethylene glycol fatty acids. Among the preferred conjugation polymers can be mentioned polysorbates comprising monopalmitate, dipalmitate, tripalmitate, monolaurate, dilaurate, trialaurate, monooleate, dioleate, trioleate, monostearate, distearate, and tristearate. Other lower fatty acids can be used. It is preferred that the average molecular weight of the polymer resulting from each combination is on the scale of about 750 to about 5,000 daltons. Alternative polymers are preferably polyethylene glycol ethers or fatty acid esters, such as fatty acids being lauric, palmitic, oleic and stearic acids, other lower fatty acids can be used and polymers ranging from 250 to 5,000 daltons by weight average molecular It is preferred to have a derivable group in the polymer, when said group may be at the end terminating with polyethylene glycol or at the end terminating with the fat portion. The derivable group can also be cited within the polymer and can therefore serve as a separator between the peptide and the polymer. Various methods for modifying sorbitan fatty acid to achieve the desired polymer will be treated in greater detail with structural illustrations. Polysorbates are sorbitol esters and their anhydrides, which are copolymerized with ethylene oxide. The structure of a representative polymer is shown below.

(Formula 1) The sum of w, x, y, z is 20 and Ri, R2 and R3 are each independently selected from the group consisting of lauric, oleic, palmitic and stearic acid radicals, or (and R2 are each hydroxyl while R3 It is a radical of lauric, palmitic, oleic or stearic acid or lower fatty acid.These polymers are commercially available and are used in pharmaceutical formulations.When a higher molecular weight polymer is desired, it can be synthesized from glycolipids such as monolaurate, mono-oleate sorbitan, sorbitan monopalmitate or sorbitan monostearate, and an appropriate polyethylene glycol. The glycolipid structures that can be used as starting reagents are described below. m = 10 to 16 (Formula 2) In the synthesis of polymers of glycolipids substituted in three positions with polyethylene glycol, a desired polyethylene glycol having two free hydroxyls at the termini is protected in a termination with a trityl group in pyridine using one mole of trityl chloride. The remaining free hydroxyl group of the polyethylene glycol is converted to tosylate or bromide. The desired glycolipid is dissolved in a suitable solvent and treated with sodium hydride. The tosylate or bromide of the protected polyethylene glycol is dissolved in inert solvent and added in excess to the glycolipid solution. The product is treated with a solution of paratoluenesulfonic acid in inert anhydrous solvent at room temperature and purified by column chromatography. The structures of the transformation are described below.

(Formula 3) M = 10 to 16 Sum of x, y, z = 8 to 240 By adjusting the molar equivalent of reagents and using the appropriate molecular weight scale of polyethylene glycol, mono- or disubstituted glycolipids of the desired molecular weight scale can be obtained by following the previous procedures.

(Formula 4) wherein each n and m can vary independently, and have any suitable appropriate value for the specific drug to be stabilized, e.g., from 1 to 16. The sugar portion of the glycolipid described above could be substituted with glycerol or aminoglycerol whose structural samples are shown immediately.

(Formula 5) In this modification, the primary alcohol was first etherified or esterified with a portion of fatty acid such as lauric, oleic, palmitic or steretic; the amino group is derived with fatty acids to form amides or secondary amino groups, as shown below.

O CH? C-ICHJ CH, CH2O (CH2) mCH3 HO-CH 'HO-CH' * CH2OH% CH2OH wherein m may have any suitable value, e.g., from 10 to 16. The remaining primary alcohol group is protected by a trityl group while the secondary alcohol group is converted with polyethylene glycol to a desired polymer. Usually, the polyethylene glycol has a leaving group in a terminal group and a methoxy group in the other terminal group. The polyethylene glycol is dissolved in inert solvent and added to a solution containing glycolipid of sodium hydride. The product is deprotected in paratoluenesulfonic acid at room temperature to give the desired polymer as described. p-TsA = Paratoluenesulfonic acid (Formula 7) It is sometimes convenient to incorporate fatty acid derivatives in different parts of the polyethylene glycol chain to achieve certain physicochemical properties similar to polysorbates that have been replaced with two / three molecules of fatty acids, v.gr. , polysorbate trioleate. The structures representing the polymers are shown in the following reaction scheme as the open chain of the polysorbate.

R = alkyl, C5 to d8; n = 5 to 120; O O X = O, S, O-, CNH-; O O O O Drug NH-C-O: NH-C O- can be -C0-, HN C- R = alkyl, from Cs to C? 8; n = from 5 to 120; m = from 2 to 15; O O X = O, S, C-O-, ONH-; O O O O ll II .. II ?? Drug NH-C-O: NH-C O- can be -CO-, HNC- R = Alkyl, from C5 to C? 8; m = from 5 to 18; n = from 2 to 15; y = from 5 to 120; and where m, n and y can vary independently within the above scales in relation to each other. (Formulas 8) In the synthesis of polymer A, it is convenient to protect the hydroxyl portions in the first and second carbons of glycerol, e.g., solquetal. The remaining hydroxyl group is converted to the sodium salt in an inert solvent and reacted with halogenated polyethylene glycol or tosylate in which one end of the polyethylene glycol has been protected as an ester. The glycerol protection is removed and the resulting two free hydroxyl groups are converted to the corresponding sodium salts. These salts are reacted in inert solvent with polyethylene glycol which has been partially derived with fatty acids. The reaction takes place after the free hydroxyl is converted to the tosylate or bromide. The polymer G is synthesized in the same manner except that the protected glycerol is first reacted with esters of fatty acids that have been halogenated in the terminal carbon of the acid. In the synthesis of polymer C, it is preferred to start with 1,3-dihalo-2-propanol. The dihalo compound was dissolved in an inert solvent and treated with the sodium salt of two moles of polyethylene glycol which has previously been derived with one mole of a fatty acid moiety. The product was purified by chromatography or dialysis. The resulting dry product was treated, in inert solvent, with sodium hydride. The sodium salt thus formed was reacted with a partially protected polyethylene glycol halo derivative. Sometimes it may be convenient to omit the sugar portion of the polymer. The resulting polymer still contains a fragment of polyethylene glycol. The membrane affinity properties of the fatty acid moiety can be retained by replacing an appropriate fatty acid with a lipophilic long chain alkane; therefore, biocompatibility is conserved. In one example of this embodiment, polyethylene glycol with two terminal free hydroxyl groups is treated with sodium hydride in inert solvent. An equivalent weight of a primary bromide derivative of a fatty acid-like portion is added to the solvent of the polyethylene glycol mixture. The desired product is extracted in inert solvent and purified by column chromatography if necessary.

