US 20030229006 A1
A therapeutic formulation comprising a microemulsion of a therapeutic agent in free and/or conjugatively coupled form, wherein the microemulsion comprises a water-in-oil (w/o) microemulsion including a lipophilic phase and a hydrophilic phase, and has a hydrophilic and lipophilic balance (HLB) value between 3 and 7, wherein the therapeutic agent may for example be selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, non-naturally occurring opioids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminease, adenosine deaminase, ribonuclease, trypsin, chymotrypsin, papain, Ara-A (Arabinofuranosyladenine), Acylguanosine, Nordeoxyguanosine, Azidothymidine, Didesoxyadenosine, Dideoxycytidine, Dideoxyinosine Floxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, or I-darubicin, Erythromycin, Vancomycin, oleandomycin, Ampicillin; Quinidine and Heparin. In a particular aspect, the invention comprises an insulin composition suitable for parenteral as well as non-parenteral administration, preferably oral or parenteral administration, comprising insulin covalently coupled with a polymer including (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the insulin, the linear polyalkylene glycol moiety and the lipophilic moiety are conformationally arranged in relation to one another such that the insulin in the composition has an enhanced in vivo resistance to enzymatic degradation, relative to insulin alone. The microemulsion compositions of the invention are usefully employed in therapeutic as well as non-therapeutic, e.g., diagnostic, applications.
1. An oligomer having a formula:
A is —C(O)ONH2, —C(O)OH, or —NH—C(O)OH;
m is 2 to 15;
n is 5 to 120;
X is —O—, —S—, —C(O)O—, or —C(O)NH—; and
R is an alkyl.
2. The oligomer of
3. The oligomer of
4. The oligomer of
5. The oligomer of
6. The oligomer of
7. The oligomer of
8. The oligomer of
9. A method of modifying the pharmaceutical characteristics of a drug comprising conjugating the drug to an oligomer of
10. A salt of an oligomer of
11. An activated form of the oligomer of
12. An N-hydroxy-succinimide ester of the oligomer of
13. A branched oligomer comprising the oligomer of
14. A protein covalently coupled to the oligomer of
15. An oligomer having a formula:
A is —C(O)ONH2, —C(O)OH, or —NH—C(O)OH;
n is 5 to 120;
X is —O—, —S—, —C(O)O—, or —C(O)NH—; and
R is an alkyl.
16. The oligomer of
17. The oligomer of
18. The oligomer of
19. The oligomer of
20. The oligomer of
21. A method of modifying the pharmaceutical characteristics of a drug comprising conjugating the drug to an oligomer of
22. A salt of an oligomer of
23. An activated form of the oligomer of
24. An N-hydroxy-succinimide ester of the oligomer of
25. A branched oligomer comprising the oligomer of
26. A protein or peptide covalently coupled to the oligomer of
27. Methyl(ethyleneglycol)7-O-hexanoic acid.
28. An N-hydroxy-succinimide ester of the methyl(ethyleneglycol)7-O-hexanoic acid of
29. A protein or peptide covalently coupled to the methyl(ethyleneglycol)7-O-hexanoic acid of
 1. Field of the Invention
 The present invention relates to microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents, and to methods of making and using same. The compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhymics, anti-coagulants, etc., and prodrugs, precursors, derivatives, and intermediates thereof.
 2. Description of the Related Art
 In the field of pharmaceutical therapeutic intervention, and the treatment of disease states and physiological conditions, a wide variety of therapeutic agents have come into use, including various proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs precursors, 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 that the peptides play in replacement therapy is so important that many research activities are being directed towards the synthesis of large quantities by recombinant DNA technology. Many of these polypeptides are endogenous molecules which are very potent and specific in eliciting their biological actions. Other non-(poly)peptidyl therapeutic agents are equally important and pharmaceutically efficacious.
 A major factor limiting the usefulness of these therapeutic substances for their intended application is that they are easily metabolized by plasma proteases when given parenterally. The oral route of administration of these substances is even more problematic because in addition to proteolysis in the stomach, the high 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 cleaved by exo and endopeptidases in the intestinal brush border membrane to yield di- and tripeptides, and even if proteolysis by pancreatic enzymes is avoided, polypeptides are subject to degradation by brush border peptidases. Any of the therapeutic agent that survives passage through the stomach is further subjected to metabolism in the intestinal mucosa where a penetration barrier prevents entry into the cells.
 In spite of 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, nutritional and drug (poly)peptides are absorbed by specific peptide transporters in the intestinal mucosa cells. These findings indicate that properly formulated therapeutic agents such as (poly)peptides and proteins may be administered by the oral route, with retention of sufficient biological activity for their intended use. If, however, 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 proteolytic enzymes and enhance their penetration capability through the intestinal mucosa, then it would be possible to utilize them properly for their intended purpose. The product so obtained would offer advantages in that more efficient absorption would result, with the concomitant ability to use lower doses to elicit the optimum therapeutic effect.
 The problems associated with oral or parenteral administration of therapeutic agents such as proteins are well known in the pharmaceutical industry, and various strategies are being used in attempts to solve them. These strategies include incorporation of penetration enhancers, such as the salicylates, lipid-bile salt-mixed micelles, glycerides, and acylcarnitines, but these frequently are found to cause serious local toxicity problems, such as local irritation and toxicity, complete abrasion of the epithelial layer and inflammation of tissue. These problems arise because enhancers are usually co-administered with the therapeutic agent and leakages from the dosage form often occur. Other strategies to improve oral delivery include mixing the therapeutic agent with protease inhibitors, such as aprotinin, soybean trypsin inhibitor, and amastatin, in an attempt to limit degradation of the administered therapeutic agent. Unfortunately these protease inhibitors are not selective, and endogenous proteins are also inhibited. This effect is undesirable.
 Enhanced penetration of therapeutic agents across mucosal membranes has also been pursued by modifying the physicochemical properties of candidate drugs. Results indicate that simply raising lipophilicity is not sufficient to increase paracellular or transcellular transport. Indeed it has been suggested that cleaving peptide-water hydrogen bonds is the main energy barrier to overcome in obtaining diffusion of peptide therapeutics across membranes (Conradi, R. A., Hilgers, A. R., Ho, N. F. H., and Burton, P. S., “The influence of peptide structure on transport across Caco-2 cells”, Pharm. Res., 8, 1453-1460, (1991)). Protein stabilization 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, N.Y., (1981)) disclosed various methods of derivatization of enzymes to provide water soluble, non-immunogenic, in vivo stabilized products.
 A great deal of work dealing with protein stabilization has been published. Abuchowski and Davis disclose various ways of conjugating enzymes with polymeric materials (Ibid.). More specifically, these polymers are dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications. The same authors, in U.S. Pat. No. 4,179,337, disclose that polyethylene glycol rendered proteins soluble and non-immunogenic when coupled to such proteins. These polymeric materials, however, did not contain fragments suited for intestinal mucosa binding, nor did they contain any moieties that would facilitate or enhance membrane penetration. While these conjugates were water-soluble, they were not intended for oral administration.
 Meisner et al., U.S. Pat. No. 4,585,754, teaches that proteins may be stabilized by conjugating them with chondroitin sulfates. Products of this combination are usually polyanionic, very hydrophilic, and lack cell penetration capability. They are usually not intended for oral administration.
 Mill et al., U.S. Pat. No. 4,003,792, teaches that certain acidic polysaccharides, such as pectin, algesic acid, hyaluronic acid and carrageenan, can be coupled to proteins to produce both soluble and insoluble products. Such polysaccharides are polyanionic, derived from food plants. They lack cell penetration capability and are usually not intended for oral administration.
 In Pharmacological Research Communication 14, 11-120 (1982), Boccu et al. disclosed that polyethylene glycol could be linked to a protein such as superoxide dismutase (“SOD”). The resulting conjugated product showed increased stability against denaturation and enzymatic digestion. The polymers did not contain moieties that are necessary for membrane interaction and thus suffer from the same problems as noted above in that they are not suitable for oral administration.
 Other techniques of stabilizing peptide and protein drugs in which proteinaceous drug substances are conjugated with relatively low molecular weight compounds such as aminolethicin, fatty acids, vitamin B12, and glycosides, are described in the following articles: R. Igarishi et al., “Proceed. Intern. Symp. Control. Rel. Bioact. Materials, 17, 366, (1990); T. Taniguchi et al. Ibid 19, 104, (1992); G. J. Russel-Jones, Ibid, 19, 102, (1992); M. Baudys et al., Ibid, 19, 210, (1992).
 The modifying compounds are not polymers and accordingly do not contain moieties necessary to impart both the solubility and membrane affinity necessary for bioavailability following oral as well as parenteral administration. Many of these preparations lack oral bioavailability.
 Another approach which has been taken to lengthen the in vivo duration of action of proteinaceous substances is the technique of encapsulation. M. Saffran et al., in Science, 223, 1081, (1986) teaches the encapsulation of proteinaceous drugs in an azopolymer film for oral administration. The film is reported to survive digestion in the stomach but is degraded by microflora in the large intestine, where the encapsulated protein is released. The technique utilizes a physical mixture and does not facilitate the absorption of released protein across the membrane.
 Ecanow, U.S. Pat. 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 may be administered by inhalation, oral, parenteral and transdermal routes. These approaches do not provide 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 entrapment of the therapeutic agent in liposomes. A review of this technique is found in Y. W. Chien, “New Drug Delivery Systems”, Marcel Dekker, New York, N.Y., 1992. Liposome-protein complexes are physical mixtures; their administration gives erratic and unpredictable results. Undesirable accumulation of the protein component in certain organs has been reported, in the use of such Uposome-protein complexes. In addition to these factors, there are additional drawbacks associated with the use of liposomes, such as cost, difficult manufacturing processes requiring complex lypophilization cycles, and solvent incompatibilities. Moreover, altered biodistribution and antigenicity issues have been raised as limiting factors in the development of clinically useful liposomal formulations.
 The use of “proteinoids” has been described recently (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. Symp. Control. Rel. Bioac. Mater., 19, 116 (1992)). Oral delivery of several classes of therapeutics has been reported using this system, which encapsulates the drug of interest in a polymeric sheath composed of highly branched amino acids. As is the case with liposomes, the drugs are not chemically bound to the proteinoid sphere, and leakage of drug out of the dosage form components is possible.
 A peptide which has been the focus of much synthesis work, and efforts to improve its administration and bioassimilation, is insulin.
 The use of insulin as a treatment for diabetes dates back to 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 from the pancreas had therapeutic effects in diabetic dogs. Treatment of a diabetic patient in that same year with pancreatic extracts resulted in a dramatic, life-saving clinical improvement. A course of daily injections of insulin is required for extended recovery.
 The insulin molecule consists of two chains of amino acids linked by disulfide bonds; the molecular weight of insulin is around 6,000. The B-cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin. Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting (“C”) peptide. 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 may exist as a monomer, dimer or a hexamer formed from three of the dimers. The hexamer is coordinated with two Zn2+ atoms. Biological activity resides in the monomer. Although until recently bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known. Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity. Until recently animal extracts provided all insulin used for treatment of the disease. The advent of recombinant technology allows commercial scale manufacture of human insulin (e.g., Humulin™ insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.).
