Glycated proteins

Abstract

Discloses herein are conjugates of a protein and one or more reducing sugars, wherein one, two, three or more target amino groups of the protein are glycated. Also disclosed are methods for the preparation of the glycated protein. Conjugates having one or more ketoamine linkages are efficiently prepared through the reaction between the protein and reducing sugars and exhibit desirable pharmaceutical properties.

Description

GLYCATED PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 509,161, filed on June 20, 2023, the content of which is herein incorporated by reference into the subject application.TECHNICAL FIELD

[0002] The present invention relates to conjugates of a reducing sugar and a protein in and methods for preparing the conjugates via glycation of the a and / or s-amino groups of the protein. In particular, the invention relates to a method of glycation of the two a-amino groups and solitary s-amino group of the protein with reducing-sugars to produce a high yield of mono-glycated, di-glycated or tri -glycated protein in which the reducing-sugar is covalently attached by a ketoamine linkage.BACKGROUND

[0003] Insulin is a small protein composed of fifty-one amino acids that is essential for the treatment of diabetes. In its native form, its effectiveness in preventing hyperglycemia is limited to 4-6 hours requiring multiple injections per day to control blood sugar levels. Since its discovery extensive research has been undertaken to develop improved insulin therapeutics in order to decrease the number of daily injections and more closely approach the natural control of glucose levels. A number of improved fast acting and basal insulins have been produced that are now in use. However, the administration of insulin and management of glucose levels are still not ideal.

[0004] Glycation, sometimes called non-enzymatic glycosylation, is the covalent attachment of a sugar to a protein by the reaction of an amino group with a reducing-sugar. Under aqueous conditions, reducing-sugars like glucose react with amino groups forming a covalent ketoamine linkage. Insulin has three amino groups and the effect of glycation of insulin is of particular interest because high in vivo concentrations of glucose in diabetics can lead to the glycation of insulin’s amino groups. However, as a result of the multilayered procedures required for preparing glycated or glycosylated insulins and the lack of clarity of the effects on activity, glycated insulin has received very little attention as a possible therapeutic for the treatment of diabetes.

[0005] A need exists for the development of novel modified insulin and other biologically important or active proteins aimed at improving their physical and pharmaceutical properties, enhancing activity and improving parenteral and oral delivery systems as well as reducing the cost of production.SUMMARY

[0006] The compounds of this patent document address the need. An aspect of the disclosure provides a glycated protein, wherein one or more target amino groups of the protein are glycated by a reducing sugar, and in some embodiments wherein the extent of glycation on one or more target amino groups of the protein can reach greater than 80%. The glycated protein may be mono-, di- or triglycated.

[0007] In some embodiments, the three amino groups are glycated with three same reducing sugars. In some embodiments, the three amino groups are glycated with two or three different reducing sugars. In some embodiments, two amino groups are glycated with two same reducing sugars. In some embodiments, two amino groups are glycated with two different reducing sugars.

[0008] The reducing sugar may be a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, the reducing sugar is selected from the group consisting of allose, altrose, glucose, gulose, iodose, mannose, galactose, talose, ribose, arabinose, xylose, lyxose, erythrose, threose, glyceraldehyde, psicose, fructose, sorbose tagatose, ribulose, xylulose, erthrulose, dihydroxyacetone, maltose, lactose, sucrose, and cellobiose.

[0009] Another aspect provides a method of glycating the protein. The method includes(a) lyophilizing an aqueous mixture comprising the protein and a reducing sugar to produce a lyophilizate; and(b) heating the lyophilizate at a temperature for a period of time in a reactor in the presence of water vapor, wherein overall pressure in the reactor ranges from about 0.001 mbar to about 100 mbar.

[0010] In some embodiments, the lyophilizate is substantially free from liquid water in step (b). In some embodiments, the water vapor is unsaturated. In some embodiments, the water vapor is generated from liquid water during the heating.

[0011] In some embodiments, after step (a) and before step (b), the method includes introducing water to the reactor and freezing the water so that it is not in contact with the lyophilizate. In some embodiments, step (b) proceeds at a temperature ranging from about 40 °C to about 150 °C.

[0012] In some embodiments, the water vapor in the reactor has a vapor pressure ranging from about 0.001 mbar to about 10 mbar. In some embodiments, the water vapor in the reactor has a vapor pressure ranging from about 0.01 mbar to about 1 mbar.

[0013] In some embodiments, the reactor is a closed system. In some embodiments, the aqueous mixture has a pH ranging from about 4 to about 11. In some embodiments, the heating continues for a period ranging from about 1 to about 72 hours.

[0014] In some embodiments, the ratio between the protein and the reducing sugar ranges from about 1: 10 (wt / wt) to about 10: 1 (wt / wt). In some embodiments, the reducing sugar comprise 1 to 10 saccharide units.

[0015] Another aspect of this patent document discloses a pharmaceutical composition comprising the compounds or glycated insulin described herein or the pharmaceutically acceptable salt, isomer, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0016] Another aspect of this disclosure provides a method of treating a disease or a condition in a subject, wherein the disease or condition is related to the deficiency or insufficiency or inadequacy of a biologically active protein. The method includes administering to the subject a therapeutically effective amount of a glycated form of the protein disclosed herein. In some embodiments, all three amino groups are glycated by a reducing sugar.

[0017] Another aspect of this disclosure provides a method of treating deficiency of a protein in a subject. The method includes administering to the subject a therapeutically effective amount of a glycated form of the protein disclosed herein.DETAILED DESCRIPTION

[0018] Various embodiments of this patent document disclose methods of preparing glycated proteins including for example insulin, glucagon-like peptide- l(GLP-l), adropin, and various hormones. The methods generally include reacting a lyophilized mixture of protein with a reducing sugar under reduced pressure in the presence of controlled water vapor pressure and temperature. As a result, one or more amino groups of the protein are covalently attached to sugars by a ketoamine linkage to provide a mono-, di-, tri-, or multi-glycated protein.

[0019] While the following text may reference or exemplify specific embodiments of a glycated protein or a method of preparation, it is not intended to limit the scope of the compound or method to such particular reference or examples. Various modifications may be made by those skilled in the art,in view of practical and economic considerations, such as pH range of the lyophilized mixture, the water vapor pressure and the temperature of the glycation process.

[0020] The articles "a" and "an" as used herein refers to "one or more" or "at least one," unless otherwise indicated. That is, reference to any element or component of an embodiment by the indefinite article "a" or "an" does not exclude the possibility that more than one element or component is present.

[0021] The “amino” refers to -NH2.

[0022] The term "subject" encompasses any animal, but preferably a mammal, e.g., human, nonhuman primate, a dog, a cat, a horse, a cow, or a rodent. More preferably, the subject is a human.

[0023] The term “excipient” refers to an inert substance that is added to a solution or mixture to promote the reaction.

[0024] The term “glycation” or “glycating” as used herein refers to the covalent attachment of a reducing sugar to a protein (e.g. insulin) by the reaction of an amino group of the protein with a carbonyl group (ketone or aldehyde) of the reducing-sugar to form a ketoamine linkage. A mono-glycated, diglycated or tri-glycated protein contain one, two, or three ketoamine linkages, respectively resulting from the glycation of the protein.

[0025] The term “unsaturated water vapor” refers to a state in which the amount of water vapor present in the reactor is less than the maximum amount the reactor can hold at a given temperature and pressure. In other words, the reactor is capable of holding more water vapor without condensing into liquid water. The term "unsaturated" indicates that there is a potential for the reactor to become saturated, meaning it can reach a point where it cannot hold any more water vapor and condensation occurs.