CH3 (CH2) mCH2Br + CH3 (CH2) mCH2 (OC2H4) nOH (Formula 9) When it is desired to form an ester ligation between the fatty acid and the polyethylene glycol, the acid chloride of the acid is treated with excess of desired polyethylene glycol in suitable inert solvent. The polymer is extracted in the inert solvent and further purified by chromatography if necessary. Or CH3 (CH2) mCOCl + HOCH2CH2 (OC2H4) nOH? CH3 (CH2) mC? CH2CH2 (OC2H4) nOH (Formula 10) In some peptide modifications, it is desired to conjugate the fatty acid portion directly to the therapeutic agent. In this case the polymer is synthesized with the derivable function placed on the fatty acid portion. A solution of mono-methoxypolyethylene glycol of appropriate molecular weight in inert solvent was treated with sodium hydride followed by the addition of solution containing the ethyl ester of a fatty acid having a leaving group on the terminal carbon of the acid. The product was purified after solvent extraction and, if necessary, by column chromatography.

O NaH CH3CH20C (CH2) mBr + HOO ^ CH ^ O H ^ XR O CH3CH2? C (CH2) srOCH2CH2 (OC2H4) nXR (Formula 11) The ester protection was removed by treating it with dilute acid or base.

(Formula 12) Where it is desired to form a carbamate linkage with the drug, the carboxyl or ether were converted to a hydroxyl group by a chemical reduction method known in the art.

HO- (CH ^ OC ^ XR (Formula 13) The functional groups that were used in the conjugation of the drug are usually at one terminal end of the polymer but, in some cases, it is preferred that the functional group be placed within the polymer. In this situation, the derivative groups serve as separators. In one example of this embodiment, a portion of fatty acid can be tested on the alpha carbon to the carboxylic group and the acid portion is esterified. The experimental procedure for said type of compound is similar to that described above, resulting in the product shown below.

CH ^ CH ^ OC ^^ XR COOH (Formula 15) When an extended separator is desired, a polyethylene glycol monoether can be converted to an amino group and treated with subclinical anhydride that has been derivatized with a fatty acid moiety. A desired polyethylene glycol having the primary amine was dissolved in sodium phosphate pH buffer at pH 8.8 and treated with a fatty acid portion of substituted succinic anhydride as shown in the following scheme. The product was isolated by solvent extraction and purified by column chromatography if necessary.

(Formula 15) It should be understood that the above reaction schemes are provided for the purposes of illustration only and that they should not be narrowly interpreted with respect to the other reactions and structures that can be beneficially used in the modification of the drug in the broad practice of the present invention. , e.g., to achieve solubility, stabilization and cell membrane affinity for parenteral and oral administration.

The reaction of the polymer with the drug to obtain covalently conjugated products is easily carried out. For the sake of brevity in the present discussion, the polymer is termed as (P) Where the polymer contains a hydroxyl group, it is first converted to an active carbonate derivative such as para-nitrophenyl carbonate. The activated derivative is then reacted with the amino residue of the drug in a short time under moderate conditions producing carbamate derivatives.

(Formula 16) The above reaction and reagent only serve as illustration and are not exclusive; other activation reagents can be used which result in the formation of urethane, or other ligatures. The hydroxyl group can be converted to an amino group using reagents known in the art. Subsequent coupling with drug through its carboxyl groups results in amide formation. Where the polymer contains a carboxyl group, it can be converted to a mixed anhydride and reacted with the amino group of the drug to create a conjugate containing an amide ligature. In another process, the carboxyl group can be treated with water-soluble carbodiimide and reacted with the drug to produce conjugates containing amide ligatures. The activity and stability of the drug conjugates can vary in many ways, using a polymer of different molecular size. The solubilities of the conjugates can vary by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition. Hydrophilic and hydrophobic characteristics can be balanced by carefully combining portions of fatty acid and polyethylene glycol. In the following some illustrative modification reactions are established for polymer-drug conjugates of the present invention.

In the above reaction scheme involving species I, J and K, the routes are shown to modify the hydrophilicity / lipophilicity balance of the conjugation polymer. The ester groups in the conjugation polymer are susceptible to hydrolysis by esterases. the conjugation polymer containing ester groups can therefore be modified to convert the ester groups to ether groups which are more resistant to hydrolysis. The reaction scheme involving the L and M species illustrates the conversion of hydroxyl groups to carboxylate groups. In this regard, the carboxyl groups will provide the carboxylate anion to conjugate amino residues of nucleosides or other drugs.

