Ultra-long-acting pharmaceutical composition containing insulin

Abstract

The present invention discloses a stable, ultra-long-acting injectable pharmaceutical composition comprising insulin glargine, collagen, and one or more pharmaceutically acceptable excipients or carriers. Furthermore, the present invention relates to a method for preparing said composition and its use for the treatment of metabolic disorders, including diabetes mellitus.

Description

Technology Field

[0001] The present invention relates broadly to the field of biopharmaceuticals, in particular to ultra-long-acting insulin glargine formulations. Specifically, the present invention relates to a stabilized injectable solution comprising insulin glargine, collagen, and one or more pharmaceutically acceptable excipients or carriers thereof. The present invention also relates to a method for preparing said composition and its use for the treatment of metabolic disorders, including diabetes mellitus. Background Technology

[0002] Diabetes is a serious medical condition characterized by the body's inability to metabolize glucose or insufficient glucose metabolism. In 2019, approximately 9.3% of the global adult population was diagnosed with diabetes, and this number is expected to increase to nearly 11% by 2045 (https: / / www.statista.com / statistics / 271464 / percentage-of-diabetics Worldwide / ).

[0003] Diabetes is a major cause of death worldwide. There are two main types of diabetes: Type 1 diabetes, in which damage to pancreatic beta cells results in insufficient insulin secretion and requires frequent administration of exogenous insulin to sustain life; and

[0004] It is caused by a lack of endogenous insulin to control blood sugar and can be managed through diet, exercise, medication, or insulin injections, but about 20% of cases are classified as type 2 diabetes, which is controlled by insulin injections.

[0005] Hypoglycemia frequently occurs due to insulin use in both types of diabetes because it is very difficult to predict the amount of endogenous insulin normally secreted by the body to regulate blood sugar. Insulin must be administered periodically to maintain blood sugar levels in diabetic patients within the normal range.

[0006] Insulin injections are prescribed to patients with diabetes. Insulin is a natural hormone secreted to regulate blood glucose levels. In healthy individuals, insulin is released from the pancreas into the bloodstream when blood sugar levels rise. Increased blood sugar levels after a meal are rapidly regulated by increased insulin secretion. Insulin plays a crucial role in converting excess blood sugar into glycogen and storing it in the liver.

[0007] Since the introduction of insulin in the 1920s, continuous efforts have been made to improve the treatment of diabetes. To avoid extreme blood sugar levels, diabetic patients often undergo a treatment method of injecting insulin multiple times with meals.

[0008] Insulin is a polypeptide composed of 51 amino acids, divided into the following two amino acid chains: a chain A with 21 amino acids and a chain B with 30 amino acids. The two chains are connected to each other through two disulfide cross-links. Insulin preparations have long been used to treat diabetes.

[0009] Traditionally, rapid-acting generic insulin preparations or their intermediate-acting insulin protamine preparations have been used to treat diabetic patients. Over time, new insulin analogs and derivatives have been developed. Insulin analogs and derivatives differ in one or more amino acid positions in the structure of human insulin or in amino acid chain length.

[0010] Many insulins, insulin analogs, and derivatives are available on the market. Commonly used insulins, insulin analogs, or insulin derivatives are classified as follows:

[0011] Rapid-acting insulin analog (bolus) : For example, insulin aspart (Novolog®); insulin lispro (Humalog®); insulin glulisine (Aprida®), rapid-acting human insulin (Viaject®). These analogs begin to act within 5 to 15 minutes after administration and remain active for 3 to 4 hours.

[0012] Short-acting insulin (bolus) : For example, regular insulin (Humulin® or Novolin®). Regular insulin begins to act within 30 minutes of administration and lasts for about 5 to 8 hours.

[0013] Intermediate-acting insulin For example, isophanin insulin, aspart protamine, and lisproprotamine belong to this group and act 1 to 3 hours after administration. The duration of action is 16 to 24 hours.

[0014] Long-acting insulin (Basal) : For example, insulin glargine, insulin degludec, and insulin detemir. These analogs begin to act within 1 to 2 hours, and the duration of action varies from about 12 hours to about 24 hours.