[0026] The term “reducing sugar” refers to any sugar that is capable of acting as a reducing agent. A reducing sugar can be any of all monosaccharides, some disaccharides, some oligosaccharides, and some polysaccharides.

[0027] The term “ketoamine” or “ketoamine linkage” refers to a moiety formed from the amino group of a protein and the carbonyl group of a sugar. As illustrated in the example scheme below, the boxed moiety is a ketoamine linkage in a conjugate from the reaction between a sugar and a protein.H2N— protein Ri proteinR1 : sugar chain ketoamineR2: H or CH20H linkage

[0028] The term "treating" or "treatment" of any disease or condition refers, in some embodiments, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In some embodiments "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In some embodiments, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In some embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder, or even preventing the same . “Prophylactic treatment” is to be construed as any mode of treatment that is used to prevent progression of the disease or is used for precautionary purpose for persons at risk of developing the condition.

[0029] The term “pharmaceutically acceptable salts” means salts of compounds which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy- 2-ene- 1 -carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, -tol uenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Non-limiting examples of acceptable organic bases includeethanolamine, diethanolamine, triethanolamine, tromethamine, and A-methylglucamine. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0030] The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or additional carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a pharmaceutical composition exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. In some embodiments, pharmaceutically acceptable salts of the compounds disclosed herein are provided.

[0031] The term "subject" encompasses any animal, but preferably a mammal, e.g., human, nonhuman primate, a dog, a cat, a horse, a cow, or a rodent. More preferably, the subject is a human.

[0032] The term “carrier” refers to a chemical compound that facilitates the incorporation of a compound into cells or tissues.

[0033] The term “therapeutically effective amount” refers to an amount of a compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0034] Chemical glycosylation of insulin has been used as a strategy to elucidate the effect of glycation on its activity. It differs from naturally occurring aqueous glycation in that a reactive chemical reagent is used to achieve a covalent attachment of the sugar and, unlike the aqueous glycation that occurs in vivo, the sugar is not attached by a ketoamine linkage.

[0035] Prior to the present disclosure, a tri -glycated insulin, in which all the amino groups were glycated through a ketoamine linkage, had never been reported and its properties and biological activity were unknown and unpredictable. Determination of the effects of naturally occurring aqueous glycation on the activity of insulin was made difficult by the fact that glycation of amino groups in water is a highly unfavorable reaction and extended periods of reaction time and high concentrations of reducing- sugar are required to achieve significant glycation. Of particular note is that even after such incubations a tri-glycated insulin with ketoamine linkages has never been reported.

[0036] The methodology previously described in literature for in-vacuo glycation of proteins applied only to the glycation of the s-amino groups of large globular proteins. While glycation was achieved, it did not appear to be quantitative. For instance, insulin is a small protein and when incubated with glucose under same in-vacuo conditions as large globular proteins, it was not as readily glycated. While large globular proteins have many s-amino groups, insulin has only one s-amino group, and two terminal a-amino groups from its A and B chains. The a-amino group differs substantially from an s-amino group in its chemical properties, namely, pKaand reactivity, and there was no evidence that the a-amino groups of insulin could be glycated in-vacuo in the same manner as s-amino groups. It was not obvious what modifications were necessary to achieve quantitative glycation of insulin and indeed significant modifications to the previously described in-vacuo glycation methodology had to be made.

[0037] An aspect of this patent document provides a method of glycating a protein (e.g. insulin, adropin, GLP-1, etc.) having one or more amino groups. The method generally includes lyophilizing an aqueous mixture of the protein and a reducing sugar or a reducing compound containing an aldehyde or a ketone group with an a-hydroxy on the carbon immediately adjacent to the aldehyde or ketone group to produce a lyophilizate; and heating the lyophilizate in a reactor in the presence of water vapor. The protein may contain one, two, three, more, five or more amino groups. In some embodiments, the protein has a molecular weight ranging from about 500 to about 1,000, from about 1,000 to about 5,000, from about 1,000 to about 10,000, from about 1,000 to about 15,000, from about 5,000 to about 10,000, from about 10,00 to about 50,000, from about 50,00 to about 100,000, from about 100,00 to about 200,000, or from about 200,00 to about 500,000 Da. In some embodiments, the protein has a molecular weight of about 500, about 800, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 10,000, about 12,000, about 15,000 Da or any range between any two of the aforementioned values. In some embodiments, the protein is insulin or GLP-1.

[0038] Each of the amino groups of the protein may independently be alpha-, beta-, gamma-, delta-, epsilon-, or zeta- amino group. In some embodiments, the protein, straight chain or branched, contains at least one, at least two, at least three, or multiple (1, 2, 3, 4, 5, 6, 7, 8 or more) a-amino group. The amino group is NH2. In some embodiments, the protein contains 1, 2 or 3 a-amino groups. In some embodiments, the protein further contains an s-amino groups. In some embodiments, the amino groups capable of being glycated in the protein consist of 1 or 2 a-amino groups and 1, 2, or more s-amino groups. In some embodiments, the amino groups capable of being glycated in the protein consist of 2 a-amino groups and 1 s-amino group. In some embodiments, the amino groups capable of being glycated in the protein consist of 1 a-amino groups and 2 s-amino groups.

[0039] Non-limiting examples of proteins that can be glycated include Adrenocorticotropic hormone (ACTH), Adropin, Amylin, Angiotensin, Atrial natriuretic peptide(ANP), Calcitonin, Cholecystokinin (CCK), Gastrin, Ghrelin, Glucagon, Glucose-dependent insulinotropic polypeptide(GIP), Glucagon-like peptide- l(GLP-l), Growth hormone, Follicle-stimulating hormone (FSH), Insulin, Leptin, Luteinizing hormone (LH), Melanocyte -stimulating hormone (MSH), Oxytocin, Parathyroid hormone(PTH), Prolactin, Renin, Somatostatin, Thyroid-stimulating hormone (TSH), Thyrotropin-releasing hormone (TRH), Vasopressin, also called arginine vasopressin (A VP) or antidiuretic hormone (ADH), Vasoactive intestinal peptide (VIP), Somatotropin (GH1), Gonadotropin Releasing Hormone 1 (GNRH1, 2), Growth Hormone Releasing Hormone (GHRH), Parathyroid Hormone Like Hormone (PTHLH), Corticotropin Releasing Hormone (CRH), Anti-Mullerian Hormone(AMH), Chorionic Somatomammotropin Hormone 1 (CSH1), ChorionicSomatomammotropin Hormone 2 (CSH2), Pro-Melanin Concentrating Hormone (PMCH), and Resistin (RETN).

[0040] The presence of liquid water may lead to undesirable side products. Accordingly, the lyophilizate in the heating step is preferably free from liquid water in the heating step. In some embodiments, lyophilizate contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1% by weight liquid water, or substantially free from liquid water. In some embodiments, the reactor contains liquid water but the liquid water is not in contact with the lyophilizate.

[0041] Without being bound by any particular theory, it is postulated that water vapor could interact with the protein-reducing-sugar lyophilizate and facilitate the proton transfers. The water vapor pressure may range from about 0.001 to about 300, from about 10 to about 200, from about 1 to about 100, from about 10 to about 100, from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 40 to about 90, from about 40 to about 80, from about 50 to about 70, from about 40 to about 60, from about 60 to about 80, from about 0.001 to about 50, from about 0.001 to about 20, from about 0.001 to about 10, from about 0.01 to about 5, from about 0.01 to about 2, from about 0.01 to about 1, from about 0.1 to about 5, from about 0.1 to about 1, or from about 0.5 to about 1 mbar. Nonlimiting examples of the water vapor pressure in the reactor include 0.001, 0.002, 0.005, 0.008, 0.01, 0.02, 0.05, 0.08, 0.10, 0.15, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.0, 1.2, 1.4, 1.6, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, and any range between any two values disclosed above (all values in mbar). In some embodiments, the water vapor is unsaturated during the heating step.