In general, various techniques can be advantageously employed to improve the stability characteristics of the polymer conjugates of the present invention, including: functionalization of the polymer with higher hydrolysis resistance groups, e.g., the previously illustrated conversion of groups from ester to ether groups; modifying the lipophilic / hydrophilic balance of the conjugation polymer, as appropriate for the drug being stabilized by the polymer at the level appropriate for the molecular weight of the drug being stabilized by the polymer. The only property of polymers derived from polyalkylene glycol of value for the therapeutic applications of the present invention is general biocompatibility. The polymers have various properties of water solubility and are non-toxic. They are non-antigenic, non-immunogenic and do not interfere with biological activities of enzymes. They have long circulation in the blood and are easily excreted from living organisms. The products of the present invention have been found useful for sustaining the biological activity of therapeutic nucleosides, peptides and other therapeutic agents and for example, can be prepared for therapeutic administration by dissolving in water or acceptable liquid medium. Administration is either by parenteral or oral route. Fine colloidal suspensions may be prepared for parenteral administration in order to produce a depot effect, or by the oral route.

In the dry lyophilized state, the drug polymer conjugates of the present invention have good storage stability; The solution formulations of the conjugates of the present invention likewise are characterized by good storage stability. The therapeutic polymer conjugates of the present invention can be used for the prophylaxis or treatment of any condition or disease state for which the constituent drug is effective. In addition, the polymer-based conjugates of the present invention can be used in the diagnosis of constituents, conditions and disease states in biological systems or specimens, as well as for diagnostic purposes in non-physiological systems. In addition, the polymer conjugates of the invention may have application in the prophylaxis or treatment of condition (s) of disease status (s) in plant systems. By way of example, the active component of the conjugate can have insecticidal, herbicidal, fungicidal and / or pesticidal efficacy arranged for use in various plant systems. In therapeutic use, the present invention contemplates a method for treating an animal subject that has or is latently susceptible to said disease condition (s) or condition (s) and in need of said treatment, comprising administering said animal an effective amount of a polymer conjugate of the present invention that is therapeutically effective for said condition or disease state. The subjects that will be treated by the polymeric conjugates of the present invention include human and non-human animal subjects (e.g., birds, dogs, cats, cows, horses) and are preferably mammalian subjects and more preferably are human subjects. Depending on the specific condition or disease state to be combated, the animal subjects may be administered polymer conjugates of the invention at any suitable therapeutically effective and safe dose, as can be readily determined within the practice of the art and without undue experimentation. The conjugates of the polymeric drug of the invention can be administered by themselves as well as the form of ethers, pharmaceutically acceptable salts and other physiologically functional derivatives thereof. The present invention also contemplates pharmaceutical formulations, both for veterinary medicinal use and for humans, which comprise as the active agent one or more polymeric conjugates of the invention. In such pharmaceutical and drug formulations, the active agent is preferably used together with one or more pharmaceutically acceptable carriers for the same and optionally any other therapeutic ingredients. The carrier (s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and unduly harming the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in an amount appropriate to achieve the desired daily dose. Formulations include those suitable for parenteral as well as non-parenteral administration and specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial administration , sub-arachnoid, bronchial, lymphatic, vaginal and intrauterine. Formulations suitable for oral and parenteral administration are preferred. When the active agent is used in a formulation comprising a liquid solution, the formulation can advantageously be administered orally or parenterally. When the active agent is used in a liquid suspension formulation or as a powder in a biocompatible vehicle formulation, the formulation can be advantageously orally administered, rectally or bronchially. When the active agent is used directly in the form of a solid powder, the active agent can be administered orally advantageously. Alternatively, it can be administered bronchially, via nebulization of the powder in a vehicle gas, to form a gaseous dispersion of the powder inspired by the patient from a breathing circuit comprising a suitable nebulizer device. The formulations comprising the active agent of the present invention can be conveniently present in the unit dosage forms and can be prepared by any of the methods well known in the art of pharmacy. Said methods generally include the step of associating the active ingredient (s) with a vehicle which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately associating the active ingredient (s) with a liquid vehicle, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms. of the desired formulation. Formulations of the present invention suitable for oral administration may be present as discrete units such as capsules, wafers, tablets, or troches, each containing a predetermined amount of the active ingredient as a powder or granules.; or a suspension in an aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion or a potion. A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which are optionally mixed with a binder, disintegrant, lubricant, inert diluent, surface active agent or surface active agent. discharge. Molded tablets comprised of a mixture of the active compound powder with a suitable vehicle can be made by molding in a suitable machine. A syrup can be made by adding the active compound to a concentrated aqueous solution of a sugar, for example, sucrose, for which it is possible to add any accessory ingredient (s). Said accessory ingredient (s) may include flavors, suitable preservatives, agents for delaying the crystallization of the sugar and agents for increasing the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol. The formulations suitable for parenteral administration! conveniently comprise a sterile aqueous preparation of the active conjugate, which is preferably isotonic with the blood of the recipient (e.g., physiological saline). Such formulations may include suspending agents and thickening agents or other microparticle systems that are designed to deliver the compound to blood components or one or more organs. The formulations may be present in the form of a dose or multiple doses. Nasal spray formulations comprise purified aqueous solutions of the active conjugate with preservatives and isotonic agents. Said formulations are preferably adjusted to a pH and isotonic state compatible with the nasal membranes of the mucosa. Formulations for rectal administration may be present as a suppository with a suitable vehicle such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acid. The ophthalmic formulations were prepared in a method similar to the nasal spray, except that the pH and isotonic factors were preferably adjusted to match that of the eye. Topical formulations comprise the active compound dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydric alcohols and other bases used for topical pharmaceutical formulations. In addition to the ingredients mentioned above, the formulations of this invention may also include one or more accessory ingredients selected from diluents, pH buffer solutions, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. . Accordingly, the present invention contemplates the provision of suitable polymers for the in vitro stabilization of drugs in solution, as a preferred illustrative application of non-therapeutic application. The polymers can be used, for example, to increase the thermal stability and resistance to enzymatic degradation of the drug The increase in the thermal stability characteristic of the drug via conjugation in the form of the present invention provides a means for improving shelf life, room temperature stability and strength of reagents and research equipment The following Examples are provided to illustrate the present invention and should not be considered as limiting thereof Example I Conjugate 1 Polysorbate trioleate p-nitrophenyl To a solution of p-nitrophenyl chloroformate (0.8 g, 4 mmol) in 50 ml of acetonitrile anhydride was added to dry polysorbate trioleate (7 g, 4 mmol) followed by dimethylaminopyridine (0.5 g, 4 mmol). Mix of reaction 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 cooled overnight in dry benzene and the additional precipitate was removed by filtration. The solvent was removed under reduced pressure and the residual benzene was removed by low pressure evacuation to give 6.4 g of p-nitrophenyl polysorbate trioleate carbonate. Coupling of insulin with activated polymer To a solution of activated polysorbate trioleate (1 g) in aqueous mixture of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) was added to a solution of bovine insulin (50 mg) in buffer solution. Phosphate at pH 8.8 01 M. The pH was maintained by the addition of 1N NaOH as necessary. The reaction mixture was stirred at room temperature for 2.5 h. After this time, the mixture was subjected to gel filtration chromatography using Sephadex G-75. Purification by elution with phosphate buffer at pH 7.00.1M and collection of fractions with an automatic fraction collector yields Conjugate 1. The polymer content was determined by trinitrobenzenesulfonic acid (TNBS) analysis and mass spectrometry and the protein concentration by the Biuret Method. A molar ratio of polymer to insulin was determined to be 1: 1. Conjugate I was also obtained using pure organic solvent (e.g., DMSO, DMF). Example II Conjugate 2 The terminal hydroxyl group of polyethylene glycol monostearate was 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 (8 mg) in 0.1 M phosphate buffer at pH 8.8. The pH was maintained by careful adjustment with 1N NaOH. After stirring for 3 hours, the reaction mixture was quenched with excess glycine and subjected to gel filtration chromatography using Sephadex G-75. The insulin / polymer conjugate was collected and cleaved. The protein content was determined by Biuret analysis, giving a quantitative yield.