 Although insulin has now been used for more than 70 years as a treatment for diabetes, few studies of its formulation stability appeared until two recent publications (Brange, J., Langkjaer, L., Havelund, S., and Vølund, A., “Chemical stability of insulin. I. 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., 2, 727-734, (1992)). In these publications, the authors exhaustively describe chemical stability of several insulin preparations under varied temperature and pH conditions. Earlier reports focused almost entirely on biological potency as a measure of insulin formulation stability. However the advent of several new and powerful analytical techniques—disc electrophoresis, size exclusion chromatography, and HPLC—allows a detailed examination of insulin's chemical stability profile. Early chemical studies on insulin stability were difficult because the recrystallized insulin under examination was found to be no more than 80-90% pure. More recently monocomponent, high-purity insulin has become available. This monocomponent insulin contains impurities at levels undetectable by current analysis techniques.
 Formulated insulin is prone to numerous types of degradation. Nonenzymatic deamidation occurs when a side-chain amide group from a glutaminyl or asparaginyl residue is hydrolyzed to a free carboxylic acid. There are six possible sites for such deamidation in insulin: GlnA5, GlnA15, AsnA18, AsnA21, AsnB3, and GlnB4. Published reports suggest that the three Asn residues are most susceptible to such reactions.
 Brange et al. (ibid) reported that in acidic conditions insulin is rapidly degraded by extensive deamidation at AsnA21. In contrast, in neutral formulations deamidation takes place at AsnB3 at a much slower rate, independent of insulin concentration and species of origin of the insulin. However, temperature and formulation type play an important role in determining the rate of hydrolysis at B3. For example, hydrolysis at B3 is minimal if the insulin is crystalline as opposed to amorphous. Apparently the reduced flexibility (tertiary structure) in the crystalline form slows the reaction rate. Stabilizing the tertiary structure by incorporating phenol into neutral formulations results in reduced rates of deamidation.
 In addition to hydrolytic degradation products in insulin formulations, high molecular weight transformation products are also formed. Brange et al. showed by size exclusion chromatography that the main products formed on storage of insulin formulations between 4 and 45° C. are covalent insulin dimers. In formulations containing protamine, covalent insulin protamine products are also formed. The rate of formulation of insulin-dimer and insulin-protamine products is affected significantly by temperature. For human or porcine insulin, (regular N1 preparation) time to formation of 1% high molecular weight products is decreased from 154 months to 1.7 months at 37° C. compared to 4° C. For zinc suspension preparations of porcine insulin, the same transformation would require 357 months at 4° C. but only 0.6 months at 37° C.
 These types of degradation in insulin may be of great significance to diabetic subjects. Although the formation of high molecular weight products is generally slower than the formation of hydrolytic (chemical) degradation products described earlier, the implications may be more serious. There is significant evidence that the incidence of immunological responses to insulin may result from the presence of covalent aggregates of insulin (Robbins, D.C. Cooper, S. M. Fineberg, S. E., and Mead, P. M., “Antibodies to covalent aggregates of insulin in blood of insulin-using diabetic patients”, Diabetes, 36, 838-841, (1987); Maislos, M., Mead, P. M., Gaynor, D. H., and Robbins, D. C., “The source of the circulating aggregate of insulin in type I diabetic patients is therapeutic 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 many 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. Responses were not significant when dimer content was in the range 0.3-0.6%. As a result it is recommended that the level of covalent insulin dimers present in formulation be kept below 1% to avoid clinical manifestations.
 Several insulin formulations are commercially available; although stability has been improved to the extent that it is no longer necessary to refrigerate all formulations, there remains a need for insulin formulations with enhanced stability. A modified insulin which is not prone to formation of high molecular weight products would be a substantial advance in the pharmaceutical and medical arts, and modifications providing this stability (and in addition providing the possibility of oral availability of insulin) would make a significant contribution to the management of diabetes.
 In addition to the in vivo usage of therapeutic agents including polypeptides, proteins, nucleosides, and other biologically active molecules, such agents also find substantial and increasing use in diagnostic reagent applications. In many such applications, these agents are utilized in solution environments wherein they are susceptible to thermal and enzymic degradation. Examples of such diagnostic agents include enzymes, peptide and protein hormones, antibodies, enzyme-protein conjugates used for immunoassay, antibody-hapten conjugates, viral proteins such as those used in a large number of assay methodologies for the diagnosis or screening of diseases such as AIDS, hepatitis, and rubella, peptide and protein growth factors 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 utilized as a reagent in kits used for the colorimetric detection of antibody or antigen in biological fluids. Although such enzyme is commercially available in various forms, including free enzyme and antibody conjugates, its storage stability in solution often is limited. As a result, alkaline phosphatase conjugates are frequently freeze-dried, and additives such as bovine serum albumin and Tween 20 are used to extend the stability of the enzyme preparations. Such approaches, while advantageous in some instances to enhance the resistance to degradation of the therapeutic and/or diagnostic agents, have various shortcomings which limit their general applicability.
 In general, the approaches of the prior art for formulating proteinaceous therapeutic agents for enhanced stability in vivo do not provide intact stability of such agents against acid and proteolytic enzymes of the gastrointestinal tract, the property desired for oral delivery of protein drugs. In spite of this fact, intensive efforts are being made in pharmaceutical and scientific organizations toward oral administration of protein drugs. These efforts have largely focused on insulin as a protein drug of choice for developing oral dosage forms, but have not been successful in yielding formulations that replace parenteral administration.
 Formulations of free insulin using different techniques have been attempted.
 Liposome entrapped insulin for oral administration and its attendant drawbacks have been described hereinabove.
 A recent approach involves formulation of insulin in a liquid medium, using absorption enhancers. U.S. Pat. No. 5,653,987 to Modi and Chandarana teaches that insulin can be formulated for oral or nasal delivery using at least two different absorption enhancers. A close examination of the enhancers described in this reference reveals that these enhancers are not pharmaceutically acceptable. In the examples provided in this patent, most contain either sodium lauryl sulphate, a detergent known to damage the lining of the gastrointestinal tract, or polyoxyethylene 9-lauryl ether, which is used in extracting protein from biological specimens, and additionally is known to be spermatocidal in character. The formulation and synthesis method of U.S. Pat. No. 5,653,987 differ fundamentally from the concept and method of the present invention. Additionally, unlike the present invention, the formulation approach of U.S. Pat. No. 5,653,987 does not address enzymatically stabilized insulin conjugates.
 Still another recent approach of free insulin formulation for oral delivery utilizes the technique of microemulsion. This formulation approach has been described by Cho, Y. W., et al, in U.S. Pat. Nos. 5,656,289 and 5,665,700, by Owen, Albert J., in U.S. Pat. No. 5,646,109 and by Desai, Ashok J., in U.S. Pat. No. 5,206,219.
 Cho, Y. W., et al teaches in U.S. Pat. No. 5,656,289 that oral proteinaceous compositions comprising oil/water emulsions can form chylomicra or provide chylomicra to sites of absorption in the gastrointestinal tract Such patent teaches that the hydrophilic phase may contain ethanol. In the disclosed examples of microemulsion preparations, a substantial amount of ethanol is needed in the hydrophilic portion of the emulsion. Ethanol, however, is known to denature many proteinaceous drugs. Further, the method of manufacturing the proteinaceous composition described by Cho et al. in U.S. Pat. No. 5,656,289 involves microfluidization, which can damage or denature protein drugs as a result of the heat generation and shear force entailed in the microfluidization process.
 In U.S. Pat. No. 5,665,700, Cho et al. teach that proteinaceous compounds can be orally delivered in a water-in-oil formulation comprising a hydrophilic phase dispersed in a lipophilic phase to form an emulsion. Microfluidization with its aforementioned attendant drawbacks is also disclosed in U.S. Pat. No. 5,665,700 for preparing the microemulsion. Free insulin (insulin that is not modified by conjugation with amphiphilic polymers, as disclosed hereinafter and in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe) is unstable in the gastrointestinal tract. In the examples provided in U.S. Pat. No. 5,665,700, most preparations disclosed by Cho et al. contain protease inhibitors. Inhibition in such formulations will, however, not be specific to insulin, and inhibitors may cause severe gastrointestinal problems as a result of inhibition of intestinal proteinaceous contents which are otherwise digestible. Further, lecithin complexation with insulin is required in Cho et al.'s microemulsion formulation. In the present invention, the use of lecithin is not essential.
 As a result of the protease inhibition resulting from Cho et al.'s formulations containing protease inhibitors, indiscrininate absorption of toxic proteinaceous material may result in the in viva use of such formulations. The formulations of the Cho et al. U.S. Pat. Nos. 5,656,289 and 5,665,700 are based on a concept and a method of manufacture that differ fundamentally from the concept and method of the herein disclosed invention.
 Another microemulsion formulation of free insulin is described in U.S. Pat. No. 5,646,109 to A. J. Owen. Owen teaches that a water-in-oil (w/o) microemulsion readily converts to an oil-in-water (o/w) emulsion by the addition of aqueous fluid to the w/o microemulsion. A close examination of the conditions necessary for convertible microemulsion in the formulation of U.S. Pat. No. 5,646,109 reveals that the resultant HLB (hydrophilic and lipophilic balance required is greater than 7. In one example described at column 22, lines 26-28 of U.S. Pat. No. 5,646,109, Owen demonstrates that his formulation containing resultant HLB of 4 produces a non-convertible microemulsion and has no effect in promoting rectal delivery of calcitonin. The use of resultant HLB of greater than 7 to prepare a convertible microemulsion for oral delivery of insulin is based on a concept that differs from the concept of herein disclosed invention, as hereinafter more fully described.
 Desai, A. J., U.S. Pat. No. 5,206,219 describes another microemulsion formulation for oral administration, in which a liquid polyol solvent and lipid cosolvent containing a proteinaceous medicament, e.g., insulin, is treated to form a microemulsion in the gastrointestinal tract at sites of absorption. Desai teaches that a vital ingredient in the formulation is a protease inhibitor. The purpose of using the inhibitor is to prevent the degradation of the proteinaceous medicament. Problems that arise from using protease inhibitors to aid in oral delivery of proteinaceous drugs have been enumerated hereinabove.
 International Publications WO 93-02664 and WO94-08610 of Constantides et al. describe compositions of w/o microemulsion formulations containing peptides and proteins using medium chain fatty acid triglycerides, surfactants and a hydrophilic phase containing proteins. The ratio of triglycerides to low HLB surfactants in these preparations can be 5:1 to 1.5:1. Constantides et al. also describe the preparation of microemulsion compositions containing salts of medium chain fatty acids (high HLB surfactant) in which the insulin loading is 0.35 mg/mL. Contrary to the approach of the invention hereinafter disclosed, a careful examination of the examples of the Constantides et al., International Publications WO 93-02664 and WO94-08610 reveals their combined HLB strength to be above 7.
 It would therefore be a substantial advance in the art, and is correspondingly an object of the present invention, to provide an improved formulation for the administration of therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs precursors, derivatives, and intermediates of the foregoing, which avoids the foregoing problems.
 It is another object of the invention to provide an improved formulation for the oral administration of protein and peptide therapeutic agents, in which the therapeutic agent is stabilized against proteolysis and degradation in the gastrointestinal tract.
 It is yet another object of the invention to provide an improved formulation for administration of therapeutic agents which are of free form and/or are conjugatively stabilized as described in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe.
 Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.
 The present invention relates to microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents, and to methods of making and using same. The compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs, precursors, derivatives, and intermediates thereof.
 The formulations of the present invention may utilize conjugation-stabilized therapeutic and/or diagnostic agent compositions, which are conjugatively stabilized as more fully described in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe, the disclosures of which are hereby incorporated herein in their entirety.
 More particularly, the formulations of the present invention may utilize covalently conjugated therapeutic and/or diagnostic complexes wherein the therapeutic and/or diagnostic agent peptide is covalently bonded to one or more molecules of a polymer incorporating as an integral part thereof a hydrophilic moiety, e.g., a linear polyalkylene glycol, and wherein said polymer incorporates a lipophilic moiety as an integral part thereof.