[0042] The overall pressure in the reactor may range for example, from about 0.01 to about 1000, from about 0.01 to about 800, from about 0.01 to about 500, from about 0.01 to about 200, from about 0.01 to about 100, from about 1 to about 100, from about 10 to about 100, from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 40 to about 90, from about 40 to about 80, from about 50 to about 70, from about 40 to about 60, from about 60 to about 80, from about 0.01 to about 10, from about 0.01 to about 5, from about 0.01 to about 2, or from about 0.01 to about 1 mbar. Nonlimiting examples of the overall pressure in the reactor include 0.01, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 20, 30, 40, 50, 80, 100, 150, 200, 300, 400, 500, 600, 800, and any range between any two values disclosed above (all values in mbar).

[0043] The water vapor can be generated in any suitable means. Under low pressure conditions an added portion of liquid water vaporizes and facilitates proton transfers in the glycation reaction without disturbing the lyophilizate. When this is carried out a substantial increase in the extent of glycation can be observed. The reactor may contain a separate compartment for holding liquid water, which vaporizes under vacuo or low pressure and heating condition. Alternatively, a suitable amount of water can be added to the reactor containing the insulin-reducing-sugar lyophilizate and frozen so that it is not in contact with the lyophilizate. Preferably, the reactor is a closed or sealed system.

[0044] Besides water vapor, other acid vapor may also serve as a proton source. Nonlimiting examples of the acid include formic acid, acetic acid, and trifluoroacetic acid. The amounts and partial pressure of the acid can be readily adjusted to meet the needs of a specific reaction. Other proton source such as alcohol (e.g. methanol, ethanol, propanol) can also be used alone or in combination with water or acid.

[0045] Various other factors may impact the outcome of the glycation process, including for example, the ratio of protein or insulin to reducing-sugar; pH of lyophilization (LpH); the amount of added liquid water or the pressure of the water vapor; incubation temperature; time of incubation; number of incubations. In cases where glycation appeared to be less than quantitative in the first incubation, as determined by free amino analysis, a second, third, fourth, fifth or additional incubation can be performed by re-dissolving the lyophilizate in water followed by re-lyophilization and repeating the incubation.

[0046] The ratio between insulin or a protein and the reducing sugar may range from about 1 : 10 to about 10: 1, from about 1 : 5 to about 10: 1, from about 1 : 2 to about 10: 1, from about 1 : 1 to about 10: 1, from about 2: 1 to about 10: 1, from about 3: 1 to about 10: 1, from about 4: 1 to about 10: 1, or from about 5: 1 to about 10: 1 (wt:wt). Nonlimiting examples of the ratio between the protein and the reducingsugar include about 15: 1, about 10: 1, about 8: 1, about 5: 1, about 2: 1, about 1: 1, about 1:2, about 1:5, about 1: 10, and any range between any two of the above disclosed values. When the aqueous mixture includes two or more reducing sugars, the above ratio also applies to each individual reducing sugar.

[0047] The ratio between protein (e.g. insulin or GLP-1) and the reducing sugar or a reducing compound may also be a molar ratio between the aldehyde group or ketone group of a reducing compound and the particular amino group of interest. For example, if only a particular a-amino groups or a particular e-amino group is to be glycated. The molar ratio between the aldehyde group or ketone group of the reducing compound and the particular amino group may range from about 1 : 3 to about 5: 1, from about 1:2 to about 2: 1, from about 1: 1 to about 3: 1, from about 1: 1 to about 2: 1, or from about 2: 1 to about 1: 1. Nonlimiting examples of the molar ratio between the aldehyde group or ketone group of the reducing compound and the particular amino group include about 3: 1, about 2: 1, about 1: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, and any range between any two of the above disclosed values. The respective amounts of the reducing compound / sugar and the protein can be calculated accordingly. When the aqueous mixture includes two or more reducing sugars, the above ratio also applies to each individual reducing sugar. When two or more amino groups in the protein (e.g. 1 a- amino and 1 a-amino groups, 2 a-amino and 1 a-amino groups, etc.) are to be glycated, the above ratios still apply and can be adjusted based on specific proteins and reducing compounds. One of ordinary skill in the art will be able to make the adjustment without undue experiments.

[0048] The pH of the initial aqueous mixture may also impact the outcome of the glycation. In some embodiments, the pH ranges from about 2 to about 12, from about 3 to about 12, from about 4 to about 11, from about 5 to about 11, from about 6 to about 11, from about 7 to about 9, from about 7 to about 11, from about 8 to about 11, from about 9 to about 11, or from about 10 to about 11. Nonlimiting examples of the pH value include about 6.0, about 6.5, about 7.0, about 7.2, about 7.5, about 7.8, about 8.0, about 8.2, about 8.5, about 8.8, about 9.0, about 9.5, and about 10.0, and any range between any two of the above disclosed values.

[0049] The temperature for the heating or incubation step may range from about 30 °C to about 250 °C, from about 30 °C to about 200 °C, from about 40 °C to about 200 °C, from about 40 °C to about 150 °C, from about 40 °C to about 100 °C, from about 50 °C to about 100 °C, or from about 60 °C to about 90 °C. Nonlimiting examples of the temperature include about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 70 °C, about 80 °C, about 90 °C, about 100 °C, about 100 °C, about 110 °C, about 120 °C, about 130 °C, about 140 °C, about 150 °C, about 160 °C,about 170 °C, about 180 °C, about 190 °C, about 110 °C, about 200 °C, and any range between any two of the above disclosed values.

[0050] Because different reducing sugars may exhibit different reactivities, the incubation temperature may be controlled or adjusted accordingly to achieve selective glycation. For instance, when glycating an 8-amino group, a lower temperature can be applied to enhance the selectivity towards e-amino over a-amino. If a mixture of reducing sugars is used, the temperature can be raised in stages so that the more reactive sugar (e.g. aldehyde containing sugar) reacts with an amino group first and the less reactive sugar (e.g. ketone-containing sugar or some polysaccharide with more bulky structures) reacts mostly at a higher temperature. The temperature control can be used together with the amounts of the reactants as described above.

[0051] The heating or incubation at an elevated temperature may continue for a period ranging from about 1 to about 72 hours, from about 1 to about 60 hours, from about 1 to about 48 hours from about 1 to about 36 hours, from about 1 to about 24 hours, from about 1 to about 12 hours, from about 1 to about 6 hours. Nonlimiting examples of the length of the heating or incubation period include about 1, about 2, about 3, about 5, about 8, about 10, about 12, about 15, about 20, about 24, about 30, about 32, about 40, about 48, about 55, about 60, about 72 hours, about 4 days, about 6 days, about 8 days, about 10 days, and any range between any two of the above disclosed values. The reaction time can be adjusted depending on the reducing sugar and / or the target amino to be glycated. When multiple sugars or multiple amino groups are involved in glycations, reactions time for each stage can be the same or different.

[0052] The above mentioned reducing compounds containing one or more aldehyde or ketone active groups with adjacent a-hydroxyl group can be used for glycation of one, two or more proteins in the same reaction mixture. With two or more such active groups, a compound can serve as a cross linker by glycating two proteins or two moieties of the same protein.

