ES2362269T3 - MONODISPERSED BLENDS AND PROCEDURES TO TREAT DIABETES. - Google Patents

Abstract

A substantially monodispersed mixture of conjugates, in which each comprises an insulin polypeptide-oligomer conjugate comprising the structure: wherein: Insulin polypeptide-X (CH2) m (OG2H4) nOR (III) X is a moiety ester, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; m is between a lower limit of 1 and an upper limit of 30; n is between a lower limit of 1 and an upper limit of 50; and R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety.

Description

Field of the Invention

The present invention relates to a substantially monodispersed mixture of conjugates, in which each comprises an insulin-oligomer polypeptide conjugate and methods of treating diabetes mellitus.

Background of the invention

There are currently 15.7 million people, or 5.9% of the population of the United States, who suffer from diabetes mellitus. Every day, approximately 2,200 people are diagnosed with diabetes and approximately 798.00 will be diagnosed this year. Diabetes is the seventh leading cause of death (sixth leading cause of death from illness) in the United States.

Diabetes mellitus, better known as diabetes, is a disease in which the body does not produce or use insulin properly, a hormone that helps the body convert sugars and other nutrients into energy. In a non-diabetic individual, insulin is produced in the pancreas, on the islets of Langerhans, in response to an increase in glucose in the intestines and / or blood. Next, insulin acts together with the liver to control glucose metabolism in the body. Although diabetes is usually thought to be a blood sugar disease, diabetes can lead to numerous fatal complications. For example, diabetes can lead to several microvascular diseases, such as retinopathy, nephropathy and neuropathy. In the United States, diabetes is the main cause of new cases of blindness in people between the ages of 20 and 74, it is the main cause of terminal nephropathy and it is the most frequent cause of lower limb amputations. Diabetic individuals are also more likely to develop life-threatening macrovascular diseases, such as heart disease and stroke.

There are several types of diabetes. Insulin-dependent diabetes mellitus (DMDI), usually called type 1 diabetes, is an autoimmune disease that affects the islets of Langerhans, destroying the body's ability to produce insulin. Type 1 diabetes can affect up to 1 million people in the United States. Non-insulin dependent diabetes mellitus (NIDDM), usually called type 2 diabetes, is a metabolic disorder resulting from the body's inability to produce enough insulin or properly use the insulin produced. Approximately 90 percent of all diabetic individuals in the United States suffer from type 2 diabetes, which is usually associated with obesity and a sedentary lifestyle.

In general, the goal of diabetes treatment is to control blood glucose levels and keep them within limits that mimic those of the non-diabetic individual, that is, reproduce the natural physiological glucose homeostasis. To date, this objective has not been fully achieved.

Normally, diabetes is treated by monitoring glucose levels in the body through blood and / or urine samples and trying to control glucose levels in the body using a combination of diet and parenteral insulin injections. Parenteral injections, such as subcutaneous and intramuscular injections, release insulin in the peripheral system. In studies such as the Diabetes Control and Complications Trial (DCCT) study, it has been shown that close control of blood glucose levels within normal limits can reduce or eliminate microvascular complications associated with diabetes. As observed in the DCCT, close control of blood glucose levels can be achieved in some individuals using three or more daily injections of insulin or by treatment with an insulin pump (continuous subcutaneous insulin infusion). If closely followed, these regimens offer the potential for some disease control. However, an increased risk of hypoglycemia was observed in the DCCT when a close control of blood glucose levels was attempted by peripheral administration. Additionally, peripheral insulin administration may result in peripheral hyperinsulinemia, which may increase the risk of various medical complications. In addition, many patients can routinely breach these regimens due to lack of comfort and / or shame associated with subcutaneous insulin administration.

In an attempt to overcome the discomfort and inconvenience of subcutaneous insulin administration, several non-invasive alternatives to injectable insulin have been proposed. For example, US patents. numbers

5,320,094 to Laube et al., 5,364,838 to Rubsamen and 5,997,848 to Patton et al. propose intrapulmonary administration of insulin using inhaler devices. The usefulness of these procedures may be limited by the requirement to direct the operation of the release device, the potential for pulmonary irritation with prolonged use and altered release to patients with pulmonary disorders. The administration of insulin in the oral mucosa has also been proposed. Although these administration procedures may have been termed "oral administration," non-injectable forms of peripheral administration with their related problems and difficulties are effective.

The administration of insulin through the nasal mucosa by an aerosol device has also been proposed. These procedures resulted in unacceptable levels of insulin absorption variability within the same patient and between doses, as well as irritation of the nasal mucosa. As with the intrapulmonary and oral mucosa procedures described above, the administration of insulin through the nasal mucosa is a non-injectable form of peripheral administration.

Although these non-injectable forms of peripheral administration can avoid the discomfort of the injectable forms, they still present the same risks of peripheral hyperinsulinemia associated with peripheral insulin injection. In addition, and perhaps most importantly, they do not release insulin in the liver in an effective way. As described above and in more detail in Goodman & Gilman’s, The Pharmacological Basis of Therapeutics 1487-1507 (9th ed. 1996), insulin acts together with the liver to control glucose metabolism in the body. To achieve a more effective treatment of diabetes mellitus, procedures that simulate the natural release of insulin in the liver should be provided.

Several procedures have been proposed that may attempt to simulate the natural release of insulin in the liver. For example, US Pat. No. 4,579,730 to Kidron et al. proposes a pharmaceutical composition with enteric coated for oral insulin administration. Although this patent states that the proposed pharmaceutical formulations have the same effect as naturally secreted insulin on blood glucose levels, insulin is not rapidly absorbed from the intestinal lumen into the bloodstream. For example, the patent reports in column 4, lines 15-16, that only 5-10% of insulin is absorbed for 60 minutes. Additionally, this formulation appears to have a limited effect on blood glucose levels within the first hour after administration. (See column 4, Table 1).

As another example, US Pat. No. 4,963,526 to Ecanow proposes an oral dosage form of insulin based on a two-phase liquid aqueous coacervate system that incorporates insulin into a coacervated phase of the system. Insulin administered using this formulation does not appear to have a rapid effect on blood glucose levels. For example, in column 9, line 52 to column 10, line 12, the patent informs the measurement of blood glucose levels three hours after administration and the observation of blood glucose levels of 66.3 % and 47.1% of initial blood levels depending on the dosage given.

As another example, US Pat. No. 4,863,896 to Geho et al. proposes procedures to treat diabetes that use a combination of peripherally administered insulin and vesicles with encapsulated insulin targeting hepatocytes (HDVI). The liver will only capture HDVI as long as the free insulin administered separately supplies the needs of the peripheral system. The patent indicates that HDVI can be administered orally; however, dosage intervals are not provided. Co-administration of HDVI and peripheral insulin is necessary because, unlike naturally produced insulin, no HDVI will pass through the liver into the peripheral system. (See column 2, lines 3-14).

As another example, US Pat. No. 5,704,910 to Humes proposes an implantable device for releasing a preselected molecule, for example a hormone such as insulin, into the systemic circulation of a mammal. The patent contemplates anchoring an insulin producing device in the portal vein before the liver. Relying on such a device to control the life-threatening disease of diabetes mellitus may not be practical. In addition, it is not clear what dose of insulin the device would administer and precisely how the required dose would be regulated.

In light of the foregoing, there is a need in the art for methods of treating diabetes mellitus that better simulate the natural release of insulin in the liver, which results in the improvement of glucose homeostasis in administration forms. of the product that are comfortable and easy for patients to use.

Summary of the Invention

The embodiments of the present invention provide compositions for use in the treatment of diabetes mellitus that effectively reproduce the natural physiological glucose homeostasis of non-diabetics. In contrast to the conventional treatment procedures described above, the embodiments of the present invention provide a rapid and effective absorption of insulin into the portal bloodstream. This administration of insulin in the portal bloodstream results in the administration of an effective amount of insulin in the liver; however, unlike the HVDI used in the conventional procedure described above, some of the insulin provided to the liver is free to pass through the liver into the peripheral system. Thus, the embodiments of the present invention allow diabetic individuals with healthy livers to metabolize glucose in a manner that more closely mimics the natural glucose metabolism in non-diabetic individuals than any of the conventional procedures described above. The embodiments of the present invention can result in rapid but controlled decreases in blood glucose levels after administration and provide effective control of postprandial glucose levels. As a result, the embodiments of the present invention may allow diabetic individuals to control glucose levels and eliminate or reduce the occurrence of many complications associated with diabetes mellitus. In addition to having the liver perform glucose homeostasis, the embodiments of the present invention may also partially or completely play the role of the liver in regulating the metabolism of lipids and amino acids.

According to a first embodiment, the present invention provides a substantially monodispersed mixture of conjugates, in which each comprises an insulin-oligomer polypeptide conjugate comprising the structure:

Insulin polypeptide-X (CH2) m (OC2H4) nOR (III)

5 in which:

X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a moiety carbonate, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; m is between a lower limit of 1 and an upper limit of 30; n is between a lower limit of 1 and an upper limit of 50; Y

R 10 is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety.

According to a second embodiment, the present invention provides a monodispersed mixture of conjugates, in which each comprises an insulin-oligomer polypeptide conjugate comprising the structure:

Insulin polypeptide-X (CH2) m (OC2H4) nOR (III)

15 in which:

X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a moiety carbonate, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; m is between a lower limit of 1 and an upper limit of 30; n is between a lower limit of 1 and an upper limit of 50; Y

R 20 is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety.

In accordance with yet another embodiment, the present invention provides the use of said mixtures for the manufacture of a medicament for the treatment of diabetes.

In yet another embodiment, the present invention provides a method of synthesizing substantially monodispersed mixtures of polymers, comprising polyethylene glycol moieties, said process comprising: reacting a substantially monodispersed mixture of compounds having the structure of Formula I:

R1 (OC2H4) m-O-X + (I)

wherein R1 is H or a lipophilic moiety; m is from 1 to 25; and X + is a positive ion with a substantially monodispersed mixture of compounds having the structure of Formula II:

30 R2 (OC2H4) nOMs (II)

wherein R2 is H or a lipophilic moiety; Ms is a mesylate residue; and n is 1 to 25, under conditions sufficient to provide a substantially monodispersed mixture of polymers comprising polyethylene glycol moieties and having the structure of Formula III:

R2 (OC2H4) m + n-OR1 (III)

In yet another embodiment, the invention provides the use of a substantially monodispersed mixture of an insulin-oligomer polypeptide conjugate, comprising the structure of Formula V:

image 1

for the preparation of an orally administrable medication for diabetes mellitus treatments.

Embodiments of the present invention provide a rapid and convenient administration of insulin to the portal vein 40 and, therefore, to the liver. Using the present invention, diabetic individuals can effectively treat blood glucose variations and complications associated with diabetes mellitus.

