HK1150970B - An orally administerable solid pharmaceutical composition and a process thereof - Google Patents

Description

Orally administrable solid pharmaceutical compositions and methods thereof

Technical Field

The present invention relates to pharmaceutical compositions of the ad hoc invention that allow for uptake via oral delivery of proteins/peptides or their conjugates, and/or cation-insulin conjugate complexes (conjugatecomplex), showing desirable pharmacokinetic properties and efficacy in diabetic efficacy models in dogs and humans. Preferred formulations comprise from 0.01% to 20% w/w of insulin, insulin compound conjugate and/or cationic insulin conjugate, from 10% to 60% w/w of one or more selected from saturated or unsaturated C₄-C₁₂The fatty acid component of a fatty acid and/or a salt of such a fatty acid, and additionally comprising an optimal amount of other pharmaceutically suitable polymeric excipients which allow to achieve improved solubility, dissolution rate and effective bioavailability of poorly water soluble compositions and a consistent in vivo release profile upon scale-up during production (scalability). Another aspect of the invention features a method of making the above-described formulation.

Background

It is increasingly being investigated that the conventional subcutaneous route of administration of insulin is replaced by oral drug delivery mechanisms that do not alter its physiological clinical activity. The problems faced in designing effective oral drug delivery systems for biological macromolecules in this area of technology are mainly due to their sensitivity to enzymatic degradation and low epithelial permeability. In addition, the structure and configuration of insulin is susceptible to changes upon exposure to formulation and process conditions, resulting in a loss of biological activity. Some approaches to address these limitations include the use of insulin analogues, administration of peptides such as amylin (amylin), glucagon-like peptides, C-peptides, inhaled forms, intranasal forms, but these do not satisfactorily address the limitations of bioavailability alone.

There remains a need in the art for pharmaceutically acceptable complexes comprising derivatized insulin conjugates with increased bioavailability or other improved pharmaceutical properties relative to existing conjugates. In addition, these improved conjugates need to be delivered in the form of a stable and extemporaneous preparation of a formulation that readily maximizes the benefit of oral delivery of the protein. The present invention addresses the limitations of insulin delivery by combining improved insulin conjugates with improved formulations with increased bioavailability.

Examples of insulin compounds include human insulin, insulin lispro, des30 insulin, natural proinsulin, artificial proinsulin, and the like. The cationic component can be, for example, a divalent metal cation selected from the group consisting of: zn + +, Mn + +, Ca + +, Fe + +, Ni + +, Cu + +, Co + + and Mg + +. The cation-insulin compound conjugate complex further includes a modifying moiety that binds (e.g., covalently or ionically) to the insulin compound to provide an insulin compound conjugate. In addition, the modifying moiety is selected such that the solubility of the insulin compound conjugate is equal to or greater than the water solubility of the corresponding unbound insulin compound, and the water solubility of the insulin compound conjugate is reduced by the addition of zinc. The modifying moiety is selected such that the solubility of the insulin compound conjugate is equal to or greater than the water solubility of the corresponding unbound insulin compound, the water solubility of the insulin compound conjugate being reduced by the addition of zinc; and the water solubility of the complex is greater than the water solubility of the insulin compound.

Examples of suitable modifying moieties and insulin conjugates that can be used to prepare the compositions can be found in U.S. Pat. nos. 7,060675, 6,303,569, 6,214,330, 6,113906, 5,985,263, 5,900,402, 5,681,811, 5,637,749, 5,612,640, 5,567,422, 5,405,877, 5,359,030, the entire disclosures of each of which are incorporated herein by reference. Further examples of such cation-insulin compound conjugate complexes are provided in U.S. patent applications US2003/083232, US 2006/0019873 and US 2006/0019874.

In contrast to the prior art, the compounds of the present invention show a reduced mitogenic potential, down toOne quarter of mitogenic potential of (a).

Insulin binds with sub-nanomolar affinity and activates its cognate Insulin Receptor (IR). Insulin also binds to the structurally related Insulin Growth Factor Receptor (IGFR), but its affinity for the insulin growth factor receptor is reduced to about one thousand times that of the insulin receptor. Thus, at physiological insulin concentrations, IGFR plays no role in mediating the effects of insulin. However, at very high insulin concentrations (diabetic patients receiving insulin), it exerts a mitogenic effect through IGFR, which transmits growth stimuli more efficiently than the insulin receptor (Lammers R et al, EMBO 1989).

One of The insulin analogues identified earlier with a modification at The aspartic acid B10 residue was aimed at providing a fast acting insulin effect, however, The modification in turn resulted in a significant increase in The mitogenicity of The analogue [ Drejer, k., The biological activity of insulin analogues from in vitro receptor binding to in vivo glucose uptake. diabetes metals Rev, 1992.8 (3): p.259-85 ]. Several studies of the binding of insulin analogs to the insulin growth factor-1 (IGF-1) receptor and the insulin receptor were conducted. Binding constants were measured and correlated with the metabolic and mitogenic potency of the insulin analogues studied [ Kurtzhals, p. et al, correlates of receptor binding and metabolic and mitogenic potential of insulin analogues designed for clinical uses, 2000.49 (6): p.999-1005 ]. According to the studies conducted, insulin lispro, insulin aspart (insulin aspart) and insulin glargine (insulin glargine) have minimal changes in binding to insulin receptors compared to normal insulin, while insulin detemir (insulin indexemir) is significantly reduced. Each of these analogs has a similar or increased dissociation rate from IGF receptors, which correlates with their mitogenic behavior.

The mitogenic potency of proteins/peptides and their conjugates, cation-peptide conjugate complexes, cation-insulin conjugate complexes is of primary concern due to increased mitogenicity and the risk of growth of human breast epithelial cells. It has been shown that the IGF-1 binding of insulin aspart is similar to that of natural human insulin. Insulin lispro and insulin glargine increase in binding affinity to IGF-1 receptor by 1.5 to 6.5 fold, respectively, suggesting that insulin glargine has a significantly greater mitogenic response.

The long-term effects of the mitogenic properties of insulin analogues, their conjugates, cation-peptide conjugate complexes, cation-insulin conjugate complexes remain an important factor to be considered. The point to be considered in the document CPMP/SWP/372/01 on non-clinical evaluation of potential carcinogenicity of insulin analogues is set forth: "Natural human insulin has a weak mitogenic effect in addition to its metabolic action. This effect becomes important for the safety of insulin analogues, as structural modifications of the insulin molecule can increase mitogenic potency, possibly leading to growth stimulation of pre-existing neoplasms. "" although enhanced insulin-like growth factor 1(IGF-1) receptor activation and/or aberrant signaling through the insulin receptor has been implicated, the mechanism or mechanisms responsible for mitogenic activity of insulin analogs remain to be elucidated. "

Because evidence that IGF-1 promotes the growth of colon, breast, prostate, and lung cancers is accumulating, there is a need to develop proteins/peptides and their conjugates, cation-peptide conjugate complexes, cation-insulin conjugate complexes that are evaluated in cell proliferation assays to have minimal mitogenic risk and are considered safe for long-term treatment.

The present invention relates to proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes that show mitogenic properties that are reduced by a factor of four compared to that of Insugen. Another aspect of the invention relates to the fact that the excipients in the pharmaceutical product do not statistically affect the mitogenic potency profile of the drug substance.

Another aspect of the invention relates to pharmaceutical compositions invented without preparation that allow the uptake of proteins/peptides or their conjugates and/or cation-insulin conjugate complexes via oral delivery, showing desirable pharmacokinetic properties and efficacy in diabetic efficacy models in dogs and humans.

WO00/50012 discloses a solid oral dosage form comprising a drug and an enhancer, wherein the enhancer is a salt of a medium chain fatty acid having a carbon chain length of about 6-20 carbon atoms. US2006/0018874 discloses a solid pharmaceutical composition formulated for oral ingestion administration having a fatty acid component and a therapeutic agent in an amount of 0.1 to 75% w/w, wherein the fatty acid component comprises a saturated or unsaturated fatty acid and/or salt.

Despite the above, there is still a need for producing a viable oral insulin formulation which can overcome the problems associated with loss of biological activity during production and at the same time show enhanced resistance to enzymatic degradation in vivo after ingestion. The present invention addresses both of these needs. Thus, the present invention solves the problem in the art of designing a highly efficient oral insulin-peptide conjugate drug delivery mechanism.

The present invention shows several advantages in terms of dosing and convenient methods of administration. Yet another advantage afforded by the present invention over prior art compositions is that the inventors propose the claimed, rationally designed oral formulation of IN-105 with other measurable components of excipients. And the process for producing the orally administrable tablet can be easily scaled up. In addition, due to this rationally designed oral formulation and its method of production, the amplification factor does not affect the in vivo drug properties or its in vivo release characteristics.

Existing oral insulin formulations exhibit low levels of stability, precluding the possibility of oral administration of such therapeutic agents. One of the most significant aspects of the present invention is characterized by the fact that the immediate release oral insulin formulations of the present invention are stable over a range of temperatures without various parameters that would adversely affect tablet stability, such as hardness, disintegration time, high molecular weight impurity accumulation and dissolution rate. The tablets thus produced show excellent stability even under accelerated stability conditions of 40 ℃/75% relative humidity. The inherent stability characteristics of the molecule and the production method are attributed to the stability of the formulation.

Another aspect of the present invention relates to an improved pharmaceutical composition of a cation-insulin conjugate complex prepared by a scalable spray drying process comprising the step of preparing an aqueous suspension of the cation-insulin conjugate complex with at least one fatty acid component and optionally one or more pharmaceutically acceptable excipients.

In a typical process for fine particles using a spray drying process, a material such as an ingredient intended to form a majority of the particles is dissolved in a suitable solvent to form a solution. Alternatively, the material intended to be spray dried may be suspended or emulsified in a non-solvent to form a suspension or emulsion. Other components, such as drugs, pharmaceutically acceptable excipients or pore formers are optionally added at this stage. The solution is then atomized to form a fine mist of droplets. The droplets are immediately passed into a drying chamber where they are contacted with a drying gas. The solvent evaporates from the droplets into the drying gas to solidify the droplets, thereby forming particles. The particles are then separated from the drying gas and collected.

Problems may be encountered in scaling up such spray drying processes, for example, from laboratory or pilot plant scale to industrial equipment scale. If the drying rate and drying capacity are not sufficiently optimized, undesirable problems may be faced, such as improper drying of the solvent particles, reduced product yield, purity, and the like. On the other hand, increasing the drying rate that is inappropriately scaled up may lead to inappropriate particle morphology and/or particle size distribution of some product particles, such as those with decisively defined performance specifications. More importantly, it can also alter the manner in which the solid-forming material precipitates upon evaporation of the solvent, thereby altering the structure (e.g., porosity) of the particles, thereby moving away from specification standards such that the particles cannot properly contain and deliver a diagnostic or therapeutic agent.

Accordingly, there is a need in the art for improved spray drying processes that result in the production of homogeneous solid amorphous dispersions of high purity, with improved flow characteristics, improved content consistency, and improved collection efficiency.

It is an object of the present invention to provide a spray-dried composition of a cation-insulin conjugate complex, and to a drying process that provides improved drying of the particles without adversely affecting product purity, yield and stability. The process of the present invention results in uniform spray-dried solid particles of the therapeutic agent of interest that are further formulated with other necessary excipients to provide an oral pharmaceutical composition of the cation-insulin conjugate complex.

Other objects and advantages of the present invention will be apparent to those skilled in the art from the following disclosure and claims.

The purpose of the invention is as follows:

the primary object of the present invention was to develop an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, diluents, lubricants, plasticizers, permeation enhancers, and solubilizers.

It is another object of the present invention to develop solid pharmaceutical compositions that can be administered orally, wherein the oral dosage form is in the form of tablets, capsules, granules, powders or sachets (sachets) or dry suspensions.

It is yet another object of the present invention to develop a method for producing an orally administrable solid pharmaceutical composition of IN-105.

It is yet another object of the present invention to develop a tablet composition of IN-105.

Yet another aspect of the invention is to develop a dose of an orally administrable solid pharmaceutical composition that achieves maximum control of postprandial blood glucose concentration within 5-60 minutes after administration in diabetic patients.

It is yet another object of the present invention to develop a stable orally administrable pharmaceutical composition of the cation-insulin conjugate complex.

It is yet another object of the present invention to develop a method for preparing amorphous spray-dried particles of a cation-insulin compound conjugate.

It is yet another object of the present invention to develop spray-dried compositions of IN-105.

It is yet another object of the present invention to develop a pharmaceutical composition comprising a cation-peptide conjugate complex wherein the excipient does not affect the mitogenic potency of the conjugate.