[0053] In some embodiments, the compound containing one or more aldehyde and / or ketone groups is a reducing sugar. Various reducing sugars can be applied to the methods disclosed herein. In some embodiments, the sugar is a monosaccharide. In some embodiments, the reducing sugar is selected from pentose, tetrose, keto hexose, keto pentose, and keto tetrose. In some embodiments, the reducing sugar is a disaccharide, a trisaccharide, or a polysaccharide. In some embodiments, the reducing sugar contains 1-50, 1-30, 1-20, 1-10, 1-8, 1-5 or 1-3 saccharide units, each unit being similar to a monosaccharide. Nonlimiting examples of the reducing sugars include allose, altrose, glucose, gulose, iodose, mannose, galactose, talose, ribose, arabinose, xylose, lyxose, erythrose, threose, glyceraldehyde,psicose, fructose, sorbose tagatose, ribulose, xylulose, erthrulose, dihydroxyacetone, maltose, lactose, sucrose, cellobiose, any polysaccharide comprised of two or more monosaccharide with one or more reducing groups.

[0054] The methods disclosed herein thus can be used to glycate one, two, three or more amino groups (e.g. alpha-, beta-, gamma-, delta-, epsilon-NH2) of insulin or a protein with reducing-sugars to produce novel biologically active mono-glycated, di -glycated or tri -glycated compounds. In some embodiments, the protein, the reducing sugar, the water vapor, the temperature, and the time are selected in their respective amounts or values so that the targeted one, two or three amino groups of the protein are glycated at a yield of more than 70%, more than 80, more than 90%, more than 95%, more than 98%, or more than 99%. In some embodiments, the protein includes three amino groups. In some embodiments, one amino group of the protein is glycated. In some embodiments, two amino groups of the protein are glycated. In some embodiments, three amino groups of the protein are glycated. In some embodiments, only three amino groups are present in the protein or can be glycated in the protein. In some embodiments, one, two or three amino groups glycated in the protein are alpha amino groups. In some embodiments, the protein is insulin.

[0055] Regarding modification to insulin, tri-glycation confers the following favorable properties to insulin. 1) rapid onset of activity; 2) prolonged activity in vivo; 3) increased resistance to proteolysis; 3) increased resistance to fibrillation; and 4) increased solubility at physiological pH values.

[0056] It has been observed that different amino groups (e.g. alpha v epsilon) of a protein may exhibit different reactivities in glycation. Meanwhile, different reducing sugars have been shown to require different incubation temperatures for in-vacuo conjugation to a protein. Therefore, when two or more reducing sugars may be present in an aqueous mixture and the subsequent lyophilizate, glycation on different amino groups can be achieved by controlling one or more of parameters selected from the amount of the reducing sugars, the ratio between the protein and the sugars, the pH range, the incubation temperature, and the length of reaction / incubation time. As a result, di-glycated, tri-glycated, or polyglycated proteins can be obtained with different reducing sugars conjugating to different amino groups.

[0057] In some embodiments, the amino-containing insulin or polypeptide is used in a limited amount and the one or more sugars are used in excess amounts. In some embodiments, after one or more cycles of reaction, the amount of the amino-containing insulin or polypeptide that has reacted to form the desired product is more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of its starting amount. In some embodiments, the one or more of the sugars, their respective amounts and the reaction conditions are selected so that,for one, two or more target amino groups to be glycated, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of the one, two or more target amino groups are glycated in the product. For instance, under conditions disclosed herein, more than 80% or more than 90% of the s-amino of insulin can be glycated in one reaction cycle. Similarly, more than 80% or more than 90% of the three aminos of insulin can be glycated in one reaction cycle. If necessary, the reaction can be repeated for one or more cycles to improve the glycation extent of the aminos.

[0058] To ensure completion of glycation of one or more amino groups, the method disclosed herein can include a step of determining the content of free amino groups for example by TNBS or OPA analysis. If the glycation of one or more amino groups is incomplete, the incubation in the presence of water vapor can be repeated one or more times. If necessary, the mixture can be lyophilized again and / or a suitable amount of water vapor can be re-introduced (e.g. via steps of freezing the water and then subjecting it to vacuo and optionally heating). An additional amount of a reducing compound or sugar can be introduced if necessary. If a high degree or yield of glycation is achieved for one or more amino group is achieved, the excess amount of sugar can be removed (e.g. via dialysis). In the event of glycating a second, a third, or further amino group in the protein with one or more different reducing compounds or sugars, they can be introduced after the determination of amino content and optional removal of excess reducing sugar in the earlier steps of glycation.

[0059] If necessary, a product can be isolated and / or purified before it is subjected to glycation of a second amino group, a third amino group, or more amino groups. For instance, after glycation of a protein, the crude product mixture can be purified to isolate a first protein (mono-glycated at a a-amino) and a second protein (mono-glycated at a a-amino). Each of the two isolated mono-glycated proteins can be separately subjected to glycation condition at a second amino group and / or third amino group with one or two reducing sugars to obtain di-glycated or tri-glycated proteins, each of which can be subsequently purified and isolated. An isolated di-glycated proteins can also be further subjected to glycation condition to prepare tri-glycated or higher order glycated proteins.

[0060] Accordingly, an aspect of the patent document includes sequentially reacting two three or more reducing sugars with insulin or a protein. The two, three or more reducing sugars may be added together to an aqueous mixture for lyophlization. By controlling the amounts of individual reducing sugars, the reaction temperature and reaction time, the sugars can react in stages with different amino groups of the protein to form di-glycated, tri-glycated, or poly-glycated proteins. Alternatively, the two, three or more reducing sugars may be added sequentially so that each sugar can react with a particularamino group in the protein. The different parameters (e.g. the ratio of protein or insulin to reducing-sugar; pH of lyophilization (LpH); the amount of added liquid water or the pressure of the water vapor; incubation temperature; time of incubation; number of incubations) can be controlled as described above.

[0061] A further aspect of the patent document relates to a glycated protein as a conjugate of a reducing sugar and a protein, wherein the conjugate comprises one, two, three or more ketoamine linkages formed from the protein and the sugars. Specific embodiments of the protein and the reducing sugar are as described above. The conjugate may also be formed from a protein and two, three or more reducing sugars and contain two, three or more ketoamine linkages. In particular, for each individual of one, two, three or more target amino groups, more than 80%, more than 90%, more than 90%, or more than 99% of the individual target amino group can be glycated. In some embodiments, the conjugate has a purity of more than 80%, more than 90%, more than 95%, or more than 99%. In some embodiments, the protein is insulin and the conjugate is a mono-, di-, or tri-glycated insulin. In some embodiments, the conjugate is a tri-glycated insulin. In some embodiments, the high purity product is obtained without purification (e.g. chromatograph column, recrystallization, etc.).

[0062] For mono-, di- or try-glycated protein or insulin, each of the three amino groups may be independently glycated by a different reducing sugar, which can be selected from for example allose, altrose, glucose, gulose, iodose, mannose, galactose, talose, ribose, arabinose, xylose, lyxose, erythrose, threose, glyceraldehyde, psicose, fructose, sorbose tagatose, ribulose, xylulose, erythrulose, dihydroxyacetone, maltose, lactose, sucrose, cellobiose, glucosyl glucosyl glucose, raffinose, and a polysaccharide comprising three or more monosaccharide with one or more reducing groups. In some embodiments, the conjugate is a di- or tri-glycated insulin, wherein each of the glycated amino groups contains a ketoamine linkage formed with an aforementioned reducing sugar. In some embodiments, the conjugate is a tri-glycated insulin, wherein the glycated amino groups contain a ketoamine linkage formed with a same or a different reducing sugar. In some embodiments, the conjugate is a tri-glycated insulin, wherein each of the three amino groups are glycated with a reducing sugar of the same structure (i.e. 3: 1 molar ratio between insulin derived moiety and the reducing sugar derived moiety in the conjugate). In some embodiments, the tri-glycated insulin has a purity of more than 80%, more than 90%, more than 95%, or more than 99%.