Brief description of the figures

Figure 1a shows insulin levels in pancreatomized dogs receiving increasing doses of orally administered HIM2 in accordance with the embodiments of the present invention;

Figure 1b shows glucose levels in pancreatomized dogs receiving increasing doses of orally administered HIM2 in accordance with the embodiments of the present invention; Figure 2 shows serum insulin levels (+/- SEM) in normal volunteers after oral administration of HIMX at a dose volume of 4 ml compared to oral placebo administration (Comparison Figure); Figure 3a shows serum insulin levels (+/- SEM) in normal volunteers after oral administration of HIMX at a dose volume of 20 ml compared to oral administration of placebo (Comparison Figure); Figure 3b shows serum glucose levels (+/- SEM) in normal volunteers after oral administration of HIMX at a dose volume of 4 ml compared to oral administration of placebo (Comparison Figure); Figure 4a shows peripheral insulin concentrations after oral administration of HIM2 in patients with type 1 diabetes in accordance with the embodiments of the present invention compared to the subcutaneous (SC) administration of 4 insulin units; Figure 4b shows peripheral glucose concentrations after oral administration of HIM2 in patients with type 1 diabetes in accordance with the embodiments of the present invention compared to oral administration of placebo; Figure 5 shows effects of two sequential doses of HIM2 administered orally in accordance with embodiments of the present invention on glucose and insulin in a fasting type 1 diabetes patient who has not been given another insulin; Figure 6 shows effects of two sequential doses of HIM2 administered orally in accordance with embodiments of the present invention on plasma glucose levels in a fasting type 1 diabetes patient; Figure 7 shows the effects on plasma glucose in patients with type 1 diabetes after oral administration of HIM2 in accordance with the embodiments of the present invention; Figure 8 shows the effects of blood glucose in patients with type 1 diabetes after oral administration of HIM2 according to embodiments of the present invention when oral administration of the same dose of HIM2 was repeated one week later; Figure 9 shows the effects on fasting blood glucose in patients with type 1 diabetes after oral administration of HIM2 with co-administration of basal (pump) insulin in accordance with embodiments of the present invention; Figure 10 shows the effects on postprandial glucose in a patient with type 1 diabetes after oral administration of HIM2 in accordance with embodiments of the present invention; Figure 11 shows the effects on postprandial glucose in patients with type 1 diabetes after oral administration of HIM2 in accordance with embodiments of the present invention; Figure 12 shows the effects on postprandial glucose in patients with type 1 diabetes after oral administration of HIM2 in accordance with embodiments of the present invention; Figure 13 shows the effects on postprandial glucose in patients with type 1 diabetes after oral administration of HIM2 in accordance with embodiments of the present invention; Figure 14 shows a reaction scheme to provide monodispersed mixtures of polyethylene glycol; Figure 15 shows a reaction scheme to provide monodispersed mixtures of an oligomer comprising a polyethylene glycol moiety; Figure 16 shows plasma insulin levels in the portal sale (VP) and vena cava (VC) after oral administration of HIM2 at 2.5 mg / kg according to embodiments of the present invention; Figure 17 shows blood glucose and plasma insulin levels in the portal sale (VP) and vena cava (VC) after oral administration of dextrose solution in fasting mice for comparative purposes; Figure 18 shows plasma insulin levels at the portal sale (VP) and vena cava (VC) after subcutaneous administration of recombinant human insulin at 25 /g / kg for comparative purposes; Figure 19 shows plasma glucose levels at the portal sale (VP) and vena cava (VC) after oral administration of HIM2 at 2.5 mg / kg (2 x ED50) in accordance with embodiments of the present invention. ; and Figure 20 shows plasma glucose levels at the portal sale (VP) and vena cava (VC) after subcutaneous administration of recombinant human insulin at 25 g / kg (2 x ED50) for comparative purposes.

Detailed description of the preferred embodiments

In non-diabetic individuals, the pancreas continuously supplies low (or baseline) levels of insulin. The liver supplies glucose to the rest of the body when the body is fasting. Normally this is called hepatic glucose production. In response to a meal, the pancreas releases elevated levels of insulin. First, insulin interacts with the liver and thus tells you to stop the production of liver glucose and begin to absorb the ingested glucose as part of the meal. Some bolus of insulin administration by the pancreas crosses the liver and interacts with other cells in the body, especially the muscle, indicating that they absorb and use glucose. Therefore, in non-diabetic individuals, the pancreas and liver function in tandem to provide glucose homeostasis.

The embodiments of the present invention can restore glucose homeostasis of a diabetic individual who possesses a healthy liver (i.e., a liver capable of performing normal glucose uptake and production except by the absence of the insulin regulatory hormone) . By activating the liver to regulate blood glucose levels, the methods of the present invention can reduce or eliminate hyperglycemia and / or hypoglycemia associated with conventional methods of treating diabetes mellitus. The embodiments of the present invention can also reduce or eliminate some, if not all, of the microvascular complications (e.g., kidney disease, retinopathy and / or neuropathy) and / or macrovascular complications (e.g., myocardial infarction and / or stroke) normally associated with diabetes mellitus. In addition, embodiments of the present invention may reduce or eliminate hyperinsulinemia associated with peripheral administration (eg, subcutaneous, intrapulmonary, intranasal, in the oral mucosa) of insulin. In addition, the embodiments of the present invention can reduce or eliminate the hyperlipidemia associated with diabetes through the activation of the liver to improve its metabolism of fatty acids. Proper activation of the liver can also restore other metabolic pathways in hepatocytes regulated by genes and related to complications associated with diabetes mellitus.

As used herein, the term "insulin drug" refers to any molecule capable of producing one or more biological responses associated with insulin (eg, regulation of glucose homeostasis in target tissues such as liver, muscles and / or fats, stimulation of the use and storage by the cell of glucose, amino acids and / or fatty acids and inhibition of catabolic processes such as the degradation of glycogen, fats and proteins), including, among other insulin polypeptides such as insulin, insulin analogs, active insulin fragments and active analogs of insulin fragments, derivatives of insulin polypeptides and insulin agonist molecules, mixtures thereof or pharmaceutical compositions containing said molecules or mixtures of said molecules.

As used herein, the term "insulin" means the insulin of one of the following species: human, cows, pigs, sheep, horses, dogs, chickens, ducks or whales, provided from natural, synthetic or natural sources. of genetic engineering. In various embodiments of the present invention, insulin is preferably human insulin.

As used herein, the term "insulin analog" means that in which one or more of the amino acids have been replaced while retaining part or all of the insulin activity. The analog is described by indicating the replacement amino acids with the replacement position as a superscript followed by a description of the insulin. For example, "ProB29 insulin, human" means that the lysine normally found in the B29 position of a human insulin molecule has been replaced with proline.

Insulin analogs can be obtained in several ways, as those skilled in the art will understand. For example, certain amino acids can be substituted with other amino acids in the structure of insulin without a noticeable loss of the ability to interact with structures such as, for example, antibody antigen binding regions or binding sites on the substrate molecules. Since the interactive capacity and nature of insulin defines its biological functional activity, certain substitutions can be made in the amino acid sequence and, in any case, remain a polypeptide with similar properties.

When making such substitutions, the hydropathic index of amino acids can be considered. It is generally understood in the art the importance of the hydropathic index of amino acids in conferring interactive biological function on a polypeptide. It has been accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting polypeptide, which, in turn, defines the interaction of the protein with other molecules, for example enzymes, substrates, receptors, DNA, antibodies, antigens and the like. . Each amino acid has been assigned a hydropathic index based on its hydrophobicity and load characteristics as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). As those skilled in the art will understand, certain amino acids may be substituted by other amino acids that have a similar hydropathic index or score and remain a polypeptide with similar biological activity, that is, continue to obtain an equivalent of the functionally biological polypeptide. In making such changes, the substitution of amino acids whose hydropathic indices are within  2 of each is preferred, particularly preferred are those within  1 of each and even more particularly preferred are those within  0.5 of each.

It is also understood in the art that the substitution of similar amino acids can be performed effectively based on hydrophilicity. U.S. Pat. 4,554,101 provides that the higher average local hydrophilicity of a protein, driven by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in US Pat. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (_3.0); aspartate (+3.0  1); glutamate (+3.0  1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5  1); Alanine (-0.5); histidine (0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). As those skilled in the art understand, an amino acid can be substituted with another that has a similar hydrophilicity value and at the same time obtain a biologically equivalent and, in particular, immunologically equivalent polypeptide. In making such changes, the substitution of amino acids whose hydrophilicity values are within  2 of each is preferred, particularly preferred are those within  1 of each and even more particularly preferred are those within  0.5 each.

Therefore, as indicated above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example their hydrophobicity, hydrophilicity, charge, size and the like. Examples of substitutions (ie, amino acids that can be exchanged without significantly altering the biological activity of the polypeptide) that take into account several of the above characteristics are well known to those skilled in the art and include, for example: arginine and lysine. ; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

As those skilled in the art will understand, insulin analogs can be prepared by various recognized peptide synthesis techniques, including, but not limited to, classical procedures (solution), solid phase procedures, semisynthetic procedures and recombinant DNA procedures.

Examples of human insulin analogs include, among others, human GlyA21 insulin; insulin GlyA21 GlnB3, human; AlaA21 insulin, human; insulin AlaA21 GlnB3, human; GlnB3 insulin, human; GlnB30 insulin, human; insulin GlyA21 GluB30, human; insulin GlyA21 GlnB3 GluB30, human; Insulin GlnB3 GluB30, human; human AspB28 insulin; LysB28 insulin, human; insulin LeuB28, human; ValB28 insulin, human; AlaB28 insulin, human; insulin AspB28 ProB29, human; human LysB28 ProB29 insulin; insulin LeuB28 ProB29, human; ValB28 ProB29 insulin, human; Insulin AlaB28 ProB29, human.

As used herein, the term "active insulin fragment" means a segment of the amino acid sequence in insulin that retains part or all of the insulin activity. Insulin fragments are cited indicating the position (s) in an amino acid sequence, followed by a description of the amino acid. For example, a fragment of "human B25-B30 insulin" would be the six amino acid sequence corresponding to positions B25, B26, B27, B28, B29 and B30 in the amino acid sequence of human insulin.

As used herein, the term "active insulin fragment analog" means a segment of the amino acid sequence found in the insulin molecule in which one or more of the segment's amino acids have been substituted for the time that conserves part or all of the insulin activity.