Description of the invention:

accordingly, the present invention provides an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, diluents, lubricants, plasticizers, permeation enhancers and solubilizers; an orally administrable solid pharmaceutical composition, wherein the oral dosage form is in the form of a tablet, capsule, granule, powder or sachet, or a dry suspension; method for producing an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, diluents, emollientsLubricants, plasticizers, penetration enhancers and solubilizers; method for producing an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a diluent, a lubricant, a plasticizer, a solubilizer, and a penetration enhancer, the method comprising the steps of: a) grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acid and/or a salt of such a fatty acid, b) granulating the fatty acid from step (a) with an organic solvent, c) air-drying the granulate from step (b), d) sieving the dried granulate to obtain a granulate having the desired particle size, e) blending the fatty acid granulate with the cation-insulin conjugate complex and other excipients, f) compressing the blended mixture for tableting; method for producing an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer, the method comprising the steps of: a) grinding of suitably saturated or unsaturated C₄-C₁₂A fatty acid and/or a salt of such a fatty acid and a binder, b) suspending the cation-insulin conjugate complex in an organic solvent using a binder to form a wet mass, c) granulating the components from step (e) using a binder, d) sieving the dried granules from step (d), e) blending the granules with other excipients, f) compressing the blended mixture for tableting; a method in which an organic solvent selected from the group consisting of isopropanol, acetone, methanol, methyl isobutyl ketone, chloroform, 1-propanol, isopropanol, acetonitrile, 1-butanol, 2-butanol, ethanol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, dichloromethane, tert-butanol, toluene, carbon tetrachloride or a combination thereof is used; 5-500mg tablet composition of a cation-insulin conjugate complex, said composition comprising saturated or unsaturatedC₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; 50mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; 100mg tablet composition of a cation-insulin conjugate complex, the composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; 150mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; 200mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; 250mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, lubricants, plasticizers and penetration enhancers; a dose of an orally administrable solid pharmaceutical composition that achieves maximum control of postprandial blood glucose concentration in diabetic patients within 5-60 minutes after administration; stable orally administrable pharmaceutical composition of cation-insulin conjugate complexes, said composition comprising a saturated or unsaturated C₄-C₁₂Fatty acid, fatAt least three of a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a diluent, a lubricant, a plasticizer, a penetration enhancer, and a solubilizer, characterized in that the composition remains stable when exposed to conditions selected from the group consisting of: (a) a temperature range of about 2-40 ℃, (b)25 ℃ ± 2 ℃/60% ± 5% Relative Humidity (RH), 30 ℃ ± 2 ℃/65% ± 5% relative humidity, 40 ℃ ± 2 ℃/75% ± 5% relative humidity for at least 6 months; a method of preparing amorphous spray-dried particles of a cation-insulin compound conjugate, the method comprising the steps of: a) preparing a solution or suspension comprising the cation-insulin compound conjugate and the fatty acid component in a solvent, b) spraying the solution into a chamber under conditions that allow removal of a substantial portion of the solvent, c) obtaining spray-dried particles of the cation-insulin compound conjugate; IN-105 spray-dried compositions; IN-105, wherein the drug is substantially amorphous and homogeneous; a spray-dried composition of IN-105 having a purity of at least 95%; a spray-dried composition of IN-105 having a purity of at least 98%; a spray-dried composition of IN-105 having a purity of at least 99%; and amorphous form of IN-105; an insulinotropic cation-peptide conjugate complex, characterized in that said conjugate shows a decrease of one fourth of mitogenicity compared to its natural counterpart (counterpart), and a pharmaceutical composition comprising a cation-conjugate complex comprising a pharmaceutically acceptable excipient, said composition being characterized in that said excipient does not affect the mitogenic potency of said conjugate.

Description of the drawings:

FIG. 1: mean plasma insulin Curve-IN-105 formulation tablets (FDT-3)

FIG. 2: normalized glucose curves in FDT-3 dosing

FIG. 3: mean plasma insulin Curve-IN-105 formulation tablets (FDT-19)

FIG. 4: normalized glucose curves in FDT-19 dosing

FIG. 5: mean plasma insulin Curve-IN-105 formulation tablets (FDT-20)

FIG. 6: glucose profiles following FDT-20 dosing

FIG. 7: plasma insulin profile

FIG. 8: glucose infusion rate curve

FIG. 9: plasma glucose profile

FIG. 10: parallelism and linearity of bioassay of different concentrations of Insugen (R) and IN105 as determined by PLA software. Data represent mean ± SEM of triplicate experiments.

FIG. 11: insugen mitogenic potency compared to IN-105 and HIM-2 was analyzed using a 4-point PLA IN the linear range.

FIG. 12: parallelism and linearity of the bioassays of Insugen (R) compared to IN105(A) and Insugen compared to HIM-2(B) at different concentrations determined by the PLA software. Data represent mean ± SEM of triplicate experiments.

FIG. 13: mitogenic activity of IN-105 analyzed IN comparison to IN-105 powder using a 4-point PLA assay IN the linear range.

FIG. 14: comparison of metabolic potency between Insugen (R), IN105 and HIM-2.

FIG. 15: parallelism and linearity of the bioassays of Insugen (R) compared to IN105(A) and Insugen compared to HIM-2(B) at different concentrations determined by the PLA software. Data represent mean ± SEM of triplicate experiments.

FIG. 16: (A) single site competition curves for Insugen (R), (B) IN105, and (C) HIM-2, respectively. The curves were determined by Graph Pad version-4 software. Data represent the mean of triplicate experiments.

Detailed description of the invention:

the present invention relates to an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, diluents, lubricants, plasticizers, permeation enhancers, and solubilizers.

IN another embodiment of the present invention, the cation-insulin conjugate complex is IN-105.

In yet another embodiment of the invention, the fatty acid component is capric acid and/or lauric acid or salts thereof.

In another embodiment of the invention, the fatty acid is sodium caprate.

In another embodiment of the present invention, the binder is selected from the group comprising polyvinylpyrrolidone, carboxymethylcellulose, methylcellulose, starch, gelatin, sugars, natural and synthetic gums or combinations thereof.

In another embodiment of the invention, the binder is polyvinylpyrrolidone.

In another embodiment of the invention, the diluent is selected from the group comprising calcium salts, cellulose or cellulose derivatives, palatinose, organic acids, sugars and sugar alcohols, pectate salts (pactatesalts) or combinations thereof.

In another embodiment of the present invention, the diluent is mannitol.

In another embodiment of the present invention, the disintegrant is selected from the group comprising cross-linked polyvinylpyrrolidone, carboxymethylcellulose, methylcellulose, cation exchange resins, alginic acid, guar gum, or a combination thereof.

In another embodiment of the present invention, the lubricant is selected from the group comprising magnesium stearate, sodium benzoate, sodium acetate, fumaric acid, polyethylene glycol, alanine and glycine.

In another embodiment of the invention, the lubricant is magnesium stearate.

In another embodiment of the present invention, the penetration enhancer is selected from the group comprising sodium dodecyl sulfate, sodium laurate, palmitoyl carnitine, phosphatidylcholine, cyclodextrin and derivatives thereof, carnitine and derivatives thereof, mucoadhesive polymers, zonulooccludin toxins, bile salts, fatty acids, or combinations thereof.

In another embodiment of the present invention, the penetration enhancer is sodium lauryl sulfate.

In another embodiment of the present invention, the penetration enhancer is beta-cyclodextrin.

In another embodiment of the present invention, the plasticizer is selected from the group consisting of polyethylene glycol, propylene glycol, acetyl citrate, triacetin, acetyl monoglyceride (ethyl monoglyceride), rapeseed oil, olive oil, sesame oil, acetyl triethyl citrate, sorbitol, diethyl oxalate, diethyl malate, diethyl fumarate, dibutyl succinate, dibutyl phthalate, dioctyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, tributyrin, triacetin, or a mixture thereof.

In another embodiment of the invention, the plasticizer is polyethylene glycol.

The invention also relates to pharmaceutical compositions for oral administration, wherein the oral dosage form is in the form of tablets, capsules, granules, powders or sachets or dry suspensions.

The invention also relates to a method for producing an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex, which comprises a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: binders, disintegrants, diluents, lubricants, plasticizers, permeation enhancers, and solubilizers.

The invention also relates to a process for the production of an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex according to claim 18, comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a diluent, a lubricant, a plasticizer, a solubilizer, and a penetration enhancer, the method comprising the steps of:

a. grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids.

b. Granulating the fatty acid from step (a) with an organic solvent.

c. Air drying the pellets from step (b).

d. The dried granules are sieved to obtain granules having the desired particle size.

e. The fatty acid particles are blended with the cation-insulin conjugate complex and other excipients.

f. The blended mixture was compressed for tableting.

The invention also relates to a process for the production of an orally administrable solid pharmaceutical composition of a cation-insulin conjugate complex according to claim 18, comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid or salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer, the method comprising the steps of:

a) grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acidsAnd/or salts of such fatty acids and binders.

b) The cation-insulin conjugate complex is suspended in an organic solvent using an adhesive to form a wet mass.

c) Granulating the components from step (b) using a binder.

d) Sieving the dried granules of step (c).

e) The granules are blended with other excipients.

f) The blended mixture was compressed for tableting.

In another embodiment of the present invention, the organic solvent used is selected from the group consisting of isopropanol, acetone, methanol, methyl isobutyl ketone, chloroform, 1-propanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethanol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, dichloromethane, tert-butanol, toluene, carbon tetrachloride, or combinations thereof.

The invention also relates to a 5-500mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a 50mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a 100mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂Fatty acid, fatty acid ester or salt thereof and is selected from the group consisting ofAt least three of the following groups of pharmaceutically acceptable excipients: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a 150mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a 200mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a 250mg tablet composition of a cation-insulin conjugate complex, said composition comprising a saturated or unsaturated C₄-C₁₂At least three of a fatty acid, a fatty acid ester or a salt thereof and a pharmaceutically acceptable excipient selected from the group comprising: a binder, a disintegrant, a lubricant, a plasticizer, and a penetration enhancer.

The invention also relates to a dose of an orally administrable solid pharmaceutical composition that achieves maximum control of postprandial blood glucose concentration in diabetic patients within 5-60 minutes after administration.

In another embodiment of the present invention, the orally administrable solid pharmaceutical composition according to any of the preceding claims, which produces a serum glucose reduction of at least 5% in a human patient within 120 minutes after oral administration.

The invention also relates to stable orally administrable pharmaceutical compositions of cation-insulin conjugate complexes comprising a saturated or unsaturated C₄-C₁₂Fatty acid, fatty acid ester or salt thereof and drug selected from the group consisting ofAt least three of a group of pharmaceutically acceptable excipients: a binder, a disintegrant, a diluent, a lubricant, a plasticizer, a penetration enhancer, and a solubilizer, characterized in that the composition remains stable when exposed to conditions selected from the group consisting of:

(a) a temperature range of about 2 to 40 c,

(b)25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.

In another embodiment of the invention, the impurity is increased by no more than 5% compared to the impurity content at the time of production.

In another embodiment of the invention, the impurity is increased by no more than 10% compared to the impurity content at the time of production.

In another embodiment of the invention, the assay shows no more than a 10% reduction of said compound in said composition.

In another embodiment of the invention, the dissolution profile is at least 75% at any of the time intervals.

In another embodiment of the invention, the time interval is in the range of 2 years.

In another embodiment of the invention, the difference between the hardness curves is at most 1kg/cm when compared to the hardness curve at production²。

In another embodiment of the invention, at least 95% ± 2% of the composition remains undegraded upon exposure to conditions selected from the group consisting of:

(a) a temperature range of about 2-8 ℃ or 25-40 ℃

(b)25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.

In another embodiment of the invention, at least 90% ± 2% of the composition remains undegraded upon exposure to a condition selected from the group consisting of:

(a) a temperature range of about 2-8 ℃ or 25-40 ℃

(b)25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.

The present invention also relates to a method of preparing amorphous spray-dried particles of a cation-insulin compound conjugate, the method comprising the steps of:

a. a solution or suspension comprising the cation-insulin compound conjugate and a fatty acid in a solvent is prepared.

b. The solution is sprayed into a chamber under conditions that allow for the removal of most of the solvent.

c. Spray-dried particles of the cation-insulin compound conjugate were obtained.

In another embodiment of the present invention, the fatty acid component is selected from the group consisting of C₄-C₁₂Fatty acids and/or salts of such fatty acids.

In another embodiment of the present invention, the fatty acid component is sodium caprate.

IN another embodiment of the invention, the cation-insulin compound conjugate is IN-105.

In another embodiment of the present invention, the solvent is water.

In another embodiment of the present invention, the solution or suspension comprising the cation-insulin conjugate and the fatty acid component in the solvent further comprises a diluent.

In another embodiment of the present invention, the diluent is mannitol.

In another embodiment of the invention, the size of the spray dried particles is from about 1 to 100 microns.

In another embodiment of the invention, the solution or suspension comprising the cation-insulin conjugate is spray dried at a temperature of 80 ℃ to 150 ℃.

In another embodiment of the present invention, the atomization pressure for spray drying is 0.5kg/cm²To 1.5kg/cm²。

In another embodiment of the invention, the spray-dried composition of the cation-insulin compound conjugate has a purity of at least 95%.

In another embodiment of the invention, the spray-dried composition of the cation-insulin compound conjugate has a purity of at least 98%.

In another embodiment of the invention, the spray-dried composition of the cation-insulin compound conjugate has a purity of at least 99%.

The present invention relates to spray-dried compositions of IN-105.

The present invention also relates to spray-dried compositions of IN-105 wherein the drug is substantially amorphous and homogeneous.

The invention also relates to a spray-dried composition of IN-105 having a purity of at least 95%.

The invention also relates to a spray-dried composition of IN-105 having a purity of at least 98%.

The invention also relates to a spray-dried composition of IN-105 having a purity of at least 99%.

The invention also relates to the amorphous form of IN-105.

The invention also relates to an insulinotropic cation-peptide conjugate complex, characterized in that said conjugate exhibits a reduced mitogenic property by a factor of four compared to its natural counterpart.