[0015] Mixed insulin For example, a mixture of NPH and regular insulin. There are several modified formulations of mixed insulin with different mixing ratios. The onset of action of these mixed formulations is approximately 30 minutes. Mixed insulin contains the same type of insulin. Two different types of insulin cannot be mixed. That is, insulin lispro cannot be mixed with insulin detemer, insulin aspart, or insulin glargine. Mixed formulations of insulin lispro can only be mixed with insulin lispro regular and insulin lispro protamine—two forms of insulin lispro.

[0016] Insulin glargine is a long-acting basal insulin administered once daily. Insulin glargine is formulated in acidic solvents and possesses an altered isoelectric point. It is isoelectric at physiological pH, precipitates upon subcutaneous injection, and forms an amorphous depot. The gradual redissolution of this subcutaneous depot is the primary mechanism of action. The long duration of action of insulin glargine (up to 24 hours) is directly related to the formation of micro-precipitates; the rate of dissociation of micro-precipitates into insulin glargine monomers; and the slow rate of absorption of insulin glargine from subcutaneous tissue. However, in many patients, insulin glargine does not persist for 24 hours. The drug precipitates after injection into subcutaneous tissue and is absorbed through slow redissolution. Although it exhibits "peakless" action, the drug is associated with inter-individual variability, and many patients exhibit a peak of action, particularly at high doses.

[0017] Another disadvantage of insulin glargine is that, unlike isophane insulin, it cannot be mixed with soluble insulin, leading to precipitation. Consequently, many patients using dual (mixed) insulin must increase their daily injection frequency or switch to basal-bolus injection therapy. Furthermore, insulin resistance, which is observed in many patients, develops with the long-term use of long-acting insulin, requiring an increase in insulin dosage. In vitro studies indicate that glargine binds preferentially to insulin-like growth factor-1 (IGF-1) receptors rather than insulin receptors. This may indicate a potential for promoting mitotic abnormalities with long-term use. Therefore, there is a need for insulin glargine formulations that not only exhibit a longer duration of action but also reduce inter-individual variability and show less mitotic abnormality.

[0018] Biomaterials are widely utilized in the drug and pharmaceutical fields for efficient drug delivery within the body. Among known biopolymers, collagen has emerged as one of the most attractive choices due to its excellent biocompatibility, biodegradability, and weak antigenicity, as well as its well-known molecular structure, biological properties, and the way it interacts with the body (Friess, 1998; Lee et al., 2001).

[0019] Collagen, the most abundant protein in mammals, is a major structural protein that accounts for about 30% of the total protein in all vertebrates. More than 90% of the extracellular protein in tendons and bones, and more than 50% of the protein in the skin, consists of collagen. The animal skin of cattle or pigs, and the Achilles tendons of cattle or horses, are currently the main sources of collagen.

[0020] To date, 28 types of collagen have been identified and described, the most common of which are as follows:

[0021] Type 1: Main component of bone tissue.

[0022] Type 2: Main component of cartilage.

[0023] Type 3: Main component of reticular fibers.

[0024] Type 4: Major component of the basement membrane, which is the epithelial secretory layer.

[0025] Type 5: Primarily found on cell surfaces, hair, and placenta.

[0026] There are many prior art works disclosing pharmaceutical compositions that include the binding affinity and combination of insulin and collagen.

[0027] U.S. Patent Application 20130225492 A pharmaceutical composition comprising a drug and collagen is disclosed, and the composition has good handling properties and sustained release. The sustained-release pharmaceutical composition comprises a drug; collagen; and at least one sugar selected from monosaccharides, disaccharides, trisaccharides, and tetrasaccharides.

[0028] U.S. Patent No. 5,922,356 B2 In the patent, it is disclosed that a sustained-release formulation containing a therapeutically active substance as an active ingredient, collagen as a drug carrier, and glycosaminoglycan as an additive is used for the treatment or prevention of a disease.

[0029] Japanese Patent 0543453 A local sustained-release formulation for promoting wound healing is disclosed, which is obtained by mixing a carrier containing collagen as an essential component with a physiologically active substance having wound healing-promoting activity.

[0030] Japanese Patent 56122317 It is disclosed that when the collagen concentration decreases, the degradation of the drug containing gelled collagen is accelerated and the drug release rate is increased.