As used herein, "insulin polypeptide derivative" refers to an insulin polypeptide, such as insulin, an insulin analogue, an active insulin fragment or an analogue of the active insulin fragment, which it has been conjugated with one or more moieties, such as acyl moieties (e.g., fatty acids) and / or oligomers, which improve the lipophilicity and / or hydrophilicity of the insulin polypeptide so that the insulin polypeptide conjugate is more lipophilic and / or more hydrophilic than the corresponding unconjugated insulin polypeptide. The hydrophilicity of an insulin polypeptide derivative can be compared with the hydrophilicity of the insulin polypeptide unconjugated by various means, as those skilled in the art will understand. For example, a given amount of said insulin polypeptide derivative can be added to water, and the resulting solution is mixed and filtered. The filtrate can be analyzed using known HPLC methods to determine the amount of conjugate present in the filtrate and, therefore, the amount of conjugate dissolved in the water. Alternatively, the filter paper can be weighed before and after filtration to determine the weight of the conjugate not dissolved in water. This weight can be used to determine the concentration of conjugate in water. The same procedure can be repeated using the unconjugated insulin polypeptide and the two concentrations can be compared. The molecule that produces the highest concentration in water is considered the most hydrophilic molecule. The lipophilicity of an insulin polypeptide derivative can be compared with the lipophilicity of the insulin polypeptide unconjugated by various means, as those skilled in the art will understand. For example, a given amount of said polypeptide derivative can be analyzed by reverse phase HPLC as will be understood by those skilled in the art. The unconjugated insulin polypeptide can be analyzed using the same reverse phase HPLC method and the elution times of the insulin polypeptide derivative and the unconjugated insulin polypeptide can be compared. The molecule with the longest elution time is considered the most lipophilic molecule.

As used herein, the term "amphiphilically balanced insulin oligomer-polypeptide conjugate" refers to a conjugate that is both more lipophilic than the unconjugated insulin polypeptide and more hydrophilic than the unconjugated insulin polypeptide. One skilled in the art will understand how to determine whether an oligomeric insulin polypeptide conjugate is amphiphilically balanced. For example, a given amount of the insulin oligomer-polypeptide conjugate can be added to water, and the resulting solution mixed and filtered. The filtrate can be analyzed using known HPLC methods to determine the amount of conjugate present in the filtrate and, therefore, the amount of conjugate dissolved in the water. Alternatively, the filter paper can be weighed before and after filtration to determine the weight of the conjugate not dissolved in water. This weight can be used to determine the concentration of conjugate in water. The concentration of the insulin oligomer-polypeptide conjugate in the water should be greater than the water concentration of the unconjugated insulin polypeptide determined using the same procedure. A given amount of the insulin-oligomer polypeptide conjugate can then be analyzed by reverse phase HPLC, as will be understood by those skilled in the art. The elution time of the insulin-oligomer polypeptide conjugate should be longer than the elution time of the unconjugated insulin polypeptide.

As used herein, the term "portal administration" means administration of all, or substantially all, a dose given to the portal vein. Portal administration can be achieved through several routes of administration, including, but not limited to, oral administration, subcutaneous administration in the peritoneal cavity, rectal administration and direct infusion into the portal vein.

As used herein, the term "peripheral administration" means administration of all,

or substantially all, a dose given to the peripheral system. Peripheral administration can be achieved by several routes of administration, including, inter alia, intrapulmonary, intranasal, by the oral mucosa and parenteral injections (eg, subcutaneous and intramuscular injections).

As used herein, the term "polyalkylene glycol" refers to linear or branched polyalkylene glycol polymers, such as polyethylene glycol, polypropylene glycol and polybutylene glycol, and includes the polyalkylene glycol monoalkyl ether. The term "polyalkylene glycol subunit" refers to a single polyalkylene glycol unit. For example, a subunit of polyethylene glycol would be -O-CH2-CH2-O-.

As used herein, the term "lipophilic" means the ability to dissolve in lipids and / or the ability to penetrate, interact and / or cross biological membranes and the expression "lipophilic moiety" or "lipophilic" means a remainder that is lipophilic and / or that, when bound to another chemical entity, increases the lipophilicity of said chemical entity. Examples of lipophilic moieties include, among others, alkyls, fatty acids, fatty acid esters, cholesteryl, adamantyl and the like.

As used herein, the term "lower alkyl" refers to substituted or unsubstituted alkyl moieties having one to five carbon atoms.

As used herein, the term "higher alkyl" refers to substituted or unsubstituted alkyl moieties having six or more carbon atoms.

As used herein, phrases such as "between X and Y" should be construed to include X and Y.

As used herein, phrases such as "between X and Y" mean "between approximately X and approximately Y" and phrases such as "from approximately X to Y" mean "from approximately X to approximately Y".

As used herein, the terms "ester moiety", "thioester moiety", "carbamate moiety", "ether moiety", "thiocarbamate moiety", "carbonate moiety", "thiocarbonate moieties", "urea moiety" and "amide residue" are used to refer to the rest cited, in any of its various possible orientations. The rest may include one or two lower alkylene moieties in addition to the aforementioned rest. For example, the term "ester moiety" refers to a moiety -OC (O) -, a moiety -C (O) -O- or any of these moieties having a lower alkylene moiety at one or both ends of the moiety .

The compositions of the present invention can be used in a method of treating diabetes mellitus in a patient in need of such treatment, which includes orally administering an effective amount of an insulin drug to the patient in order to treat diabetes mellitus in the patient, in which the effective amount of the insulin drug is administered so as to provide a concentration of the insulin drug in the blood of the portal vein between about 10, 200 or 4000 and 600, 800 or 1000 U / ml in about 60 minutes from the administration, more preferably in about 15 or 30 minutes from the administration. The procedures can provide concentrations of the insulin drug in the bloodstream that are up to 100 times the basal levels of insulin usually present.

The effective amount of the insulin drug is preferably between about 0.05, 0.1 or 0.2 and 2.5 or 10 mg per kilogram of the patient's body weight. More preferably, the effective amount of the insulin drug is between about 0.3 and 1 mg per kilogram of the patient's body weight. When the dosage of the insulin drug is too low, desirable activation of the liver is not achieved. When the dosage is too high, an excessive amount of the insulin drug can pass through the liver in the peripheral system, potentially resulting in a state of hypoglycemia.

Oral administration of an effective amount of an insulin drug preferably provides rapid decreases in fasting peripheral blood glucose concentrations. Preferably, peripheral blood glucose concentrations decrease by approximately 10, 15 or 25 percent in approximately 5, 15 or 30 minutes after administration.

Preferably, embodiments of the present invention provide a rapid release of the insulin drug to the peripheral system. Oral administration of an effective amount of the insulin drug provides a maximum concentration of insulin in peripheral blood, preferably in about 60 minutes after administration, more preferably in about 30 minutes after administration, and even more preferably in about 15 minutes after administration. administration. The rapid achievement of a maximum glucose concentration counteracts an increase in postprandial glucose that simulates the natural pancreatic pattern of insulin administration at mealtime. Preferably, the administered insulin drug clears from the bloodstream in about 3 or 4 hours and, more preferably, clears from the bloodstream in about 2 hours.

Oral administration of an effective amount of the insulin drug stabilizes the concentration of peripheral glucose. For example, the concentration of peripheral blood glucose can be maintained at +/- approximately 5, 10, 20 or 50 percent of an average peripheral glucose concentration. The average peripheral glucose concentration can be determined in a period of time of approximately 30, 60, 90 or 240 minutes starting at approximately 15, 30 or 60 minutes from the administration.

Oral insulin administration procedures to a patient suffering from diabetes mellitus reduce the patient's hepatic glucose production. Preferably, the hepatic glucose production is reduced by at least about 25, 35, 50, 75, 90 or 95 percent when compared to the hepatic glucose production of the patient without administration and, more preferably, is reduced by approximately 100 percent when compared to the liver glucose production of the patient without administration. This reduction in hepatic glucose production occurs, preferably, in approximately 30, 60 or 90 minutes after administration. Hepatic glucose production is preferably determined by measuring peripheral glucose levels in a period of time in which peripheral insulin levels are the baseline levels, or close to them. Preferably, the period of time is between about 1 and 4 hours, more preferably between about 1 and 2 hours, and, more preferably, about 1.5 hours.

Oral administration of an effective amount of insulin controls an increase in glucose that is normally associated with the ingestion of a meal (i.e., the postprandial increase in glucose levels). The postprandial increase in glucose levels can be partially or completely controlled by methods of the present invention. Preferably, at least about 25 percent of postprandial glucose is absorbed in the liver, more preferably at least about 40 percent is absorbed and, even more preferably, at least about 55 percent is absorbed. Preferably, postprandial glucose absorption occurs in approximately 120 minutes from the ingestion of the food and, more preferably, occurs in approximately 15 or 30 minutes from the ingestion of the food.

Oral administration of the insulin drug can occur at various times during the day. Preferably, the insulin drug is administered during, or about, the time of the meal (eg, within one hour). In some embodiments, the insulin drug is administered less than about an hour before eating a meal. Preferably, the insulin drug is administered less than about 30 minutes before eating a meal and, more preferably, it is administered less than about 20 minutes before eating a meal. In other embodiments, the insulin drug is administered less than one hour after ingesting a meal and, preferably, it is administered less than about 30 minutes after ingesting a meal. In yet other embodiments, the insulin drug is administered at the same time as a meal. The insulin drug administered at the same time as food intake may be less preferred because it may require higher doses and result in a variability from one dose to another for a given patient.

Insulin drug administration may occur before one or more meals a day. Additionally, the insulin drug can be administered several times in addition to at mealtime, for example before sleeping four or more hours (eg, at bedtime at night) and / or after waking up from four or more hours of sleep (e.g., getting up in the morning). Administration of the insulin drug according to the methods of the present invention before sleeping four or more hours can provide effective glucose homeostasis during all or part of the soil period, which prevents or reduces the probability of the phenomenon of Dawn (Dawn Phenomenon), which usually occurs in individuals with type 1 diabetes mellitus and is characterized by the appearance of a hypoglycemic episode during a period of sleep.

In accordance with embodiments of the present invention, an insulin drug is preferably administered at appropriate doses and frequencies so that liver activation is achieved and / or maintained so as to effect glucose homeostasis. For example, the insulin drug can be administered uninterruptedly (that is, it is administered at least once a day) or cyclically (that is, it is administered for one or more consecutive days, followed by one or more consecutive days without administration. ). Uninterrupted administration may be desirable when it is necessary to administer one or more doses per day in order to achieve and / or maintain liver activity to control / help control glucose levels in the bloodstream. When uninterrupted administration is used, it may be possible to use lower doses of insulin.