In another embodiment of the invention, the conjugate reduces cell proliferation to at least 20% ± 5% of its natural counterpart in vitro and/or in vivo.

In another embodiment of the invention, the conjugate reduces cell proliferation to at least 2% ± 0.5% of its natural counterpart in vitro and/or in vivo.

In another embodiment of the invention, the conjugate exhibits a metabolic efficiency similar to its natural counterpart.

The invention also relates to a pharmaceutical composition comprising a cation-peptide conjugate complex, said composition comprising a pharmaceutically acceptable excipient, said composition being characterized in that said excipient does not affect the mitogenic potency of said conjugate.

It is a primary object of the present invention to provide immediate release formulations comprising proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes that exhibit desirable pharmacokinetic properties and efficacy in a human diabetic potency model upon oral delivery. The current state of the art acknowledges the instability of unmodified insulin in the gastrointestinal tract and efforts have been made to circumvent these problems by developing formulations that are reasonably designed for immediate release.

The cation-peptide conjugate complex, cation-insulin conjugate complex of the present invention is characterized by showing reduced mitogenic potency. The conjugate according to the invention is advantageous in the fact that it has a relatively low or no risk of inducing a mitogenic response even after long-term use and can therefore be safely used for the long-term treatment and management of diabetes. Said therapeutic compounds of the invention are characterized by exhibiting a reduction of mitogenic potency by a factor of four when compared to Insugen (R).

According to an important aspect of the invention, said excipients in said drug product do not affect the mitogenic characteristics of said drug substance, so that said formulation or composition has a relatively reduced mitogenic potency compared to the insulin therapies currently on the market.

It is another object of the present invention to provide immediate release formulations comprising proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes that exhibit stability at a range of temperatures for time intervals up to 12 months. The formulations developed by the present invention are stable at room temperature and also show stability at high temperatures.

According to one aspect of the invention, the pharmaceutical compositions so produced are evaluated under different temperature test conditions and over a time interval of up to 12 months.

A preferred embodiment of the present invention relates to an orally deliverable formulation comprising 0.01% to 20% w/w of insulin, an insulin compound conjugate and/or a cationic insulin conjugate, 10% to 60% w/w of one or more compounds selected from saturated or unsaturated C₄-C₁₂A fatty acid component of a fatty acid and/or a salt of such a fatty acid, 10% to 60% w/w diluent, 1% to 20% disintegrant, 0.01% to 5% binder and 0.01% to 5% adsorbent, including but not limited to other suitable pharmaceutically acceptable carriers.

Accordingly, one aspect of the present invention relates to a formulation comprising at least one biologically active compound selected from the group consisting of: derivatized insulin conjugates, insulin analogs, insulin complexes, including but not limited to other therapeutic agents having insulin-like activity.

In yet another aspect, the present invention provides a method of producing the orally deliverable formulation described above under optimal operating conditions using suitable excipients that enhance the resistance of the therapeutically active agent to degradation.

In yet another preferred aspect, the method of producing an orally deliverable tablet having resistance to enzymatic degradation comprises the steps of: proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes:

1. grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids.

2. Granulating the fatty acid from step (1) with an organic solvent.

3. Air drying the pellets from step (2).

4. The dried granules were sieved to obtain granules having a particle size of about 250 microns.

5. The fatty acid particles, the cation-insulin compound conjugate, and other excipients are blended.

6. The blended mixture was compressed for tableting.

In accordance with the above objects and others, the present invention is directed, in part, to an oral solid dosage form comprising a modified insulin in a dose to achieve maximum control of postprandial blood glucose concentration in a diabetic patient within 20-30 minutes after administration.

One skilled in the art will readily appreciate that the dosage level may vary with the particular compound, the severity of the symptoms, and the subject's susceptibility to side effects. The preferred dosage of a given compound can be readily determined by one skilled in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound. The dosage unit form may be a liquid or a solid, such as a tablet, capsule, or granule, including a powder or sachet.

The invention also relates to the extemporaneous spray drying process which results in the production of 1-100 micron spray dried particles containing the therapeutic agent together with at least one pharmaceutically acceptable excipient.

The method comprises (a) preparing a composition comprising a therapeutic agent and optionally a diluent at a concentration of 0.001% to 20% w/w, optionally 10% to 60%w/w of one or more compounds selected from the group consisting of saturated or unsaturated C₄-C₁₂A fatty acid component of a fatty acid and/or a salt of such a fatty acid, (b) spray drying the resulting slurry to form a spray dried powder.

The subsequent aspect of the invention relates to combining other pharmaceutical excipients with the spray-dried powder, which is compressed into tablet form.

The ability of the method of the invention to provide, for example, uniform solid particles with a therapeutic agent gives conditions that help to provide an increase in the efficiency of evaporation relative to the heat input provided. The method of making such powdered particles described herein may include, for example, preparing an aqueous suspension or solution of the bioactive material, forming a mixture of the solution or suspension, spraying the ultra-fine droplets by reducing the pressure of the mixture, and drying the droplets into powdered particles by exchanging the spraying gas with a drying gas, for example, by spraying into a drying chamber of a spray drying apparatus.

Other objects and advantages of the present invention will be apparent to those skilled in the art from the following disclosure and claims.

The invention further provides methods of treating diabetes, impaired glucose tolerance, early stage diabetes, and late stage diabetes in animals, preferably humans, and methods of achieving glucose homeostasis comprising administering one or more unit doses of a dosage form of an immediate release formulation comprising said protein/peptide, conjugates thereof, and/or cation-polypeptide conjugate complexes, with a suitable excipient that enhances the resistance of said therapeutically active agent to degradation.

Defining:

in describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out herein.

"cation-insulin compound conjugate" includes any insulin compound component. The insulin compoundMay be, for example, a mammalian insulin compound, such as human insulin, or an insulin compound derivative or analog. The therapeutic agent relates IN particular to the molecule IN-105. IN-105 is formed by bonding a structural formula of CH at-amino acid lysine at position B29 of insulin B chain₃O-(C₄H₂O)₃-CH₂-CH₂-insulin molecules of amphoteric oligomers of-COOH. The molecules may be singly bound at positions a1, B1, and B29, doubly bound at different combinations of positions a1, B1, and B29, or triply bound at different combinations of positions a1, B1, and B29.

By "therapeutically effective amount" is meant an amount of insulin comprised within a dosage form of the present invention which is sufficient to effectively achieve clinically relevant control of blood glucose concentration during the dosing interval in human diabetic patients in the fasting or fed state.

As used herein, "diluent"/"filler"/"bulking agent" is an inert substance added to increase the volume of the formulation to make a tablet of a practical size that can be used for compression. Common diluents contemplated for use in the present invention include sugar alcohols, organic acids, galen IQ, palatinose, cellulose and cellulose derivatives, calcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, sugar powder, silicon dioxide and the like.

As used herein, a "binder" is an agent used to impart cohesiveness to the powder material. Binders, or sometimes referred to as "granulating agents," impart cohesiveness to the tablet formulation, which ensures that the tablet is intact after compression, and improves free-flowing properties by formulating particles with the desired hardness and size. Materials commonly used as binders include carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, starch; gelatin; sugars such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums such as gum arabic, sodium alginate, extracts of carrageenan, pantoea, ghatti, isapolhsks, Veegum (Veegum), microcrystalline cellulose, microcrystalline glucose, amylose, and larch arabinogalactan, and the like. Polyvinylpyrrolidone is used in the context of the present invention.

As used herein, a "disintegrant" is a substance that promotes breakage or promotes disintegration after administration of a tablet. Materials used as disintegrants are chemically classified as starches, clays, celluloses, algins (algins) or gums. Other disintegrants include Velcro HV, methylcellulose, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, alginic acid, guar gum, citrus pulp, cross-linked polyvinylpyrrolidone, carboxymethylcellulose, and the like.

The "lubricant" may be selected from: magnesium stearate, sodium benzoate, sodium acetate, fumaric acid, polyethylene glycol (PEG) with molecular weight higher than 4000, alanine and glycine. Preferred lubricants in the context of the present invention are magnesium stearate or sodium stearate.

"magnesium stearate" means a compound of magnesium mixed with solid organic acids derived from fats and consisting essentially of magnesium stearate and magnesium palmitate in varying proportions. Which is used as a pharmaceutically necessary substance (lubricant) in the production of compressed tablets.

"permeation enhancer" refers to any compound that increases membrane permeability and facilitates transport of a drug across the biological membrane, thereby improving the bioavailability of the delivered therapeutic agent. Suitable membrane permeation enhancers include surfactants such as sodium lauryl sulfate, sodium laurate, palmitoyl carnitine, polyoxyethylene lauryl ether-9 (laureth-9), phosphatidylcholine, cyclodextrin and derivatives thereof, bile salts such as sodium glycocholate, sodium deoxycholate, sodium taurocholate and sodium fusidate, chelating agents including EDTA, citric acid and salicylates, and fatty acids (e.g., oleic acid, lauric acid, acyl carnitines, mono-and di-glycerides), L-carnitine and derivatives, mucoadhesive polymers, ZOT (zonula occludens toxin), or combinations thereof.

"plasticizer" is selected from: acetyl citrate, triacetin, acetyl monoglyceride, canola oil, olive oil, sesame oil, acetyl triethyl citrate, glyceryl sorbitol, diethyl oxalate, diethyl malate, diethyl fumarate, dibutyl succinate, dibutyl phthalate, dioctyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, tributyrin, glyceryl triacetate, polyethylene glycol, propylene glycol, and mixtures thereof. The plasticizer is most preferably polyethylene glycol. The plasticizer may be present in an amount up to 40% w/w based on the film-forming polymer. A typical plasticizer used in the context of the present invention is PEG (polyethylene glycol).

"polyalkylene glycol or" PAG "refers to substituted or unsubstituted, linear or branched polyalkylene glycol polymers such as polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG), and combinations thereof (e.g., linear or branched polymers including two or more different PAG subunits such as a combination of two or more different PAG units selected from PEG, PPG, and PBG subunits), and includes monoalkyl ethers of polyalkylene glycols. The term PAG subunit refers to a single PAG unit, e.g., "PEG subunit" refers to a single unit of polyethylene glycol, e.g., - (CH 2O) -, "PPG subunit" refers to a single unit of polypropylene glycol, e.g., - (CH 2O) -, and "PBG subunit" refers to a single unit of polytetramethylene glycol, e.g., - (CH 2O) -. PAG and/or PAG subunits also include substituted PAGs or PAG subunits, e.g., PAGs including alkyl side chains such as methyl, ethyl, or propyl side chains or carbonyl side chains, and PAGs including one or more branched PAG subunits such as heteropeg or heteropeg.

The "solubilizing agent" can be any substance that enhances the water solubility of the drug.

As used herein, "pharmaceutically acceptable carrier" includes one or more agents selected from the group consisting of: carbohydrates and modified carbohydrates and derivatives thereof, polyethylene glycol and/or polypropylene glycol and derivatives thereof, inorganic fillers or lubricants, fatty acids and esters and salts thereof, preservatives and coating agents.

As used in this specification, an "effective amount" refers to an amount of any agent that is non-toxic but sufficient to provide the desired local or systemic effect and to participate in any pharmaceutical processing at a reasonable benefit/risk ratio. For example, an effective amount of lubricant refers to an amount sufficient to act to lubricate the composition for tableting purposes without providing any adverse effects.

By "organic solvent" in the context of the present invention is meant any solvent of non-aqueous origin, including liquid polymers and mixtures thereof. Organic solvents suitable for use in the present invention include: acetone, methanol, methyl isobutyl ketone, chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethanol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, dichloromethane, tert-butanol, toluene, carbon tetrachloride, or combinations thereof.

The "test" may be a laboratory test that finds and measures the amount of a particular substance.

An "impurity" may be defined as a component that is not part of the original formulation, which may cause impurities in the quality or condition of the composition, resulting substantially from any one or combination of the following characteristics:

a. contamination or stain.

b. Lack of consistency or uniformity; and (5) adulteration.

c. Something that makes something else impure; inferior components or additives.

The "dissolution test" is provided to determine compliance with the dissolution requirements described in monograph for tablet or capsule dosage forms alone. Tablet dissolution is a standardized method for measuring the rate of drug release from a dosage form. The basic effects of the dissolution test can be summarized as having attributes such as: a) optimization of efficacy during drug development and stability evaluation, b) routine evaluation of production quality to ensure consistency between product batches, c) evaluation of "bioequivalence", i.e. obtaining the same biological activity from individual batches of products obtained from the same or different manufacturers.

Dissolution may be carried out by dissolving the tablet in an optimal volume of about 37.0 + -0.5 deg.C medium and using a rotational speed of about 50 rpm. Dissolution was measured at various time intervals starting from 15 minutes. A variety of media that can be used to perform the dissolution test include water, buffers, and acidic media with a pH of 1-9, more preferably a pH of 2-7.

"hardness" is generally measured as the force required to break a tablet in a diameter compression test. The test involved placing the tablet between two anvils until the tablet broke. The crushing strength at which the tablets broke was recorded. "hardness" is sometimes referred to as tablet crushing strength. Several devices suitable for measuring tablet hardness include the Stokes (Monsanto) tester, Strong-Cobb tester, Pfizer tester, Erweka tester, Heberlein tester (or Schleuniger) tester, key tester and van der Kamp tester. The unit of hardness measurement is Kg/cm²。

"disintegration" is defined as the state in which any residue of the unit remaining on the screen of the test device, excluding insoluble coatings or fragments of the capsule shell, is a soft substance without any significant firm core.