[0031] Yaoi and his group, 1991 The invention discloses the binding efficiency of insulin and collagen by analyzing the ability of eight extracellular matrix proteins—fibronectin, vitronectin, laminin, and collagen types 1, 2, 3, 4, and 5—to bind to insulin.

[0032] Manolache and his group, 2016 Disclosed in [the text] is a gel complex comprising a collagen gel, zinc oxide, and insulin glargine at pH 7.4 and the corresponding matrix.

[0033] U.S. Patent 6,468,959 A dry pellet is disclosed in an oral, buccal, sublingual, or nasal administration formulation comprising at least one peptide agent having a given net charge dispersed in a matrix comprising a component selected from the group consisting of gelatin, fractionated gelatin, collagen hydrolysate, cross-linked gelatin, and mixtures thereof, wherein the component(s) of the hydrophilic molecule group possess an opposite net charge sufficient to form a pseudocoazervate together with the peptide agent.

[0034] U.S. Patent Application 20140213963A biocompatible insulin delivery device is disclosed comprising an insulin reservoir, a glucose-responsive plug that seals the reservoir, and optionally a protective microporous membrane that covers the exposed surface of the glucose-responsive plug and further seals the reservoir, wherein the plug comprises a polymer matrix having a stimulus-responsive component and an inorganic component applied to change the porosity of the plug in response to a stimulus, and the plug functions to release insulin from the reservoir in response to high blood glucose concentrations and prevent insulin from being released from the reservoir in response to low blood glucose concentrations.

[0035] Compositions containing insulin and collagen are known in the prior art. The compositions of the prior art are in the form of gels, pellets, or implants. Administering insulin via gel or implant requires a special syringe or device, which reduces ease of administration. Ease of administration is important for diabetic patients, as they require repeated insulin injections. The administration of gels or implants causes pain at the injection site and frequent swelling, causing discomfort to patients and resulting in low compliance. Furthermore, the special syringes or devices required for administering these medications increase the total cost of the product, making them unsuitable for many patients. Oral administration of insulin via pellets exhibits significant variability in absorption rates. In the case of oral administration of insulin, pharmacokinetic responses are often unpredictable. The problem to be solved

[0036] Therefore, there is a need for insulin glargine and collagen formulations that are not only easy to administer using standard pens and syringes but also possess increased bioavailability, a longer duration of action, and reduced immunogenicity compared to commercially available insulin glargine compositions of the same dose.

[0037] None of the prior art has introduced an injectable formulation comprising insulin glargine, collagen, and one or more pharmaceutically acceptable excipients having a pH of 2 to 5, which has a long duration of action and greater bioavailability than insulin glargine formulations commercially available at the same concentration. means of solving the problem

[0038] As used herein, the term "insulin(s)" includes mammalian insulin, insulin analogs, or derivatives.

[0039] The term "insulin analog or derivative" as used in the present invention includes analogs or derivatives of naturally secreted insulin molecules, that is, analogs or derivatives of insulin in which the structure of human insulin or animal insulin is modified to alter their physicochemical, pharmacokinetic, or pharmacodynamic properties.

[0040] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers. The composition has a pH of 2 to 5, is a solution at administration, and forms a depot at a physiological pH immediately after administration, and the composition exhibits increased bioavailability and a longer duration of action compared to an injectable composition containing an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof, among commercially available pharmaceuticals having the same dose.

[0041] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising insulin glargine or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; and the pharmaceutical composition exhibits increased bioavailability and a longer duration of action compared to a commercially available injectable pharmaceutical composition comprising insulin glargine of the same concentration or same dose.

[0042] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising insulin glargine or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; and the pharmaceutical composition exhibits increased bioavailability and a duration of action of 24 hours or more.

[0043] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising insulin glargine or a physiologically acceptable salt thereof; human collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; and the pharmaceutical composition exhibits increased bioavailability and a longer duration of action compared to a commercially available injectable composition comprising insulin glargine of the same concentration or same dose.

[0044] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; the pharmaceutical composition exhibits increased bioavailability and a longer duration of action compared to a commercially available injectable pharmaceutical composition comprising the 'insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof' having the same dose; and the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a stabilizer, an isotonic agent, a surfactant, a buffer, a preservative, an antioxidant, an acidifier, a pH adjuster, an organic solvent, an aqueous solvent, a solubilizer, and combinations thereof.