[0063] Nonlimiting examples of glycated proteins of this patent document include the following.

[0064] Table 1. examples of glycation for proteins

[0065] Further examples of glycated proteins of this patent document include the following. In examples disclosed in this patent document where a glycated protein contain one or more sugar moieties, the examples are intended to cover all combinations of sugar distribution. For instance, for a di-glycated insulin with sugars (glucose and fructose) conjugated to a-B chain amino and e-amino, the example encompasses (1) a di-glycated insulin with glucose conjugated to a-B chain amino and fructose conjugated to e-amino amino, and (2) a di-glycated insulin with fructose conjugated to a-B chain amino and glucose conjugated to s-amino amino.

[0066] Table 2. examples of glycated proteins

[0067] Another aspect of the present disclosure provides a pharmaceutical composition containing a therapeutically effective amount of the above-described conjugate and a pharmaceutically acceptable carrier.

[0068] The pharmaceutical composition may also contain one or more physiologically acceptable surface -active agents, additional carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, and coating assistants, or a combination thereof; and a composition disclosed herein. Acceptable additional carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety. Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoring agents, and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid, and esters of p- hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, microcrystalline cellulose, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methylacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents. Other agents that can be included in the composition include for example, polyols (e.g. polyethylene glycol or polypropylene glycols). The molecular weight of the polyols may range, for example, from 500 to 100,000, from 1,000 to 100,000, from 1,000 to 50,000, or from 1,000 to 5,000.

[0069] The pharmaceutical compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredient(s), as in combination therapy, or suitable carriers or excipient(s). In some embodiments, a dosage form includesthose forms in which the compound is administered per se. In addition, a dosage form may include a pharmaceutical composition. In any case, the dosage form may comprise a sufficient amount of the compound to treat a disease as part of a particular administration protocol, as would be understood by those of skill in the art. Techniques for formulation and administration of the compounds of the instant application may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, 18th edition, 1990.

[0070] The pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee -making, levigating, emulsifying, encapsulating, entrapping or tabletting processes.

[0071] Pharmaceutical compositions may be formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, diluents, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington’s Pharmaceutical Sciences, above.

[0072] Another aspect of this disclosure provides a method of treating deficiency or insufficiency or inadequacy of a biologically active protein in a subject or improving the availability (or bioavailability), solubility and / or stability of the protein. A reference or standard level of the protein can be readily established based on the level in a healthy subject. Nonlimiting examples of the protein are as disclosed in the first column of Table 1. Glycated proteins disclosed herein exhibit higher solubility and improved stability in comparison with their respective non-glycated parent proteins. By administering the subject in need thereof a therapeutically effective amount of the glycated protein disclosed herein, the deficiency of the protein can be treated. Meanwhile, the bioavailability and sufficiency of the protein can also be improved by converting it into a glycated form for administration. In some embodiments, the method includes determining that the subject has a deficiency level of lower than the reference or standard by at least 2%, by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, or by at least 80%. In some embodiments, the glycated protein or its composition and its amount is selected so that the bioavailability of the non-glycated is improved by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, or by at least 80% when the protein is administered in its glycated form (same routes of administration for glycated and non-glycated proteins).

[0073] Another aspect of this disclosure provides a method of treating a disease in a subject. The disease is associated with the deficiency or insufficiency or inadequacy of a biologically active protein. In some embodiments, the method includes determining that the subject has a deficiency level of lower than the reference or standard by at least 2%, by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, or by at least 80%. Nonlimiting examples of the protein are as disclosed in Table 1. The method includes administering to the subject in need a therapeutically effective amount of a conjugate or a pharmaceutically acceptable salt, isomer, or pharmaceutical composition thereof. Specific embodiments of the conjugate are as described above. Nonlimiting examples of the disease include diabetes, obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications.

[0074] The compositions or pharmaceutical compositions described herein may be administered to the subject by any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as rectal, vaginal, intraurethral, intraocular, intranasal, or intraauricular, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracap sularly, intraspinally, intrastemally, or the like, including infusion pump delivery; as well as (d) administration topically; as deemed appropriate by those of skill in the art for bringing the active compound into contact with living tissue.

[0075] Pharmaceutical compositions suitable for administration include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. In some embodiments, a therapeutically effective amount of a compound or conjugate is an amount effective to treat a disease or condition, for example, in a mammalian subject (e.g., a human). The therapeutically effective amount of the conjugates disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but willdepend on such factors as weight, diet, concurrent medication, and other factors which those skilled in the medical arts will recognize. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0076] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

[0077] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Typically, dosages may be about 10 microgram / kg to about 100 mg / kg body weight, preferably about 100 microgram / kg to about 10 mg / kg body weight. Alternatively, dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art.

[0078] The exact formulation, route of administration and dosage for the pharmaceutical compositions can be chosen by the individual physician in view of the patient’s condition, (see e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1, p. 1). In some embodiments, the dose range of the composition administered to the patient can be from about 0.5 to about 1000 mg / kg of the patient’s body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. In instances where human dosages for compounds have been established for at least some conditions, those same dosages, or dosages that are about 0.1% to about 500%, more preferably about 25% to about 250% of the established human dosage may be used. Where no human dosage is established, as will be the case for newly discovered pharmaceutical compositions, a suitable human dosage can be inferred from EDso or IDso values, or other appropriatevalues derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.

[0079] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0080] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of about 0. 1 mg to 2000 mg of the active ingredient, preferably about 1 mg to about 500 mg, e.g. 5 to 200 mg. In other embodiments, an intravenous, subcutaneous, or intramuscular dose of the active ingredient of about 0.01 mg to about 100 mg, preferably about 0.1 mg to about 60 mg, e.g. about 1 to about 40 mg is used. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free acid. In some embodiments, the composition is administered 1 to 4 times per day. Alternatively, the compositions may be administered by continuous intravenous infusion, preferably at a dose of up to about 1000 mg per day. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly aggressive diseases or infections. In some embodiments, the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.

[0081] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety, which are sufficient to maintain the antibiotic effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0082] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

[0083] In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0084] The amount of composition administered may be dependent on the subject being treated, on the subject’s weight, the severity of the infection, the manner of administration and the judgment of the prescribing physician.

[0085] Compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of the compound may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Of course, human clinical trials can also be used to determine the efficacy of a compound in humans.

[0086] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0087] In some embodiments, in the pharmaceutical industry, it is standard practice to provide substantially pure material when formulating pharmaceutical compositions. Therefore, in someembodiments, “substantially pure” refers to the amount of purity required for formulating pharmaceuticals, which may include, for example, a small amount of other material that will not affect the suitability for pharmaceutical use. In some embodiments, the substantially pure compound or conjugate contains at least about 96% of the compound by weight, such as at least about 97%, 98%, 99%, or 100% of the compound or conjugate.

[0088] Examples

[0089] Example 1

[0090] Materials

[0091] Human Insulin was purchased from Sigma Aldrich and used without further purification.

[0092] D-Glucose was purchased from Alfa Aesar, D-Ribose, D-Erythrose, D-Fructose andMaltose were purchased from Sigma Aldrich and used without further purification. D-glucose-l-13C99 atom %13C was purchased from Sigma Aldrich.

[0093] All other chemicals, reagents and solvents used were high purity preparations obtained from commercial sources.

[0094] 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (10-well, 30 pl) were purchased from Bio-Rad.