The term "spray drying" is used to refer conventionally and broadly to a process of separating a liquid mixture into droplets (atomization) and rapidly removing solvent from the mixture in a spray drying apparatus in which there is a strong driving force for evaporation of solvent from the droplets. The spray drying process and spray drying equipment are generally described in Perry's chemical Engineers' Handbook, pages 20-54 to 20-57 (six Edition 1984). For a more detailed description of the spray-drying process and equipment see review by Marshall: "Atomization and Spray-Drying," 50chem. Eng.Prog.monogram. series 2(1954) and Masters, Spray-Drying Handbook (fourth edition 1985).

The term "drying" as used herein relates to the droplets or particles formed after removal of the solvent from the droplets or particles.

The term "particle" as used herein includes microparticles, submicron particles and macroparticles. Typically, the particles have a diameter or longest dimension of about 100 nanometers to 5 millimeters. The particles may be spheres, capsules, irregular shapes, crystals, powders, agglomerates, or aggregates.

The present invention contemplates the use of coating agents, which may include nonfunctional coatings (insta-coat, kollicoat ir) or enteric coatings such as cellulose-based polymers, film coatings or polymethyl acrylates and other coatings known to those of ordinary skill in the art.

The present invention, in one broad combined aspect, relates to a formulation comprising a covalently bound complex of a therapeutic agent, wherein the therapeutic agent is covalently bound to one or more molecules of a polymer that incorporates hydrophilic moieties, e.g., polyalkylene glycol moieties, and lipophilic moieties, e.g., fatty acid moieties, as integral parts of the polymer. In a preferred aspect, the therapeutic agent is covalently bound by covalent bonding to one or more molecules of a linear polyalkylene glycol polymer in which lipophilic moieties, such as fatty acid moieties, are incorporated as integral parts of the polymer.

The modified insulin conjugates used in the present invention are thus developed by attaching amphiphilic low molecular weight polymers or oligomers containing a hydrophilic polyethylene glycol moiety with the alkyl chain being lipophilic. The attachment alters the solubility of the drug molecule and stabilizes the protein or peptide against enzymatic degradation in the gastrointestinal tract. The conjugate drug is absorbed across the GI wall more efficiently than the drug in its native state. Upon crossing to reach the blood stream, the bond between the hydrophilic and hydrophobic chains is hydrolyzed, leaving the highly active insulin-PEG compound circulating in the blood, thereby advantageously altering the pharmacokinetics of the drug.

The invention relates IN particular to the molecule IN-105. IN-105 is formed by bonding a structural formula of CH at-amino acid lysine at position B29 of insulin B chain₃O-(C₄H₂O)₃-CH₂-CH₂-insulin molecules of amphoteric oligomers of-COOH.

According to one of the most important aspects of the invention, said cation-peptide conjugate complexes, cation-insulin conjugate complexes of the invention are characterized by exhibiting reduced mitogenic potency. The conjugate according to the invention is advantageous in the fact that it has a relatively low or no risk of inducing a mitogenic response even after long-term use and can therefore be safely used for the long-term treatment and management of diabetes. Said therapeutic compounds of the invention are characterized by exhibiting a reduction of mitogenic potency by a factor of four when compared to Insugen (R).

According to an important aspect of the invention, said excipients in said drug product do not affect the mitogenic characteristics of said drug substance, so that said formulation or composition has a reduced mitogenic potency compared to the insulin therapies currently on the market.

By "natural counterpart" is meant an unmodified peptide/protein that exhibits a basic in vitro or in vivo biological activity similar to the modified counterpart, i.e., the molecule is the peptide/polypeptide prior to undergoing any type of modification.

According to standard terminology, the term "mitogenic" defines a substance that stimulates the division of cells that otherwise have very limited division (i.e., without the influence of the substance).

The invention of the substances provided by the present invention is revealed in the fact that, according to specific experience and experiments, the professional believes that insulin itself is considered a mitogenic substance [ DeMeyts P; the structural aspect of The tissue and tissue-tissue factor-1 receptor binding and negative biological activity and recurrence to microbial conversion method, diabetes 37: S135-S148, 1994], Ish-Shalom D, Christoffersesen CT, Vorwerk P, Sacerdoti-Sierra N, Shymko RM, Naor D and De Meyts P; a mitogenic properties of instruments and instruments media by the instrument receiver. Diabetologia 40: S25-S31 and the proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes of the invention showed a decrease of one-fourth of mitogenic potency by inducing measurable proliferation assays of Balb 3T3-a31 cells in vitro.

According to one aspect of the invention, exemplary compounds of the invention should be said to exhibit "reduced mitogenicity" over that of instgen (r) if they detectably induce lower levels of cell proliferation as measured by alamar blue dye adsorbed by the cells. Those skilled in the art will recognize other suitable methods that may be used and the present invention is in no way limited to a particular proliferation assay.

According to one aspect of the invention, exemplary compounds of the invention should be said to exhibit substantially "non-mitogenic" properties compared to instgen (r) if they detectably induce lower levels of cell proliferation as measured by alamar blue dye adsorbed by the cells. Those skilled in the art will recognize other suitable methods that may be used and the present invention is in no way limited to a particular proliferation assay.

Colorimetric in vitro bioassay was used to quantify mitogenic activity [ Okajima t., Nakamura k., Zhang h., Ling n., Tanabe t., Yasuda t., and rosenfeld r.g., sensitive colorimetric biology for insulin-like Growth Factor (IGF) Stimulation of cellpromotion and Glucose regulation: use in students of IGFAnalogs.End6Criniology, 1992, Vol.120: 2201-2210.]

Biological tests are often analyzed with the aid of parallel wire methods. The log values of the doses are plotted on the horizontal axis, while the corresponding responses are represented on the vertical axis. For individual responses to each treatment, the standard formulation is represented by blue triangles and the sample formulation by green squares.

With the help of the parallel profile method, the following assumptions were examined for statistical authenticity:

1. the dose response relationship is linear for the standard and sample formulations.

2. The dose-response curve has a pronounced slope.

3. The dose response curves for the standard and sample formulations are parallel.

The parallel profile method has several advantages over conventional single point testing. Due to the examination of the above-mentioned assumptions,

1. linear dose-response correlation was not only hypothesized but also confirmed

2. Dose-independent relative efficacy was obtained.

The reproducibility of the mitogenic potency was analyzed on a linear portion of a curve consisting of at least four adjacent points by using a 4-parameter logistic curve fit. The results show that IN105 shows statistically lower mitogenic potency than Insugen (R) by a factor of four.

According to an important aspect of the invention, the mitogenic potency of the IN-105 molecule IN powder form and IN dialysis solution form is the same. IN-105 had mitogenic decreases to one fourth of Insugen (R).

According to yet another important aspect of the present invention, the mitogenic potency of the HIM2 molecule was reduced to one thirty-one times that of Insugen (R).

Without limitation, exemplary derivatives of the invention, such as IN-105, reduce IN vitro cell proliferation to at least 20%, or at least 25%, or at least 30%, at least 35%, at least 40% of its native counterpart. More preferably to a level of at least 30% of its natural counterpart.

Without limitation, exemplary derivatives of the invention, such as HIM-2, reduce cell proliferation in vitro to at least 2%, or at least 2.5%, or at least 3%, at least 3.5% of its natural counterpart. More preferably to a level of at least 3.8%, most preferably to a level of at least 4%.

According to yet another aspect of the invention, the evaluation of insulin binding to its cognate receptor was performed to compare the relative binding affinities of the drug forms Insugen (R), IN-105 and HIM-2. The most important aspect of the present invention relates to the fact that the metabolic activity of the exemplary compounds of the present invention remains unaffected and undamaged, despite a significantly reduced mitogenic potency compared to its natural counterpart.

The effect of insulin on cells is as metabolic as dependent on the ability of insulin to bind to the insulin receptor and the metabolic assays exemplified in the present invention help determine the effect of insulin on glucose uptake by differentiated adipocytes, providing data for evaluating and comparing the metabolic efficiency of exemplary drug substances of the present invention.

Extraction of drug substances from pharmaceutical products:

insugen (R) and exemplary compounds of the invention such as IN105 and HIM2 were used to prepare Zn-free and excipient-free drug substances. The vial was purged with glacial acetic acid (pH 3.4). The clear solution was loaded onto a C8 silica reverse phase column and separated using a gradient elution containing 250mM acetic acid and 100% ethanol. Fractions were collected during elution and pooled according to greater than or equal to 99% purity. The combined eluates were dialyzed against 10mM Tris, pH 8.0 for 15 hours using a 1KD cut-off membrane. Finally, the dialyzed zinc-free and excipient-free insulin obtained at pH 8.0 was analyzed by analytical RP-HPLC.

HepG2 cells were obtained from ATCC. Dulbecco's Modified Eagle Medium (DMEM), heat-inactivated Fetal Bovine Serum (FBS), 100X penicillin-streptomycin solution, and 100X HEPES salt solution were purchased from Invitrogen. Bovine serum albumin, sodium hydroxide, Triton-X100 and sodium bicarbonate were obtained from Sigma Aldrich.

Radiolabelled recombinant human insulin was purchased from Immunotech (Beckman Coulter) and had a specific activity of 2200Ci/mmole (Catalog No. A36474).

The method comprises the following steps:

in the presence of 5% CO₂In humidified conditions at 37 ℃, HepG2 cells were maintained in DMEM buffered with 10mM HEPES supplemented with 10% FBS and 1X penicillin-streptomycin solution. For the experiments, HepG2 cells were trypsinized and seeded at a density of 400,000 cells/well in 24-well plates. After 3 days of incubation, radioligand binding assays (1, 2) were performed using the cells.

Prior to performing the assay, the medium was removed and the cells were washed twice with binding buffers (DMEM, 2.2mg/ml sodium bicarbonate, 1mg/ml bovine serum albumin and 50mM HEPES) to remove any traces of growth factors present in the medium. Competitive binding experiments were started in duplicate using a fixed amount of radioligand (0.325nM) and varying concentrations of cold insulin drug substance (from 10 "13M to 10" 5M). The final reaction volume was brought to 1 ml. The plates were then incubated at 15 ℃ in the presence of a shaker set at 60rpm (2). The following day, all media from wells was discarded and each well was washed twice with ice-cooled binding buffer.

Each well received 1ml of solubilizer (0.5M sodium hydroxide, 0.5% Triton-X100). Solubilized cell pellets were transferred to radioimmunoassay tubes and bound radioactivity was read in gamma particle counting tubes (Stratec BioMedical Systems, Germany). The apparatus was calibrated and found to be 80% efficient.

Binding affinity calculation: for the normalization of counts per minute (CPM values) at the different concentrations of insulin used, the percentage of binding was calculated using the following equation:

binding% (CPM sample-CPM blank)/(CPM control-CPM blank) × 100.

Wherein the CPM control is the average CPM of wells containing cells with radiolabeled insulin but without any added cold insulin, the CPM blank is the average CPM of wells without radiolabeled insulin and without cold insulin, and the CPM samples are the average CPM of wells with radiolabeled insulin and with different concentrations of cold insulin.

Analysis of competitive binding curves:

competitive binding assays measure the binding of a single concentration of labeled ligand in the presence of different concentrations of unlabeled ligand. Competitive binding assays receptor number and avidity were determined by using the same compounds as the labeled ligand and unlabeled ligand.

Experiments were performed with a single concentration of radioligand. Incubation is carried out until equilibrium is reached, typically with different concentrations of unlabeled compound spanning about six orders of magnitude.

The upper part of the curve is the plateau at a value equal to the radioligand binding in the absence of competing unlabeled drug. This is the total binding. The lower part of the curve is the platform equal to non-specific binding (NS). The difference between the top and bottom platforms is specific binding.

The Y-axis can be expressed as cpm or converted to more useful units such as fmol binding/mg protein or number of binding sites/cell. It is possible to normalize the data from 100% (no competitor) to 0% (non-specific binding at the highest concentration of competitor).

The concentration of unlabeled drug that produces half of the radioligand binding between the top and bottom plateaus is called IC₅₀(50% inhibitory concentration), also known as EC₅₀(50% effective concentration). IC (integrated circuit)₅₀Is the concentration of unlabeled drug that blocks half of the specific binding.

If the labeled ligand and unlabeled ligand compete for a single binding site, the steepness of the competitive binding curve is determined by the mass action rule.

Non-linear regression was used to fit competitive binding curves to determine log (IC)₅₀). Log (IC) is typically determined using Graph padPrism software and using the single-site competition model equation₅₀)。

To measure IC₅₀(concentration of unlabeled drug blocking 50% of the specific binding of radioligand), the nonlinear regression problem must be able to determine the 100% (total binding) plateau and the 0% (non-specific binding) plateau. Using data collected in a wide range of concentrations of unlabeled drug, the curve has clearly defined a bottom plateau and a top plateau, and the program will fit all three values (two plateaus and IC) without problems₅₀)。

A rationally formulated composition is bioavailable when immediately released. In combination therapeutics of the above-described type of association, the polymeric component may be suitably structured, modified or suitably functionalized to impart the ability to the conjugate of the association in a selected manner.

In one aspect, the invention provides fatty acid compositions having one or more saturated or unsaturated C4, C5, C6, C7, C8, C9, or C10 fatty acids and/or salts of such fatty acids. Preferred fatty acids are caprylic acid, capric acid, myristic acid and lauric acid. Preferred fatty acid salts are the sodium salts of caprylic acid, capric acid, myristic acid and lauric acid.