[0045] One aspect of the present invention provides a method for treating type 1 and type 2 diabetes in a patient, comprising administering a stable, ultra-long-acting injectable pharmaceutical composition comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers. The pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; exhibits increased bioavailability and a longer duration of action compared to an injectable pharmaceutical composition comprising the 'insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof' having the same dose, and the pharmaceutical composition is administered 2 to 3 times per week.

[0046] One aspect of the present invention provides a method for preparing a stable ultra-long-acting injectable solution comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers having a pH of 2 to 5, and the method comprises the following steps:

[0047] a) A step of dissolving accurately weighed zinc-containing insulin glargine crystals in injectable water with the help of several μL of 1M HCl;

[0048] b) a step of preparing a buffer with a pH of 2 to 5;

[0049] c) A step of dissolving an isotonic agent in a buffer;

[0050] d) a step of diluting the concentrated insulin glargine of step (A) with the buffer of step b);

[0051] e) a step of adding collagen to the solution of step d) while stirring constantly;

[0052] f) A step of adjusting the final pH of the solution to 2 to 5. Effects of the invention

[0053] The present invention discloses a stable, ultra-long-acting injectable solution comprising insulin glargine, collagen, and one or more of the excipients thereof at a pH of 2 to 5. The present invention also discloses a method for preparing said composition and its use for the treatment of metabolic disorders including diabetes mellitus. Brief explanation of the drawing

[0054] Figure 1 shows the standard curve of insulin glargine in the presence of collagen. Figure 2 illustrates a simulation of plasma insulin concentrations after administering Gla-100 or Gla-300 once daily according to the prior art (Reference: Modelling of Subcutaneous Absorption of Long-Acting Insulin Glargine in Type 1 Diabetes, Michele et al.: IEE Trans Biomed Eng 202; 67(2): 624-631.) Figure 3 shows an ‘in vitro model of insulin glargine release’ used in the present invention. Specific details for implementing the invention

[0055] While studying other formulations to prepare a pharmaceutical composition containing insulin glargine that not only exhibits a duration of more than 24 hours but is also easy to administer, has higher bioavailability, and lower immunogenicity, the inventors found that when human type I collagen is mixed with insulin glargine at an acidic pH, the formation of M-1 and M-2, which are soluble metabolites of insulin glargine at physiological pH, is significantly reduced (M-1 and M-2 are insulin metabolites that are produced when exposed to proteases in physiological saline to form a composition similar to that of interstitial fluid). Since the said metabolites of insulin glargine carry the blood glucose-lowering activity, any drug formulation capable of reducing the formation of said metabolites is directly correlated with the sustained release and dissolution profile of the drug, and these in vitro M-1 and M-2 formation kinetics are interpreted as having an improved pharmacological effect, with a longer half-life of the circulating active drug, as long as a pharmaceutical depot is formed at the injection site.

[0056] The composition of the present invention is acidic and forms a depot at physiological pH after administration in vivo. The inventors observed that at the injection site (physiological pH), human type I collagen molecules, which are triple helix proteins, bind to insulin and form an additional barrier against insulin glargine already crystallized at the physiological pH of 7.4 at the injection site to inhibit the action of proteases in the interstitial fluid within the extracellular matrix at the injection site. A two-compartment model was proposed for insulin glargine absorbed through systemic blood circulation. The first compartment is where the precipitate is redissolved to form a hexamer, followed by the conversion of the hexamer into a monomer, and the second compartment is where it is transported into the blood through the vascular capillaries of the subcutaneous tissue. Through this, it was confirmed that the release of insulin is controlled by the additional barrier of collagen at the physiological pH of 7.4. Furthermore, it was confirmed that collagen inhibits proteases that degrade insulin at the injection site. This protease inhibition increases the bioavailability of insulin in the blood, implying that collagen can cooperate with insulin to help regulate blood glucose levels. Therefore, the pharmaceutical composition of the present invention exhibits superior bioavailability compared to commercially available compositions containing the same concentration of insulin glargine.

[0057] The pharmaceutical composition of the present invention is a solution because both insulin glargine and collagen are soluble at an acidic pH. The solution of the present invention is easy to administer using a conventional syringe or pen and does not require any special syringe or device for administration. Easy injection administration improves patient compliance. Furthermore, since the pharmaceutical composition of the present invention can be easily administered via existing pens and syringes, it is cost-effective and does not impose an additional burden on the patient.