Cyclic administration may be desirable when liver activity persists for one or more days after insulin administration. For example, the insulin drug may be administered for one or more days, followed by a period of one or more days during which the insulin drug is not administered. Cyclic administration does not have to follow a uniform administration pattern. For example, a cyclic administration regime may employ four days of administration, followed by one day without administration, followed by two days of administration, followed by three days without administration. Cyclic administration can be used to treat type 1 or type 2 diabetes mellitus; however, cyclic administration may be more beneficial in the treatment of type 2 diabetes mellitus.

In other embodiments, the methods of treating diabetes mellitus in a patient in need thereof include administering to the patient an effective amount of a first insulin drug orally, as described in the various previous embodiments and administering to the peripheral system of the patient an effective amount of a second insulin drug. Preferably, peripheral administration is performed by parenteral injection. More preferably, peripheral administration is performed by continuous subcutaneous insulin injection (ISCI), as those skilled in the art will understand. The dose of the ISCI is preferably selected to provide a basal level of insulin in the body. The dose of ISCI may be between about 0.1 and 3 units (U) per hour and, preferably, between about 0.5 and 1.5 U / hour. The first and second insulin drugs may be the same or different.

In yet other embodiments, the methods of treating diabetes mellitus in a patient in need thereof include administering to the patient an effective amount of a first insulin drug orally, as described in the various previous embodiments and administering continuously. in the vein it carries a basal amount of a second insulin drug. This administration can be achieved by ISCI which is administered in the peritoneal cavity. It is believed that these procedures may mimic the bolus of insulin introduced into the portal vein through the pancreas of a non-diabetic individual after ingestion of a meal, as well as mimic the basal level of insulin provided by the pancreas continuously in non-diabetic individuals. . The first insulin drug and the second insulin drug may be the same or different.

The insulin drug of the embodiments described above is an insulin polypeptide derivative. The insulin polypeptide derivative is an acylated insulin polypeptide or an insulin-oligomer polypeptide conjugate. Acylated insulin polypeptides are insulin polypeptides that have been derived with one or more acyl containing moieties, such as fatty acid moieties and / or arylacil moieties. The remaining fatty acid may be a saturated or unsaturated, linear or branched fatty acid residue, such as, among others, caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, oleic acid, elaidic acid, linoleic, linolenic, arachidic and arachidonic acid, or a fatty acid derivative such as a fatty acid-aryl derivative (e.g., phenylacetyl)

or a fatty acid cycloalkyl derivative (eg, cyclohexylacetyl or cyclohexylpropionyl). Arilacil remains include, among others, benzoyl. The insulin-oligomer polypeptide conjugate is an insulin polypeptide conjugated to an oligomer, such as a polyalkylene glycol moiety or a polyalkylene glycol-containing moiety. The polypeptide insulin derivatives according to the embodiments of the present invention can be synthesized using methods that are known to those skilled in the art.

In accordance with embodiments of the present invention, the insulin-oligomer polypeptide conjugate is an amphiphilically balanced insulin-oligomer polypeptide conjugate. The amphiphilically balanced insulinoligomer polypeptide conjugate preferably comprises an insulin polypeptide coupled to an oligomer comprising a hydrophilic moiety coupled to a lipophilic moiety. Preferably, the insulin polypeptide is insulin or an insulin analog. More preferably, the insulin polypeptide is human insulin or a human insulin analog. Even more preferably, the insulin polypeptide is human insulin. The hydrophilic moiety can be coupled to the lipophilic moiety through a hydrolysable or non-hydrolysable bond, or there may be one or more intermediate moieties that couple the hydrophilic moiety to the lipophilic moiety.

The hydrophilic moiety of the amphiphilically balanced insulin-oligomer polypeptide conjugate is a hydrophilic moiety, as understood by those skilled in the art, including, among others, polyalkylene glycols such as polyethylene glycol or polypropylene glycol, polyoxyethylene polyols, copolymers thereof and block copolymers. thereof, as long as the hydrophilicity of the block copolymers is maintained. Preferably, the hydrophilic moiety is a polyalkylene glycol moiety. The rest of polyalkylene glycol has at least 1, 2, 3, 4, 5, 6 or 7 subunits of polyalkylene glycol. Preferably, the rest of the polyalkylene glycol has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. The remaining polyalkylene glycol has, more preferably, between a lower limit of 2, 3, 4, 5 or 6, and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 polyalkylene glycol subunits. Even more preferably, the rest of the polyalkylene glycol has a lower limit of 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11 or 12 subunits of polyalkylene glycol. Even more preferably, the polyalkylene glycol moiety has between a lower limit 4, 5 or 6 and an upper limit of 6, 7 or 8 subunits of polyalkylene glycol. The rest of polyalkylene glycol has, more preferably, 7 subunits of polyalkylene glycol. The polyalkylene glycol moiety of the oligomer is preferably a lower alkyl polyalkylene glycol moiety, such as a polyethylene glycol moiety, a polypropylene glycol moiety or a polybutylene glycol moiety. When the polyalkylene glycol moiety is a polypropylene glycol moiety, the moiety preferably has a uniform structure (ie, not random). An example of a polypropylene glycol residue having a uniform structure is as follows:

image 1

This uniform structure of polypropylene glycol can be described as having only one methyl substituted carbon atom adjacent to each oxygen atom in the polypropylene glycol chain. Such uniform polypropylene glycol moieties may exhibit both lipophilic and hydrophilic characteristics.

5 The lipophilic moiety of the amphiphilic insulin-oligomer polypeptide conjugate is a lipophilic moiety as understood by those skilled in the art. The lipophilic moiety has at least 1, 2, 3, 4, 5 or 6 carbon atoms. Preferably, the lipophilic moiety has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 carbon atoms. The lipophilic moiety has, more preferably, between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms. Even more preferably, the lipophilic moiety has between a lower limit 3, 4, 5, 6, 7, 8 or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms Even more preferably, the lipophilic moiety has between a lower limit of 3, 4, 5, 6 or 7 and an upper limit of 6, 7, 8, 9 or 10 carbon atoms. More preferably, the lipophilic moiety has 6 carbon atoms. Preferably, the lipophilic moiety is selected from the group consisting of alkyl moieties.

15 saturated or unsaturated, linear or branched, saturated or unsaturated fatty acid residues, linear or branched, cholesterol and adamantane. Examples of alkyl moieties include, among others, saturated linear alkyl moieties, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched saturated alkyl moieties, such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derived from the above saturated alkyl moieties, which include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. Examples of fatty acid residues include, among others, unsaturated fatty acid residues, such as, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; and saturated fatty acid residues, such as acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate

25 and cerotato.

The oligomeric portion of the amphiphilically balanced insulin-oligomer polypeptide conjugate may comprise one or more other moieties as will be understood by those skilled in the art, including, but are not limited to, additional hydrophilic moieties, spacer moieties, linker moieties and termination moieties. The various moieties in the oligomer are covalently coupled to each other by hydrolysable or non-hydrolysable bonds.

The oligomeric portion of the amphiphilically balanced insulin-oligomer polypeptide conjugate may comprise one or more additional hydrophilic moieties (i.e. moieties in addition to the hydrophilic moiety), including, among others, sugars, polyalkylene glycols and polyamine / PEG copolymers. Adjacent polyalkylene glycol moieties will be considered the same moiety if they are linked by ether bonds. For example, the rest

—O — C2H4 — O — C2H4 — O — C2H4 — O — C2H4 — O — C2H4 — O — C2H4—

35 is a simple polyethylene glycol moiety having six subunits of polyethylene glycol. If this moiety were the only hydrophilic moiety in the oligomer, the oligomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered different moieties if they are linked by a different bond to an ether bond. For example, the rest

image 1

it is a polyethylene glycol moiety that has four polyethylene glycol subunits and an additional hydrophilic moiety that has two polyethylene glycol subunits. Preferably, the oligomers according to embodiments of the present invention comprise a polyalkylene glycol moiety and no additional hydrophilic moiety.

The oligomeric portion of the amphiphilically balanced insulin-oligomer polypeptide conjugate may comprise one or more spacer moieties as will be understood by those skilled in the art. Spacers can be used

45 to, for example, separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the insulin polypeptide, to separate a first hydrophilic moiety or lipophilic moiety from a second hydrophilic moiety or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety of a linker moiety. Preferably, the spacer moieties are selected from the group consisting of sugar, cholesterol and glycerin moieties. The sugar moieties may be several sugar moieties as will be understood by those skilled in the art, including, among others, monosaccharide moieties and disaccharide moieties. Preferred monosaccharide moieties have between 4 and 6 carbon atoms.

The oligomeric portion of the amphiphilically balanced insulin polypeptide-oligomer conjugate may comprise one or more linker moieties that are used to bind the oligomer with the insulin polypeptide as will be understood by those skilled in the art. The linking moieties are preferably selected from the group consisting of alkyl and fatty acid moieties. The alkyl linker moiety may be a saturated or unsaturated, linear or branched alkyl moiety as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl , undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl , vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. The alkyl linker moiety may have a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms and preferably has between 1, 2, 3, 4 or 5 and 8, 9, 10, 11 or 12 carbon atoms. The fatty acid linker moiety may be a saturated or unsaturated, linear or branched fatty acid moiety, as will be understood by those skilled in the art, including, but not limited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, docosahexaenoate; acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate and cerotate. The fatty acid linker moiety may have a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 carbon atoms and preferably has between 1, 2, 3, 4 or 5 and 8, 10, 12, 14 or 16 carbon atoms.

The oligomeric portion of the amphiphilically balanced insulin polypeptide-oligomer conjugate may comprise one or more termination moieties at one or more ends of the oligomer, which are not bound to the insulin polypeptide. Preferably, the termination moiety is an alkyl or alkoxy moiety. Preferably, the alkyl or alkoxy moiety has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 carbon atoms. More preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3, 4, 5, 6 or 7 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms Even more preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3, 4 or 5 and an upper limit of 5, 6, 7, 8, 9 or 10 carbon atoms. Even more preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3 or 4 and an upper limit of 5, 6 or 7 carbon atoms. The alkyl moiety may be a saturated or unsaturated, linear or branched alkyl moiety as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhex propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. The alkoxy moiety may be various alkoxy moieties, including, but not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadexyloxy, octa nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propyl pentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy, 2-butenyloxy, 2 ethyloxy, 1-propynyloxy, and 2-propynyloxy. The termination moiety is, more preferably, a lower alkyl moiety, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl or tert-pentyl, or a lower alkoxy moiety, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, secbutoxy, tert-butoxy, pentyloxy or tert-pentyloxy. More preferably, the termination moiety is methyl or methoxy. Although the termination moiety is preferably an alkyl or alkoxy moiety, it should be understood that the termination moiety may be several moieties as will be understood by those skilled in the art, including, but are not limited to, sugars, cholesterol, alcohols and fatty acids.