The modifying moiety may comprise other hydrophilic polymers. Examples include poly (ethoxylated polyols) such as poly (ethoxylated glycerol), poly (ethoxylated sorbitol) and poly (ethoxylated glucose); polyvinyl alcohol ("PVA"); dextran; carbohydrate-based polymers, and the like. The polymers may be homopolymers or random copolymers or terpolymers of block copolymers and monomers based on the above polymers, linear or branched.

The total amount of cation-insulin compound conjugate to be used can be determined by one skilled in the art. The amount of the therapeutic agent is an amount effective to achieve the purpose of the particular active agent. The amount in the composition is a therapeutically effective dose, i.e., a pharmacologically or biologically effective amount. However, when the composition is used in a dosage unit form such as a capsule, tablet or liquid, the amount may be less than the pharmacologically or biologically effective amount, as the dosage unit form may comprise multiple delivery agent/biologically or chemically active agent compositions or may comprise an isolated pharmacologically or biologically effective amount. The total effective amount can then be administered in increasing units comprising the entire pharmacologically or biologically or chemically active amount of the biologically or pharmacologically active agent.

In certain preferred embodiments, the pharmaceutical composition contained in one or more dosage forms comprises from about 5mg to about 800mg of the delivery agent, preferably from about 10mg to about 600mg, more preferably from about 10mg to about 400mg, more preferably from about 25mg to about 200mg, most preferably about 75mg, 100mg or 150 mg. More preferably, the composition provides a peak plasma insulin concentration within about 15 minutes to about 60 minutes after oral administration to a diabetic person, and more preferably within about 10 to 20 minutes after oral administration to a diabetic person.

For the purposes of the present invention, a dosage form of the present invention comprising a therapeutically effective amount of insulin may comprise one or more unit doses (e.g., tablets, capsules, powders, semisolids, oral sprays, sublingual tablets (e.g., gelcaps or films)) to achieve the therapeutic effect. Further for the purposes of the present invention, a preferred embodiment of the dosage form is an oral dosage form.

In some cases, the complexed insulin compound conjugates exhibit a prolonged or otherwise altered pK profile relative to a technically acceptable control, such as a corresponding uncomplexed insulin compound conjugate. The pK curve can be evaluated using standard in vivo experiments, for example in mice, rats, dogs or humans. The assay described herein for assessing said properties of the cation-insulin compound conjugate complex is an aspect of the present invention.

In a preferred aspect, the method of producing an orally deliverable tablet comprising at least one biologically active compound selected from the group consisting of proteins/peptides, conjugates thereof and/or cation-polypeptide conjugate complexes (having resistance to enzymatic degradation) comprises the steps of:

1. grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids.

2. Granulating the fatty acid from step (1) with an organic solvent.

3. Air drying the pellets from step (2).

4. The dried granules are sieved to obtain granules having the desired particle size.

5. The fatty acid granules, the cation-insulin compound conjugate, the disintegrant, the binder, and other excipients are blended.

6. Tablet compression, polishing and packaging.

In a further preferred aspect, the method for producing an orally deliverable tablet comprises the following steps, wherein the tablet contains at least one active compound selected from the group consisting of: proteins/peptides, their conjugates and/or cation-polypeptide conjugate complexes (resistant to enzymatic degradation):

1. grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids such as sodium caprate, PVP-K-30.

2. IN-105 was suspended IN an organic solvent using PVPK-30 as the binder to form a wet mass.

3. The resulting components were granulated using PVP-K30 as a binder.

4. The dried sodium caprate granules were sieved through a # 45 (355 micron) sieve.

5. The sodium caprate particles are blended with other excipients.

6. Tablet compression, polishing and packaging.

According to the most important aspect of the invention, the term "stable", when applied to the compositions of the invention, means for the purposes of the present invention that the formulation remains undegraded or does not degrade to an unacceptable degree even after exposure of the formulation to storage conditions selected from: (i) a temperature range of about 2-8 ℃, 25 ℃,30 ℃ and 40 ℃, (ii)25 ℃/60% Relative Humidity (RH), 30 ℃/65% relative humidity, 40 ℃/75% relative humidity for at least one year, or any combination of the conditions. In the context of the present invention, undegraded or not degraded to an unacceptable degree means that the dosage component remains intact within applicable stability limits during the test.

According to one aspect of the invention, the stability test is performed for 1 to 12 months, preferably 1 month, preferably 2 months, preferably 3 months, preferably 4 months, preferably 5 months, preferably 6 months, preferably 7 months, preferably 9 months, preferably 11 months, and most preferably 12 months. The present invention takes into account the stability properties of the dosage forms to remain compatible for a period of up to about 2 years.

The stability properties of the pharmaceutical compositions of the present invention are evaluated under different temperature test conditions, such as at lower temperatures of 2-8 ℃, ambient room temperature conditions of 25-30 ℃ and slightly higher temperatures of 40 ℃. It is clearly understood by those skilled in the art that the temperature conditions and the stability established in the temperature conditions may not be strictly limited to the temperature conditions tested herein. The present invention allows for the extension of the stability properties to be shown during a wider range of test conditions to be established at lower and higher temperatures. The composition and its stability properties are not affected by many variations at temperatures of 2-40 ℃.

The stability attributes of the pharmaceutical compositions of the present invention were evaluated under different relative humidity test conditions, such as RH conditions 25 ℃/60% Relative Humidity (RH), 30 ℃/65% relative humidity, 40 ℃/75% relative humidity, or a combination of conditions as indicated herein. It is clearly understood by those skilled in the art that the relative humidity conditions, and the stability established in the relative humidity conditions, may not be strictly limited to the relative humidity conditions tested herein. The present invention allows for the extension of said stability properties to be shown during test conditions established over a wide range of relative humidities not explicitly disclosed herein. The composition and its stability attributes are not affected by many variations at relative humidity of 25 ℃/60% -40 ℃/75%.

According to one aspect of the invention, the composition of the invention is characterized in that at least 95% ± 2% of said composition remains undegraded when exposed to conditions selected from:

(a) a temperature range of about 2-8 ℃ or 25-40 ℃,

(b)25 ℃/60% Relative Humidity (RH), 30 ℃/65% relative humidity, 40 ℃/75% relative humidity for at least 6 months.

According to one aspect of the invention, the composition of the invention is characterized in that at least 95% ± 2% of said composition remains undegraded when exposed to conditions selected from:

(a) a temperature range of about 2-8 ℃ or 25-40 ℃,

(b)25 ℃/60% Relative Humidity (RH), 30 ℃/65% relative humidity, 40 ℃/75% relative humidity for at least 6 months.

According to yet another aspect of the invention, the tested different stability attributes of the tablet composition include, but are not limited to, hardness, disintegration time, dissolution profile and chromatographic purity.

Thus, various embodiments of the present invention exhibit several advantages in terms of dosing and convenient methods of administration. Yet another advantage afforded by the present invention over prior art compositions is that the inventors propose that the claimed, rationally designed IN-105 oral formulations with other excipients and the manufacturing process of such orally administrable tablets can be easily scaled up without affecting the IN vitro or IN vivo release profile. In addition, due to the rationally designed oral formulation and the method of production thereof, the amplification factor does not affect the in vivo drug properties or the in vivo release characteristics thereof.

One of the important aspects of the present invention relates to spray drying the composition to obtain a homogeneous, amorphous mixture of the cation-insulin conjugate complex.

Various parameters can be optimized to adjust the average size of the droplets. Droplet size can be affected by, for example: adjusting the pressure near the supercritical fluid or the high pressure gas pressure; adjusting the suspension or dissolution pressure, adjusting the flow rate of the suspension or solution, selecting the internal diameter of the nozzle tube, adjusting the temperature of the drying gas, adjusting the pressure inside the particle formation vessel, changing the concentration of the components of the suspension or solution, and the like. For example, the suspension or solution can be supplied to the mixing chamber at about 0.5ml/min to about 40ml/min to be sprayed from a nozzle opening of 100 micron internal diameter; a lower rate forms smaller droplets and a faster rate forms larger droplets. In the method, droplets are preferably formed in a mass median diameter range of about 1 micron to about 200 microns.

In the present invention, the temperature at which spray drying is effected is generally between about 80 ℃ to about 300 ℃, preferably between about 100 ℃ to about 180 ℃. Temperature control may be particularly important for maintaining the stability of product particles formed from or containing highly heat sensitive substances.

The pharmaceutical compositions obtained using certain embodiments of the present invention may be administered to any animal that may experience the beneficial effects of the compounds of the present invention. Of the most importance of such animals are humans, although the invention is not intended to be so limited.

The method of making particles of a powdered formulation according to the present invention may comprise, for example, preparing an aqueous suspension or solution of a biologically active material and a polyol and optionally one or more other compatible pharmaceutical excipients, forming a mixture of the solution or suspension with a pressurised gas, spraying the ultra-fine droplets by reducing the pressure of the mixture, and drying the droplets into powdered particles by exchanging the spraying gas with a drying gas, for example, by spraying into a drying chamber of a spray drying apparatus.

The term "solvent" refers to a liquid in which the material forming the majority of the spray-dried particles is dissolved, swirled or emulsified, for delivery to the atomizer of the spray dryer and evaporated into the drying gas, whether or not the liquid is a solvent or non-solvent for the material. The choice of solvent depends on the form of the bulk material (bulk material) and the material to be fed to the atomizer, for example, whether the material is to be dissolved, swirled or emulsified in the solvent. The most preferred solvent in the context of the present invention is water.

When a spray drying method is employed, the raw material in the form of a solution comprising at least one active ingredient selected from the group consisting of a cation-insulin compound conjugate in combination with a diluent is spray dried to produce particles.

The particle size of the drug solution is determined by the nebulizer used to spray the polymer solution, the nebulization pressure, the flow rate, the ingredients used, their concentration, the type of solvent, the viscosity, the nebulization temperature (inlet and outlet temperatures), the molecular weight of the therapeutic agent. Generally, the higher the molecular weight, the larger the particle size, assuming the same concentration.

One aspect of the present invention relates to a comparative dog clamp study (dog clamp study) of two experimental formulations of cationic-insulin compound conjugate oral compositions prepared by direct compression method and spray drying method.

The following measurements were performed: glucose infusion rate, plasma insulin concentration, and plasma glucose level (to allow assessment of endogenous insulin compound release). The rate of glucose infusion required to maintain euglycemia provides an indication of the effect of the insulin compound. Glucose infusion rates and plasma insulin levels were evaluated and compared.

Experimental formulations prepared by spray drying methods showed consistent levels of drug absorption and resulting glucose infusion rates with no loss of stability or biological activity.

The experimental formulation I (formulation 862) had the following composition

Composition (I)	Amount (mg)/tablet
		IN-105	6
Decanoic acid sodium salt	150
		Mannitol	150
Explotab (croscarmellose sodium)	25

The present invention is further illustrated in detail in the following examples. It will be clear that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

The invention will be better understood from the following examples. However, one of ordinary skill in the art will readily appreciate that these embodiments are merely illustrative of the present invention, the scope of which is defined by the claims.

The following examples represent preferred embodiments of the present invention.

Example I:

the pharmaceutical formulations of the present invention were prepared and tested. Thus, the required amount of ground sodium caprate was accurately weighed into a planetary mixer and granulated with 700ml isopropanol. The amount of isopropanol added to convert the powder mixture into particulate form was calculated. The wet mass was scraped every 5 minutes so that the mixture did not adhere to the walls of the planetary mixer. The wet mass was passed through an 18# sieve in a wet granulator and air dried overnight in the dark.

Water content of the pellets: sample weight 0.662 g; the water content is 2.27%

The appropriate amounts of IN-105, sodium caprate particles, Kollidon CL and pearlitol were weighed and passed through a # 60 sieve and mixed IN a double cone mixer at a speed of 12 r.p.m. for 20 minutes. After 20 minutes of homogeneous mixing, the blend was lubricated for 3 minutes using an aerosol and magnesium stearate at a speed of 88-90 r.p.m.

Table: i is

Composition (I)	Each tablet (mg)	For 1000g
			IN-105*	5.8207	17.27
Sodium caprate granules	150.000	445.10
			Kollidon CL	33.700	100.00
Pearlitol SD 200	144.1093	427.62
			Aerosil 200 Pharma	1.685	5.00
Magnesium stearate	1.685	5.00
				In total-337 mg	In total-1000 g

Tablets prepared according to example I were tested in six healthy male beagle dogs on an empty stomach for 26 hours. Each tablet was dosed with 20ml of water. Blood samples were collected for measuring blood glucose levels and plasma insulin levels. As shown in fig. 1 and 2, the tablet resulted in a rapid increase in plasma insulin levels, exhibiting a Cmax of about 100mU/ml, with a corresponding decrease in plasma glucose concentration of about 35% at a Tmax of 20 minutes from the time of administration.

The tablets so produced were subjected to stability studies according to ICH guidelines. Long-term studies were conducted at 2-8 ℃ and 25 ℃ and accelerated stability studies were conducted at 30 ℃/65% RH and 40 ℃/75% RH.