Example III Conjugate 3 Tetrahydro-2- (12-bromododecanoxy) -2H-pyran To a solution of 12-bromo-1-dodecanol (1 mol) in dichloromethane containing pyridinium p-toluenesulfonate (P-TSA) was added dihydropyran ( moles). The reaction mixture was stirred for 24 hours and then washed twice with water and dried over anhydrous MgSO 4. The dichloromethane was removed under reduced pressure. If necessary, the resulting product is purified by chromatography on silica gel. Coupling of polyethylene glycol to the tetrahydropyran derivative The tetrahydropyran derivative described above, dissolved in dry benzene, was added to a solution of polyethylene glycol (1 mole) in dry benzene containing NaOH (1 ml). The reaction mixture was stirred at room temperature for 24 hours. After that time the mixture was eluted through a column of silica gel with benzene. If necessary, further purification is carried out by column chromatography. The protective tetrahydropyran group is removed by treatment with p-TSA at room temperature. The final product is purified, if necessary, by column chromatography. The hydroxyl group of the polymer is activated by reaction with p-nitrophenyl chloroformate as described above. The conjugation with insulin was carried out as described for Conjugate 1.

EXAMPLE IV Comparative studies using bovine insulin above were carried out in polymer-insulin conjugates and on natural insulin to determine their relative stability and activity in animal models. In animal studies, the efficacy of polymer-insulin to decrease blood level was compared to that of native insulin. Male and female mice averaging 25 g in weight fasted overnight and were used in groups of five for each treatment carried out in several phases over a period of two days. Each test animal received a single dose of native insulin (Group 1, 100 μg / kg, subcutaneously); native insulin (Group 2, 1.5 mg / kg, orally by priming); Conjugate 1) Group 3, 100 μg / kg, orally); or Conjugate 1 (Group 4, 100 μg / kg, subcutaneously) at time 0. An additional group (Group 5) did not receive insulin of any kind but was confronted with glucose 30 minutes before scheduling the sampling times. The animals were fasted during the night before treatment and for the duration of the study. All test materials were prepared in saline with pH regulated phosphate, pH 7.4. Thirty minutes before the scheduled sampling times of 0.5, 1, 2, 4, 8 and 24 hours after the insulin treatment, the animals were confronted with a glucose bolus dose (5 g / kg, as a 50% solution). % given orally) so that each animal received only one dose of insulin or Conjugate 1 and one glucose challenge. At the scheduled sample time, blood was collected from the tail vein and analyzed immediately for glucose content using a One-Touch Digital Glucose Meter (Life Sean). The results of the test are shown in Figure 1, for Groups 1-5. Blood glucose levels for group 1 animals (native, subcutaneous insulin) were approximately 30% of the control animals (Group 5, untreated) at the time point in the 30th minute. This hypoglycemic effect was delayed only 3.5 hours in animals of Group 1. The oral insulin administered orally (Group 2) decreased blood glucose levels to a maximum of 60% of the control, this maximum response occurred 30 minutes after treatment with insulin. In contrast, glucose levels in animals in Group 3 (Conjugate 1, 100 μg / kg, p.o.) were decreased with an apparent delayed onset of hypoglycemic activity. The hypoglycemic activity in animals of Group 3 was greater than that of Group 2 animals although the dose of insulin administered to Group 3 was only one fifteenth of that given to Group 2. At all time points after 3 hours, glucose levels were lower for Group 3 animals than for any bull treatment group, the largest difference being in the sampling units of four to eight hours. The glucose levels in the animals of Group 4 (Conjugate 1, 100 μg / kg, s.c.) followed the same course as those for animals of Group 1 during the first four hours of the study. After four hours, the Group 2 glucose levels remained above the control levels (untreated, Group 5) while the Group 4 glucose levels fell, at eight hours, to 62% of the levels of the Group 5 and remained below the levels of Group 5. Example V A study of the efficacy of insulin was carried out in male and female albino mice using as insulin test materials in unconjugated form, and Conjugate 1. A The objective of this study was to determine if insulin in the form of Conjugate 1 is capable of acting on blood glucose levels in the same way as insulin when administered subcutaneously. A second objective was to determine if the insulin complex of Conjugate 1, unlike free insulin, is capable of acting to lower the blood glucose level when administered orally. The results are shown in Figure 2. where "Insulin Complex" denotes Conjugate 1. Blood samples from the base line were obtained for serum glucose analysis of 10 albino mice not fasted (5 male and 5 male). females); the values of the base line in figure 2 are denoted by the symbol "O". Three additional groups (five males and five females each) were fasted overnight and loaded with glucose alone orally by priming (5 g / kg of body weight). Ten animals were sacrificed in each of the three periods to obtain blood samples for glucose analysis: 30, 60 and 120 minutes after dosing. A commercial insulin and Conjugate 1 were each administered either orally (po) or parenterally (s.) To groups of fasting mice (five males and five females, for slaughter and blood analysis in each of the three periods) provide different treatment regimens. The treatment, administration routes and symbols shown for the results in Figure 2, included: (i) glucose (5 g / kg p.o.), symbol: "•"; (ii) insulin (100 μg / kg, s.c.) and glucose (5 g / kg p.o.), symbol: "V"; (V) Conjugate 1 (100 μg / kg, sc and glucose (5 g / kg po), symbol ""; (v) Conjugate 1 (250 mg / kg, sc) and glucose (5 g / kg po), symbol: "" "(vi) Conjugate 1 (1.5 mg / kg, sc) and glucose (5 g / kg po), symbol:"? "In these tests of Conjugate 1, the concentration of the protein in the solution administered was 0.1 mg protein / ml solution, for comparison purposes, a covalently modified insulin-polymer conjugate was included, having a protein concentration in the administered solution of 0.78 mg protein / ml solution, (vii) Conjugate 1 modified (100 μg / kg, sc) and glucose (5 g / kg po) symbol: "?" Insulin was administered 15 minutes before loading the glucose.The glucose was orally administered by priming to the whole group of the animal base line at a dose of 5 g / kg (10 mg / kg of a 50% w / v solution in normal saline) When the insulin was orally administered by priming, it was given at a dose e 1.5 mg / kg (18.85 ml / kg of a 0.008% solution in normal saline). When the insulin was administered subcutaneously, it was given at a dose of 100 μg / kg (2.5 ml / kg of a 0.004% w / v solution in normal saline). When the polymer-insulin complex of Conjugate 1 was orally administered by priming, it was given at a dose of 1.56 mg / kg (2.0 ml / kg undiluted test material). When the polymer-insulin complex of Conjugate 1 was administered subcutaneously, it was given at a dose of 100 μg / kg (1.28 ml / kg of a 1:10 dilution of the received 0.78 mg / ml solution). The modified conjugate 1 contained 0.1 ml of insulin / ml and was dosed at a rate of 1.0 ml / kg to obtain a dose of 100 μg / kg. Glucose was measured using the Gemini Centrifuge Analyzer and glucose reagent kits were purchased. The analysis was a coupled enzymatic assay based on the reaction of glucose and ATP catalyzed by hexokinase, coupled with the glucose-6-phosphate dehydrogenase reaction to give NADH. The duplicate samples were analyzed and the mean value was reported. The dilution (1: 2 or 1: 4) of some serum samples was necessary in order to determine the very high glucose concentration present in certain samples. After glucose loading, the glucose in the medium serum rose to a high level in 30 minutes, declined in 60 minutes and was below the base line in 120 minutes. If commercial insulin was administered subcutaneously (100 μg / kg of body weight), it was highly effective in preventing an increase in blood glucose, however, if insulin was given orally (at a dose of 2.5 mg / kg) there was no effect on Elevation of blood glucose This was expected, since insulin, a protein, is easily hydrolyzed in the digestive tract and was not absorbed intact in the bloodstream When Conjugate 1 was given subcutaneously at a dose of 100 or 250 μg / kg, was highly effective to restrict the elevation in blood glucose after the glucose load.The mean values of serum glucose at 250 μg / kg of Conjugate 1 were lower, although not significantly, in 30 minutes, significantly lower in 60 minutes and in 120 minutes it was returned to the base line. With free insulin at 100 μg / kg and conjugate 1 at 100 μg / kg, the glucose level remained below the base line in 120 minutes. Modified Conjugate 1 administered at 100 μg / kg produced a significant reduction in blood glucose in 30 minutes. Example VI Preparation of Para-nitrophenyl Carbonate of Polysorbate Monopalmitate The polysorbate monopalmitate was first dried by the azeotropic method using dry benzene. To a solution of the dried polymer (2 g, 2 mmoles) in 10 ml of dry pyridine was added para-nitrophenyl chloroformate (0.6 g, 3 mmol). The mixture was stirred at room temperature for 24 hours. The reaction mixture was quenched on ice and diluted with dry benzene and filtered through filter aid. This procedure was repeated and finally the solvent was removed in the rotary evaporator. The traces of the solvent were removed in vacuo. The yield of the product is 1.8 g. EXAMPLE VII Preparation of Polysorbate Monopalmitate Conjugate with Insulin According to the conjugation reaction procedure described previously of Example 1 but using polysorbate monopalmitate in the amount of 1 g of insulin in the amount of 80 mg, with separation by CLAR from the reaction product, a conjugate was obtained covalently linked to insulin-polysorbate monopalmitate. EXAMPLE VIII Preparation of Polysorbate Monopalmitate Conjugate with Insulin According to the conjugation reaction procedure described previously of Example 1 but using polysorbate monopalmitate in the amount of 1 g of insulin in the amount of 80 mg, with separation of CLAR from the reaction product, a conjugate was obtained covalently linked to insulin-polysorbate monopalmitate.