[0058] As used herein, the term "pharmaceutical composition" means a mixture containing a therapeutic compound to be administered to mammals, e.g., humans, to prevent, treat, or control a specific disease or condition affecting mammals.

[0059] As used herein, "collagen" refers to a fibrous protein that constitutes bone, cartilage, tendons, and other connective tissues. As used herein, "collagen" includes, but is not limited to, collagen types 1, 2, 3, 4, and 5, human collagen, and processed or modified collagen.

[0060] As used herein, the term “buffer” means a solution containing a weak acid and its salt or a weak base and its salt that is resistant to changes in pH. As used herein, the term “buffer” includes, but is not limited to, phosphate, acetate, citrate, arginine, glycylglycine, or tris (i.e., 2-amino-2-hydroxymethyl-1,3-propanediol) buffers and corresponding salts and combinations thereof.

[0061] As used herein, “preservatives” refers to compounds that may be used to prevent the growth of fungi and other microorganisms. As used herein, “preservatives” include, but are not limited to, benzoic acid, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetypiridinium chloride, chlorobutanol, phenol, phenyl ethyl alcohol, 2-phenoxyethanol, phenyl mercury nitrate, thimerosal, metacresol, and combinations thereof.

[0062] As used herein, “isotonic agent” refers to a compound that is physiologically acceptable, prevents the circulation of water across the tissue cell membranes in contact with the formulation, and imparts tonicity suitable for the formulation. An “isotonic agent” is a compound such as glycerin, which is commonly used for this purpose at known concentrations. Other available possible isotonic modifiers include salts, e.g., sodium chloride, dextrose, lactose, and combinations thereof.

[0063] As used in this specification, "pH adjuster" means a combination of acid and alkali. As used in this specification, "pH adjuster" may be selected from the group consisting of o-phosphoric acid, citric acid, acetic acid, succinic acid, lactic acid, gluconic acid, tartaric acid, 1,2,3,4-butanetetracarboxylic acid, fumaric acid, or malic acid. The alkali is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium hydroxide, ammonium hydroxide, magnesium oxide, calcium hydroxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicate, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, or triethanolamine, and combinations thereof.

[0064] As used herein, “solubilizing agent” refers to a substance capable of solubilizing or partially solubilizing therapeutic compounds and / or polymers. Suitable “solubilizing agent” as used herein includes, but is not limited to, polysorbates and poloxamers, nonionic and ionic surfactants, food acids and bases (e.g., sodium bicarbonate), polyhydric alcohols, alcohols, and combinations thereof.

[0065] As used herein, the term “acidifying agent” refers to a compound that provides a proton or hydrogen ion and / or accepts an electron. Suitable acidifying agents include, but are not limited to, formic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, tartaric acid, diarizoic acid, glutamic acid, lactic acid, maleic acid, succinic acid, acetic acid, citric acid, or citric anhydride, and combinations thereof, and such acidifying agents include in the form of particulate solids.

[0066] Suitable “organic solvents” used in this specification are selected from the group consisting of N-methylpyrrolidone (NMP), dichloromethane, dimethylformamide (DMF), dimethylacetamide (DMAC), acetonitrile, tetrahydrofuran, dioxane, methanol, ethanol, isopropanol, tert-butanol, and combinations thereof, but are not limited thereto.

[0067] Suitable "water-soluble solvents" as used in this specification include water, water for injection, and mixtures of water and alcohol.

[0068] As used herein, "surfactants" refer to fatty acid esters or ethers with polyhydric alcohols such as glycerol and sorbitol (Span®, Tween®, particularly Tween® 20 and Tween®80, Myrj®, Brij®, Cremophore® or Poloxamer, Pluronics® and Tetronics®), polysorbates (Tween™), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethylamine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohol polyoxyethylene sorbitan, octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salts (sodium deoxycholate, sodium cholate), and polyoxyl It includes, but is not limited to, castor oil (Cremophor™), nonylphenol ethoxylate (Tergitol™), cyclodextrin, lecithin, methylbenzethonium chloride (Hyamine™), and combinations thereof.