 In one particular aspect, the present invention relates to a formulation including a physiologically active therapeutic agent covalently coupled with a polymer comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the therapeutic agent, linear polyalkylene glycol moiety, and the lipophilic moiety are conformationally arranged in relation to one another such that the physiologically active therapeutic agent in the formulation has an enhanced in vivo resistance to enzymatic degradation, relative to the physiologically active therapeutic agent alone (i.e., in an unconjugated form devoid of the polymer coupled thereto).
 In another aspect, the invention relates to a formulation including a physiologically active therapeutic agent composition of three-dimensional conformation comprising a physiologically active therapeutic agent covalently coupled with a polysorbate complex comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the physiologically active therapeutic agent, the linear polyalkylene glycol moiety and the lipophilic moiety are conformationally arranged in relation to one another such that (a) the lipophilic moiety is exteriorly available in the three-dimensional conformation, and (b) the physiologically active therapeutic agent in the physiologically active therapeutic agent composition has an enhanced in vivo resistance to enzymatic degradation, relative to the physiologically active therapeutic agent alone.
 In a further aspect, the invention relates to formulations including a multiligand conjugated therapeutic agent complex comprising a triglyceride backbone moiety, having:
 a bioactive therapeutic agent covalently coupled with the triglyceride backbone moiety through a polyalkylene glycol spacer group bonded at a carbon atom of the triglyceride backbone moiety; and
 at least one fatty acid moiety covalently attached either directly to a carbon atom of the triglyceride backbone moiety or covalently joined through a polyalkylene glycol spacer moiety.
 In such multiligand conjugated therapeutic agent complex, the α and β carbon atoms of the triglyceride bioactive moiety may have fatty acid moieties attached by covalently bonding either directly thereto, or indirectly covalently bonded thereto through polyalkylene glycol spacer moieties. Alternatively, a fatty acid moiety may be covalently attached either directly or through a polyalkylene glycol spacer moiety to the a and a′ carbons of the triglyceride backbone moiety, with the bioactive therapeutic agent being covalently coupled with the β-carbon of the triglyceride backbone moiety, either being directly covalently bonded thereto or indirectly bonded thereto through a polyalkylene spacer moiety. It will be recognized that a wide variety of structural, compositional, and conformational forms are possible for the multiligand conjugated therapeutic agent complex comprising the triglyceride backbone moiety, within the scope of the foregoing discussion.
 In such a multiligand conjugated therapeutic agent complex, the bioactive therapeutic agent may advantageously be covalently coupled with the triglyceride modified backbone moiety through alkyl spacer groups, or alternatively other acceptable spacer groups, within the broad scope of the invention. As used in such context, acceptability of the spacer group refers to steric, compositional, and end use application specific acceptability characteristics.
 In yet another aspect, the invention relates to a formulation including a polysorbate complex comprising a polysorbate moiety including a triglyceride backbone having covalently coupled to a, a′ and β carbon atoms thereof functionalizing groups including:
 (i) a fatty acid group; and
 (ii) a polyethylene glycol group having a physiologically active moiety covalently bonded thereto, e.g., a physiologically active moiety is covalently bonded to an appropriate functionality of the polyethylene glycol group.
 Such covalent bonding may be either direct, e.g., to a hydroxy terminal functionality of the polyethylene glycol group, or alternatively, the covalent bonding may be indirect, e.g., by reactively capping the hydroxy terminus of the polyethylene glycol group with a terminal carboxy functionality spacer group, so that the resulting capped polyethylene glycol group has a terminal carboxy functionality to which the physiologically active moiety may be covalently bonded.
 The invention relates to a further aspect to a formulation including a stable, aqueously soluble, conjugated therapeutic agent complex comprising a physiologically active therapeutic agent covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety. In such complex, the physiologically active therapeutic agent may be covalently coupled to the physiologically compatible polyethylene glycol modified glycolipid moiety by a labile covalent bond at a free amino acid group of the therapeutic agent, wherein the labile covalent bond may be scissionable in vivo by biochemical hydrolysis and/or proteolysis. The physiologically compatible polyethylene glycol modified glycolipid moiety 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 such complex, the physiologically compatible polyethylene glycol modified glycolipid moiety may suitably comprise a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids, and polyethylene glycol esters of fatty acids, wherein the fatty acids for example 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 may illustratively comprise a peptide, protein, nucleoside, nucleotide, antineoplastic, agent, antibiotic, anticoagulant, antiarrhythmic agent, antiviral agent, or prodrugs, precursors, intermediates, or derivatives thereof.
 For example, the therapeutic agent may comprise peptide selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalmic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, non-naturally occurring opioids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminease, adenosine deaminase ribonuclease, trypsin, chemotrypsin, and papain.
 As other examples, the therapeutic agent may comprise: An antiviral such as: Ara-A (Arabinofuranosyladenine), Acylguanosine, Nordeoxyguanosine, Azidothymidine, Dideoxyadenosine, or Dideoxycytidine; an anti-cancer agent such as Dideoxyinosine Floxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, or I-darubicin; and antibiotic such as Erythormycin, Vancomycin, oleandomycin, or Ampicillin; an antiarrhythmic such as Quinidine; or an anticoagulant such as Heparins.
 In another aspect, the present invention relates to an oral administration dosage form for the mediation of insulin deficiency, comprising a formulation including a pharmaceutically acceptable carrier and a stable, aqueously soluble, conjugated insulin complex comprising insulin or proinsulin covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
 In a further aspect, the invention relates to a method of treating insulin deficiency in a human or non-human mammalian subject exhibiting such deficiency, comprising orally administering to the subject an effective amount of a conjugated insulin composition comprising a stable, aqueously soluble, conjugated insulin complex comprising insulin covalently or proinsulin covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
 The term “peptide” as used herein is intended to be broadly construed as inclusive of polypeptides per se 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 number average molecular weights. As used herein, the term “covalently coupled” means that the specified moieties are either directly covalently bonded to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties. The term “conjugatively coupled” means that the specified moieties are either covalently coupled to one another or they are non-covalently coupled to one another, e.g., by hydrogen bonding, ionic bonding, Van der Waals forces, etc. The term “free” in reference to a therapeutic agent means that the therapeutic agent is not conjugatively coupled in the specific formulation. The term “therapeutic agent” means an agent which 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
 As used herein, the term “hydrophilic and lipophilic balance” or “HLB” means a value determined in accordance with the method described in P. Becher et al., “Nonionic Surfactant, Physical Chemistry,” Marcel Dekker, New York (1987), pages 439-456. The HLB value is an empirical value on an arbitrary scale that is conveniently and widely used in surfactant chemistry to provide a measure of the polarity of a surfactant or mixture of surfactants, as for example is referenced in U.S. Pat. No. 5,646,109. As noted in such patent at column 16, lines 24-27 thereof, HLB values are commonly provided by surfactant suppliers, and HLB values for a mixture of surfactants are calculated on a volumetric basis. See also U.S. Pat. No. 5,206,219 at column 5, lines 3-17, giving illustrative examples of HLB values for various esters of polyethylene glycol, polyethylene fatty acid esters and polyoxyethylated fatty acids.
 The invention thus comprehends various compositions for therapeutic (in vivo) application, wherein the therapeutic agent component of the conjugated therapeutic agent complex is a physiologically active, or bioactive, therapeutic agent. In such therapeutic agent-containing compositions, the conjugation of the therapeutic agent component to the polymer comprising hydrophilic and lipophilic moieties may be direct covalent bonding or indirect (through appropriate spacer groups) bonding, and the hydrophilic and lipophilic moieties may also be structurally arranged in the polymeric conjugating structure in any suitable manner involving direct or indirect covalent bonding, relative to one another. Thus, a wide variety of therapeutic agent species may be accommodated in the broad practice of the present invention, as necessary or desirable in a given end use therapeutic application.
 In another aspect, covalently coupled therapeutic agent compositions such as those described above may utilize therapeutic agent components intended for diagnostic or in vitro applications, wherein the therapeutic agent is for example a diagnostic reagent, or a complement of a diagnostic conjugate for immunoassay or other diagnostic or non-in vivo applications. In such non-therapeutic applications, the complexes of the invention are highly usefully employed as stabilized compositions which may for example be formulated as hereinafter more fully described with compatible solvents or other solution-based formulations to provide stable compositional forms which are of enhanced resistance to degradation.
 In the foregoing therapeutic and non-therapeutic (e.g., diagnostic) applications, the present invention relates in one broad compositional aspect to formulations including covalently conjugated therapeutic agent complexes wherein the therapeutic agent is covalently bonded to one or more molecules of a polymer incorporating as an integral part of said polymer a hydrophilic moiety, e.g., a polyalkylene glycol moiety, and a lipophilic moiety, e.g., a fatty acid moiety. In one preferred aspect, the therapeutic agent may be covalently conjugated by covalent bonding with one or more molecules of a linear polyalkylene glycol polymer incorporated in which, as an integral part thereof is a lipophilic moiety, e.g., a fatty acid moiety.
 In another broad aspect, the present invention relates to formulations of non-covalently conjugated therapeutic agent complexes wherein the therapeutic agent is non-covalently associated with one or more molecules of a polymer incorporating as an integral part thereof a hydrophilic moiety, e.g., a polyalkylene glycol moiety, and a lipophilic moiety, e.g., a fatty acid moiety. The polymer may be variously structured and arranged analogous to description of the polymer in the covalently conjugated therapeutic agent complexes described above, but wherein the therapeutic agent is not bonded to the polymer molecule(s) in a covalent manner, but is nonetheless associated with the polymer, as for example by associative bonding, such as hydrogen bonding, ionic bonding or complexation, Van der Waals bonding, micellular encapsulation or association (of the specific therapeutic agent), etc., or alternatively wherein the therapeutic agent is in a free form in the formulation, unassociated with any conjugating polymer.
 In associatively conjugated therapeutic agent compositions of the above-described type, the polymer component may be suitably constructed, modified, or appropriately functionalized to impart the ability for associative conjugation in a selectively manner (for example, to impart hydrogen bonding capability to the polymer viz-a-vis the therapeutic agent), within the skill of the art.
 The formulations of therapeutic agent component(s) and polymeric moiety/(ies) within the broad scope of the present invention may for example utilize a therapeutic agent component for therapeutic (e.g., in vivo) applications, as well as non-therapeutic therapeutic agent components, e.g., for diagnostic or other (in vitro) use.
 The formulations of the present invention advantageously comprise a microemulsion of a therapeutic agent in free and/or conjugatively coupled form, wherein the microemulsion has a hydrophilic and lipophilic balance (HLB) value of between 3 and 7, more preferably from about 5 to about 6.5 and most preferably from about 5 to about 6. The microemulsion suitably comprises a water-in-oil (w/o) emulsion of such character having a clear optical character.
 In a specific aspect, the present invention relates to an oral dosage form of insulin. Such insulin may be in the free state and/or conjugatively coupled with an amphiphilic polymer (the conjugative coupling may be covalent coupling and/or associative (non-covalent) coupling).
 The present invention also relates to a method by which the insulin conjugates and/or free insulin may be prepared in microemulsion formulation.
 The present invention provides a safe and effective oral dosage form applicable to free insulin and/or insulin conjugates, as well as to other therapeutic agents, e.g., proteinaceous medicaments, in free form and/or modified by amphiphilic polymers for the purpose of enhancing their stability for oral administration. Unmodified therapeutic agents that are amphiphilic in character can also be usefully employed in the broad practice of the present invention.