EXAMPLE VIII Preparation of Enzyme-Polymer Conjugates The coupling of alkaline phosphatase (AP) to the polymer was carried out using the same procedure as described for Conjugate 1 in Example I. In addition, to determine whether a high or low ratio of polymer to protein could be more advantageous, the conjugates were prepared using 140 moles of polymer / mole of enzyme and 14 moles of polymer / mole of enzyme. The number of polymeric groups per conjugated AP molecule are 30 and 5, respectively, for the high and low polymer ratios. The following procedure was used to obtain approximately 5 groups / alkaline phosphatase molecule: 4.1 mg (without salt) was dissolved in sodium bicarbonate. 0.05M. To this solution was added activated polymer (0.75 mg) in water / dimethyl sulfoxide and the solution was stirred for 3 to 12 hours at room temperature. The resulting reaction mixture was dialyzed against a salt solution (NaCl 0.3 N) in dialysis tubing (PM reduction of 12,000-14,000) for 12 hours with 4 to 6 changes of dialysis solution. The same procedure was used for the high ratio. The total protein concentration of the dialyzed material was determined by the Biuret method. Activity Measurement and Stability Study The phosphatase analysis was carried out according to the method of A. Voller et al., Bulletin WHO, 53, 55 (1976). An aliquot (50 microliters) was added to microwells and mixed with 200 microliters of substrate solution (10 g / L, 4-nitrophenyl phosphate in 20% ethanolamine pH buffer, pH 9.3) and incubated at room temperature. environment for 45 minutes. The reaction was stopped in 50 microliters of 3M NaOH. Absorbance was measured at 405 nm in a microplate reader. The phosphatase activity was compared to that of the native enzyme under various conditions. Diluted solutions containing similar concentrations of alkaline phosphatase and alkaline phosphatase-polymer conjugates were stored at various temperatures. The enzymatic activity was tested periodically. The two polymers tested at 5 ° C, 15 ° C, 35 ° C and 55 ° C were compared with the control alkaline phosphatase stored at 5 ° C. As can be seen in Table A, the initial enzymatic activity of both polymers was about three times higher than the control. Both polymer-enzyme conjugates had increased thermal stability over the native enzyme. This is especially evident for the conjugate characterized by the higher ratio of polymer to enzyme.

Table A EXAMPLE IX Conjugate 1A To a solution of insulin (50 mg) in pH buffer solution of 0.05 M sodium bicarbonate pH 9.2 was added an activated polymer solution (1 g) in water / dimethyl sulfoxide and stirred for 3 hours. hours at room temperature. The pH of the mixture was maintained by careful adjustment with 1N NaOH. The reaction mixture was then dialyzed against phosphate saline of pH 7.0 0.1M. The reaction mixture was then dialysed against pH 7.0 0.1M phosphate solution. The purified product was lyophilized. The protein content (48 mg) was determined by Biuret analysis. The number of polymer chains bound to insulin was determined by TNBS analysis giving a ratio of two moles of polymer to one mole of insulin. EXAMPLE X Synthesis of OT? QCAraCMP Synthesis of Tributyl AraCMP; A mixture of 200 mg of AraCMP (0.62 mmole), 1 ml of pyridine, 161 μl of tri-n-butylamine (0.67 mmole) and 1.1 ml of butyric anhydride (6.2 mmole) was stirred for 21 hours at room temperature. The methanol (1 ml) was added to the reaction mixture to destroy the unreacted butyric anhydride and stirred for 1 hour. The reaction mixture was then stirred with water (0.5 ml) for 24 hours at room temperature to remove the butyric phosphate binding. After roto-evaporating the solvent, the Ara CMP tributyl product was extracted into 10 ml of chloroform and the organic layer was washed with 3 x 15 ml of water. The chloroform layer was dried over MgSO 4 and evaporated to dryness. This product was used in the next step without fer purification.