[0069] The "antioxidant" as used in this specification may be selected from the group consisting of ascorbate (sodium / acid), sodium bisulfite, butylated hydroxyanisole (bha), butylated hydroxytoluene (bht), cysteine / cysteine ​​hydrochloride, sodium dithionite (sodium hydrosulfite, sodium sulfoxylate), gentisic acid, gentisic acid ethanolamine, glutamate monosodium, glutathione, sodium formaldehyde sulfoxylate, potassium metabisulfite, sodium metabisulfite, methionine, monothioglycerol (thoglycerol), propyl gallate, sodium sulfite, tocopherol alpha, alpha tocopherol hydrogen succinate, sodium thioglycolate, and combinations thereof.

[0070] As used herein, "one or more stabilizers" include, but are not limited to, surfactants, antioxidants, preservatives, solubilizers, esterase inhibitors, and combinations thereof. One or more stabilizers are fatty acid esters and ethers with polyhydric alcohols such as glycerol and sorbitol (Span®, Tween®, particularly Tween® 20 and Tween®80, Myrj®, Brij®, Cremophore® or Poloxamer, Pluronics® and Tetronics®), polysorbate (Tween™), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethylamine oxide, cetyltrimethylammonium bromide (CTAB), glycerol, polyethoxylated alcohol polyoxyethylene sorbitan, octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salts (sodium deoxycholate, sodium cholate), polyoxyl Castor oil (Cremophor™), Nonylphenol ethoxylate (Tergitol™), Cyclodextrin, Lecithin, Methylbenzethonium chloride (Hyamine™), Benzoic acid, Zinc oxide, Butylparaben, Ethylparaben, Methylparaben, Propylparaben, Sodium benzoate, Sodium propionate, Benzalkonium chloride, Polyvinyl alcohol, Benzethonium chloride, Benzyl alcohol, Cetylpyridinium hydrochloride, Chlorobutanol, Phenol, Phenylethyl alcohol, 2-Phenoxyethanol, Phenylmercury nitrate, Thimerosal, Metacresol, Ascorbate (Sodium / Acid), Sodium bisulfite, Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), Cysteine / Cysteinate HCl, Sodium dithionite (Sodium hydrosulfite, Sodium sulfoxylate), Genticic acid, ethanolamine gentiic acid, monosodium glutamate, glutathione, sodium formaldehyde sulfoxylate, potassium metabisulfite, sodium metabisulfite, methionine, monothioglycerol (thioglycerol), propyl gallate, sodium sulfite, tocopherol alpha, alpha-tocopherol hydrogen succinate,It is selected from sodium thioglycolate, esterase inhibitors, such as pancreatic secretion inhibitors, protease inhibitors, and serine esterase inhibitors, such as aprotinin, and combinations thereof.

[0071] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; the pharmaceutical composition exhibits increased bioavailability and a longer duration of action compared to a commercially available injectable pharmaceutical composition containing the 'insulin analog or derivative having an isoelectric point of 5 to 8.5' having the same dose; and the 'insulin analog or derivative having an isoelectric point of 5 to 8.5' is insulin glargine. Insulin glargine is present in a concentration range of 100 IU to 1,000 IU / ml. In a preferred embodiment of the present invention, insulin glargine is present in a concentration range of 100 IU to 300 IU per ml.

[0072] One aspect of the present invention provides a stable, ultra-long-acting injectable pharmaceutical composition comprising: an insulin analog or derivative having an isoelectric point of 5 to 8.5, or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers, wherein the pharmaceutical composition has a pH of 2 to 5; the pharmaceutical composition is a solution at administration and forms a depot at a physiological pH immediately after administration; the pharmaceutical composition exhibits increased bioavailability and a longer duration of action compared to a commercially available injectable pharmaceutical composition comprising the 'insulin analog or derivative having an isoelectric point of 5 to 8.5' having the same dose; and wherein the collagen present in the composition is human collagen. The human collagen used in the present invention is obtained from recombinant DNA technology. In a preferred embodiment of the present invention, it is preferable to use human collagen type 1 or to use a rapid human collagen solution prepared by slowly removing salts from an acidic collagen solution using acetic acid and EDTA and adding acidic water for injection. Meanwhile, collagen should be used in a concentration range of 3 to 10 mg / ml.