[0095] SDS-PAGE was performed in a Mini-PROTEAN Tetra Vertical Electrophoresis Cell. The compositions of sample buffer were 60 mmol / L, Tris-HCl, pH 6.8, 2% (w / v) SDS, 10% (w / v) glycerol, and 0.01% (w / v) Coomassie brilliant blue G-250. The running buffer contains 25 mmol / L, Tris, 192 mmol / L, glycine, and 0. 1% (w / v) SDS, which were the same as those of the standard Laemmli method (Laemelli, U.K., Nature 227, 680-685, 1970). The samples (10 ml of loading volume per well, containing 1 pg of protein) were applied to each well with a microsyringe. The gel was then subjected to electrophoresis under constant voltage of 100 V. For insulin detection, the gel was stained with 0.1% Coomassie brilliant blue G-250 in 10% acetic acid and 30% methanol for 50 min and then destained in an aqueous solution of 15% methanol and 15% acetic acid.

[0096] In-Vacuo Glycation of Lyophilized Human Insulin

[0097] Typically, 1. 11 mg pentose, or 1.33.mg hexose, or 2.66 mg disaccharide and 4.00 mg Insulin (insulimreducing-sugar (3: 1 wt / wt) was dissolved in 2.0 ml of de-ionized water in a 10 ml Schlenk flask with a ground glass stopcock. The pH was adjusted to pH 8.0 by the addition of 0.2 N NaOH and the solution shell frozen in liquid nitrogen or dry ice / acetone and lyophilized. The top of theSchlenk flask with the lyophilizate was immersed in liquid nitrogen and water (0.50 pl) was added and frozen so that it was not in contact with the lyophilizate. The Schlenk tube was evacuated to 0.2-0.3 mbar and sealed by closing the stopcock. The flask was then incubated in an oven or oil bath at 65°C for 24 h. The extent of glycation was monitored by determination of the free amino content by TNBS or OPA analysis. In cases where the glycation was less than quantitative a second incubation was carried out by re-dissolving the lyophilizate in 4 ml of water and repeating the incubation as described above. Triglycated insulin is the only product and isolation is achieved by dialysis to remove the excess sugars followed by lyophilization.

[0098] Orthopthaldeyde (OPA) Assay

[0099] The OPA solution consisted of the following chemicals diluted to 50 ml in a volumetric flask with de-ionized water: 0.50 mg sodium tetraborate; 2.5 ml 20% (wt / wt) SDS; 40 mg of OPA (in 1 ml methanol); and 100 pL 2-mercaptoethanol. This OPA reagent was prepared freshly every time before use. A small aliquot of 40 pL of an insulin solution at a concentration of 2 mg / mL was added to 3.0 ml of OPA reagent in a 4.0 ml quartz cuvette; the solution was mixed briefly by inversion and incubated for 2 min at ambient temperature. The sample was excited at 340 nm and the emission intensity of the fluorescence was measured from 360 nm to 600 nm in a QM-4 SE spectrometer from Photon Technology International (PTI) equipped with double excitation and emission monochromators. The intensity at 430 nm was used for calculation of reaction conversion.

[0100] Trinitnobenzene Sulphonic Acid (TNBS) Assay

[0101] Insulin samples were dissolved in the reaction buffer (0.10 M NaHCCE, pH 8.5) at a concentration of 0.20 mg / mL. To 0.5 m of the insulin solution, 0.25 mLO.03% (w / v) solution of TNBS was added and mixed. This solution was incubated at 37°C for two hours. Before measurement, 0.125 ml of 20% SDS and 0.125 ml of 1 M HC1 were added to each sample. The absorption at 350 nm of each sample was measured with a Cary series UV-Vis-NIR spectrophotometer from Agilent Technologies.

[0102] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0103] SDS-PAGE was performed in a Mini-PROTEAN Tetra Vertical Electrophoresis Cell. The compositions of sample buffer were 60 mmol / L, Tris-HCl, pH 6.8, 2% (w / v) SDS, 10% (w / v) glycerol, and 0.01% (w / v) Coomassie brilliant blue G-250. The running buffer contains 25 mmol / L, Tris, 192 mmol / L, glycine, and 0. 1% (w / v) SDS, which were the same as those of the standard Laemmli method (Laemelli, U.K., Nature 227, 680-685, 1970). The samples (10 pl of loading volume per well, containing 1 pg of protein) were applied to each well with a microsyringe. The gel was then subjectedto electrophoresis under constant voltage of 100 V. For insulin detection, the gel was stained with 0.1% Coomassie brilliant blue G-250 in 10% acetic acid and 30% methanol for 50 min and then destained in an aqueous solution of 15% methanol and 15% acetic acid.

[0104] Fast Protein Liquid Chromatography (FPLC)

[0105] The glycated or native insulin was dissolved in 10 mM pH 7.4 sodium phosphate buffer at the concentration of 2.0 mg / mL. A 0.5 mL sample was injected into the Amersham AKTA purifier FPLC system controlled by UNICOR software and equipped with a Superdex 75 Increase 10 / 300 GL column from Cytiva, P-903 Pump, UV-900 Detector, pH / C-900 Monitor and Frac-900 / 901 Fraction Collector. The column was eluted with 10 mM pH 7.4 sodium phosphate buffer at a flow rate of 0.8 mL / min at 22 °C. The eluent was continuously monitored at 280 nm. The column was calibrated with molecular weight standards.Nuclear Magnetic Resonance (NMR)

[0106] 13C-NMR spectra were obtained using a 400 MHz Bruker AvIII HD instrument. In 0.5 mL 8.0 M reference.

[0107] Differential Scanning Calorimetry (DSC)

[0108] Data collection was performed using a VP -DSC differential scanning microcalorimeter (MicroCai, LLC, Northampton, MA). All protein scans were performed with 10 mM pH 7.4 phosphate buffer in the reference cell from 20 to 110 °C at a scan rate of 1.5°C / min. All samples and references were degassed immediately before use. A buffer-buffer reference scan was subtracted from each sample scan prior to concentration normalization. Data analysis was carried out using Origin 7.0 (OriginLab, Northampton, MA).

[0109] Proteolytic Stability

[0110] The increase in free amino groups due to proteolysis was used to quantify the extent of proteolysis in the insulin samples on incubation with pancreatic proteases. To increase the sensitivity of the assay, advantage was taken of the fact that the s-amino groups of the pancreatic proteases could be blocked without loss of activity so that no free amino groups were present at the start of the assay.[oni] A solution containing 2.0 mg / ml of tri-glucosylated insulin, tri-ribosylated insulin and tri-maltosylated insulin in 10 mM pH 7.4 PBS buffer was prepared. To a 500 pL aliquot ofthis solution, 500 pL of a solution containing 40 pg of chymotrypsin, 40 pg elastase and 40 pg trypsin in PBS pH 7.4, where all the enzymes’ amino groups were blocked, was added. The glycated-insulin to enzyme ratiofor each enzyme was 251 (wt / wt). All samples were incubated in at 37°C, and 40 pL aliquots were taken out at specified time points and free amino content determined by OPA assay.

[0112] Biological Activity

[0113] Male and female nonobese diabetic / severe combined immunodeficiency (NOD / SCID) mice ages 8-10 weeks were injected with low dose STZ (35mg / kg) dissolved in citric acid buffer by intraperitoneal injection for 5 days (Day 0-Day 4). At Day 0, mice have a non-fasted blood glucose level of 3-6mmol / L. Blood glucose is measured on Day 10 to assess hyperglycemia caused by STZ. Mice that have become hyperglycemic (>12mmol / L) are randomly assigned into an experimental group. The biological activity of all glycated insulins was tested by blood glucose depression.

[0114] Example 2

[0115] In order to determine the optimal conditions for quantitative glycation, the following conditions were varied in turn for each of the reducing sugars: the wt:wt ratio of insulin to reducing-sugar; pH of lyophilization (LpH); sub-microliter quantity of added water; incubation temperature; time of incubation; number of incubations.