Conjugation of OT ^ C (polyoxyethylene f10] cetyl ether) to tributyl AraCMP TPSCI (1.3 mmoles) was dissolved in 6 ml of anhydrous chloroform and added to tributyl AraCMP and stirred for 40 minutes at room temperature. The resulting activated TBS tributyl AraCMP was added to 876 mg of OT10 (1.2 mmol) in 2.1 ml of pyridine and stirred at room temperature for 4! hours. The CLD of the reaction mixture in THF: methanol (10: 0.75 v / v) showed complete disappearance of tributylated AraCMP. The solvent was evaporated and the product was suspended in 6 ml of water and extracted in 2 x 6 ml of chloroform. The butyl groups of the conjugated OT10 AraCMP were deprotected by stirring the chloroform layer with 4.5 ml of 2.0 M ammonia solution in methanol overnight at room temperature. Purification of AraCMP QTio Conjugate The solvent was rotoevaporated in the above reaction mixture and sulfuric acid was precipitated from TPS with 15 ml of water. The pH of the aqueous layer was reduced to 1-2 and the product was extracted in 6x35 ml of chloroform. The HPLC of the chloroform layer on a C8 analytical column in isopropanol-water-0.1% TFA showed a hydrophobic nucleoside product. The 31P NMR of the product shows the binding of a polymeric residue to the phosphate portion and the 1 H NMR shows the polymeric resonances as well as the nucleoside resonances. The product was purified on the C-8 column with a gradient of isopropanol-water-0.1% TFA. Industrial Applicability The compositions of therapeutic agents stabilized by conjugation of the invention can be readily employed to administer a variety of therapeutic agents to patients in need thereof, including therapeutic agents that are metabolically impaired by plasma proteases when administered parenterally and including therapeutic agents that somehow decompose on oral administration by proteolysis in the stomach, or are somehow metabolically degraded in the intestinal mucosa. For these therapeutic agents susceptible to degradation, the conjugation thereof with a lipophilic / hydrophilic complexing portion according to the present invention achieves a substantial improvement in the efficacy and bioavailability characteristics of said therapeutic agents when performing the treatment. The conjugation stabilization compositions of the invention are applicable to a wide variety of therapeutic agents including therapeutic agents such as insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone. erythropoietin, hypothalactic release factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, opioids not present in nature, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, papain, Ara-A (Ababirioufranosiladenina), Acylguanosine, Nordeoxyguanosine, Azidothymidine, Dideoxydenosine, Dideoxycytidine, Dideoxy-inosine Fluxoridine, 6-mercaptopurine, Doxorubicin, Daunorubicin, or l-Darubicin, Erythromycin, Vancomycin, Oleandericin, Ampicillin; Quinidine and Heparin.

Claims (36)