 The present invention differs from the composition and method of preparation of other known microemulsions. As discussed in the “Background of the Invention” section hereof, the current state of the art recognizes the instability of protein drugs in the gastrointestinal tract and has endeavored to circumvent such problem by formulating protein drugs with protease inhibitors.
 Such protease inhibitors by their intrinsic character function to protect harmful proteinaceous material in the tract from degradation, and the added protease inhibitors thereby interfere with natural and desirable physiological digestive processes. The presence of protease inhibitors in oral formulations may therefore have quite serious consequences to the health and well-being of the patient. The present invention contemplates the use of therapeutic formulations in which the therapeutic agent(s) can be usefully and effectively stabilized without protease inhibitors and their attendant problems.
 The microemulsion formulation of the present invention provides a dosage form for the safe and effective oral administration of (i) the free form of therapeutic agents such as insulin, (ii) amphiphilic conjugates of such therapeutic agents, or (iii) mixtures of such free form therapeutic agents and their conjugates.
 The method of the invention for preparing microemulsion formulations useful as oral dosage forms of therapeutic agents such as insulin, utilizes emulsification techniques in a novel fashion with respect to the selection of oil, water and cosurfactant components, to provide a microemulsion having a specific HLB character useful for oral drug delivery. The oil component of the microemulsion formulation of the invention-appropriately comprises a pharmaceutically acceptable oil, preferably of a food grade quality.
 In reference to insulin, the selection of oil, water and cosurfactant constituents to yield a microemulsion having an HLB value of less than 7 provides a stable formulation for incorporating free form insulin and/or insulin conjugates for effective oral administration of the insulin.
 In the insulin formulations of the invention, particularly preferred insulin forms include the so-called hex-insulin mixture hereinafter disclosed, which is amphiphilic and able to distribute well in oil, surfactant and water. The hex-insulin mixture is formed from insulin conjugated with a hexyl polymer which forms a mixture of mono-, di- and tri-conjugates of insulin as well as free insulin as the conjugative reaction product (the mono-, di- and tri-prefixes referring to the number of polymer moieties attached to the insulin molecule).
 The hex-insulin mixture is more stable than unconjugated insulin against proteolytic digestion (Table B). In closed loop assay determinations (FIGS. 3, 4), the hex-insulin mixture is better absorbed than unconjugated insulin and gives better glucose reduction than insulin (on an insulin weight basis). In the microemulsion formulation of the present invention, the concentration of the hex-insulin mixture in the formulation is able to be increased above the concentration possible for free zinc insulin alone, and the formulation of both the hex-insulin mixture and free zinc insulin in the microemulsion compositions of the present invention shows improved absorption of the hex-insulin mixture in relation to absorption of zinc insulin (FIG. 6). Accordingly, the hex-insulin mixtures of the invention are highly efficacious in providing superior absorption of the insulin component and concomitant reduction of blood glucose in therapeutic use.
 When used in free form in the formulations of the present invention, the insulin may be used in any suitable form, such as in salt forms such as zinc insulin, sodium insulin, etc. or other pharmaceutically acceptable forms of insulin.
 While the invention is described hereinafter primarily with respect to the preferred oral administration use of the formulation of the invention, the formulation of the invention may also be used for parenteral administration or any other suitable mode of adminstration.
 Other aspects, features, and modifications of the invention will be more fully apparent from the ensuing disclosure and appended claims.
FIG. 1 is a graph of serum glucose, in mg/dL, as a function of time, in minutes, for administration of insulin per se and in complexed forms.
FIG. 2 is a graph of serum glucose, in mg/dL, as a function of time, in hours, for administration of insulin in various forms.
FIG. 3 is a graph of ELISA assay results for native insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight.
FIG. 4 is a graph of ELISA assay results for insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight, wherein the insulin is administered as a conjugate, insulin-hexyl-PEG7-OMe.
FIG. 5 is a graph of percent of pre-dose blood glucose level as a function of time, for free insulin, and for conjugated insulin at respective doses of 1, 3 and 10 milligrams of insulin per kilogram of body weight, against a control of phosphate buffered solution (PBS) and bovine serum albumen (BSA) in various Sprague Dawley rats.
FIG. 6 is a graph of insulin and blood glucose levels in pancreatomized dogs as a function of time, after dosing with a microemulsion insulin formulation representative of the present invention.
 The disclosures of the following United States Patents are hereby incorporated herein in their entirety: U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe.
 Modification of therapeutic agents with non-toxic, non-immunogenic polymers affords certain advantages. If modifications are made in such a way that the products (polymer-therapeutic agent conjugates) retain all or most of their biological activities the following properties may result: epithelial penetration capability may be enhanced; the modified therapeutic agent may be protected from proteolytic digestion and subsequent abolition of activity; affinity for endogenous transport systems may be improved; chemical stability against stomach acidity may be imparted; and the balance between lipophilicity and hydrophobicity of the polymers may be optimized. Proteinaceous substances endowed with the improved properties described above can be effective as replacement therapy following either oral or parenteral administration. Other routes of administration, such as nasal and transdermal, are potentially advantageously employed with the modified therapeutic agent
 In non-therapeutic (e.g., diagnostic) applications, conjugation-stabilization of diagnostic 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 bonded to a polymer as hereinafter disclosed. The resultingly covalently conjugated agent is resistant to environmental degradative factors, including solvent- or solution-mediated degradation processes. As a result of such enhanced resistance to degradation, the shelf life of the active ingredient is able to be significantly increased, with concomitant reliability of the therapeutic agent-containing composition in the specific end use for which same is employed.
 The covalent conjugation of therapeutic agents with polymers in the manner of the present invention effectively minimizes hydrolytic degradation, and achieves in vitro and in vivo stabilization.
 Analogous benefits are realized when therapeutic, diagnostic, or reagent species are non-covalently, associatively conjugated with polymer molecule(s) in the manner of the present invention.
 Additionally, the formulation of the present invention may utilize the therapeutic agent in a free form, as previously described.
 The formulations of the invention may utilize the therapeutic agent in any suitable combination or permutation of the foregoing forms (covalently conjugated, associatively conjugated, and/or free form (unconjugated)).
 Utilizing a peptide covalently bonded to the polymer component as an illustrative embodiment of the form of the therapeutic agent used in the practice of the invention, the nature of the conjugation, involving cleavable covalent chemical bonds, allows for control in terms of the time course over which the polymer may be cleaved from the peptide (insulin). This cleavage may occur by enzymatic or chemical mechanisms. The conjugated polymer-peptide complex will be intrinsically active. Full activity will be realized following enzymatic cleavage of the polymer from the peptide. Further, the chemical modification will allow penetration of the attached peptide, e.g., insulin, through cell membranes.
 In a preferred aspect of the present invention when the therapeutic agent is employed in a conjugated form, membrane penetration-enhancing properties of lipophilic fatty acid residues are incorporated into the body of the conjugating polymer. In this respect, again utilizing insulin as the peptide of interest, fatty acid polymer derivatives of insulin improve the intestinal absorption of insulin: carbamylation of the amino groups of PheB1 and LysB29 with long-chain fatty acid polymers yield compounds which provide some degree of hypoglycemic activity. This derivatization increases the stability of insulin in intestinal mucosa and, its absorption from the small intestine.
 While the ensuing description is primarily 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 not thus limited, but rather extends to any species which are useful in free form or covalently or associatively conjugatable, for use in microemulsion formulations in the manner of the invention, including, but not limited to: the following peptide species: calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoictin, hypothalmic releasing factors, prolactin, thyroid stimulating hormone, endorphins, antibodies, hemoglobin, soluble CD-4, clotting factors, tissue plasminogen activator, enkephalins, vasopressin, non-naturally occurring opioids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminease, adenosine deaminase ribonuclease, trypsin, chemotrypsin, and papain, alkaline phosphatase, and other suitable enzymes, hormones, proteins, polypeptides, enzyme-protein conjugates, antibody-hapten conjugates, viral epitopes, etc.; antivirals such as: Ara-A (Arabinofuranosyladenine), Acylguanosine, Nordeoxyguanosine, Azidothymidine, Dideoxyadenosine, and Dideoxycytidine; anti-cancer agents such as Dideoxyinosine Floxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, and I-darubicin; and antibiotic such as Erythormycin, Vancomycin, oleandomycin, and Ampicillin; antiarrhythmics such as Quinidine; and anticoagulants such as Heparins.
 In the practice of the present invention when the therapeutic agent is employed in a conjugated form, suitable polymers for conjugation with the therapeutic agents are employed so as to obtain the desirable characteristics enumerated above. Such modified therapeutic agents may be employed for sustained in vivo delivery of the therapeutic agent.
 The present invention provides microemulsion formulations for delivery of therapeutic agents orally in their active form. The invention may also be practiced with amphiphilic prodrugs that are therapeutically effective by oral or parenteral administration.
 Within the broad scope of the present invention, microemulsion formulations are provided including agents useful for therapeutic applications, as well as immunoassay, diagnostic, and other non-therapeutic (e.g., in vitro) applications. The formulations of the present invention may be employed to provide stabilized peptide and nucleoside compositions variously suitable for in vivo as well as non-in viva applications, wherein the peptide and nucleoside agents may be conjugated and/or unconjugated in character.
 Within the broad scope of the present invention, when the therapeutic agent employed in the formulation is conjugated, a single polymer molecule may 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 utilize a variety of polymers as conjugating agents for a given therapeutic agent; combinations of such approaches may also be employed. Further, it will be recognized that the conjugating polymer(s) may utilize any other groups, moieties, or other conjugated species, as appropriate to the end use application. By way of example, it may be useful in some applications to covalently bond to the polymer a functional moiety imparting UV-degradation resistance, or antioxidant character, or other properties or characteristics to the polymer. As a further example, it may be advantageous in some applications to functionalize the polymer to render same reactive or cross-linkable in character, to enhance various properties or characteristics of the overall conjugated material. Accordingly, the polymer may contain any functionality, repeating groups, linkages, or other constituent structures which do not preclude the efficacy of the conjugated composition for its intended purpose.
 Illustrative polymers that may usefully be employed achieve these desirable characteristics are described herein below in an exemplary reaction scheme. In covalently bonded peptide applications, the polymers may be functionalized and then coupled to free amino acid(s) of the peptide(s) to form labile bonds which permit retention of activity with the labile bonds intact. Removal of the bond by chemical hydrolysis and proteolysis then enhances the peptidal activity.
 The polymers utilized in the invention, when the therapeutic agent employed in the formulation is conjugated, may suitably incorporate in their molecules constituents such as edible fatty acids (lipophilic end), polyethylene glycols (water soluble end), acceptable sugar moieties (receptor interacting end), and spacers for therapeutic agent attachment. Among the polymers of choice, polysorbates are particularly preferred and are chosen to illustrate various embodiments of the invention in the ensuing discussion herein. The scope of this invention is of course not limited to polysorbates, and various other polymers incorporating above-described moieties may usefully be employed in the broad practice of this invention. Sometimes it may be desirable to eliminate one of such moieties and to retain others in the polymer structure, without loss of objectives. When it is desirable to do so, the preferred moieties to eliminate without losing the objectives and benefits of the invention are the sugar and/or the spacer moieties.
 It is preferred to operate with conjugating polymers whose molecular weights are in the range of between 120 and 10,000 daltons.
 In the practice of the present invention, polyalkylene glycol residues of C2-C4 alkyl polyalkylene glycols, preferably polyethylene glycol (PEG), are advantageously incorporated in the polymer systems of interest.