[0116] Table 3. the in-vacuo incubation conditions and the extent of glycation achieved for representative reducing-sugars.* Four separate determinations. % Glycation = 100% - % free aminoFree amino determined by TNBS analysis.

[0117] The extent of glycation observed for the aldo monosaccharides demonstrates that under appropriate conditions they readily in-vacuo glycate all the amino groups of insulin to produce triglycated insulins. It was observed that the rate of glycation at an incubation temperature of 65°C increases in the order triose>tetrose>pentose>hexose. In the case of aldo pentoses and especially aldo tetroses, extended time of incubation results in the formation of advanced glycation end products (AGEs). In such cases, the glycation conditions can be adjusted to eliminate such formationby adjusting the various incubation conditions. Insulin could also be glycated with the keto hexose fructose but required three incubations at a higher temperature than the aldo hexoses. Insulin could be glycated with maltose but required three incubations and a much higher incubation temperature of 90°C. In summary, the results demonstrate that the revised in-vacuo glycation methodology developed for the in-vacuo glycation of insulin can be employed with aldo and keto monosaccharides and reducing disaccharides to achieve quantitative glycation of the free amino groups.

[0118] Further evidence of quantitative glycation and homogeneity of the tri-glucosylated and tri-maltosylated insulins is provided by SDS gel electrophoresis. Both triglycated insulins show one distinct band which moves slower than native insulin. There is no evidence of any native insulin or partially glycated insulin in these preparations.

[0119] 13C-NMR spectrum confirmed tri-glucosylated insulin prepared by in-vacuo glycation with l-13C-glucose. Three prominent resonances at 53.1, 53.4 and 53.7 ppm are observed. For glucose covalently attached to a protein by a ketoamine linkage, a resonance in the region of 53 ppm is expected. The observation that three such resonances are observed in this region is in accord with the evidence from free amino analysis (Table 1) and SDS-PAGE that a tri-glucosylated insulin has been produced in which the glucose is attached by a ketoamine linkage.

[0120] Depending on conditions, insulin in solution can exist as a mixture of associated states in the form of hexamers, tetramers, dimers and as a monomer in sufficiently dilute solution. The insulin monomer is the biologically active form of insulin. However, the dimer has a dissociation constant of approximately 10'5M and therefore the monomer is the predominant form only at concentrations below 10'6M. As tri -glycated insulin has not been prepared previously, it was not known how this would affect its association state compared to native insulin.

[0121] The association state of the tri-glycated insulin was investigated by molecular-sieve chromatography using a Superdex 75 in a fast protein chromatography system. Solutions of native insulin and tri-glycated insulins (2.0 mg / mL) were prepared in 10 mM PBS pH 7.4 and a 0.5 mb sample was injected. The elution profdes of native insulin, tri-glucosylated, tri-ribosylated and tri-maltosylated insulin were obtained. At 2.0 mg / ml (3.44 xlO'4M) insulin exists primarily as a dimer (MW = 11,600 daltons) and this is supported by the elution volume which was found to be near that of ribonuclease (MW = 13,700). All three tri-glycated insulins have a significantly larger elution volume than native insulin indicating that they are predominantly monomeric at concentrations where insulin is in an associated state.

[0122] The thermograms for native insulin and tri-glucosylated insulin were obtained. While both unfold approximately over the same temperature range, the notable difference is that native insulin has a biphasic profile while the tri-glucosylated insulin is monophasic. This is consistent with the molecular-sieve results showing that native insulin is dimeric and the tri-glucosylated insulin is monomeric.

[0123] Isoelectric Point

[0124] The isoelectric point of the tri-glucosylated insulin was estimated by dissolving 2.0 mg in 1.0 ml of water at pH 3.0. Titration of the solution was performed by the addition of 0.5 pl of 0.20 N NaOH with stirring. Three titrations were carried out and the pH at which the solution turned cloudy was noted and then noted again when the solution became clear. The solution turned cloudy at pH 3.97 and turned clear at pH 5.37. The midpoint gives an estimated isoelectric point of 4.67. In comparison, the solution of native insulin turned cloudy at pH 4.62 and clear at pH 6.08 giving an estimated isoelectric point of pH 5.35 in good agreement with the value of 5.4 reported for human insulin.

[0125] The tri-glucosylated insulin is much more soluble than native insulin at neutral pH values.The attachment of three hydrophilic glucosyl residues is expected to give increased solubility and the lower isoelectric point enhances this effect imparting a high solubility at physiological pH values.

[0126] Fibrillation

[0127] Native, tri-glucosylated, tri-ribosylated and tri-maltosylated insulins were dissolved in 10 mM phosphate buffer pH 7.4 at a concentration at 1.0 mg / mL. These solutions were left in the dark at 22°C and monitored by Dynamic Light Scattering (DLS) time points over a period of 20 days. Each sample had three replicates. After 20 days the Z value, a measure of the particle size of aggregates, is significantly reduced for all three glycated insulins in comparison to native insulin. Insulin has the undesirable property that it undergoes aggregation and the formation of fibrils. The DLS results indicate that the tri-glycated insulins are more resistant to fibrillation than native insulin.

[0128] Proteolytic Stability

[0129] The proteolytic stability of tri-glucosylated insulin was determined by the increase in free amino groups generated by proteolysis of peptide bonds on incubation with a mixture of pancreatic proteases containing equal amounts of trypsin, chymotrypsin and elastase. A high ratio of each proteolytic enzyme to the glycated in insulin (1:25 wt / wt) was used to ensure that extensive proteolysis will occur with any susceptible peptide bond. Figure 7 shows that all three tri-glycated insulins generate much less free amino groups when incubated with the three pancreatic proteolytic enzymes and thereforeless proteolysis has occurred. After 7 hours the glycated insulins have undergone very limited proteolysis demonstrating that glycation of all three amino groups substantially increases the resistance of insulin to proteolytic degradation.

[0130] Example 3

[0131] Biological Activity

[0132] The biological activity of the tri-glucosylated, tri-ribosylated and tri-maltosylated insulins were determined by their ability to lower blood glucose levels in mice that had been treated with Streptozotocin (STZ) to induce hyperglycemia. Mice were injected with 0.3 units of native insulin and 1.25 units of each of the tri -glycated units.

[0133] The effect of insulin, tri-glucosylated insulin, tri-ribosylated insulin and tri-maltosylated insulin on blood glucose levels in vivo on day 17 after treatment with STZ was evaluated.

[0134] Compared to untreated controls, human insulin treated mice maintained significantly reduced blood glucose levels for Ih post-treatment (n=3 per treatment group). Mice treated with tri-maltosylated insulin had blood glucose levels that remained significantly reduced compared to controls for 5 h post-treatment, while in tri-ribosylated insulin treated mice the glucose in the blood remained significantly reduced for 7 h post-treatment and tri-glucosylated insulin treated mice glucose remained significantly reduced for 9 h post-treatment.

[0135] Based on reports in the literature, it appears to be the general consensus that glycation of insulin results in impairment of its biological activity. In these studies, insulin was glycated under aqueous conditions and only the production of mono-glycated or di-glycated insulin was reported. The results obtained for tri-glycated insulins are not in accord with these conclusions. Tri-glycated insulins are fully active in lowering blood sugar levels and, in addition, have a prolonged activity compared to native insulin.

[0136] Example 4

[0137] The glycation procedure used to prepare tri-glycated insulin was applied to GLP-1. The conditions which optimize the glycation were determined as described for insulin. D-glucose was chosen as representative of monosaccharides and maltose as representative of larger reducing sugars. As show in Table 2, the conditions to achieve extensive glycation were very similar to those determined for the glycation insulin. As in the case of insulin, extensive glycation was achieved in a single 24 hour in-vacuo incubation and subsequent incubations achieved complete glycation of the free amino groups.