In accordance with other embodiments of the present invention, the insulin drug administered in accordance with the methods of treating diabetes mellitus in a patient in need of such treatment described above is an insulin-oligomer polypeptide conjugate comprising the structure of Formula I:

Insulin polypeptide-B-Lj-Gk-R-G’m-R’-G "n-T (I)

in which:

B is a binding residue; L is a linker moiety; G, G ’and G" are individually selected spacer moieties; R is a lipophilic moiety and R ’is a polyalkylene glycol moiety, or R’ is the lipophilic moiety and R is the rest of polyalkylene glycol; T is a remainder of termination; Y j, k, m and n are 0 or 1 individually.

In accordance with these embodiments of the present invention, the insulin polypeptide is preferably insulin.

or an insulin analog. More preferably, the insulin polypeptide is human insulin or a human insulin analog, and, even more preferably, the insulin polypeptide is human insulin. The insulin-oligomer polypeptide conjugate of Formula I is preferably an amphiphilically balanced insulin-oligomer polypeptide conjugate.

In accordance with these embodiments of the present invention, the polyalkylene glycol moiety has at least 1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. Preferably, the rest of the polyalkylene glycol has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. The remaining polyalkylene glycol has, more preferably, between a lower limit of 2, 3, 4, 5 or 6, and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 polyalkylene glycol subunits. Even more preferably, the rest of the polyalkylene glycol has a lower limit of 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11 or 12 subunits of polyalkylene glycol. Even more preferably, the polyalkylene glycol moiety has between a lower limit 4, 5 or 6 and an upper limit of 6, 7 or 8 subunits of polyalkylene glycol. The rest of polyalkylene glycol has, more preferably, 7 subunits of polyalkylene glycol. The polyalkylene glycol moiety of the oligomer is preferably a lower alkyl polyalkylene glycol moiety, such as a polyethylene glycol moiety, a polypropylene glycol moiety or a polybutylene glycol moiety. When the polyalkylene glycol moiety is a polypropylene glycol moiety, the moiety preferably has a uniform structure (ie, not random). An example of a polypropylene glycol residue having a uniform structure is as follows:

image 1

This uniform structure of polypropylene glycol can be described as having only one carbon atom

Methyl substituted adjacent to each oxygen atom in the polypropylene glycol chain. Such uniform polypropylene glycol moieties may exhibit both lipophilic and hydrophilic characteristics.

In accordance with those embodiments of the present invention, the lipophilic moiety is a lipophilic moiety as understood by those skilled in the art. The lipophilic moiety has at least 1, 2, 3, 4, 5 or 6 carbon atoms. Preferably, the lipophilic moiety has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms. The lipophilic moiety has, more preferably, between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms. The lipophilic moiety has, even more preferably, between a lower limit of 3, 4, 5, 6, 7, 8 or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Even more preferably, the lipophilic moiety has a lower limit of 3, 4, 5, 6 or 7 and an upper limit of 6, 7, 8, 9 or 10 carbon atoms. More preferably, the lipophilic moiety has 6 carbon atoms. Preferably, the lipophilic moiety is selected from the group consisting of saturated or unsaturated, linear or branched alkyl moieties, saturated or unsaturated, linear or branched fatty acid moieties, cholesterol and adamantane. Examples of alkyl moieties include, among others, saturated linear alkyl moieties, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched saturated alkyl moieties, such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derived from the above saturated alkyl moieties, which include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. Examples of fatty acid residues include, among others, unsaturated fatty acid residues, such as, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate,

35 linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; and saturated fatty acid residues, such as acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate and cerotate.

In accordance with those embodiments of the present invention, the spacer residues G, G 'and G ", are spacer moieties as understood by those skilled in the art. The spacer moieties are preferably selected from the group consisting of sugar moieties, cholesterol and glycerin The sugar moieties can be several sugar moieties as will be understood by those skilled in the art, including but not limited to monosaccharide moieties and disaccharide moieties. Preferred monosaccharide moieties have between 4 and 6 carbon atoms. , the oligomers of these embodiments do not include spacer moieties (ie, k, n and n are preferably 0).

In accordance with these embodiments of the present invention, the binding moiety, B, may be several binding moieties

45 as understood by those skilled in the art, including, among others, an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety

or a covalent bond; When the binding moiety is a carbamate moiety or an amide moiety, the nitrogen moiety of the moiety is preferably provided by an amino moiety of the insulin polypeptide, such as the  amino moiety in the LysB29 position of human insulin. The binding moiety is preferably an ester moiety, an ether moiety, a carbamate moiety, a carbonate moiety, an amide moiety, or a covalent bond. The binding moiety is, more preferably, an ester moiety, a carbamate moiety, a carbonate moiety or an amide moiety. The binding moiety is, even more preferably, an amide moiety having the nitrogen portion of the amide moiety provided by an amino moiety of the insulin polypeptide.

In accordance with those embodiments of the present invention, the linker moiety, L, may be several linker moieties such

55 as those skilled in the art understand. The linking moieties are preferably selected from the group consisting of alkyl and fatty acid moieties. The alkyl linker moiety may be a saturated or unsaturated, linear or branched alkyl moiety as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl , undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl , vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2propyl. The alkyl linker moiety may have a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms and preferably has between 1, 2, 3, 4 or 5 and 8, 9, 10, 11 or 12 carbon atoms. The fatty acid linker moiety may be a saturated or unsaturated, linear or branched fatty acid moiety, as will be understood by those skilled in the art, including, but not limited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, docosahexaenoate; acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate and cerotate. The fatty acid linker moiety may have a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29 or 30 carbon atoms and preferably has between 1, 2, 3, 4 or 5 and 8, 10, 12, 14 or 16 carbon atoms.

In accordance with these embodiments of the present invention, the termination moiety, T, is preferably an alkyl or alkoxy moiety. Preferably, the alkyl or alkoxy moiety has a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms. More preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3, 4, 3, 4, 5, 6 or 7 and an upper limit of 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 carbon atoms. Even more preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3, 4 or 5 and an upper limit of 5, 6, 7, 8, 9 or 10 carbon atoms. Even more preferably, the alkyl or alkoxy moiety has between a lower limit of 1, 2, 3 or 4 and an upper limit of 5, 6 or 7 carbon atoms. The alkyl moiety may be several saturated or unsaturated, linear or branched alkyl moieties as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexylpenyl vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. Examples of alkoxy moieties can be various alkoxy moieties, including, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy , nonadecyloxy, eicosiloxi, isopropoxy, sec-butoxy, tertbutoxy, 2-methylbutoxy, tert-pentyloxy, 2-methylpentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propilpentiloxi, vinyloxy, allyloxy, 1buteniloxi, 2-butenyloxy, ethynyloxy , 1-propynyloxy, and 2-propynyloxy. The termination moiety is, more preferably, a lower alkyl moiety, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl or tert-pentyl, or a lower alkoxy moiety, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy or tercpentyloxy. More preferably, the termination moiety is methyl or methoxy. Although the rest of the termination is preferably an alkyl or alkoxy moiety, it should be understood that the rest of the termination may be several moieties as will be understood by those skilled in the art, including, inter alia, sugar moieties, cholesterol, alcohols and moieties. of fatty acid.

In yet other embodiments, the insulin drug administered according to the methods of treating diabetes mellitus in a patient in need of such treatment described above is an insulin-oligomer polypeptide conjugate comprising the structure of Formula II:

Insulin polypeptide-X (CH2) mY (C2H4O) nR (II)

in which:

X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; it is preferably an ester moiety, an ether moiety, a carbamate moiety, a carbonate moiety, an amide moiety or a covalent bond; it is more preferably an ester moiety, a carbamate moiety, a carbonate moiety or an amide moiety; and it is even more preferably an amide moiety. When X is an amide moiety or a carbamate moiety, an amino group of the insulin polypeptide is preferably the nitrogen portion of the amide or carbamate moiety; And it is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; it is preferably an ester moiety, an ether moiety, a carbamate moiety, a carbonate moiety, an amide moiety or a covalent bond; it is more preferably an ester moiety, an ether, a carbamate moiety, a carbonate moiety or an amide moiety; and it is even more preferably an ether moiety. m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30, is more preferably between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, is even more preferably between a lower limit of 3, 4, 5, 6, 7, 8 or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and is even more preferably between a lower limit of 3, 4, 5, 6 or 7 and an upper limit of 6, 7, 8, 9 or 10; n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or According to the invention, the insulin drug is an insulin-oligomer polypeptide conjugate comprising the structure of Formula III:

Insulin polypeptide-X (CH2) m (OC2H4) nOR (III)

in which:

5 X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; it is preferably an ester moiety, an ether moiety, a carbamate moiety, a carbonate moiety, an amide moiety or a covalent bond; it is more preferably an ester moiety, a carbamate moiety, a carbonate moiety or an amide moiety; and it is even more preferably an amide moiety. When X is an amide moiety or a carbamate moiety, an amino group of the

Insulin polypeptide is preferably the nitrogen portion of the amide or carbamate moiety; m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30, is more preferably between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, is even more preferably between a limit

15 lower than 3, 4, 5, 6, 7, 8 or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and is even more preferably between a lower limit of 3, 4, 5, 6 or 7 and an upper limit of 6, 7, 8, 9 or 10; n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, is more preferably between a lower limit of

20 2, 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, is even more preferably between a lower limit of 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11 or 12 polyalkylene glycol subunits, it is even more preferably between a lower limit of 4, 5 or 6 and an upper limit of 6, 7 or 8 subunits of polyalkylene glycol, and is, more preferably, 7; Y; R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety.

The alkyl moiety may be a saturated or unsaturated, linear or branched alkyl moiety as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl , undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl -propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. The rest of the alkyl is, more

Preferably, a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tert-butyl, pentyl or tert-pentyl. The alkyl moiety is, even more preferably, a C1 to C3 alkyl. The alkyl moiety is, more preferably, methyl. The remaining fatty acid may be a saturated or unsaturated, linear or branched fatty acid residue, as will be understood by those skilled in the art, including, but not limited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linoleate, arachidonate ,

35 eicosapentaentoate, docosahexaenoate; acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate and cerotate. The rest of the sugar may be several sugar residues as those skilled in the art will understand. Likewise, the rest of the alcohol can be several alcohol residues as those skilled in the art will understand.

In accordance with these embodiments of the present invention, the insulin polypeptide is preferably insulin.

40 or an insulin analog. More preferably, the insulin polypeptide is human insulin or a human insulin analog, and, even more preferably, the insulin polypeptide is human insulin. The insulin-oligomer polypeptide conjugate of Formula III is preferably an amphiphilically balanced insulin-oligomer polypeptide conjugate.