 The presence of these PEG residues will impart hydrophilic properties to the polymer and to the corresponding polymer-therapeutic agent conjugates. Certain glycolipids are known to stabilize therapeutic agents such as proteins and peptides. The mechanism of this stabilization probably involves association of the glycolipid fatty acid moieties with the hydrophobic domain of the peptide or protein; aggregation of the protein or peptide is thus prevented. It also is known that aggregated peptides are poorly absorbed in the small intestine compared to native peptides. The invention therefore contemplates polymer-peptide conjugation products in which the peptide, e.g., insulin, is conjugated with either the hydrophilic or hydrophobic residue of the polymer. The fatty acid portion of the polymer is provided to associate with the hydrophobic domain of the peptide and thus prevent aggregation in solution. The resulting polymer-peptide conjugates thus will be: stabilized (to chemical and enzymatic hydrolysis); water-soluble, due to the PEG residue; and, by virtue of the fatty acid-hydrophobic domain interactions, not prone to aggregation.
 Polyalkylene glycol derivatization has a number of advantageous properties in the formulation of polymer-therapeutic agent conjugates in the practice of the present invention, as associated with the following properties of polyalkylene glycol derivatives: improvement of aqueous solubility, while at the same time eliciting no antigenic or immunogenic response; high degrees of biocompatibility; 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 as conjugating components of the inventive formulation thus comprise lipophilic and hydrophilic moieties, rendering the resulting polymer-drug conjugate highly effective (bioactive) in oral as well as parenteral and other modes of physiological administration. As used hereinafter, the terms “drug” and “therapeutic agent” are used interchangeably.
 Set out below as illustrative examples of polymer-therapeutic agent conjugates of the present invention are the formulae of covalently bonded conjugates denoted for ease of subsequent reference as Conjugate 1, Conjugate 2, and Conjugate 3, wherein “drug” is insulin or other therapeutic agent, and specific values of m, n, w, x, and y will be described in the ensuing discussion.
 w+x+y+z=20; and
 R=oleic acid:
 or other fatty acid radicals from C4-C20.
 wherein: m and n each are independently from 1 to 125.
 wherein: m and n each are independently from 1 to 125.
 Conjugate 1 features commercially available polysorbate monooleate at the center of the polymeric system, a sugar derivative used in many pharmaceutical applications. Lipophilic and absorption enhancing properties are imparted by the fatty acid chain, while the polyethylene glycol (PEG) residues provide a hydrophilic (hydrogen bond accepting) environment. Drug is attached through a carbamate linkage adjacent to the PEG region of the polymer.
 In Conjugate 2 the sugar residue is excluded, but drug is once again attached to the polymer through a carbamate bond adjacent to the hydrophilic PEG region of the polymer. The lipophilic fatty acid region of the polymer is thus some distance from the point of attachment to drug, e.g. insulin.
 The arrangement described above for Conjugate 2 is reversed in the case of Conjugate 3. Once more the sugar residue is excluded, but in this structure the lipophilic fatty acid residue is closest to the point of attachment to drug and the hydrophilic PEG region is distant from the point of attachment, which again is through a carbamate bond.
 Varied alignments of hydrophilic and lipophilic regions relative 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 which provide lipophilic and hydrophilic domains to the drug. In Conjugates 1, 2, and 3 the point of attachment of the carbamate bond between the polymers is preferably the amine function.
 In the general practice of the invention, various methods of coupling the polymers to the therapeutic agent, e.g., peptide, nucleoside, etc. are available and are discussed more fully hereinafter.
 The polymers utilized in therapeutic agent conjugation in accordance with the invention are designed to incorporate good physical characteristics that enable them to achieve the desired objectives. Absorption enhancers, while enabling penetration of the drug through the cell membrane, do not improve the stability characteristics of the drug, and in vivo applications may therefore utilize the polymer-drug conjugates of the invention in formulations devoid of such penetration enhancers. One aspect of the present invention therefore relates to the incorporation of fatty moiety within the polymer, to mimic penetration enhancers. The microemulsion formulations of the invention are preferably devoid of any protease inhibitors. Protease inhibitors have been used in the prior art to enhance the stability of proteinaceous therapeutic agents, as hereinearlier discussed, but are desirably absent in the microemulsion formulations of the present invention.
 In the covalently conjugated polymer-therapeutic agent conjugates of the present invention, the drug may be covalently attached to the water-soluble polymer by means of a labile chemical bond. This covalent bond between the drug and the polymer may be cleaved by chemical or enzymatic reaction. The polymer-drug product retains an acceptable amount of activity; full activity of the component drug is realized when the polymer is completely cleaved from the drug. Concurrently, portions of polyethylene glycol are present in the conjugating polymer to endow the polymer-drug conjugate with high aqueous solubility and prolonged blood circulation capability. The modifications described above confer improved solubility, stability, and membrane affinity properties on the drug. As a result of these improved characteristics the invention contemplates parenteral and oral delivery of both the active polymer-drug species and, following hydrolytic cleavage, bioavailability of the drug per se, in in vivo applications.
 The polymers used in the embodiment described below can be classified as polyethylene glycol modified lipids and polyethylene glycol modified fatty acids. Among preferred conjugating polymers may be mentioned polysorbates comprising monopalmitate, dipalmitate, tripalmitate, monolaurate, dilaurate, trialaurate, monooleate, dioleate, trioleate, monostearate, distearate, and tristearate. Other lower fatty acids can be utilized. The number average molecular weight of polymer resulting from each combination is preferred to be in the range of from about 750 to about 5,000 daltons. Alternative polymers of preference are polyethylene glycol ethers or esters of fatty acids, such fatty acids being lauric, palmitic, oleic, and stearic acids, and other lower fatty acids can be utilized, with the polymers ranging from 250 to 5,000 daltons in number average molecular weight. It is preferred to have a derivatizable group in the polymer, where such group can be at the end terminating with polyethylene glycol or at the end terminating with fatty moiety. The derivatizable group may also be situated within the polymer and thus may serve as a spacer between the peptide (or other therapeutic agent) and the polymer.
 Several methods of modifying fatty acid sorbitan to achieve the desired polymer will be discussed in further detail with structural illustrations. Polysorbates are esters of sorbitols and their anhydrides, which are copolymerized with ethylene oxide. Shown below is the structure of a representative polymer.
 The sum of w, x, y, z is 20 and R1, R2 and R3 are each independently selected from the group consisting of lauric, oleic, palmitic and stearic acid radicals, or R1 and R2 are each hydroxyl while R3 is lauric, palmitic, oleic or stearic acid radical, or lower fatty acid. These polymers are commercially available and are used in pharmaceutical formulations. Where a higher molecular weight polymer is desired, it may be synthesized from glycolipids such as sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate or sorbitan monostearate, and an appropriate polyethylene glycol. Structures of glycolipids which may be used as starting reagents are depicted below.
 In the synthesis of glycolipid polymers substituted in three positions with polyethylene glycol, a desired polyethylene glycol having two free hydroxyls at the termini is protected at one terminus with a trityl group in pyridine using one mole of trityl chloride. The remaining free hydroxyl group of the polyethylene glycol is converted to either tosylate or bromide. The desired glycolipid is dissolved in a suitable inert 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 solution of glycolipid. The product is treated with a solution of para-toluenesulfonic acid in anhydrous inert solvent at room temperature and purified by column chromatography. The structures of the transformation are depicted below.
 By adjusting the molar equivalent of reagents and using the appropriate molecular weight range of polyethylene glycol, mono or disubstituted glycolipids of the desired molecular weight range can be obtained by following the above procedures.
 wherein each n and m may vary independently, and have any suitable value appropriate to the specific drug being stabilized, e.g., from 1 to 16.
 The sugar portion of the glycolipid described above can be substituted with glycerol or aminoglycerol whose structural formulae are shown below.
 In this modification, the primary alcohol is first etherified or esterified with a fatty acid moiety such as lauric, oleic, palmitic or stearic; the amino group is derivatized with fatty acids to form amides or secondary amino groups, as shown below.
 wherein m may have any suitable value, e.g., from 10 to 16.
 The remaining primary alcohol group is protected with a trityl group while the secondary alcohol group is converted with polyethylene glycol to a desired polymer. Usually, the polyethylene glycol bears a leaving group at one terminal and a methoxy group at the other terminal. The polyethylene glycol is dissolved in inert solvent and added to a solution containing glycolipid and sodium hydride. The product is deprotected in para-toluenesulfonic acid at room temperature to give the desired polymer as depicted.
 Sometimes it is desirable to incorporate fatty acid derivatives in different parts of the polyethylene glycol chain to achieve certain physicochemical properties similar to polysorbates that have been substituted with two/three molecules of fatty acids, e.g., polysorbate trioleate.
 Structures representing the polymers are shown in the reaction scheme below as the open chain of the polysorbate.
 and wherein m, n, and y may be independently varied within the above ranges, relative to one another.
 In the synthesis of polymer A, it is desirable to protect the hydroxyl moieties on the first and second carbon of glycerol, e.g. solketal. The remaining hydroxyl group is converted to the sodium salt in an inert solvent and reacted with halogenated or tosylated polyethylene glycol 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 derivatized with fatty acids. Reaction takes place after the free hydroxyl is converted to the tosylate or bromide.
 Polymer G is synthesized in the same manner except that the protected glycerol is first reacted with esters of fatty acids which have been halogenated at the terminal carbon of the acid.
 In the synthesis of polymer C, it is preferable to start with 1,3-dihalo-2-propanol. The dihalo compound is dissolved in inert solvent and treated with the sodium salt of two moles of polyethylene glycol which has been previously derivatized with one mole of a fatty acid moiety. The product is purified by chromatography or dialysis. The resulting dry product is treated, in inert solvent, with sodium hydride. The sodium salt thus formed is reacted with a halo derivative of partially protected polyethylene glycol.
 Sometimes it may be desired to omit the sugar portion of the polymer. The resulting polymer still contains a polyethylene glycol fragment. The membrane affinity properties of the fatty acid moiety may be retained by substituting a fatty acid proper with a lipophilic long chain alkane; biocompatibility is thus preserved. In one instance of this embodiment the polyethylene glycol with two terminal free hydroxyl groups is treated with sodium hydride in inert solvent. One equivalent weight of a primary bromide derivative of a fatty acid-like moiety is added to the polyethylene glycol solvent mixture. The desired product is extracted in inert solvent and purified by column chromatography if necessary.
 Where it is desired to form an ester linkage 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 inert solvent and further purified by chromatography if necessary.
 In some modifications of peptides, it is desired to conjugate the fatty acid moiety directly to the therapeutic agent. In this case the polymer is synthesized with the derivatizable function placed on the fatty acid moiety. A solution of mono-methoxypolyethylene glycol of appropriate molecular weight in inert solvent is treated with sodium hydride followed by the addition of solution containing the ethyl ester of a fatty acid bearing a leaving group at the terminal carbon of the acid. The product is purified after solvent extraction and if necessary, by column chromatography.
 The ester protection is removed by treating with dilute acid or base.
 Where it is desired to form a carbamate bond with the drug, the carboxyl or ester is converted to a hydroxyl group by a chemical reduction method known in the art
HO—(CH2)m—(OC2H4)nXR (Formula 13)
 The functional groups that are used in the drug conjugation are usually at a terminal end of the polymer, but in some cases, it is preferred that the functional group is positioned within the polymer. In this situation, the derivatizing groups serve as spacers. In one instance of this embodiment, a fatty acid moiety may be brominated at the carbon alpha to the carboxylic group and the acid moiety is esterified. The experimental procedure for such type of compound is similar to the one outlined above, resulting in the product shown below.