[0073] The composition of the present invention is useful for the treatment of metabolic disorders. The metabolic disorders are selected from the group consisting of insulin resistance syndrome, diabetes mellitus, hyperlipidemia, fatty liver disease, cachexia, obesity, atherosclerosis, and arteriosclerosis. In a preferred embodiment of the present invention, the composition of the present invention is useful for the treatment of type 1 and type 2 diabetes mellitus.

[0074] The composition of the present invention exhibits a longer duration of action, i.e., a duration of 24 hours or more, compared to commercially available compositions of the same concentration or dose. The composition of the present invention may be administered 1 to 3 times per week.

[0075] One aspect of the present invention discloses a method for preparing a stable ultra-long-acting injectable solution comprising insulin glargine or a physiologically acceptable salt thereof; collagen; and one or more pharmaceutically acceptable excipients or carriers having a pH of 2 to 5, wherein the method comprises the following steps:

[0076] a) a step of accurately weighing zinc-containing insulin glargine, adding it to water for injection, and solubilizing it by adding several μL of 1M HCl;

[0077] b) a step of preparing a buffer with a pH of 2 to 5;

[0078] c) A step of dissolving an isotonic agent in a buffer;

[0079] d) a step of diluting the concentrated insulin glargine of step a) with the buffer of step b);

[0080] e) a step of adding collagen to the solution of step d) while stirring constantly; and

[0081] f) A step of adjusting the final pH of the solution to 2 to 5.

[0082] Another aspect of the present invention provides a method for controlling the release of an insulin analog, derivative, or metabolite having an isoelectric point of 5.8 to 8.5 from a pharmaceutical composition. The method comprises the steps of formulating insulin glargine with collagen and one or more pharmaceutically acceptable salts at a pH of 2 to 5, and precipitating the composition at a physiological pH immediately after administration to create an additional barrier of collagen around the insulin. The additional barrier controls the release of the insulin glargine precipitate as an insulin glargine hexamer and the release from the hexamer to the monomer, as well as protecting the insulin from protease degradation.

[0083] Embodiments are further defined in the following examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

[0084] Example 1: Preparation of unit composition (100 IU / ml)

[0085] Insulin Glargine Composition (100 IU / ml Concentration) 1 Vial (1,000 IU / 10 ml) Preparation Composition Serial number ingredient sheep 1. Insulin glargine 1,000 IU (36.4 mg) 2. Rapid Polymerization Collagen (RPC) 30-50 mg 3. Zinc (Zn) 250-350 mcg 4. m-cresol 2.5-3.0 mg 5. 85% Glycerol 15-25 mg 6. Polysorbate 20 15-25 mcg 7. Injectable water Maximum volume of 10ml

[0086] Ultra-long-acting insulin glargine could be manufactured by first preparing a rapid polymerization collagen solution and then using it as an excipient.

[0087] A. Preparation of Rapid Polymerizing Collagen (RPC) Solution

[0088] A collagen solution was prepared by dissolving 2 g of commercially available recombinant human type 1 collagen in 50 ml of 1 M acetic acid. The collagen solution was dialyzed using a 0.5 M acetic acid + 50 mM EDTA solution, and 0.25 M acetic acid was added to dilute the collagen to a concentration of 2 mg / ml. The collagen was precipitated at its isoelectric point (pH 7). The precipitate was washed twice with water for injection. The washed precipitate was suspended in water for injection and dissolved by adding a minimum volume of 1 N HCl. Water for injection was added to dilute the collagen to a concentration of 40 mg / ml. The solution was sterile filtered through a 0.2 micron filter. Subsequently, the solution was placed in a sterile silicone-treated glass vial and stored at 2 to 8°C.

[0089] B. Evaluation of the rapid polymerization properties of the above-prepared solution (polymerization kinetics of the RPC solution in a PBS environment)

[0090] 4.5 ml of phosphate-buffered saline was distributed into five glass test tubes. 0.5 ml of the rapid polymerization collagen solution prepared above was added to the glass test tubes to test the polymerization properties, and the test results were as follows.