In yet other embodiments, the insulin drug is an insulin-oligomer polypeptide conjugate comprising the structure of Formula IV:

image 1

in which:

m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30, is more preferably between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, is even more preferably between a lower limit of 3, 4, 5, 6, 7, 8 or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and is even more preferably between a lower limit of 3, 4, 5, 6 or 7 and an upper limit of 6, 7, 8, 9 or 10; n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and an upper limit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, is more preferably between a lower limit of 2, 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, is even more preferably between a lower limit of 3, 4, 5 or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11 or 12

5 polyalkylene glycol subunits, is even more preferably between a lower limit of 4, 5 or 6 and an upper limit of 6, 7 or 8 polyalkylene glycol subunits, and is, more preferably, 7; and R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety. The alkyl moiety may be a saturated or unsaturated, linear or branched alkyl moiety as will be understood by those skilled in the art, including, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,

10 nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl -ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl and 2-propynyl. The alkyl moiety is, more preferably, a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tert-butyl, pentyl or tert-pentyl. The alkyl moiety is, even more preferably, a C1 to C alkyl

15 C3. The alkyl moiety is, more preferably, methyl. The remaining fatty acid may be a saturated or unsaturated, linear or branched fatty acid residue, as will be understood by those skilled in the art, including, but not limited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linoleate, arachidonate , eicosapentaentoate, docosahexaenoate; acetate, caproate, caprylate, caprate, laurate, myristate, palmitate, stearate, arachididate, behenate, lignocerate and cerotate. The rest of sugar can be several sugar remains

20 as those skilled in the art will understand. Likewise, the rest of the alcohol can be several alcohol residues as those skilled in the art will understand.

In accordance with these embodiments of the present invention, the insulin polypeptide is preferably insulin.

or an insulin analog. More preferably, the insulin polypeptide is human insulin or a human insulin analog, and, even more preferably, the insulin polypeptide is human insulin. The conjugate

The insulin-oligomer polypeptide of Formula IV is preferably an amphiphilically balanced insulin-oligomer polypeptide conjugate.

In another embodiment, the insulin drug is an insulin-oligomer polypeptide conjugate comprising the structure of Formula V:

image 1

In accordance with these embodiments of the present invention, the insulin polypeptide is preferably insulin.

or an insulin analog. More preferably, the insulin polypeptide is human insulin or a human insulin analog, and, even more preferably, the insulin polypeptide is human insulin. When the insulin polypeptide in the structure of Formula V is human insulin and the oligomer is conjugated to the human insulin lysine B29, this insulin-oligomer conjugate is referred to herein as HIM2. Is

It is preferable to use a substantially monodispersed or monodispersed mixture of insulinoligomer conjugates. The insulin-oligomer polypeptide conjugate of Formula V is amphiphilically balanced when the insulin polypeptide is insulin.

HIM2 can be synthesized by various procedures, as those skilled in the art will understand. Preferably, HIM2 is synthesized using proinsulin as a starting material. For example, HIM2 has been synthesized as follows: Recombinant proinsulin having a leader peptide (PM 10,642 Dalton) was obtained from Biobras, of Belo Horizonte, Brazil. A 2.32 x 10-3 mmol portion of the proinsulin was dissolved in 10 ml of DMSO. To the solution was added 324 µl of triethylamine. The resulting solution was allowed to stir for 5 minutes and then a solution of activated methylheptaethylene glycol (oligomer (PEG7) -hexyl) (9.30 x 10-3 mmol) in acetonitrile was added. The evolution of the conjugation reaction (acylation) was monitored by HPLC. When the reaction appeared to be complete, it was quenched by the addition of 3.54 ml of 5% aqueous solution of trifluoroacetic acid. Then, the reaction mixture was processed and exchanged in 100 mM Tris-HCl buffer, at pH 7.6, to provide a mixture of the product. An aliquot of the Tris-HCl solution of the product mixture was analyzed by HPLC to determine the concentration of the polypeptide. A solution of trypsin (treated with TPCK; bovine pancreas) in 100 mM Tris-HCl buffer was prepared, at pH 7.6. A solution of 50 carboxypeptidase B (from porcine pancreas) in 100 mM Tris-HCl buffer was prepared, at pH 7.6. Then, the product mixture (0.424 mol / ml) was allowed to react with trypsin (5.97 x 10-4 mol / ml) and carboxypeptisase B (1.93 x 10-4 mol / ml). After 30 minutes, the reaction was quenched by the addition of 1.58 ml of 1% trifluoroacetic acid in acetonitrile. Majority products were identified by retention time in HPLC (with respect to retention times of known reference standards) and mass spectrum analysis. In this way insulin (10%) and

Insulin conjugated with LysB29-Hexyl-PEG7-oligomer (84%).

In the various embodiments of the insulin-oligomer polypeptide conjugates described above, the oligomer is covalently coupled to the insulin polypeptide. In some embodiments, the oligomer is coupled to the insulin polypeptide using a hydrolysable bond (eg, an ester or carbonate bond). A hydrolysable coupling can provide an insulin-oligomer polypeptide conjugate that acts as a prodrug. In other embodiments, the oligomer is coupled to the insulin polypeptide using a non-hydrolyzable bond (eg, a carbamate, amide or ether bond).

Insulin-oligomer polypeptide conjugates employed in the various embodiments described above can be synthesized by various procedures as will be understood by those skilled in the art. The oligomers according to embodiments of the present invention are substantially monodispersed and, preferably, are monodispersed.

A monodispersed mixture of insulin polypeptide-polypeptide conjugates can be synthesized using, for example, methods described in US Pat. No. 5,359,030 to Ekwuribe; U.S. Patent nº

5,438,040 to Ekwuribe; U.S. Patent No. 5,681,811 to Ekwuribe; or U.S. Pat. No. 6,309,633 to Ekwuribe et al. using a mixture of monodispersed polyethylene glycol (PEG) as a starting material. Such monodispersed PEG mixtures can be provided by, for example, procedures described in Yiyan Chen & Gregory L. Baker, Synthesis and Properties of ABA Amphiphiles, 64 J. Org. Chem. 6870-6873 (1999) and in Gérard Coudert et al., A Novel, Unequivocal Synthesis of Polyethylene Glycols, Synthetic Communications, 16 (1): 19-26 (1986). A preferred method of synthesizing monodispersed mixtures of PEG to be used to form monodispersed mixtures of insulin-oligomer polypeptide conjugates includes reacting a monodispersed mixture of compounds having the structure of Formula I:

R1 (OC2H4) n-O-X + (I)

wherein R1 is H or a lipophilic moiety; n is from 1 to 25; and X + is a positive ion with a monodispersed mixture of compounds having the structure of Formula II:

R2 (OC2H4) m-OMs (II)

wherein R2 is H or a lipophilic moiety; and m is 1 to 25,

under sufficient conditions to provide a monodispersed polymer blend comprising polyethylene glycol moieties and having the structure of Formula III:

R2 (OC2H4) m + n-OR1 (III)

Examples of reaction schemes are provided in Figures 14 and 15.

The procedures described above can be carried out using a pharmaceutical composition comprising an insulin drug as described above and a pharmaceutical carrier. The vehicle must, of course, be acceptable in the sense of being compatible with any of the other ingredients of the pharmaceutical composition and should not be harmful to the subject. The carrier may be a solid or a liquid, or both, and, preferably, is formulated with the insulin drug as a single dose formulation, for example a tablet, which may contain from about 0.01 or 0.5% to about 95 % by weight of the insulin drug. The pharmaceutical compositions can be prepared by any of the well-known pharmacy techniques, including, but not limited to, mixing the components, optionally including one or more auxiliary ingredients. See, for example, Remington, The Science And Practice of Pharmacy (9th Ed. 1995).

Pharmaceutical compositions suitable for oral administration may be presented in the form of small units, such as capsules, sachets, lozenges or tablets, in which each contains a predetermined amount of the mixture of insulin-oligomer polypeptide conjugates; in the form of powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an emulsion of oil in water or water in oil. Such formulations may be prepared by any suitable pharmacy procedure that includes the step of associating the insulin drug and a suitable vehicle (which may contain one or more auxiliary ingredients as indicated above).

In addition to an insulin drug, solid pharmaceutical compositions for oral administration according to embodiments of methods of the present invention may comprise several other ingredients as understood by those skilled in the art, including, but not limited to, one or more of the ingredients described in National Formulary 19, pages 2404-2406 (2000). For example, solid pharmaceutical formulations may include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; binding agents such as starches, gum arabic, microcrystalline cellulose, cellulose, methylcellulose and syrup; anti-caking agents such as calcium silicate; coating agents such as methacrylates and shellac rubber; preservatives such as methyl- and propylhydroxybenzoates; sweetening agents or flavoring agents. Polyols, buffers and inert fillers can also be used. Examples of polyols include, but are not limited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose and the like. Suitable buffers include, but are not limited to, phosphate, citrate, tartrate, succinate and the like. Other inert fillers that can be used encompass those known in the art and are useful in the manufacture of various dosage forms. If desired, solid formulations may include other components such

5 as volume forming agents and / or granulation agents and the like. Solid pharmaceutical compositions can be provided by various procedures as will be understood by those skilled in the art.

Solid dosage units for oral administration can be prepared by various procedures as will be understood by those skilled in the art. For example, the insulin drug may be mixed with a pulverulent solid carrier such as, for example, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with a talented antifriction agent such as, for example , magnesium stearate, calcium stearate and polyethylene glycol waxes. The mixture can then be compressed into tablets. Tablets for oral use can be prepared as follows, although other techniques can be used. The solid substances are ground or sieved to a desired particle size and the bonding agent is homogenized and suspended in a suitable solvent. The active ingredient and auxiliary agents are mixed with the binding agent solution. 15 The resulting mixture rewetts to form a uniform suspension. Normally, humidification causes the particles to aggregate slightly and the resulting mass is pressed through a stainless steel sieve having a desired size. The layers of the mixture are then dried in controlled drying units for a certain period of time to achieve a desired particle size and consistency. The granules of the dried mixture are filtered to remove any dust. To this mixture are added agents of

20 disintegration, antifriction and anti-adhesives.

Finally, the mixture is compressed into tablets using a machine with the appropriate dies and molds to obtain the desired tablet size. The person skilled in the art can select the operating parameters of the machine.

If coated tablets are desired, the cores prepared above can be coated with a solution

25 sugar concentrate, which may contain gum arabic, gelatin, talc, titanium dioxide, or with a lacquer dissolved in volatile organic solvent or a mixture of solvents. Additionally, the coating can be carried out in aqueous or non-aqueous media using various excipients, including, but not limited to, dispersed methylcellulose, dispersed ethylcellulose, dispersed methacrylates or mixtures thereof. Several dyes can be added to this coating in order to distinguish between tablets with different active compounds or with different amounts

30 of the active compound present.