[0138] Table 4. In Vacuo Glycation of CLP-1* Free amino determined by OPA analysis.% Glycation = 100% - % free amino

[0139] GLP-1 (3.298 kDa) is a significantly smaller polypeptide than insulin (5.808 kDa) composed of 30 amino acids as compared to 51 amino acids in insulin. Like insulin it has three free amino groups available for glycation. Given that GLP-1 can be readily glycated under the same conditions as insulin demonstrates that in-vacuo glycation used for insulin can be applies to other polypeptides.

[0140] All references cited herein are incorporated herein by reference in their entireties. It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described. Rather, the scope of the present invention is defined by the claims which follow. It should further be understood that the above description is only representative of illustrative examples of embodiments. The description has not attempted to exhaustively enumerate all possible variations. The alternate embodiments may not have been presented for a specific portion of the invention, and may result from a different combination of described portions, or that other undescribed alternate embodiments may be available for a portion, is not to be considered a disclaimer of those alternate embodiments. It will be appreciated that many of those un-described embodiments are within the literal scope of the following claims, and others are equivalent.

Claims

WE CLAIM1. A glycated protein, wherein one, two, three or more target amino groups of the protein are glycated by one or more reducing sugars, preferably more than 80% of the one, two, three or more target amino groups are glycated.

2. The glycated protein, wherein three target amino groups of the protein are glycated.

3. The glycated protein of any of the preceding claims, wherein three target amino groups are glycated with three same reducing sugars.

4. The glycated protein of any of the preceding claims, which has a purity of greater than 95%.

5. The glycated protein of any of the preceding claims, wherein the one or more reducing sugars are mono-saccharides.

6. The glycated protein of any of the preceding claims, wherein at least one of the one or more reducing sugars is a di-saccharide or polysaccharide.

7. The glycated protein of any of the preceding claims, wherein the protein has a molecular weight ranging from about 1500 to about 8000 Da.

8. The glycated protein of any of the preceding claims, wherein the one or more reducing sugars are selected from the group consisting of allose, altrose, glucose, gulose, iodose, mannose, galactose, talose, ribose, arabinose, xylose, lyxose, erythrose, threose, glyceraldehyde, psicose, fructose, sorbose tagatose, ribulose, xylulose, erthrulose, dihydroxyacetone, maltose, lactose, sucrose, and cellobiose.

9. The glycated protein of any of the preceding claims, wherein three amino groups are glycated with two or three different reducing sugars.

10. The glycated protein of any of the preceding claims, wherein the protein is selected from the group consisting of Adrenocorticotropic hormone (ACTH), Adropin, Amylin, Angiotensin, Atrial natriuretic peptide(ANP), Calcitonin, Cholecystokinin (CCK), Gastrin, Ghrelin, Glucagon, Glucose-dependent insulinotropic polypeptide(GIP), Glucagon-like peptide- l(GLP-l), Growth hormone, Follicle-stimulating hormone (FSH), Insulin, Leptin, Luteinizing hormone (LH), Melanocyte -stimulating hormone (MSH), Oxytocin, Parathyroid hormone(PTH), Prolactin, Renin, Somatostatin, Thyroid-stimulating hormone (TSH), Thyrotropin-releasing hormone (TRH), Vasopressin, also called arginine vasopressin (AVP) or anti-diuretic hormone (ADH), Vasoactive intestinal peptide (VIP), Somatotropin (GH1), Gonadotropin Releasing Hormone 1 (GNRH1, 2), Growth Hormone Releasing Hormone(GHRH), Parathyroid Hormone Like Hormone (PTHLH), Corticotropin Releasing Hormone (CRH), Anti-Mullerian Hormone(AMH), Chorionic Somatomammotropin Hormone 1 (CSH1), Chorionic Somatomammotropin Hormone 2 (CSH2), Pro-Melanin Concentrating Hormone (PMCH), and Resistin (RETN), wherein preferably the protein is selected from insulin, and GLP-1.

11. A method of glycating a protein, comprising:(a) lyophilizing an aqueous mixture comprising the protein and a reducing sugar to produce a lyophilizate;(b) heating the lyophilizate at a temperature for a period of time in a reactor in the presence of water vapor, wherein overall pressure in the reactor ranges from about 0.001 mbarto about 100 mbar;(c) optionally determining the extent of glycation and / or formation advanced glycation end product and optionally adjusting pressure of the water vapor; and(d) optionally repeating steps (a) and (b).

12. The method of claim 11, the lyophilizate is substantially free from liquid water in step (b).

13. The method of any one of claims 11-12, wherein the water vapor is unsaturated, preferably the water vapor has a vapor pressure ranging from about 0.001 mbarto about 10 mbar, more preferably the water vapor has a vapor pressure ranging from about 0.01 mbar to about1 mbar, most preferably the water vapor has a vapor pressure ranging from about 40 mbar to about 80 mbar.

14. The method of any one of claims 11-13, further comprising after step (a) and before step (b), introducing water to the reactor and freezing the water so that it is not in contact with the lyophilizate.

15. The method of any one of claims 11-14, wherein step (b) proceeds at a temperature ranging from about 40 °C to about 150 °C.

16. The method of any one of claims 11-15, wherein the reactor is a closed system.

17. The method of any one of claims 11-16, wherein the aqueous mixture has a pH ranging from about 7 to about 9.

18. The method of any one of claims 11-17, wherein the protein is selected from the group consisting of Adrenocorticotropic hormone (ACTH), Adropin, Amylin, Angiotensin, Atrial natriuretic peptide(ANP), Calcitonin, Cholecystokinin (CCK), Gastrin, Ghrelin, Glucagon, Glucose-dependent insulinotropic polypeptide(GIP), Glucagon-like peptide-l(GLP-l), Growth hormone, Follicle-stimulating hormone (FSH), Insulin, Leptin, Luteinizing hormone (LH), Melanocyte-stimulating hormone (MSH), Oxytocin, Parathyroid hormone(PTH), Prolactin, Renin, Somatostatin, Thyroid-stimulating hormone (TSH), Thyrotropin-releasing hormone (TRH), Vasopressin, also called arginine vasopressin (A VP) or anti-diuretic hormone (ADH), Vasoactive intestinal peptide (VIP), Somatotropin (GH1), Gonadotropin Releasing Hormone 1 (GNRH1, 2), Growth Hormone Releasing Hormone (GHRH), Parathyroid Hormone Like Hormone (PTHLH), Corticotropin Releasing Hormone (CRH), Anti-Mullerian Hormone(AMH), Chorionic Somatomammotropin Hormone 1 (CSH1), Chorionic Somatomammotropin Hormone 2 (CSH2), Pro-Melanin Concentrating Hormone (PMCH), and Resistin (RETN), wherein preferably the protein is selected from insulin, and GLP-1.

19. A pharmaceutical composition, comprising a therapeutically effective amount of the glycated protein of claim 1 and a pharmaceutically acceptable carrier.

20. A method of treating a disease or a condition associated with deficiency of a protein in a subject, comprising administering to the subject a therapeutically effective amount of a glycated protein of any one of claims 1-10 or the pharmaceutical composition of claim 19, preferably wherein the disease is selected from the group consisting of diabetes, obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, and diabetic complications.

21. A method of treating deficiency of a protein, comprising administering to a subject in need thereof a therapeutically effective amount of the glycated protein of any one of claims 1-10 or the pharmaceutical composition of claim 19.

Images