Stability of 5mg IN-105 tablet at 2-8 deg.C

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	Test%	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15min	NMT 3.0％	NLT 93％	(mg)	(w.r.t Label Claim)	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102％	99.1％
								1	1.58	1.50	0.052	94.8	5.07	101％	92.6％
3	1.51	1.50	0.059	94.5	5.07	101％	104.9％
								6	1.42	1.44	0.065	94.5	5.14	103％	103.7％
9	1.38	1.41	0.056	94.7	5.1	102％	99.3％
								12	1.40	1.45	0.060	94.4	5.08	102％	95.6％

Stability of 5mg IN-105 tablets at 25 ℃ 60% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	Test of％	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	(w.r.t Label Claim)	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	99.1
								1	1.70	1.40	0.13	94.8	5.06	101.0	77.2
2	1.63	1.38	0.26	94.0	5.04	101.0	85.6
								3	1.66	1.36	0.23	94.5	5.09	102.0	95.9
6	1.84	1.34	0.19	94.7	4.71	94.0	84.7
								9	1.59	1.33	0.19	94.0	4.69	94.0	85.2

12

1.74

1.38

0.25

94.3

4.85

97.0

85.6

Stability of 5mg IN-105 tablets at 30 ℃ 65% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	Test%	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	(w.r.t Label Claim)	NLT 75.0%, at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	99.1
								1	1.69	1.48	0.06	95.2	5.10	102.0	84.1
2	1.55	1.50	0.059	94.8	5.12	102.0	82.6
								3	1.55	1.50	0.070	94.6	5.13	102.0	100.4
6	1.63	1.44	0.130	94.3	4.92	98.0	94.3

Stability of 5mg IN-105 tablets at 40 ℃ 70% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	Test%	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15min	NMT 3.0％	NLT 93％	(mg)	(w.r.t Label Claim)	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	99.1
								1	1.58	1.43	0.110	94.2	4.84	97.0	86.0
2	1.54	1.45	0.130	93.8	4.79	96.0	90.0
								3	1.51	1.48	0.150	93.9	4.64	93.0	79.0
6	1.42	1.53	0.200	93.3	4.75	96.0	78.0

Example 2:

the milled sodium caprate was weighed accurately and placed in a planetary mixer and mixed for 2 minutes. The weighed required amount of IN-105 was added to isopropanol with 0.35% w/w polyvinylpyrrolidone to form a suspension and stirred well for 30 minutes using a magnetic stirrer. During granulation, the appropriate amount of isopropanol was added.

The granules were mixed in a planetary mixer for 15 minutes and passed through a 14# sieve and dried in a hot air oven at 30 ℃ for 2 hours.

Table 2:

composition (I)	Quantity (each piece)	Quantity (g)
			Decanoic acid sodium salt	150mg	192.67
IN-105	5.7078mg	7.33
			PVP-K 30	5.45mg	7.00

The planetary mixer speed was 4 for 15 min. IPA was added in a volume of 200ml

0.7g (0.35%) PVP +7.33g IN-105 was suspended IN 80ml IPA. The remaining 6.3g of PVP was suspended in 40ml of IPA. The remaining amount of IPA was added to form a hard wet mass.

The blending process comprises the following steps:

accurately weighed amounts of IN-105+ sodium caprate + PVP K-30 granules, Pearlitol and Kollidon CL were passed through a # 45 sieve and mixed IN an octagonal mixer. After uniform mixing, the blend is lubricated with Aerosil and magnesium stearate, stirred well and compressed into tablets.

Table 3:

composition (I)	Each tablet (mg)	For 50g (g)
			IN-105	5.7078	0.92
Sodium caprate granules	150.00	24.19
			PVP K-30	5.45	0.88
Pearlitol	114.742	18.51
			Kollidon CL	31	5.00
Aerosil 200 Pharma	1.55	0.25
			Magnesium stearate	1.55	0.25
	A total of-310 mg	In total-50 g

Tablets prepared according to example 2 were tested in six healthy male beagle dogs on an empty stomach for 26 hours. Each tablet was dosed with 20ml of water. Blood samples were collected for measuring blood glucose levels and plasma insulin levels. As shown in fig. 3 and 4, the tablet resulted in a rapid increase in plasma insulin levels, exhibiting a Cmax of about 75mU/ml, a Tmax of 20 minutes from administration. This resulted in a corresponding decrease in plasma glucose concentration of about 35% from baseline.

The tablets so produced were subjected to stability studies according to ICH guidelines. Long-term studies were conducted at 2-8 ℃ and 25 ℃ and accelerated stability studies were conducted at 30 ℃/65% RH and 40 ℃/75% RH.

Stability of 5mg IN-105 tablet at 2-8 deg.C

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	(w.r.t Label Claim	NLT 75.0%, at 15min
0	1.88	2.10	0.13	95.3	5.34	107.0	94.0％
								1	1.58	1.50	0.052	94.8	5.07	102.0	90.0％
3	1.51	1.50	0.059	94.5	5.07	102.0	102.0％
								6	1.42	1.44	0.065	94.5	5.14	103.0	86.0
9	1.38	1.41	0.056	94.7	5.21	104.0	95.0
								12	1.40	1.45	0.060	94.4	5.18	104.0	89.0

Stability of 5mg IN-105 tablets at 25 ℃ 60% RH

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.88	2.1	0.13	95.3	5.34	107.0	94.0％
								1	1.60	1.40	0.13	94.8	5.26	105.0	89.0
2	1.60	1.38	0.26	94.0	5.22	105.0	91.0
								3	1.52	1.36	0.23	94.5	5.29	106.0	94.0
6	1.48	1.34	0.19	94.7	5.17	103.0	90.0
								9	1.45	1.33	0.19	94.0	5.13	103.0	103.0
12	1.48	1.38	0.25	94.3	5.20	104.0	87.0

Stability of 5mg IN-105 tablets at 30 ℃ 65% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.88	2.1	0.13	95.3	5.34	107.0	94.0％
								1	1.61	1.48	0.06	95.2	4.97	99.0	90.0％
2	1.58	1.50	0.059	94.8	4.92	98.0	102.0％
								3	1.51	1.50	0.070	94.6	4.88	98.0	86.0％
6	1.42	1.44	0.130	94.3	4.79	96.0	79.0％

Stability of 5mg IN-105 tablets at 40 ℃ 75% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 90％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.88	2.1	0.13	95.3	5.34	107.0	94.0％
								1	1.58	2.0	0.17	94.2	5.09	102.0	96.0％
2	1.54	2.3	0.19	93.7	5.06	101.0	84.0％
								3	1.51	2.2	0.16	93.9	5.01	100.0	89.0％
6	1.42	1.9	0.29	93.3	4.91	98.0	92.0％

Example 3:

accurately weighed amounts of IN-105, sodium caprate + beta-cyclodextrin complex, sodium bicarbonate, and Kollidon CL were screened through a 45# sieve and mixed IN a polyethylene bag. After intimate mixing, the blend is lubricated with Aerosil and magnesium stearate, mixed thoroughly and compressed into tablets.

Table 4:

composition (I)	Each tablet (mg)	For 50g (g)
			IN-105	5.7078	1.30
β Cyclodextrin	40.38	9.18
			Sodium caprate granules	75.00	17.05
Sodium bicarbonate	74.71	16.98
			Kollidon CL	22	5.00
Aerosil 200 Pharma	1.1	0.25
			Magnesium stearate	1.1	0.25
	Total-220 mg	In total-50 g

Stability of 5mg IN-105 tablet at 2-8 deg.C

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	4.99	99.0	95.0％
								1	1.58	1.50	0.052	94.8	4.99	99.0	91.0％
3	1.51	1.50	0.059	94.5	5.01	100.0	85.0％
								6	1.42	1.44	0.065	94.5	4.96	99.0	101.0％
9	1.38	1.41	0.056	94.7	4.93	99.0	92.0％
								12	1.40	1.45	0.060	94.4	4.97	99.0	89.0％

Stability of 5mg IN-105 tablets at 25 ℃ 60% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	4.99	99.0	95.0％
								1	1.60	1.40	0.13	94.8	5.06	101.0	89.0％
2	1.60	1.38	0.26	94.0	4.93	99.0	91.0
								3	1.52	1.36	0.23	94.5	4.92	98.0	101.0
6	1.48	1.34	0.19	94.7	4.95	99.0	87.0
								9	1.45	1.33	0.19	94.0	4.91	98.0	90.0
12	1.48	1.38	0.25	94.3	4.87	97.0	85.0

Stability of 5mg IN-105 tablets at 30 ℃ 65% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	4.99	99.0	95.0％
								1	1.61	1.48	0.06	95.2	5.00	100.0	94.0％
2	1.58	1.50	0.059	94.8	4.81	96.0	92.0％
								3	1.51	1.50	0.070	94.6	4.83	96.0	84.0％
6	1.42	1.44	0.130	94.3	4.72	94.0	90.0％

Stability of 5mg IN-105 tablets at 40 ℃ 75% RH

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	4.99	99.0	95.0％
								1	1.58	1.50	0.110	94.2	4.88	98.0	90.0
2	1.57	1.48	0.130	93.6	4.92	94.0	81.0
								3	1.51	1.50	0.150	93.9	4.77	95.0	79.0
6	1.42	1.44	0.200	93.3	4.64	92.8	76.0

Example 4:

accurately weighed amounts of IN-105, sodium lauryl sulfate, Kollidon CL, and Pearlitol were sieved through a # 45 sieve and mixed IN an octagonal mixer. After intimate mixing, the blend is lubricated with Aerosil and magnesium stearate, mixed thoroughly and compressed into tablets.

Table 5:

composition (I)	Each tablet (mg)	For 50g (g)
			IN-10560#	5.7078	1.90
Sodium dodecyl sulfate	50.0	16.67
			Kollidon CL	15.00	5.00
Pearlitol SD 200	77.7922	25.93
			Aerosil 200 Pharma	0.75	0.25
Magnesium stearate	0.75	0.25
				Total-150 mg	In total-50 g

Tablets prepared according to example 4 were tested in six healthy male beagle dogs on an empty stomach for 26 hours. Each tablet was dosed with 20ml of water. Blood samples were collected for measuring blood glucose levels and plasma insulin levels. The results obtained are shown in fig. 5 and 6. The tablets so produced were subjected to stability studies according to ICH guidelines. Long-term studies were conducted at 2-8 ℃ and 25 ℃ and accelerated stability studies were conducted at 30 ℃/65% RH and 40 ℃/75% RH.

Stability of 5mg IN-105 tablet at 2-8 deg.C

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.64	1.62	0.064	95.7	5.16	103.2	97.6％
								1	1.61	1.58	0.068	95.2	5.24	104.8	98.4％
2	1.62	1.56	0.072	95.5	5.20	104.0	97.2％
								3	1.59	1.49	0.070	95.3	5.01	100.0	95.0％
6	1.59	1.54	0.076	94.8	4.94	98.8	92.7％
								9	1.55	1.42	0.082	94.2	5.10	102.0	88.4％
12	1.50	1.46	0.090	94.1	4.91	98..2	90.0％

Stability of 5mg IN-105 tablets at 25 ℃ 60% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.64	1.62	0.064	95.7	5.16	103.2	97.6％
								1	1.62	1.66	0.070	95.1	5.26	105.2	90.0％
2	1.60	1.54	0.074	95.0	5.14	102.8	94.1％
								3	1.64	1.59	0.072	95.2	4.97	99.6	89.1％
6	1.57	1.56	0.081	94.6	4.82	96.4	92.0％
								9	1.49	1.42	0.086	94.1	4.98	99.6	96.0％
12	1.44	1.47	0.092	93.7	4.87	97.4	98.4％

Stability of 5mg IN-105 tablets at 30 ℃ 65% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.64	1.62	0.064	95.7	5.16	103.2	97.6％
								1	1.62	1.62	0.072	95.3	5.10	102.0	98.6％
2	1.62	1.60	0.076	94.8	5.01	100.2	92.4％
								3	1.50	1.46	0.082	94.4	4.84	96.8	94.0％
6	1.44	1.29	0.094	93.6	4.79	95.8	88.4％

Stability of 5mg IN-105 tablets at 40 ℃ 70% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t. Label Claim	NLT 75.0% at 15min
0	1.64	1.62	0.064	95.7	5.16	103.2	97.6％
								1	1.58	1.59	0.072	95.1	5.08	102.0	92.0％
2	1.52	1.54	0.079	94.5	4.92	98.4	94.5％
								3	1.42	1.48	0.086	94.0	4.86	97.2	89.7％
6	1.38	1.46	0.102	93.2	4.64	92.8％	90.1％

Example 5:

sodium caprate, sodium bicarbonate and PEARLITOL granules were prepared using PVP K-30 (2% W/W) as binder and PEG 6000 (1% W/W) as plasticizer, as well as IN-105 added during granulation.

Accurately weighed amounts of ground sodium caprate, sodium bicarbonate and Pearlitol were placed in a planetary mixer and dry blended for 5 minutes. At the same time, PVP K30 was dissolved IN IPA and IN-105 was suspended. PEG 6000 was dissolved in water (5% v/v). The solution was stirred well using a magnetic stirrer. Sodium caprate, sodium bicarbonate and Pearlitol were granulated using PVPK and PEG solutions as granulating agents. The process was carried out for 20 minutes. The wet mass formed was passed through a # 18 sieve in a wet granulator and dried in LAF.

IPA addition volume-130 ml.

100 ml-IN-105 + PVP k.6ml-PEG IN water. The speed of the planetary mixer is 2-4, which lasts 20 min.

Speed of the wet granulator-200 rpm.