Soft gelatin capsules can be prepared, in which the capsules contain a mixture of the active ingredient and a liquid, such as vegetable oil. Hard gelatin capsules may contain granules of the active substance in combination with a solid, a powdered carrier, such as, for example, lactose, sucrose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives or gelatin .

35 Dry powder capsules can be manufactured by various procedures understood by those skilled in the art. An embodiment of a dry powder capsule that can be administered orally according to embodiments of the present invention is as follows:

Component

% (p / p)

HIM2

1.11

Sodium colato

13.29

Capric acid

5.13

Lauric acid

5.13

Tris (hydroxymethyl) aminomethane

41.04

Sodium phosphate

30.97

Sodium hydroxide

1.03

Liquid pharmaceutical compositions that can be administered orally according to embodiments of

Methods of the present invention may various liquid pharmaceutical compositions that include the insulin drug as an active ingredient as will be understood by those skilled in the art, including, among others, solutions or suspensions in aqueous or non-aqueous liquids, and oil-in-water emulsions or of water in oil. In addition to the active insulin drug, the liquid pharmaceutical formulation may comprise several ingredients, including, but not limited to, absorption enhancers, buffering agents, polyhydric alcohols, polyalkylene oxides

45 and flavoring agents. Absorption enhancers may be various absorption enhancers as will be understood by those skilled in the art, including but not limited to bile acids such as, among others, colic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid and taurocholic acid and / or pharmaceutically acceptable salts (e.g., salts of earth metals) thereof, and fatty acids such as, among others, caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, acid

5 pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, oleic acid, eaidic acid, linoleic acid, arachidic acid, arachidonic acid and mixtures thereof.

The buffering agents may be various buffering agents as will be understood by those skilled in the art, including, but not limited to, tris (hydroxymethyl) aminomethane, triethanolamine, sodium phosphate, citric acid and mixtures thereof.

The polyhydric alcohols can be various polyhydric alcohols as will be understood by those skilled in the art, including, among others, glycerol. The polyalkylene glycols can be various polyalkylene glycols as will be understood by those skilled in the art, including, but are not limited to, polyethylene glycol and polypropylene glycol.

The flavoring agents can be various flavoring agents as will be understood by those skilled in the art, including, among others, natural or artificial flavors and sweeteners.

Embodiments of liquid pharmaceutical formulations that can be administered orally according to 15 embodiments of methods of the present invention include the following:

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Tris (hydroxymethyl) aminomethane

250 mM 3.03

Sodium phosphate

250 mM 3.00

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Tris (hydroxymethyl) aminomethane

500 mM 6.05

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Tris (hydroxymethyl) aminomethane

250 mM 3.03

Triethanolamine

 250 mM 3.73

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Citric acid

500 mM 9.60

Triethanolamine

 250 mM 3.73

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Citric acid

500 mM 9.60

Tris (hydroxymethyl) aminomethane

250 mM 3.03

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pHh to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Citric acid

500 mM 9.60

Sodium phosphate

250 mM 3.00

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

2 % 2.00

Citric acid

350 mM 6.72

Tris (hydroxymethyl) aminomethane

350 mM 4.24

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Lauric acid

2 % 2.00

Citric acid

350 mM 6.72

Triethanolamine

 350 mM 4.24

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Propylene glycol

twenty % 20.00

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

one % 2.00

Capric acid

0.5 % 0.50

Lauric acid

0.5 % 0.50

Triethanolamine

 350 mM 5.22

Tris (hydroxymethyl) aminomethane

350 mM 4.24

Sodium phosphate

350 mM 4.20

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

one % 2.00

Capric acid

0.5 % 0.5

Lauric acid

0.5 % 0.5

Citric acid

350 mM 6.72

Tris (hydroxymethyl) aminomethane

350 mM 4.24

Triethanolamine

 350 mM 5.22

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Component

 Conc. unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

one % 2.00

Capric acid

0.5 % 0.50

Lauric acid

0.5 % 0.50

Citric acid

175 mM 3.36

Triethanolamine

350 mM 5.22

Tris (hydroxymethyl) aminomethane

350 mM 4.24

Sodium phosphate

175 mM 4.20

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

adjust pH to 7.8 - -

Water

c.s. - -

Although the embodiments described above of pharmaceutical formulations comprise HIM2 as an active ingredient, it is understood that the active ingredient may be several other insulin drugs described herein. The active ingredient is preferably an insulin-oligomer polypeptide conjugate as described above.

The present invention will be described below with reference to the following examples. It should be appreciated that these examples are for purposes of illustrative aspects of the present invention and do not limit the scope of the invention as defined in the claims.

Examples

Example 1

5 Numerous studies with pancreatomized and normal fasting dogs have shown that orally administered conjugated insulin is rapidly absorbed in a dose-dependent manner and is associated with dose-dependent concomitant hypoglycemic effects. Figures 1a and 1b demonstrate these effects after several oral doses of HIM2 in pancreatomized dogs. At the highest dose of HIM2 studied, 1.0 mg / kg, all dogs required glucose rescue for marked symptomatic hypoglycemia.

10 Example 2 (Comparative Example)

A phase I study was conducted in healthy volunteers. In this study an earlier, less purified (and therefore less active) form of conjugated insulin (hexyl-insulin mixture, HIMX) was used. HIMX is approximately five times less active than the more purified HIM2; therefore, a dose of 2.4 mg / kg of HIMX is approximately equivalent to one of 0.5 mg / kg of HIM2. Fasting HIMX was administered at four different dose levels, after which insulin and glucose concentrations in peripheral blood were measured. As shown in Figure 2, insulin concentrations reached maximum levels (Cmax) in the physiological range 10-15 minutes after the oral dose of HIMX (dose volume 4 ml) and were dose dependent. When a larger dose volume (20 ml) was used for the same doses, the resulting Cmax of insulin at all doses was significantly higher, demonstrating a marked influence of the formulation volume on the

20 absorption. Figure 3b shows the plasma glucose values observed after the 20 ml dose volume, confirming the hypoglycemic effects dependent on the dose of conjugate insulin observed previously in dogs. No adverse effects of HIMX were observed in this study with healthy volunteers.

Example 3

The objectives of this study in patients with type 1 diabetes were: 1) to determine the safety and tolerance of

25 increasing doses of HIM2 administered orally; 2) estimate the kinetics of HIM2 after oral dose compared to subcutaneous doses with regular insulin and placebo; and 3) evaluate the pharmacodynamics of HIM2 compared to subcutaneous insulin using blood glucose levels as a marker of the drug's effect.

This was a randomized, double-blind study, with three doses plus active control (subcutaneous insulin), double simulation, placebo-controlled, five-period and cross-group design. After a fast

30 overnight, during which euglycemia was maintained by intravenous administration of insulin, insulin was then discontinued and, after a 2-hour wash period, single doses of oral placebo or HIM2 were administered to patients (0 , 15, 0.3 and 0.6 mg / kg) and regular subcutaneous insulin (4 IU) or placebo insulin, randomly, as shown in Table 1 below. Then, plasma glucose and insulin levels were measured in series over the next four hours.

35 Table 1

Study Period

Route of administration and treatment

Oral

Subcutaneous

Placebo

 0.15 mg / kg HIM2 0.3 mg / kg HIM2 0.6 mg / kg HIM2  Placebo  Recombinant human insulin (4 IU)

one

P1, P2 P3, P4, P5, P6, P7, P8 P3, P4, P5, P6, P7, P8 P1, P2

2

P3, P4 P1, P2 P5, P6, P7, P8 P1, P2, P5, P6, P7, P8 P3, P4

3

P5, P6 P7, P8 P1, P2, P3, P4 P1, P2, P3, P4, P7, P8 P5, P6

4

P7, P8 P1, P2, P3, P4, P5, P6   P1, P2, P3, P4, P5, P6 P7, P8

5

P1, P2, P3, P4, P5, P6, P7, P8    P1, P2, P3, P4, P5, P6, P7, P8

HIM2 was well tolerated and no signs of a dose-related increase in adverse events were observed. No patient had symptoms of hypoglycemia. After oral administration of HIM2, mean plasma insulin levels increased in a dose-dependent manner and reached a maximum within 15 minutes of dose administration, returning to baseline levels in 20 minutes (Figure 4a). With the dose of 0.6 mg / kg, insulin levels increased markedly in 7 of 8 (88%) patients. By comparison, 4 IU of regular insulin administered subcutaneously produced a large insulin peak that did not reach a maximum average concentration (Cmax) UP to 66 minutes after injection. In this group of patients, HIM2 seemed to prevent or mitigate the significant increase in plasma glucose levels seen in patients.

10 diabetics when all insulin therapy is withdrawn (Figure 4b). Additionally, in these patients a dose-dependent glucose stabilizing effect was observed, reflecting an evident decrease in hepatic glucose production after administration of the HIM2 dose (Figure 4b). The effects of glucose lasted up to 120 minutes, which was significantly longer than the expected duration from the duration of increases in plasma insulin levels.

15 Single doses of HIM2 at 0.15, 0.3 and 0.6 mg / kg were well tolerated by patients with type 1 diabetes and no signs of a dose-related increase in adverse events were observed. There were increases related to the dose of plasma insulin levels, with a rapid achievement of Cmax values in 15 minutes and a return to baseline values in 90-120 minutes. The expected increase in plasma glucose levels after withdrawal of any other insulin treatment was attenuated or prevented and the effects on

20 glucose persisted for substantially longer than expected from the duration of increases in plasma insulin levels. These results indicated that orally administered HIM2 is likely to influence peripheral glucose levels by a combination of decreased hepatic glucose secretion and peripheral insulin-like activity. A potential role of HIM2 has been suggested in the treatment of diabetes by reproducing physiological hepatic portal release.

25 Example 4

In a second early phase II study, a sequential 2-dose escalation scheme was used in patients with type 1 diabetes. As in the first study with patients with type 1 diabetes, patients fasted overnight and stayed in a euglycemic state by intravenous insulin administration. In the morning, after withdrawal of intravenous insulin and a 2-hour wash period, patients received 30 doses of 0.6, 0.8 or 1.0 mg / kg of HIM2 in groups of six patients at each level. of dose. Peripheral glucose and insulin levels were determined at regular intervals after dose administration. A second dose of HIM2 was administered 2 hours after the first dose, in order to assess the effects of a "priming" dose of oral HIM2 on liver glucose production and peripheral insulin concentrations. It was found that the liquid formulation used in this study irritated the oropharynx in the first six subjects who

35 received the drug. Therefore, the study was interrupted while a capsule formulation was developed as a replacement. Upon restarting the Studio, a semi-solid dosage formulation was used in two capsule sizes.