1. CLAIMS 1. A composition comprising a stable and conjugated covalently bound therapeutic agent with one or more molecules of a polymer that is not present in nature, said polymer comprising a lipophilic portion and a hydrophilic polymeric portion, thus imparting lipophilic and hydrophilic characteristics. balanced to the composition so that the composition is soluble in pharmaceutically acceptable solvent and can interact with biological membranes.
2. 2. A composition according to claim 1, wherein the therapeutic agent comprises a peptide.
3. 3. A composition according to claim 1, wherein the therapeutic agent comprises a nucleoside.
4. 4. A composition comprising a therapeutic agent coupled stably and conjugatively with one or more molecules of a polymer that is not present in nature comprising a lipophilic portion and a portion of hydrophilic polymer wherein the therapeutic agent is soluble in aqueous and active solvents in prophylaxis or treatment of conditions or disease states in a mammalian subject.
5. 5. A peptide composition according to claim 4, wherein the polymer that is present in nature comprises (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety.
6. 6. A composition according to claim 1, wherein the therapeutic agent is a diagnostic peptide used to reactively determine the presence or absence of a condition or component in vitro or in vivo.
7. 7. A composition according to claim 1, wherein the therapeutic agent is active in a plant or component thereof.
8. A composition according to claim 1, wherein the polymer that is not present in nature comprises (i) a linear polyalkylene glycol portion and (ii) a lipophilic portion.
9. 9. A composition according to claim 1, wherein the therapeutic agent comprises an antineoplastic agent.
10. A composition according to claim 1, wherein: the polymer that is not present in nature comprises (i) a linear polyalkylene glycol portion and (ii) a lipophilic portion; the therapeutic agent comprises a physiologically active agent selected from the group consisting of peptides, proteins, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics and anticoagulants; and the physiologically active agent, the linear polyalkylene glycol portion and the lipophilic portion are arranged conformationally in relation to one another so that the physiologically active agent in the composition has an increased resistance in vivo to enzymatic degradation, relative to the physiologically active agent alone.
11. 11. A physiologically active therapeutic agent composition comprising a physiologically active therapeutically active agent covalently coupled to one or more molecules of a polymer comprising (i) a linear polyalkylene glycol portion and (ii) a lipophilic portion, wherein the therapeutic agent physiologically active, the linear polyalkylene glycol portion and the lipophilic portion are arranged conformationally in relation to one another so that the physiologically active therapeutic agent in the composition of the physiologically active therapeutic agent has increased resistance in vivo to enzymatic degradation, in relation to the physiologically active agent alone.
12. 12. A three-dimensional conformational physiologically active therapeutic agent composition comprising a physiologically active therapeutic agent coupled with a polysorbate complex comprising (i) a linear polyalkylene glycol portion and (ii) a lipophilic portion, wherein the therapeutic agent The physiologically active portion, the linear polyalkylene glycol portion and the lipophilic portion are disposed confomationally in relation to the bull so that (a) the lipophilic portion is externally available in the three-dimensional conformation and (b) the physiologically active therapeutic agent in the The composition of the physiologically active therapeutic agent has an increased resistance in vivo to enzymatic degradation, in relation to the physiologically active therapeutic agent alone.
13. 13. A multiligand conjugated therapeutic agent complex comprising a portion of the triglyceride base structure, having: a bioactive therapeutic agent covalently coupled to the portion of the triglyceride base structure through a group of triglycerides polyalkylene glycol bonded to a carbon atom of the portion of the triglyceride base structure; and at least a portion of fatty acid covalently linked directly to a portion of the triglyceride base structure or covalently linked through a polyalkylene glycol spacer portion.
14. A multiligand conjugated peptide complex according to claim 13, wherein the carbon atoms a 'and β of the bioactive portion of triglycerides have portions of fatty acids directly covalently attaching thereto or covalently linked in a manner indirectly to them through polyalkylene glycol separating portions.
15. 15. A complex of multiligand conjugated peptides according to claim 13, wherein the fatty acid portion is covalently linked either directly or through a polyalkylene glycol separating portion to coals a and a of the structure portion. of the triglyceride base, with the bioactive therapeutic agent being covalently coupled to the β-carbon of the portion of the triglyceride base structure, covalently linked directly thereto or indirectly linked thereto through a separating portion of polyalkylene.
16. 16. A polysorbate complex comprising a portion of polysorbate including a triglyceride base structure having a group of fatty acids covalently coupled to one of the carbon atoms a, a 'and β thereof and having a group of polyethylene glycol covalently coupled to one of the carbon atoms a, a 'and β thereof, wherein the polysorbate moiety has an active physiological nucleoside covalently attached thereto.