The water content of the granules.

Sample weight-0.525 g.

The water content is between 1 and 1.99 percent.

Table 6:

composition (I)	Amount/tablet (mg)	For 250g batches
			IN-105	10.6952	8.60
Decanoic acid sodium salt	150	120.55
			Sodium bicarbonate	90	72.33
Pearlitol 200SD	60.37	48.52
			PVP k-30(2％w/w)	6.22	5
PEG 6000(1％w/w)	3.11	2.5

Table 7:

composition (I)	Each tablet (MG)	For 200g (G)
			IN-105	10.6952	5.94
Decanoic acid sodium salt	150.00	83.33
			Sodium bicarbonate	90.00	50.00
Pearlitol 200 SD	60.37	33.54
			PVP K 30	6.22	3.46
PEG 6000	3.11	1.73
			Kollidon CL	36.00	20.00
Aerosil 200 Pharma	1.8	1.00
			Magnesium stearate	1.8	1.00
	A total of-360 mg	In total-200 g

Accurately weighed amounts of IN-105+ sodium caprate + sodium bicarbonate + Pearlitol + PVP K30 and PEG particles were passed through a 35# sieve and mixed with Kollidon CL IN a double cone mixer. After intimate mixing, the blend is lubricated using Aerosil and magnesium stearate, mixed thoroughly and compressed into tablets.

The tablets so produced were subjected to stability studies according to ICH guidelines. Long-term studies were conducted at 2-8 ℃ and 25 ℃ and accelerated stability studies were conducted at 30 ℃/65% RH and 40 ℃/75% RH.

Stability of 5mg IN-105 tablet at 2-8 deg.C

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	96.0％
								1	1.58	1.50	0.052	94.8	5.07	102.0	92.0％
3	1.5l	1.50	0.059	94.5	5.07	102.0	86.7％
								6	1.42	1.44	0.065	94.5	5.14	103.0	90.3％
9	1.38	1.41	0.056	94.7	5.1	103.0	98.0％
								12	1.40	1.45	0.060	94.4	5.08	102.0	89.2％

Stability of 5mg IN-105 tablets at 25 ℃ 60% RH

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	96.0％
								1	1.60	1.40	0.13	94.8	5.06	102.0	102.0％
2	1.60	1.38	0.26	94.0	5.04	101.0	93.1％
								3	1.52	1.36	0.23	94.5	5.09	102.0	91.2％
6	1.48	1.34	0.19	94.7	4.71	94.0	84.6％
								9	1.45	1.33	0.19	94.0	4.69	94.0	81.9％

Stability of 5mg IN-105 tablets at 30 ℃ 65% RH

Test interval	Hardness of	Disintegration time	HMWP	Purity of chromatography	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	96.0％
								1	1.61	1.48	0.06	95.2	5.10	104.0	89.2％
2	1.54	1.52	0.070	94.4	5.08	104.0	92.0％
								3	1.51	1.50	0.070	94.6	5.13	104.0	89.0％
6	1.42	1.44	0.130	94.3	4.71	94.2	84.7％

Stability of 5mg IN-105 tablets at 40 ℃ 75% RH

Test interval	Hardness of	Disintegration time	HMWP	Degree of chromatographic purity	Test of	% test	Dissolution of
								Moon cake	NMT 5.0 Kg/cm2	NMT 15 min	NMT 3.0％	NLT 93％	(mg)	w.r.t Label Claim	NLT 75.0% at 15min
0	1.58	1.49	0.053	95.3	5.08	102.0	96.0％
								1	1.58	1.50	0.110	94.2	4.84	97.0	98.0％
2	1.50	1.48	0.12	93.6	4.78	96.0	87.6％
								3	1.51	1.50	0.150	93.9	4.64	88.0	84.7％
6	1.42	1.44	0.200	93.3	4.75	96.0	80.0％

These are the preferred dose-proportional agents that provide the desired drug release profile. These formulations provide a conceivably substantially consistent release of IN-105.

Example 6: 1.663g of IN-105 and 50g of sodium caprate were dissolved IN water by adjusting the pH to 8.28 using 10% sodium hydroxide. The solution was taken for spray drying. The solution was spray dried at 80 ℃ in the current manner. The atomization pressure used was 1.5kg/cm²And the flow rate of the material supplied to the spray dryer was 2m 1/min. The purity of the spray dried sample was about 98% and a total of 16g of granules and 1.5g of fines were recovered from the total expected 52 g.

The spray-dried powder is mixed with other formulation excipients such as mannitol, explotab, colloidal silicon dioxide and magnesium stearate and compressed into tablets. These tablets were then tested for pharmacokinetic and pharmacodynamic responses in a dog clamp model.

Example 7: a material slurry containing IN105 at a concentration of 35g/l was used to obtain a spray-dried sample. The excipients used were mannitol and sodium caprate. To a mixture of 1050mg sodium decanoate and 1015mg mannitol was added 20ml of a solution containing 1ml of the slurry and mixed thoroughly. The solution was spray dried at 80 ℃ in the current manner. The atomization pressure used was 1.5kg/cm²The flow rate of the material supplied to the spray dryer was 2 ml/min. The recovery of the sample was about 60% and the purity of the spray dried sample was about 98%.

Example 8: a material slurry containing IN105 at a concentration of 35g/l was used to obtain a spray-dried sample. The excipients used were mannitol and sodium caprate. To a mixture of 1050mg sodium decanoate and 945mg mannitol was added 20ml of a solution containing 3ml of the slurry and mixed thoroughly. The solution was spray dried at 80 ℃ in the current manner. The atomization pressure used was 1.2kg/cm²The flow rate of the material supplied to the spray dryer was 2 ml/min. The recovery of the sample was about 60% and its purity was about 98.6%.

Example 9: a material slurry containing IN105 at a concentration of 35g/l was used to obtain a spray-dried sample. The excipients used were mannitol and sodium caprate. To a mixture of 1050mg sodium caprate and 1015mg mannitol was added 20ml of a slurry containing 1ml of the materialSolution and mix well. The solution was spray dried at 120 ℃ in the current manner. The atomization pressure used was 1.5kg/cm²The flow rate of the material supplied to the spray dryer was 2 ml/min. The recovery of the sample was about 60% and its purity was about 99.5%.

Example 10: a material slurry containing IN105 at a concentration of 35g/l was used to obtain a spray-dried sample. The excipients used were mannitol and sodium caprate. To a mixture of 1050mg sodium decanoate and 945mg mannitol was added 20ml of a solution containing 3ml of the slurry and mixed thoroughly. The solution was spray dried at 120 ℃ in the current manner. The atomization pressure used was 1.2kg/cm²The flow rate of the material supplied to the spray dryer was 2 ml/min. The recovery of the sample was about 60% and its purity was about 98.3%.

Example 11: a slurry containing IN105 at a concentration of 11.5g/l was used to obtain a spray-dried sample. The excipients used were mannitol and sodium caprate. To a mixture of 3000mg sodium caprate and 2885mg mannitol was added 50ml of a solution containing 10ml of the slurry and mixed thoroughly. The solution was spray dried at 150 ℃ in the current manner. The atomization pressure used was 0.86kg/cm²The flow rate of the material supplied to the spray dryer was 4 ml/min. The purity of the sample was about 94.71%.

Two experimental formulations screened by dog grip studies [ formulation-862, tablets prepared by direct compression method ] and [ formulation-872, tablets prepared by spray drying method ] showed: formulation-872, prepared by a spray drying process, exhibits a comparable level of consistency in drug absorption and resulting glucose infusion rate, with no loss of stability or bioactivity, providing a more economical, commercially viable and scalable method of producing the cation-insulin compound conjugate composition.

Table 8:

table 9:

table 10:

example 12:

DS formulated as Insugen (R) and IN105 IN10 mM Tris, pH 8, which had been dialyzed overnight, was provided and its concentration quantified by HPLC.

Watch-11

Table 11: dialyzed insulin concentrations for mitogenic experimental studies

3T3-A31 cells were obtained from ATCC (mouse fibroblasts, ATCC CCL-163). Dulbecco's Modified Eagle Medium (DMEM), heat inactivated Fetal Bovine Serum (FBS) and Amar blue dye were purchased from Invitrogen. IGF1 and 1MHEPES solution were obtained from SigmaAldrich. In the presence of 5% CO₂3T3-a31 cells were maintained in DMEM buffered with 10mM HEPES supplemented with 10% FBS under humidified conditions at 37 ℃. For the assay, 3T3-A31 cells were trypsinized with 0.25% trypsin-EDTAAnd (4) carrying out enzymolysis. The number of cells was counted by hemocytometer using trypan blue stain. 10,000 cells/well were seeded in 96-well plates in DMEM buffered with 10mM HEPES supplemented with 0.5% FBS. Different concentrations of each insulin were added to the wells in triplicate. After 20 hours incubation with different concentrations of growth factors, alamar blue dye (10% v/v) was added and the plates were incubated in a 37 ℃ incubator for another 4 hours. Fluorescence was measured using a 96-well plate reader (excitation wavelength 530 nm; emission wavelength 590 nm).

Table 12: comparison of mitogenic potency ratios of Insugen and IN-105 compared to HIM-2 from PLA, using a 4-point PLA assay IN the Linear Range

INSUGEN	IN-105	HIM-2
			1	0.237	0.044
1	0.256	0.021
			1	0.286	0.048
Mean number of	0.259±0.020	0.0376±0.020

Example 13:

the DS formulated as instgen (r) and IN105 at 10mm tris, pH 8 after overnight dialysis, the concentration of which was quantified by HPLC, is provided IN table 13.

Table 13: dialyzed concentrations for metabolic studies

3T3-L1 cells were obtained from ATCC. Dulbecco's Modified Eagle Medium (DMEM), heat-inactivated Fetal Bovine Serum (FBS), penicillin-streptomycin solution (10X), and 1M HEPES solution were purchased from Invitrogen. Dexamethasone, isobutylmethylxanthine, 4-aminoantipyrine, N-ethyl N-sulfopropyl-m-toluidine and glucose oxidase/peroxidase reagent were obtained from SigmaAldrich.

In the presence of 5% CO₂3T3-L1 cells were maintained in DMEM buffered with 10mM HEPES supplemented with 10% FBS, 100U/100. mu.g penicillin/streptomycin (maintenance medium) under humidified conditions at 37 ℃. In order to perform the metabolic assay, 3T3-L1 cells must be differentiated into adipocytes. 3T3-L1 cells were trypsinized with 0.25% trypsin-EDTA. The number of cells was counted by hemocytometer using trypan blue stain. 25,000 cells/well were seeded in maintenance medium in 96-well plates. The cells were allowed to fuse for the next two days. On day 3, cells were replaced with differentiation medium (0.5 mM dexamethasone and 0.25 μ M isobutylmethylxanthine in maintenance medium) for another four days. On day 7, the differentiation medium was replaced back to the maintenance medium for 3 days. On day 9, cells were washed with 1 × PBS and prepared at different concentrations in low glucose medium assay buffer (supplemented with 0.5% FBS, 2mM L-glutamine and 1 × Pen-Strep) in 15mL centrifuge tubesOf the respective insulin. The dilutions were added in triplicate to the wells. After 22 hours incubation with different concentrations of insulin, the 96-well plates were removed from the incubator for glucose evaluation. Glucose was evaluated with the aid of a GOPOD reagent, which converts glucose in the medium to gluconic acid and hydrogen peroxide. The hydrogen peroxide formed reacts with the substrate (4-aminoantipyrine and N-ethyl N-sulfopropyl-m-toluidine) to give a violet product which can be read at 550 nm. And (3) calculating metabolic activity: for data presentation, the% glucose in the medium was measured by considering the value of "no insulin" as 100%.

The results of this experiment show that the metabolic potency of Insugen (R), IN105 and HIM-2 are all similar and that the potency lies within the acceptable limits (0.8-1.2) for the PLA analysis, establishing that Insugen and IN-105 are metabolically equivalent, as shown IN FIG. 14. FIG. 14 shows that the metabolic efficacy of Insugen (R) compared to IN105 and of Insugen (R) compared to HIM-2 is the same. Insugen (R) had a metabolic potency ratio of 1.166 compared to IN105 and 1.149 compared to HIM-2. The data represent the mean ± SEM of triplicate experimental determinations. FIG. 15 shows the parallelism and linearity of the bioassays performed at different concentrations of Insugen (R) and IN105, as determined by the PLA software. The data represent the mean ± SEM of triplicate experimental values.

Table 14: comparison of the metabolic potency ratio of Insugen compared to IN-105 compared to HIM-2 was obtained using a 4-point PLA assay IN the linear range.

INSUGEN	IN-105	HIM-2
			1	1.121	1.19
1	1.2	1.104
			1	1.179	1.153
Mean number of	1.166±0.041	1.149±0.043

Example 14:

glacial acetic acid (pH 3.4) was used to purge multiple insulin vials. The clear solution was then loaded onto a C8 silica reverse phase column and separated using a gradient elution containing 250mM acetic acid and 100% ethanol. Fractions were collected during elution and the fractions were combined according to a purity of greater than or equal to 99%. The eluted pooled fractions were dialyzed for 15 hours. Dialysis was performed using a 1KD cut-off membrane versus 10mm tris, pH 8.0. Finally, the dialyzed Zn-free and excipient-free insulin obtained at pH 8.0 was analyzed by analytical RP-HPLC, and the overnight dialyzed Zn-free DS formulated as insugen (r), IN105 and HIM-2 IN10 mM Tris, pH 8 and the concentration quantitatively determined by HPLC are provided IN table 15.