HIM2 3 mg capsule formulation: HIM2 15 mg capsule formulation:

Component

MS number mg / capsule Percentage Weight

HIM2 as a protein

236  3.00 1.6 4.35

Polyethylene Glycol 400

027 30.00 16.2 43.5

USP water

012 1.5 0.8 2.18

Labrasol

 240 117.00 63.2 169.95

Syloid 244

238 33.00 17.8 47.85

Monobasic Sodium Phosphate

 239 0.09 0.05 0.13

Dibasic sodium phosphate

237 0.41 0.23 0.59

Total

185 100 268.25

Size: “3” green jelly capsules

Component

MS number mg / capsule Percentage Weight

HIM2 as a protein

 236 15.00 3.2 10.2

Polyethylene Glycol 400

027 75.0 16.0 51.00

USP water

012 3.8 0.8 2.58

Labrasol

 240 292.0 62.1 198.56

Syloid 244

238 83.0 17.6 56.44

Monobasic Sodium Phosphate

 239 0.2 0.04 0.14

Dibasic sodium phosphate

237 1.0 0.21 0.68

Total

470 100 319.60

Size: “O EL” green gelatin capsules

The results of this study demonstrate that (a) the new capsule formulation is well tolerated, (b) no side effects were observed and (c) the effects on blood glucose varied from stabilization to 5 moderate decreases with respect to values Baseline one to two hours after administration (Figure 5). There was no significant difference in the magnitude of glucose responses in the dose range used, as seen in Figure 6, which shows the average responses to the lowest and highest doses used in the study. Patients did not experience hypoglycemia with any dose. These results suggest that oral HIM2, at the doses used, influences glucose levels mainly affecting the secretion of

10 hepatic glucose and no significant effects on peripheral insulin levels.

Example 5

In two additional studies with patients with type 1 diabetes using the capsule formulation, the influence of food on the absorption and hypoglycemic activity of HIM2 has been investigated, as well as the effects of HIM2 when administered concomitantly with a conventional baseline regimen ( provided by continuous subcutaneous insulin infusion or ISCI). Preliminary results of the first study indicate that a conventional meal ingested at the same time as the dose of HIM2 may attenuate the absorption of HIM2 and hypoglycemic effects. However, if HIM2 is administered 30 minutes before ingestion of a conventional meal, absorption and effects on glucose are substantially preserved. The second study evaluates the absorption and effects of HIM2 when administered to patients with fasting type 1 diabetes who are maintained with basal insulin by ISCI, followed by fasting or a conventional meal. Preliminary results in a limited number of patients indicate that HIM2 not only reduces fasting glucose levels, but also attenuates or prevents the expected post-pandial increases in plasma glucose concentrations (Figures 7 and 8). A first study in patients with type 2 diabetes has shown similar effects of HIM2 on fasting blood glucose levels in a limited number of patients. In others

25 studies are investigating the potential of HIM2 to attenuate postprandial hyperglycemia in patients with type 2 diabetes. In a study with 12 patients with type 2 diabetes, a single oral dose (without a holder) is administered followed by a meal. In another study with 24 patients with type 2 diabetes, 3 doses / day (without holder) are given with meals for 3 days. Also, a late dose is administered at night to check the possible effects on hyperglycemia.

30 Example 6

CF-1 male mice (n = 5-10 per group) were fasted overnight and then given HIM2 (1.25 or 2.5 mg / kg, orally, in a formulation that was sample in the following table, 10 ml / kg) or human recombinant insulin (12.5 or 25 /g / kg), abdominal sc, in 1% acetate buffer, at pH 4.1)

Component

 Conc. Unity % (p / v)

HIM2

2.5 mg / ml 0.25

Sodium colato

3 % 3.00

Oleic acid

one % 2.00

Capric acid

0.5 % 0.5

Lauric acid

0.5 % 0.5

Citric acid

350 mM 6.72

Tris (hydroxymethyl) aminomethane

350 mM 4.24

Triethanolamine

 350 mM 5.22

Sucralose

 0.2 % 0.20

Strawberry aroma

0.4 % 0.4

Sodium hydroxide

Adjust pH to 7.8 - -

Water

c.s. - -

After 15, 20 or 60 minutes, blood samples were obtained in heparinized syringes, with anesthesia, from the portal vein (VP) and vena cava (VC) of each animal. A drop of blood was placed in a blood glucose meter for the measurement of blood glucose levels. The remaining sample was centrifuged to separate the plasma for the determination of immunoreactive insulin by ELISA (ALPCO).

As illustrated in Figure 16, large increases in insulin (up to 2789 /U / ml) were observed in VP 15 minutes after oral administration of HIM2 and insulin concentration decreased to 221 1U / ml at 30 minutes of administration. This profile resembles that of first-stage insulin secretion in response to

10 a meal, as illustrated in Figure 17, showing blood glucose and plasma insulin levels in the portal vein and vena cava after oral administration of dextrose solution in fasting mice. As further illustrated in Figure 16, maximum insulin levels in the VC were 31 to 36% of those in the VP.

In contrast, as illustrated in Figure 18, subcutaneous insulin administration produced 2.42.6 times higher concentrations in VC (i.e., periphery) (up to 186 186U / ml, 15 minutes after administration) than in the VP.

15 Likewise, there was a slower return to baseline values (74 /U / ml), 30 minutes after administration).

Despite the marked differences in insulin levels, HIM2 did not produce correspondingly greater decreases in blood glucose levels, compared to decreases in blood glucose levels produced by subcutaneous administration of insulin in these mice.

20 diabetics For example, as illustrated in Figure 18, oral administration of HIM2 resulted in a decrease in blood glucose levels to approximately 31 mg / ml in VC compared to a decrease in blood glucose levels to approximately 23 mg / ml in the VC illustrated in Figure 19 for subcutaneous administration of insulin. These results seem to indicate that oral administration of HIM2 has less tendency to induce hypoglycemia than subcutaneous administration of insulin.

25 In untreated mice, blood glucose levels were higher in the VC than in the VP. This difference between VP-VC was reversed by oral administration of HIM2, but was not reversed by subcutaneous administration of insulin. These results seem to indicate that oral administration of HIM2 provides greater suppression of hepatic glucose secretion than subcutaneous administration of insulin.

Claims (20)

1. A substantially monodispersed mixture of conjugates, in which each comprises an insulin-oligomer polypeptide conjugate comprising the structure: Insulin polypeptide-X (CH2) m (OG2H4) nOR (III) in which: X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a moiety carbonate, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; m is between a lower limit of 1 and an upper limit of 30; n is between a lower limit of 1 and an upper limit of 50; Y R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety. 2. A monodispersed mixture of conjugates, in which each comprises an insulin-oligomer polypeptide conjugate comprising the structure: Insulin polypeptide-X (CH2) m (OC2H4) nOR (III) wherein: X is an ester moiety, a thioester moiety, an ether moiety, a carbamate moiety, a thiocarbamate moiety, a carbonate moiety, a thiocarbonate moiety, an amide moiety, a urea moiety or a covalent bond; m is between a low limit of 1 and an upper limit of 30; n is between a lower limit of 1 and an upper limit of 50; Y R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety or a fatty acid moiety.

3.

 The mixture of claim 1 or 2, wherein R is an alkyl moiety.

Four.

 The mixture of claim 1 or 2, wherein R is a fatty acid residue.

5.

 The mixture of claim 1 or 2, wherein the insulin polypeptide is human insulin and the oligomer is covalently linked to LysB29 of human insulin.

6.

 A pharmaceutical composition, comprising the mixture of any of claims 1-5 in a pharmaceutically acceptable carrier.

7.

 The use of the mixture of any of claims 1-5 for the manufacture of a medicament for the treatment of diabetes.

8.

A method of synthesizing a substantially monodispersed mixture of polymers, comprising polyethylene glycol moieties, said process comprising:

Reacting a substantially monodispersed mixture of compounds having the structure of Formula I: R1 (OC2H4) m-OX + (I) wherein R1 is H or a lipophilic moiety; m is from 1 to 25; and X + is a positive ion with a substantially monodispersed mixture of compounds having the structure of Formula II: R2 (OC2H4) nOMs (II) wherein R2 is H or a lipophilic moiety; Ms is a mesylate residue; and n is 1 to 25, under conditions sufficient to provide a substantially monodispersed mixture of polymers comprising polyethylene glycol moieties and having the structure of Formula III: R2 (OC2H4) m + n-OR1 (III)

9.

 The process according to claim 8, wherein the polymer mixture of Formula III is a monodispersed mixture.

10.

 A monodispersed mixture of polymers comprising polyethylene glycol moieties, said polymers synthesized by the method of claim 8.

eleven.

 The process of claim 8, wherein R2 is a lipophilic moiety selected so that the polymers of Formula III are substantially water insoluble.

12.

 The use of a substantially monodispersed mixture of an insulin-oligomer polypeptide conjugate, comprising the structure of Formula V:

for the preparation of an orally administrable medication for diabetes mellitus treatments.

13.

 The use according to claim 12, wherein the insulin polypeptide is insulin.

14.

 The use according to claim 13, wherein the insulin is human insulin.

fifteen.

 The use of claim 13 or 14, wherein the oligomer is attached to the lysine in the B29 position of insulin.

16.

 The use of any of claims 12-15, wherein the conjugate of Formula V is amphiphilically balanced.

17.

 The use of claim 12, wherein the insulin polypeptide is an insulin analog selected from the group consisting of human GlyA21 insulin; insulin GlyA21 GlnB3, human; AlaA21 insulin, human; insulin AlaA21 GlnB3, human; GlnB3 insulin, human; GlnB30 insulin, human; insulin GlyA21 GluB30, human; insulin GlyA21 GlnB3 GluB30, human; Insulin GlnB3 GluB30, human; human AspB28 insulin; LysB28 insulin, human; insulin LeuB28, human; ValB28 insulin, human; AlaB28 insulin, human; insulin AspB28 ProB29, human; human LysB28 ProB29 insulin; insulin LeuB28 ProB29, human; ValB28 ProB29 insulin, human; Insulin AlaB28 ProB29, human.

18.

 The use of any of claims 12-17, wherein the conjugate of Formula V is present as a monodispersed mixture.

19.

 The use of any of claims 12-19 in which the amount of conjugates in the medicament is such that it provides a concentration of the insulin polypeptide in the blood of the portal vein between 10 and 1000 U / ml in approximately 60 minutes after administration.

twenty.

 Use of any of claims 12-18, wherein the amount of conjugates in the medicament is such that it stabilizes the concentration of peripheral glucose in the treated subject to approximately +/- 50 percent of an average glucose concentration measured for approximately one hour starting approximately 30 minutes after administration.