17. 17. A polysorbate complex according to claim 16, wherein the nucleoside is an antiviral nucleoside.
18. 18. A polysorbate complex according to claim 16, wherein the physiologically active portion is covalently bound to the polyethylene glycol group.
19. 19. A conjugate, aqueous soluble, stable therapeutic agent complex, comprising a stabilizing coupled therapeutic agent and conjugatively to a modified glycolipid portion of polyethylene glycol.
20. 20. A complex according to claim 19, wherein the therapeutic agent is covalently coupled to the modified glycolipid portion of polyethylene glycol by a labile covalent bond in a group of free amino acids of the therapeutic agent.
21. 21. A complex according to claim 19, wherein the modified glycolipid portion of polyethylene glycol comprises a polysorbate polymer.
22. 22. A complex according to claim 21, wherein the polysorbate polymer comprises ether groups of fatty acids selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, mono-oleate, dioleate, trioleate, monostearate, distearate and tristezate.
23. 23. A complex according to claim 19, wherein the modified glycolipid portion of polyethylene glycol comprises a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids and polyethylene glycol esters, wherein the fatty acids comprise a fatty acid selected from the group consisting of lauric, palmitic acids. oleic and stearic.
24. 24. A complex according to claim 19, wherein the therapeutic agent comprises an agent selected from the group consisting of Ara-A (Arabinofuranosyladenine). Acylguanosine, Nordeoxyguanosine, Azidothymidine, Dideoxydadenosine Floxuridine, 6-mercaptopurine, Doxorubicin, Daunorubicin, or l-Darubicin, Erythromycin, Vnacomycin, Oleandomycin, Ampicillin; Quinidine and Heparin.
25. 25. A complex according to claim 19, comprising a polysorbate moiety including a triglyceride base structure having a fatty acid group covalently coupled to one of the carbon atoms of a, a 'and β thereof and having a polyethylene glycol group covalently coupled to one of the carbon atoms a, a 'and β thereof, wherein the polysorbate portion has an active physiological nucleoside covalently attached thereto.
26. 26. A complex according to claim 25, wherein the therapeutic agent is covalently coupled to the structure of the triglyceride base in β-carbon atom thereof.
27. 27. A complex according to claim 19, wherein the therapeutic agent is a nucleoside.
28. A complex according to claim 25, wherein the structure of the triglyceride base comprises a biocompatible bivalent separator grouping between the β-carbon atom and one of the carbon atoms of a, a 'of the base structure .
29. 29. A polysorbate complex comprising a portion of polysorbates including a triglyceride base structure that covalently couples to carbon atoms independently selected from carbon atoms of a, a 'and ß thereof, functionalizing groups including: i) a fatty acid group; and (ii) a polyethylene glycol group having a physiologically active nucleoside portion covalently attached thereto.
30. 30. A polysorbate complex according to claim 29, wherein the physiologically active nucleoside portion is covalently linked to a terminal functionality of the polyethylene glycol group.
31. 31. A therapeutic agent composition according to claim 1, wherein the polymer has a molecular weight of from about 500 to about 10,000 daltons.
32. 32. A therapeutic agent composition according to claim 1, wherein the therapeutic agent is selected from the group consisting of Ara-A (Arabinofuranosyladenine). Acylguanosine, Nordeoxyguanosine ,. Azidothymidine, Dideoxydenosine Floxuridine, 6-mercaptopurine, Doxorubicin. Daunorubicin, or l-darubicin, Erythromycin, Vnacomycin. oleandomycin, Ampicillin; Quinidine and Heparin.
33. 33. A composition according to claim 1, wherein the therapeutic agent comprises azidothymidine.
34. 34. A polymer-therapeutic agent conjugate comprising a conjugation polymer selected from the group consisting of polymers of the formulas wherein the sum of w, x, y, z is from 4 to 100 and Ri, R2 and R3 are each independently selected from the group consisting of tauric, oleic, palmitic and stearic acid and C5-C18 alkyl radicals or R1 and R2 are each hydroxyl while R3 is a lauric, palmitic, oleic or stearic acid radical; where: m = from 10 to 16, and the sum of x, y, z = 8 to 240 where m has a value of 10 to 16, n + m is 8-240 or when more than one n is present, the sum of all n + m is 8-240 and where the sugar portion of the glycolipid it is optionally substituted with glycerol or aminoglycerol where x = 0 and where: m and n are specified before; and wherein said polymer is conjugatively coupled to a therapeutic agent.
35. 35. A method of prophylactically or interventionally treating a potential or developed condition or disease state in a human or non-human mammal subject with an effective therapeutic agent therefor, comprising administering to the subject an effective amount of a conjugated therapeutic agent composition comprising a stable, aqueous, soluble, conjugated therapeutic agent complex comprising said therapeutic agent coupled to a modified glycolipid portion of a physiologically compatible polyethylene glycol.
36. 36. A method for prolonging the activity of a therapeutically active agent in an in vivo or in vitro system. Comprising conjugatively coupling said therapeutic agent with one or more molecules of a polymer that is not present in nature comprising a lipophilic moiety and a hydrophilic polymer moiety to give a polymer-therapeutic agent composition conjugatively coupled and intoducing the polymer-agent composition therapeutic conjugatively coupled to the system in vivo or in vitro.