Watch 15

HepG2 cells were obtained from the ATCC (ATCC accession No. HB-8065). Dulbecco's Modified Eagle Medium (DMEM), heat-inactivated Fetal Bovine Serum (FBS), 100X penicillin-streptomycin solution, and 100X HEPES salt solution were purchased from Invitrogen. Bovine serum albumin, sodium hydroxide, Triton-X100 and sodium bicarbonate were obtained from Sigma Aldrich.

Radiolabelled recombinant human insulin was purchased from Perkin Elmer and has a specific radioactivity of 2200Ci/mmole (Catalog No. NEX420).

In the presence of 5% CO₂In humidified conditions at 37 ℃, HepG2 cells were maintained in DMEM buffered with 10mM HEPES supplemented with 10% FBS and 1X penicillin-streptomycin solution. For the experiments, HepG2 cells were trypsinized and seeded at a density of 400,000 cells/well in 24-well plates. After 3 days of incubation, radioligand binding assays (1, 2) were performed using the cells.

Prior to performing the assay, the medium was removed and the cells were washed twice with binding buffers (DMEM, 2.2mg/ml sodium bicarbonate, 1mg/ml bovine serum albumin and 50mM HEPES) to remove any traces of growth factors present in the medium. Competitive binding experiments were started in duplicate using a fixed amount of radioligand (0.325nM) and varying concentrations of cold insulin drug substance (from 10 "12M to 10" 7M). The final reaction volume was brought to 1 ml. The plates were then incubated overnight at 15 ℃ using a shaker set at 100rpm (2). The following day, all media from wells was discarded and each well was washed twice with ice-cooled binding buffer.

Each well received 1ml of solubilizer (0.5M sodium hydroxide, 0.5% Triton-X100). Solubilized cell pellets were transferred to radioimmunoassay tubes and bound radioactivity was read in gamma particle counting tubes (Stratec BioMedical Systems, Germany). The device is considered to be 80% efficient by calibration.

Binding affinity calculation: for the normalization of counts per minute (CPM values) at the different concentrations of insulin used, the percentage of binding was calculated using the following equation:

binding% (% CPM sample-CPM blank)/(CPM control-CPM blank) x100

Wherein the CPM control is the average CPM of wells containing cells with radiolabeled insulin but without any added cold insulin, the CPM blank is the average CPM of wells without radiolabeled insulin and without cold insulin, and the CPM samples are the average CPM of wells with radiolabeled insulin and with different concentrations of cold insulin.

Table 16: comparison of binding affinities, expressed as EC50 values, using their 95% confidence intervals.

	EC50(M)	95％CI，EC50(M)
			Insugen(R)	1.246X10-10	(1.005，1.544)X10-10
IN105	1.185X10-10	(0.863，1.626)X10-10
			HIM-2	1.157X10-10	(0.896，1.495)X10-10

The foregoing description and examples have been provided for the purpose of illustration only. They are not to be construed as unnecessarily limiting since modifications and variations thereof within the spirit of the claims will become apparent to those skilled in the art upon reading the present disclosure.

The foregoing description of the preferred embodiments and best mode of the invention known to the applicant at the time of filing the present application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, and to enable others of ordinary skill in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.

Claims (48)

1. An orally administrable solid pharmaceutical composition of IN-105, said composition comprising 0.01% to 20% w/w IN-105, 10% to 60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizer, wherein the lubricant is magnesium stearate.
2. 2. The pharmaceutical composition of claim 1, wherein the fatty acid component is capric acid and/or lauric acid or salts thereof.
3. 3. The pharmaceutical composition of claim 1, wherein the fatty acid is sodium caprate.
4. 4. The pharmaceutical composition of claim 1, wherein the binder is selected from the group consisting of polyvinylpyrrolidone, carboxymethylcellulose, methylcellulose, starch, gelatin, sugars, natural gums, synthetic gums, and combinations thereof.
5. 5. The pharmaceutical composition of claim 4, wherein the binder is polyvinylpyrrolidone.
6. 6. The pharmaceutical composition of claim 1, wherein the diluent is selected from the group consisting of calcium salts, cellulose derivatives, palatinose, organic acids, sugars, sugar alcohols, pectates, silicon dioxide, and combinations thereof.
7. 7. The pharmaceutical composition of claim 1, wherein the diluent is mannitol.
8. 8. The pharmaceutical composition of claim 1, wherein the disintegrant is selected from the group consisting of crosslinked polyvinylpyrrolidone, carboxymethylcellulose, methylcellulose, cation exchange resins, alginic acid, guar gum, and combinations thereof.
9. 9. The pharmaceutical composition of claim 1, wherein the penetration enhancer is selected from the group consisting of sodium lauryl sulfate, sodium laurate, palmitoyl carnitine, phosphatidylcholine, cyclodextrin derivatives, carnitine derivatives, mucoadhesive polymers, zonula occludens toxin, bile salts, fatty acids, and combinations thereof.
10. 10. The pharmaceutical composition of claim 9, wherein the penetration enhancer is sodium lauryl sulfate.
11. 11. The pharmaceutical composition of claim 9, wherein the penetration enhancer is beta-cyclodextrin.
12. 12. The pharmaceutical composition of claim 1, wherein the plasticizer is selected from the group consisting of polyethylene glycol, propylene glycol, acetyl citrate, triacetin, acetyl monoglyceride, rapeseed oil, olive oil, sesame oil, acetyl triethyl citrate, glyceryl sorbitol, diethyl oxalate, diethyl malate, diethyl fumarate, dibutyl succinate, dibutyl phthalate, dioctyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, tributyrin, triacetin, and mixtures thereof.
13. 13. The pharmaceutical composition of claim 12, wherein the plasticizer is polyethylene glycol.
14. 14. The pharmaceutical composition of claim 1, wherein the oral dosage form is in the form of a tablet, capsule, granule, powder or sachet, or a dry suspension.
15. 15. A method of producing an orally administrable solid pharmaceutical composition of IN-105 comprising 0.01% to 20% w/w IN-105, 10% to 60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizing agent, wherein the lubricant is magnesium stearate, the method comprising the steps of:

    a. grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids;

    b. granulating the fatty acid from step (a) with an organic solvent;

    c. air drying the pellets from step (b);

    d. sieving the dried granules to obtain granules having a desired particle size;

    e. blending the fatty acid granules with IN-105 and other excipients; and

    f. the blended mixture was compressed for tableting.
16. 16. A method of producing an orally administrable solid pharmaceutical composition of IN-105 comprising 0.01% to 20% w/w IN-105, 10% to 60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizing agent, wherein the lubricant is magnesium stearate, the method comprising the steps of:

    a grinding of suitably saturated or unsaturated C₄-C₁₂Fatty acids and/or salts of such fatty acids and binders;

    b suspending IN-105 IN an organic solvent using a binder to form a wet mass;

    c granulating the components from step (b) using a binder;

    d sieving the dried granules of step (c);

    e blending the granules with other excipients; and

    f compressing the blended mixture for tableting.
17. 17. The process of claim 15 or 16, wherein the organic solvent used is selected from the group consisting of isopropanol, acetone, methanol, methyl isobutyl ketone, chloroform, 1-propanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethanol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, dichloromethane, tert-butanol, toluene, carbon tetrachloride, and combinations thereof.
18. An orally administrable solid pharmaceutical composition of IN-105 IN the form of a 5-500mg tablet, said composition comprising 0.01-20% w/w IN-105, 10-60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizer, wherein the lubricant is magnesium stearate.
19. 19. A tablet of an orally administrable solid pharmaceutical composition having IN-105 according to claim 1, wherein the amount of IN-105 is 50 mg.
20. 20. A tablet of an orally administrable solid pharmaceutical composition having IN-105 according to claim 1, wherein the amount of IN-105 is 100 mg.
21. 21. A tablet of an orally administrable solid pharmaceutical composition having IN-105 according to claim 1, wherein the amount of IN-105 is 150 mg.
22. 22. A tablet of an orally administrable solid pharmaceutical composition having IN-105 according to claim 1, wherein the amount of IN-105 is 200 mg.
23. 23. A tablet of an orally administrable solid pharmaceutical composition having IN-105 according to claim 1, wherein the amount of IN-105 is 250 mg.
24. 24. The pharmaceutical composition of claim 1, which is presented in a dose that achieves maximum control of postprandial blood glucose concentration in a diabetic patient within 5-60 minutes after administration.
25. 25. The pharmaceutical composition of claim 1, which results in at least a 5% reduction in serum glucose within 120 minutes after oral administration in a human patient.
26. A stable orally administrable solid pharmaceutical composition of IN-105 comprising 0.01% to 20% w/w IN-105, 10% to 60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizer, wherein the lubricant is magnesium stearate,

    characterized in that the composition remains stable when exposed to conditions selected from the group comprising:

    a.2-40 deg.C; and

    25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.
27. 27. The stable orally administrable solid pharmaceutical composition of claim 26, characterized in that the impurities in the composition are increased by no more than 5% compared to the impurity content at the time of production.
28. 28. The stable orally administrable solid pharmaceutical composition of claim 26, characterized in that the impurities in the composition are increased by no more than 10% compared to the impurity content at the time of production.
29. 29. The stable orally administrable solid pharmaceutical composition of claim 26, characterized IN that the concentration of IN-105 IN the composition is reduced by no more than 10%.
30. 30. The pharmaceutical composition according to claim 1, characterized in that the dissolution profile of the solid pharmaceutical composition is at least 75% at any time interval of 0-12 months.
31. 31. The pharmaceutical composition of claim 30, wherein the time interval is in the range of 2 years.
32. 32. The stable orally administrable solid pharmaceutical composition of claim 26, wherein the difference between the hardness curves of the compositions is at most 1kg/cm when compared to the hardness curve at the time of production²。
33. 33. The stable orally administrable solid pharmaceutical composition according to claim 26, characterized in that at least 95% ± 2% of the composition remains undegraded upon exposure to conditions selected from the group comprising:

    (a) the temperature range of 2-8 ℃ or 25-40 ℃,

    (b)25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.
34. 34. The stable orally administrable solid pharmaceutical composition according to claim 26, characterized in that at least 90% ± 2% of the composition remains undegraded upon exposure to conditions selected from the group comprising:

    g.temperature range of 2-8 deg.C or 25-40 deg.C,

    h.25 ℃. + -. 2 ℃/60%. + -. 5% Relative Humidity (RH), 30 ℃. + -. 2 ℃/65%. + -. 5% relative humidity, 40 ℃. + -. 2 ℃/75%. + -. 5% relative humidity for at least 6 months.
35. 35. A method of preparing amorphous spray-dried particles comprising 0.01% to 20% w/w IN-105, 10% to 60% w/w saturated or unsaturated C₄-C₁₂Fatty acid, fatty acid ester or salt thereof, 10-60% w/w of diluent, 1-20% w/w of disintegrant, 0.5% w/w of lubricant, and optionally at least one compound selected fromThe following pharmaceutically acceptable excipients: a binder, a plasticizer, a penetration enhancer, and a solubilizing agent, wherein the lubricant is magnesium stearate, the method comprising the steps of:

    a. preparing a solution or suspension comprising IN-105 and a fatty acid component IN a solvent;

    b. spraying the solution into a chamber under conditions that allow for removal of a substantial portion of the solvent; and

    c. spray dried particles of IN-105 were obtained.
36. 36. The method of claim 35, wherein the fatty acid component is sodium caprate.
37. 37. The method of claim 35, wherein the solvent is water.
38. 38. The method of claim 35, wherein the diluent is mannitol.
39. 39. The method of claim 35, wherein the spray dried particles are 1-100 microns in size.
40. 40. The method of claim 35, wherein the solution or suspension comprising IN-105 is spray dried at a temperature ranging from 80 ℃ to 150 ℃.
41. 41. The method of claim 35, wherein the atomization pressure for spray drying is 0.5kg/cm²To 1.5kg/cm²。
42. 42. The method of any of claims 35-41, wherein the spray-dried composition of IN-105 has a purity of at least 95%.
43. 43. The method of any of claims 35-41, wherein the spray-dried composition of IN-105 has a purity of at least 98%.
44. 44. The method of any of claims 35-41, wherein the spray-dried composition of IN-105 has a purity of at least 99%.
45. 45.IN-105, which comprises 0.01-20% w/w IN-105, 10-60% w/w saturated or unsaturated C₄-C₁₂A fatty acid, a fatty acid ester or a salt thereof, 10% to 60% w/w of a diluent, 1% to 20% of a disintegrant, 0.5% w/w of a lubricant, and optionally at least one pharmaceutically acceptable excipient selected from: a binder, a plasticizer, a penetration enhancer, and a solubilizing agent, wherein the lubricant is magnesium stearate characterized in that it exhibits a one-third reduction in mitogenicity as compared to its natural counterpart.
46. 46. The insulinotropic pharmaceutical composition according to claim 45, which reduces cell proliferation in vitro and/or in vivo to at least 20% ± 5% of its natural counterpart.
47. 47. The insulinotropic pharmaceutical composition according to claim 45, which reduces cell proliferation in vitro and/or in vivo to at least 2% ± 0.5% of its natural counterpart.
48. 48. The pharmaceutical composition of claim 45, wherein said excipient does not affect mitogenic potency of said composition.