 When an extended spacer is desired, a polyethylene glycol monoether may be converted to an amino group and treated with succinic anhydride that has been derivatized with a fatty acid moiety. A desired polyethylene glycol bearing primary amine is dissolved in sodium phosphate buffer at pH 8.8 and treated with a substituted succinic anhydride fatty acid moiety as shown in the scheme below. The product is isolated by solvent extraction and purified by column chromatography if necessary.
 It is to be understood that the above reaction schemes are provided for the purposes of illustration only and are not to be limitingly construed in respect of the reactions and structures which may be beneficially utilized 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 readily carried out For the purpose of brevity in discussion herein, the polymer is referred to 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 then is reacted with the amino residue of the drug in a short period of time under mild conditions producing carbamate derivatives.
 The above reaction and reagent only serve as illustration and are not exclusive; other activating reagents resulting in formation of urethane, or other, linkages can be employed. The hydroxyl group can be converted to an amino group using reagents known in the art. Subsequent coupling with drug through their 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 bond. In another procedure, the carboxyl group can be treated with water-soluble carbodimide and reacted with the drug to produce conjugates containing amide bonds.
 The activity and stability of the drug conjugates can be varied in several ways, by using a polymer of different molecular size. Solubilities of the conjugates can be varied by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition. Hydrophilic and hydrophobic characteristics can be balanced by careful combination of fatty acid and polyethylene glycol moieties.
 Set out below are some illustrative modification reactions for polymer-drug conjugates of the present invention.
 In the above reaction scheme involving species I, J and K, routes are demonstrated for modifying the hydrophilicity/lipophilicity balance of the conjugating polymer. Ester groups in the conjugating polymer are susceptible to hydrolysis by esterases; the conjugating polymer containing ester groups therefore may be modified to convert the ester groups to ether groups which are more hydrolysis-resistant in character. The reaction scheme involving L and M species illustrates the conversion of hydroxyl groups to carboxylate groups. In this respect, the carboxyl groups will provide carboxylate anion for conjugating amino residues of nucleosides or other drugs.
 In general, various techniques may be advantageously employed to improve the stability characteristics of the polymer conjugates of the present invention, including: the functionalization of the polymer with groups of superior hydrolysis resistance, e.g., the previously illustrated conversion of ester groups to ether groups; modifying the lipophilic/hydrophilic balance of the conjugating polymer, as appropriate to the drug being stabilized by the polymer; and tailoring the molecular weight of the polymer to the appropriate level for the molecular weight of the drug being stabilized by the polymer.
 The unique property of polyalkylene glycol-derived polymers of value for therapeutic applications of the present invention is general biocompatibility. The polymers have various water solubility properties and are not 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 in sustaining the biological activity of therapeutic nucleosides, peptides, and other therapeutic agents, and may be prepared for therapeutic administration by microemulsion formulation as hereinafter more fully described. Administration preferably is by either the parenteral or oral route.
 In the dry, lyophilized state, the drug-polymer conjugates of the present invention have good storage stability; and solution formulations of the conjugates of the present invention are likewise characterized by good storage stability.
 The microemulsion formulations of the present invention may be employed for the prophylaxis or treatment of any condition or disease state for which the drug constituent thereof is efficacious.
 In addition, the microemulsion formulations of the invention may be employed for the diagnosis of constituents, conditions, or disease states in biological systems or specimens, as well as for diagnosis purposes in non-physiological systems.
 Further, the microemulsion formulations of the invention may have application in prophylaxis or treatment of condition(s) or disease state(s) in plant systems. By way of example, the active component of the formulation may have insecticidal, herbicidal, fungicidal, and/or pesticidal efficacy amenable to usage in various plant systems.
 In therapeutic usage, the present invention contemplates a method of treating an animal subject having or latently susceptible to such condition(s) or disease state(s) and in need of such treatment, comprising administering to such animal an effective amount of a microemulsion formulation of the present invention which is therapeutically effective for said condition or disease state.
 Subjects to be treated by the polymer conjugates of the present invention include both human and non-human animal (e.g., bird, dog, cat, cow, horse) subjects, and preferably are mammalian subjects, and most preferably human subjects.
 Depending on the specific condition or disease state to be combated, animal subjects may be administered the microemulsion formulation of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, and without undue experimentation.
 The microemulsion formulations of the invention may comprise the drug component per se as well as, or alternatively, such drug component in the form of pharmaceutically acceptable esters, salts, or other physiologically functional derivatives thereof.
 The present invention also contemplates pharmaceutical formulations, both for veterinary and for human medical use, which comprise as the active agent one or more therapeutic agent(s).
 In such microemulsified pharmaceutical and medicament formulations, the active agent preferably may be utilized together with one or more pharmaceutically acceptable carrier(s) therefor 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 not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.
 The 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, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration. Formulations suitable for oral and parenteral administration are preferred.
 Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or other containment structures, each containing a predetermined amount of the formulation; or in the emulsified form per se, for ingestion in a predetermined amount.
 A syrup may be made by adding a sugar, for example sucrose, to the microemulsion, together with any accessory ingredient(s). Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.
 Formulations suitable for parenteral administration conveniently comprise the microemulsion in a form which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution).
 The formulations may be presented in unit-dose or multi-dose form.
 Nasal spray formulations comprise the purified emulsion with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucus membranes.
 Formulations for rectal administration may be presented as an enema formulation.
 Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.
 Topical formulations may comprise the emulsion in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.
 In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.
 In formulations of the present invention, the conjugating polymers described herein may be employed for in vitro stabilization of drugs in therapeutic as well as non-therapeutic applications. The polymers may be employed for example to increase the thermal stability and enzymic degradation resistance of the drug. Enhancement of the thermal stability characteristic of the drug via conjugation in the manner of the present invention provides a means of improving shelf life, room temperature stability, and robustness of research reagents and kits.
 The present invention provides pharmaceutically acceptable compositions in the form of a microemulsion whose HLB value is between 3 and 7. The formulation may be prepared with surfactant components whose combined HLB strength in the dosage form is less than 7. The formulation may for example contain a mixture of surfactants in the lipid phase, and an aqueous phase containing a high HLB surfactant. The presence of additional triglycerides, other than the low HLB surfactant, is optional, but not essential.
 The lipid phase of the microemulsion of the invention can contain oil-soluble drugs, short chain, medium chain and long chain fatty acid triglycerides, monoglycerides of fatty acids, diglycerides of fatty acids, and propylene glycol esters of fatty acids, such as propylene glycol alginate.
 The low HLB surfactant of the lipid phase can be any suitable surfactant. Examples include Sorbitan oleate (Arlacel 80), monoolein/propylene glycol (Arlacel 186), C8/C10 fatty acid mono- and diglycerides from coconut oil, soy lecithin, egg phosphatides, citric acid esters of monoglycerides, lactic acid esters of monoglycerides, diacetyl tartaric acid esters of monoglycerides, succinic acid esters of monoglycerides, sucrose fatty acid esters, polyglycolyzed glycerides of oleic acids (e.g., Labrafil M1944), polyglycolyzed glycerides of lineleic acid (e.g., Labrafil M 2125), polyglycerol esters of fatty acids, including both long chain and medium chain fatty acids, e.g., polyglyceryl decaoleate (such as Caprol PGE 860), and polyglyceryl esters of mixed fatty acids such as Caprol ET (polyglyceryl-6 mixed fatty acids).
 The hydrophilic phase may contain in addition to water and water-soluble drugs, one or more polyhydroxy compounds such as carbohydrates, polyethylene glycols, and propylene glycol, in any ratio from 0-100% w/w of the aqueous phase. The hydrophilic phase may further contain polyoxyethylene(2) sorbitan oleate (Tween 80), polyoxyethylene(20 glycerol trioleate, C8/C10 polyglycolyzed glycerides from coconut oil (e.g., Labrafac CM 10, and Labrasol), Tween 60, Cremophor EL (ethoxylated castor oil), Cremophor RH 20 (ethoxylated castor oil), and sucrose fatty esters such as sucrose stearate.
 The broad range for the amount of high HLB surfactant that can be present in the composition can be 0.2%-50% w/w. The preferred range for such high HLB surfactant is 0.2-10% w/w. The broad range for the lipid phase containing surfactants is 20-90% w/w. A preferred concentration is around 70% w/w and the water content of the aqueous phase can be 2-30% w/w with the preferred range being 10-20% w/w. The preferred range of polyhydroxy solvents of the hydrophilic phase is 0-20% w/w.
 Optional ingradients for the preparation of these emulsions include stabilizers such hydroxy propyl methyl cellulose, hydroxy propyl cellulose, carboxymethyl cellulose, and carboxy cellulose, and organic acids such as polyacrylic acids and their partial esters can be beneficially used in combination.
 Other ingredients such as antioxidants and antimicrobial agents can be used in the formulation. The presence of unsaturated fatty acids in the lipid may prone them susceptible to oxidation. Oil soluble butylated hydroxy anisole, Vitamin-E, and propyl gallate can be used in the formulation in a concentration of 0.1 to 1.2% w/w. Antimicrobial preservatives, such as parabens and benzalkonium chloride, may also be used to preserve the microemulsion, e.g., in a concentration of 0.1-0.5% w/w.
 The microemulsion of the present invention can be prepared by simply mixing by hand or by mild vortexing of the formulation ingradients. The order of addition is not crucial, but in cases where the components are viscous the dilution of viscous ingradients with another oil or surfactant may be helpful. Preparation of protein stock solutions containing high concentrations of proteins are convenient to prepare the formulations as compared to dissolving protein powders directly in the emulsion formulation volume. In addition, highly accurate determinations of protein loading in the emulsion can be made by HPLC trace of the stock solution.
 As an illustrative methodology for the preparation of the microemulsion formulations of the invention, an aqueous solution containing lipid and lipid-soluble low HLB surfactant is prepared. The high HLB surfactant is mixed with the lipid phase. Slow addition of appropriate quantities of the aqueous solution to the lipid/surfactant mixture results in a clear transparent solution. Occasional gentle shaking may be desirable during the addition. The resulting formulation then can be processed for packaging and end use. For example, formulations containing less than 20% w/w of the hydrophilic phase can be made, and the formulation introduced to soft gel capsules, such as enteric-coated soft gel capsules, as unit dosage forms. Alternatively, the formulation can be given as a liquid in the prescribed or desired dose.
 The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.
 Conjugate 1
 Polysorbate Trioleate p-nitrophenyl Carbonate
 To a solution of p-nitrophenylchloroformate (0.8 g, 4 mmole) in 50 mL of anhydrous acetonitrile is added dry polysorbate trioleate (7 g, 4 mmole) followed by dimethylaminopyridine (0.5 g, 4 mmole). The reaction mixture is stirred at room temperature for 24 hours Solvent is removed under reduced pressure and the resultant precipitate is diluted with dry benzene and filtered through Celite. The residue is refrigerated overnight in dry benzene and the additional precipitate is removed by filtration. Solvent is removed under reduced pressure and residual benzene is removed by evacuation at low pressure to yield 6.4 g of polysorbate trioleate p-nitrophenyl carbonate.
 Coupling of Insulin with Activated Polymer
 To a solution of activated polysorbate trioleate (1 g) in aqueous mixture of dimethylsulfoxide (DMSO) or dimethylformamide (DMF) is added a solution of bovine insulin (50 mg) in 0.1 M pH 8.8 phosphate buffer. pH is maintained by addition of 1N NaOH as necessary. The reaction mixture is stirred at room temperature for 2.5 h. After this time the mixture is subjected to gel filtration chromatography using Sephadex G-75. Purification by elution with 0.1M pH 7.0 phosphate buffer and collection of fractions with an automated fraction collector yields Conjugate 1. The polymer content is determined by trinitrobenzenesulfonic acid (TNBS) assay and mass spectrometry, and the protein concentration by Biuret Method. A molar ratio of polymer to insulin is determined to be 1:1. Conjugate 1 is also obtained by using pure organic solvent (e.g., DMSO, DMF).
 Conjugate 2
 The terminal hydroxyl group of polyethylene glycol monostearate is activated by reaction with p-nitrophenyl chloroformate as described above. To a solution of the activated polymer (1 g) in distilled water is added bovine insulin (80 mg) dissolved in 0.1M phosphate buffer, at pH 8.8. The pH is maintained by careful adjustment with 1N NaOH. After stirring for 3 hours, the reaction mixture is quenched with excess glycine and subjected to gel filtration chromatography using Sephadex G-75. Insulin/polymer conjugate is collected and lyophilized. Protein content is determined by Biuret assay, giving a quantitative yield.
 Conjugate 3
 Tetrahydro-2-(12-bromododecanoxy)-2H Pyran
 To a solution of 12-bromo-1-dodecanol (I mole) in dichloromethane containing pyridinium p-toluenesulfonate (P-TSA) is added dihydropyran (2 moles). The reaction mixture is stirred for 24 hours and then washed twice with water and dried over anhydrous MgSO4. The dichloromethane is removed under reduced pressure. If necessary the resulting product is purified by chromatography on silica gel.
 Coupling of Polyethylene Glycol to the Terahydropyran Derivative
 The tetrahydropyran derivative described above, dissolved in dry benzene, is added to a solution of polyethylene glycol (1 mole) in dry benzene containing NaH (1 mole). The reaction mixture is stirred at room temperature for 24 hours. After that time the mixture is eluted through a silica gel column with benzene. Additional purification by column chromatography, if necessary, is performed. 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-nitrophenylchloroformate as described hereinabove. Conjugation with insulin is carried out as described for Conjugate 1.
 Comparative studies using bovine insulin were conducted on polymer-insulin conjugates and on native insulin to determine their relative stability and activity in animal models. In the animal studies, the efficacy of the polymer-insulin in lowering the blood level was compared to that of native insulin. Female and male albino mice averaging 25 g in weight were fasted overnight and used in groups of five for each treatment conducted in several phases over a period of two days.
 Each test animal received a single dose of either native insulin (Group 1, 100 μg/kg, subcutaneously); native insulin (Group 2, 1.5 mg/kg, orally by gavage); 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) received no insulin of any kind but was challenged with glucose 30 minutes before scheduled sampling times. Animals were fasted overnight before treatment and for the duration of the study. All test materials were prepared in phosphate buffered saline, pH 7.4. Thirty minutes before scheduled sampling times of 0.5, 1, 2, 4, 8 and 24 hours following treatment with insulin, animals were challenged with a bolus dose of glucose (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 immediately analyzed for glucose content using a One Touch Digital Glucose Meter (Life Scan). The results of the test are shown in FIG. 1, for Groups 1-5.
 Blood glucose levels for Group 1 animals (native insulin, subcutaneous) were approximately 30% of control (Group 5, untreated) animals at the 30 minute time point. This hypoglycemic effect lasted only 3.5 hours in Group 1 animals. Native insulin administered orally (Group 2) lowered blood glucose levels to a maximum of 60% of control, this maximum response occurring 30 minutes after treatment with the insulin. In contrast the glucose levels in animals in Group 3 (Conjugate 1, 100 μg/kg, p.o.) were lowered with an apparent delayed onset of hypoglycemic activity. The hypoglycemic activity in Group 3 animals was greater than that in Group 2 animals even though 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 other treatment group, the largest difference being at the four to eight hour sampling points. Glucose levels in Group 4 animals (Conjugate 1, 100 μg/kg, s.c.) followed the same course as those for Group 1 animals for the first four hours of the study. After four hours Group 2 glucose levels remained above control (untreated, Group 5) levels whereas Group 4 glucose levels dropped, at eight hours, to 62% of Group 5 levels, and remained below Group 5 levels.
 An insulin efficacy study was conducted on male and female albino mice using as test materials insulin in unconjugated form, and Conjugate 1. One objective of this study was to determine whether 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 whether the insulin complex of Conjugate 1, unlike free insulin, is capable of acting to decrease the blood glucose level when administered orally. The results are shown in FIG. 2, wherein “Insulin Complex” denotes Conjugate 1.
 Baseline blood samples were obtained for serum glucose analysis from 10 fasted untreated albino mice (5 males and 5 females); baseline values in FIG. 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 gavage (5 g/kg body weight). Ten animals were sacrificed at each of three time periods to obtain blood samples for glucose analysis: 30, 60 and 120 minutes after dosing. A commercial insulin and Conjugate 1 were each administered both orally (p.o) and parenterally (s.c.) to groups of fasted mice (five males and five females, for sacrifice and blood analysis at each of the three time periods), to provide different treatment regimens. The treatment, administration routes, and symbols shown for results in FIG. 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: “-” (iii) insulin (1.5 mg/kg, p.o.) and glucose (5 g/kg p.o.), symbol: “-”; (iv) Conjugate 1 (100 μg/kg, s.c.) and glucose (5 g/kg p.o.), symbol “ ”; (v) Conjugate 1 (250 μg/kg, s.c.) and glucose (5 g/kg p.o.), symbol: “▪”; (vi) Conjugate 1 (1.5 mg/kg, s.c.) and glucose (5 g/kg p.o.), symbol: “Δ”. In these tests of Conjugate 1, the concentration of the protein in the administered solution was 0.1 mg protein/mL solution; for comparison purposes, a modified covalently bonded insulin-polymer conjugate, having a protein concentration in the administered solution of 0.78 mg protein/mL solution, was included, (vii) modified Conjugate 1 (100 g/kg, s.c.) and glucose (5 g/kg p.o.), symbol: “Δ”.
 The insulin was administered 15 minutes prior to glucose loading.
 Glucose was administered orally by gavage to all but the baseline group of animals at a dose of 5 g/kg (10 mg/kg of a 50% w/v solution in normal saline). When insulin was administered orally by gavage, it was given at a dose of 1.5 mg/kg (18.85 mL/kg of a 0.008% w/v solution in normal saline). When insulin was administered subcutaneously, it was given at a dose of 1001 g/kg (2.5 mL/kg of a 0.004% w/v solution in normal saline). When the Conjugate 1 polymer-insulin complex was administered orally by gavage, it was given at a dose of 1.56 mg/kg (2.0 mL/kg of the undiluted test material). When the Conjugate 1 polymer-insulin complex was administered subcutaneously, it was given at a dose of 100 μg/kg (1.28 mL/kg of a 1:10 dilution of the 0.78 mg/mL solution received) or 250 g/kg (3.20 mL/kg of a 1:10 dilution of the solution received). The modified Conjugate 1 contained 0.1 mL insulin/mL and was dosed at a rate of 1.0 mL/kg to obtain a 100 μg/kg dose.
 Glucose was measured using the Gemini Centrifugal Analyzer and purchased glucose reagent kits. The assay was a coupled enzymatic assay based on the reaction of glucose and ATP catalyzed by hexokinase, coupled with the glucose-6-phosphate dehydrogenase reaction, yielding NADH. Duplicate samples were analyzed and the mean value reported. 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, mean serum glucose rose to a high level at 30 minutes, declined at 60 minutes, and was below baseline at 120 minutes. If commercial insulin was administered subcutaneously (100 μg/kg body weight, it was highly effective in preventing the increase in blood glucose. However, if insulin was given orally (at a high dose of 1.5 mg/kg) there was no effect on the rise of blood glucose. This was expected, since insulin, a protein, is readily hydrolyzed in the digestive tract and is not absorbed intact into the bloodstream.
 When Conjugate 1 was given subcutaneously at either 100 or 250 μg/kg dosage, it was highly effective in restricting the rise in blood glucose after glucose loading. Mean serum glucose values were significantly lower after the 100 g/kg dose of Conjugate 1 at both 30 and 60 minutes than they were after 100 μg/kg of free insulin. Mean serum glucose at 250 μg/kg of Conjugate 1 was lower, though not significantly, at 30 minutes, significantly lower at 60 minutes and at 120 minutes was returned to the baseline. With both free insulin at 100 μg/kg and Conjugate 1 at 100 μg/kg, the glucose level remained below baseline at 120 minutes.
 The modified Conjugate 1 administered at 100 μg/kg produced a significant reduction in blood glucose at 30 minutes.
 Preparation of Para-Nitrophenyl Carbonate of Polysorbate Monopalmitate
 Polysorbate monopalmitate is first dried by the azeotropic method using dry benzene. To a solution of the dry polymer (2 g, 2 mmole) in 10 mL of dry pyridine is added para-nitrophenylchloroformate (0.6 g, 3 mmol). The mixture is stirred at room temperature for 24 hours. The reaction mixture is chilled in ice and diluted with dry benzene and filtered through filter aid. This procedure is repeated and finally the solvent is removed at the rotary evaporator. Traces of solvent are removed in vacuo. The yield of the product is 1.8 g.
 Preparation of Polysorbate Monopalmitate Conjugate with Insulin
 In accordance with the previously described conjugation reaction procedure of Example I but using polysorbate monopalmitate in the amount of 1 g and insulin in the amount of 80 mg, with HPLC separation of the reaction product, an insulin-polysorbate monopalmitate covalently bonded conjugate is obtained.
 Preparation of Enzyme-polymer Conjugates
 Coupling of alkaline phosphatase (AP) to 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 would be more advantageous, conjugates were prepared using 140 moles of polymer/mole of enzyme and 14 moles of polymer/mole of enzyme. The number of polymer groups per molecule of conjugated AP are 30 and 5, respectively, for the high and low ratios of polymer.
 The following procedure was employed to obtain about 5 groups/molecule of alkaline phosphatase: 4.1 mg (salt free) was dissolved in 0.05M sodium bicarbonate. 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 (0.3N NaCl) in dialysis tubing (MW cutoff 12,000-14,000) over 12 hours with 4 to 6 changes of dialysis solution. The same procedure was used for the high ratio. Total protein concentration of dialyzed material was determined by Biuret method.
 Activity Measurement and Stability Study
 The phophatase assay was performed according to the method of A. Voller et al, Bulletin WHO, 53, 55 (1976). An aliquot (50 microliter) was added to microwells and mixed with 200 microliter of substrate solution (10 g/L, 4-nitrophenylphosphate in 20% ethanolamine buffer, pH 9.3) and incubated at room temperature for 45 minutes. The reaction was stopped by 50 microliter of 3M NaOH. The absorbence was measured at 405 nm in a micro plate reader.
 Phosphatase activity was compared with that of native enzyme under various conditions.
 Dilute 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 to the control alkaline phosphatase stored at 5° C.
 As can be seen from Table A, the initial enzymatic activity of both polymers was about three-fold higher than the control. Both polymer-enzyme conjugates had enhanced thermal stability over the native enzyme. This is especially evident for the conjugate characterized by the higher ratio of polymer to enzyme.
 Conjugate 1A
 To a solution of insulin (50 mg) in 0.05 M sodium bicarbonate buffer of pH 9.2 is added a solution of activated polymer (1 g) in water/dimethylsulfoxide and stirred for 3 hours, at room temperature. The pH of the mixture is maintained by careful adjustment with 1N NaOH. The reaction mixture then is dialyzed against 0 μM pH 7.0 phosphate buffer. Purified product is lyophilized. Protein content (48 mg) is determined by Biuret assay. The number of polymer chains linked to insulin is determined by TNBS assay, giving a ratio of two moles of polymer to one mole of insulin.
 Synthesis of OT10CAraCMP
 Synthesis of tributylAraCMP:
 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 anhydrous butyric-anhydride (6.2 mmole) was stirred for 21 hours at room temperature. Methanol (1 ml) was added to the reaction mixture to destroy 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 cleave the butyric phosphate bond. After rotoevaporating the solvent, the product, tributylAraCMP was extracted into 10 ml of chloroform and the organic layer was washed with 3×15 ml of water. The chloroform layer was dried over MgSO4 and evaporated to dryness. This product was used in the next step without further purification.
 Conjugation of OT10C (polyoxyethylene cetylether) to tributylAraCMP.
 TPSCI (1.3 mmole) was dissolved in 6 ml of anhydrous chloroform and added to tributylAraCMP and stirred for 40 minutes at room temperature. The resulting TPS activated tributylAraCMP was added to 876 mg of OT10 (1.2 mmole) in 2.1 mL of pyridine and stirred at room temperature for 4½ hours. TLC 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 water and extracted into 2×6 ml of chloroform.
 The butyl groups of the OT10 conjugated AraCMP were deprotected by stirring the chloroform layer with 4.5 ml of 2.0M ammonia solution in ethanol overnight at room temperature.
 Purification of OT10Conjugated AraCMP
 Solvent was rotoevaporated from the reaction mixture above and TPS sulfonic acid was precipitated with 15 ml water. The pH of the aqueous layer was reduced to 1-2 and the product was extracted into 6×35 ml of chloroform. HPLC of the chloroform layer on an analytical C8 column in isopropanol-water-0.1% TFA showed a hydrophobic nucleoside product. 31PNMR of the product shows attachment of a polymeric residue to the phosphate moiety and 1H NMR shows the polymeric as well as the nucleoside resonances. The product was purified on C-8 column with isopropanol-water 0.1% TFA gradient.
 A microemulsion formulation according to the invention was prepared with the composition shown below.
 The propylene glycol content of the above emulsion can be lowered by reducing combined HLB strength of the emulsion. At the same time, the drug loading in the formulation can be increased, as shown in the following Example.
 A microemulsion formulation was prepared with the composition identified in the following table:
 The aqueous phase of the above microemulsion can have polyhydroxylic alcohols such as manitol which will reduce the amount of propylene glycol and the high HLB surfactant essential to form a similar water in oil (w/o) microemulsion.
 A microemulsion formulation according to the invention was prepared with the composition shown below.
 Alternatively, the aqueous phase of this type microemulsion can accommodate a mixture of polyethylene glycol 300 and polyethylene glycol 400 in addition to propylene glycol in the aqueous phase. as in the following formulation of Example XIV.
 A microemulsion formulation according to the invention was prepared with the composition shown below.
 Alternatively, an alcohol free w/o microemulsion of similar combined HBL strength can be formed using propylene glycol mono and diesters of medium chain fatty acids (Captex 200) and sodium octanoate (anionic surfactant). The high protein load in the microemulsion formulation is an added advantage, as shown in the following Example XV.
 A microemulsion formulation according to the invention was prepared with the composition shown below.
 The formulation of similar HLB strength microemulsions containing high protein load can be obtained without the use of anionic surfactant or ethoxylated nonionic surfactant. For example decaglycerol mono and dioleate (Caprol 860), a high HLB surfactant (HLB=11) can be used along with capmul MCM and lecithin to form a microemulsion containing high protein load, as in the following Example XVI.
 A microemulsion formulation according to the invention was prepared with the composition shown below.
 By suitable choice of excipients which are solids at room temperature, a solid or semi-solid microemulsion can be prepared. For example Whitepsol HI 5 (solid C8/C10 triglyceride), Imwitor 308 (C10 mono and diglycerides), Egg lecithin (solid at RT) and Gelucire 44/14, a solid dosage form of desired HLB can be obtained.
 The liquid microemulsions of the above examples can be encapsulated in a suitable solid hard fat and delivered in a capsule as a unit dosage form. The capsules can be enteric-coated and targeted for absorption in the intestine. Alternatively, the liquid emulsions can be filled in a soft gel capsule and delivered for absorption in the intestine. These soft gel capsules can also be enteric-coated for absorption in specific areas of the intestine.
 Alternatively, the protein or other therapeutic agent can be dispersed in a mixture of lipid and surfactant in accordance with the present invention and filled in a soft gel capsule as a pre-emulsion concentrate. These concentrates can self-emulsify under biological environment exposure conditions to form the desired final emulsion.
 Synthesis of Hexyl polymer and Hex-Insulin Conjugate
 A. Preparation of Methyl(ethyleneglycol)7-O-ethyl Hexanoate:
 A solution of methyl(ethyleneglycol)7-OH (25 g: 7142 mmol) in dry tetrahydrofuran (THF) (52 mL) was added dropwise to a stirred suspension of NaH (3.057 g: 76.4 mmol, of 60% oil dispersion) in dry THF (71 mL). During the addition, the reaction temperature was kept below 20° C. After addition, the resulting solution was allowed to stir for 3 h. At the end of this period, 1-bromo ethyl hexanoate (15.93 g: 71.4 mmol), dissolved in dry THF (20 mL) was added dropwise, and the mixture was continued to stir for 6-8 hours. The solvent was removed under reduced pressure and the residue was treated with cold water (100 mL), and extracted with ethyl acetate (3×30 mL). The combined organic layer was washed sequentially with water (1×20 mL), brine (1×20 mL), dried over MgSO4 and the solvent was evaporated at reduced pressure to give methyl(ethyleneglycol)7-O-ethyl hexanoate as a pale yellow oil. The oil was chromatographed on silica gel column and eluted firstly with ethyl acetate-hexane (1:1) which removed mineral oil and bromo ethylhexanoate along with other smaller fractions of polymer. Later elution with dicholoromethane-methanol (80:20) yielded pure methyl(ethyleneglycol)7-O-ethyl hexanoate.
 B. Preparation of Methyl(ethyleneglycol)7-O-hexanoic Acid:
 The ester (25 g; 50.813 mmol) was stirred with 1N NaOH (76.21 mL) at room temperature for 3 h. The aqueous layer was cooled to 0° C. by adding ice and acidified with 6N HCl to pH 2-3 and extracted with dichloromethane (3×25 mL). The combined organic layer was washed with water (1×20 mL), brine (1×20 mL), dried over MgSO4, filtered and evaporated to leave an oil. The product was purified by column chromatography on silica gel. Elution with pure ethyl acetate removed the impurity. Further elution with dichloromethane-methanol (80:20) gave pure methyl(ethylenaglycol)7-O-hexanoic acid.
 C. Activation: Preparation of Methyl(ethyleneglycol)7-O-hexanoic Acid-N-hydroxy-succinimide Ester:
 To a stirring solution of methyl(ethyleneglycol)7-O-hexanoic acid (25 g: 53.87 mmol) in dry methylene chloride (134.69 mL) under nitrogen atmosphere, solid N-hydroxysuccinimide (6.20 g; 53.87 mmol) was added. The stirring solution, solid EDC (11.36 g; 59.26 mmol) were added in portion and the solution was stirred at room temperature for 4 h. At the end of this period the solution was treated with water (2×20 mL), 1N HCl (1×20 mL), water (1×20 mL), 1N sodium bicarbonate (2×20 mL), water (1×20 mL), 1N HCl (1×20 mL), water (1×20 mL), and brine. The organic layer was dried over MgSO4, filtered and evaporated under reduced pressure at room temperature to give pure activated ester.
 D. Conjugation of Methyl(ethyleneglycol)7-O-hexanoic Acid NHS to Insulin
 Insulin was dissolved in dimethyl sulfoxide at 25° C. and reacted with 1.2 molar equivalent of activated methyl(ethyleneglycol)7-O-hexanoic acid for 45 minutes at 25° C. The product mixture (see Table B) containing monoconjugate (42±2.5%, aveg. M.W 6200), diconjugate (39±2.5%, Ave. M.W 6334), triconjugate (4±2.5%, Avge. M.W 7074) and unreacted insulin (12.5±2.5%, Avge. M.W 5734) was purified by dialysis on a 3500 MW cut off membrane at 5° C. The solution was lyophilized and protein content, product distribution and purity was determined by reverse phase HPLC. The molecular weights of products were determined by MALDI(TOF)/MS. The product mixture was analyzed for moisture, acetic acid and traces of bromo impurity.
 Composition of Hex-Insulin Mixture
 The hex-insulin mixture contains mainly mono and disubstituted insulin conjugates. Triconjugate and free insulin are present as the minor components. The specification of the components present in the product is given in Table B below and the stability characteristics of the hex-insulin mixture are shown in Table C below, in comparison with native insulin
FIG. 3 is a graph of ELISA assay results for native insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight. Two rats were dosed at 10 milligrams insulin per kilogram of body weight (Rats C and I) and two rats were dosed at 30 milligrams insulin per kilogram of body weight (Rats K and L). The data show that the baseline level of the NRS pool is about 3.5 nanograms of insulin per milliliter.
FIG. 4 is a graph of ELISA assay results for insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight, wherein the insulin is administered as a conjugate, insulin-hexyl-PEG7-OMe.
FIG. 5 is a graph of percent of pre-dose blood glucose level as a function of time, for free insulin, and for conjugated insulin at respective doses of 1, 3 and 10 milligrams of insulin per kilogram of body weight, against a control of phosphate buffered solution (PBS) and bovine serum albumen (BSA).
 The foregoing data show that the native insulin concentration in the NRS pool is flat throughout the timecourse, while the conjugate has a high dose peak in 30 minutes with 40 ng/mL insulin.
 There is a rise in insulin in the intermediate dose but the peak falls below the level of the NRS pool.
 The data therefore show that the conjugate exhibits very good activity in the CLA and this corresponds with an increase in serum insulin to 40 ng/mL that peaks in 30 minutes.
FIG. 6 is a graph of insulin and blood glucose levels as a function of time, after dosing of canine subjects with a microemulsion insulin formulation representative of the present invention. The respective curves shown in the graph include native Zinc insulin; glucose ( ), hex-insulin; glucose (▪); native Zinc insulin; insulin (Δ); and hex-insulin; insulin (Δ).
 These data each represent the average of several determinations performed on two pairs of dogs on two separate days.
 Glucose data were normalized to the baseline glucose levels (0 minutes). Dogs were dosed orally with one of two emulsion formulations (native Zinc insulin or Hex-insulin mixture). The dose was adjusted to 1.0 mg/kg. Samples were collected at 30 minute intervals. The data were corrected for Hex-insulin reactivity in the BMI ELISA and Pharmacia RIA tests.
 As shown in FIG. 6, dogs dosed with Hex-Ins showed a slightly greater glucose depression than dogs dosed with native insulin Maximal glucose depression was from 9-180 minutes, with a return to baseline by 360-480 minutes. In contrast, the insulin levels in the Hex-Ins group were about 4 fold higher than the native insulin group. Peak activity of insulin immunoreactivity in the serum occurred within 30 minutes of dosing, and returned to baseline in all cases with 120-240 minutes.
 While the invention has been described herein, with certain features, and embodiments it will be recognized that the invention may be widely varied, and that numerous other modifications, variations, and other embodiments are possible, and such modifications, variations, and other embodiments are to be regarded as being within the spirit and scope of the invention.