[0091] Time observation

[0092] No change at 0 minutes

[0093] 1.0 min Milky white begins to appear

[0094] 5.0 min clear solution gel with visible interface

[0095] 10-minute transparent depot

[0096] Fibril formation in 20 minutes

[0097] The collagen prepared above began to form a gel within 1 minute at physiological pH and salt concentration, and formed complete fibrils within 20 minutes in an environment similar to subcutaneous cells.

[0098] C. Preparation of 5 vials (5,000 IU / 50ml) of ultra-long-acting insulin glargine (100 IU / ml concentration)

[0099] 182 mg of insulin glargine (5,000 IU) was suspended in 25 ml of water for injection, and then dissolved by adding a minimum volume of 1 M HCl. To the insulin glargine solution, ZnCl2 corresponding to 1.5 mg Zn, 13.5 mg m-cresol, 0.1 g 85% (w / v) glycerol, 0.1 mg polysorbate 20, and 200 mg RPC (5.0 ml) were added. A few drops of 1 M HCl were added to this solution to adjust the pH to 4. Water for injection was added to make the volume of the solution 50 ml. The solution was sterilely filtered by passing it through a sterile 0.2 micron filter. The filtrate was aseptically dispensed into 5 sterile silicone-treated glass vials, 10 ml each.

[0100] Example 2: Preparation of high-concentration unit composition (300 IU / ml)

[0101] Insulin Glargine Composition (300 IU / ml concentration) 1 Vial (3,000 IU / 10 ml) Preparation Composition Serial number ingredient sheep 1. Insulin glargine 3,000 IU (109.2 mg) 2. Rapid Polymerization Collagen (RPC) 30-50 mg 3. Zinc (Zn) 750-1,050 mcg 4. m-cresol 2.5-3.0 mg 5. 85% Glycerol 15-25 mg 6. Polysorbate 20 15-25 mcg 7. Injectable water Maximum volume of 10 ml

[0102] High-concentration ultra-long-acting insulin glargine could be manufactured by first preparing a rapid polymerization collagen solution and then using it as an excipient.

[0103] A. Preparation of Rapid Polymerization Collagen (RPC) Solution : The RPC solution was prepared as described in A of Example 1.

[0104] B. Preparation of 5 vials (15,000 IU / 50 ml) of the ultra-long-acting insulin glargine (300 IU / ml concentration) of Example 2

[0105] 546 mg of insulin glargine, 15,000 IU, was dissolved as described above and in Example 1 B. To the insulin glargine solution, ZnCl2 corresponding to 4.5 mg Zn, 13.5 mg m-cresol, 0.1 g 85% glycerol, 0.1 mg polysorbate 20, and 200 mg RPC (5.0 ml) were added. The pH of the solution was adjusted by adding a few drops of 1 M HCl. Water for injection was added to make the volume of the solution 50 ml. The solution was sterile filtered by passing it through a sterile 0.2 micron filter. The filtrate was aseptically dispensed into 5 sterile silicone-treated glass vials, 10 ml each.

[0106] Example 3: Preparation of diluted solutions for standard curve testing of unit composition (100 IU / ml) and high-concentration unit composition (300 IU / ml)

[0107] A. Preparation of a diluted solution of insulin glargine composition (100 IU / mL)

[0108] A diluted solution was prepared by sequentially adding 1.5 mg of ZnCl2, 13.5 mg of meta-cresol, 0.1 g of 85% glycerol, 0.1 mg of polysorbate 20, and 200 mg of RPC (5.0 ml) to 25 ml of water for injection. The pH was adjusted to 4 by adding a minimum amount of 1 M hydrochloric acid, and then water for injection was added to make the total volume 50 ml. This diluted solution was used as a blank control for insulin glargine injection solution 100 IU / ml.

[0109] B. Preparation of a diluted solution of insulin glargine composition (300 IU / ml)

[0110] A diluted solution was prepared by sequentially adding 4.5 mg of ZnCl2, 13.5 mg of meta-cresol, 0.1 g of 85% glycerol, 0.1 mg of polysorbate 20, and 200 mg of RPC (5 ml) to 25 ml of water for injection. The pH was adjusted to 4 by adding a minimum amount of 1 M hydrochloric acid, and then water for injection was added to make the total volume 50 ml. This diluted solution was used as a blank control for insulin glargine injection solution 300 IU / ml.

[0112] Example 4: Preparation of test solution and standard curve for HPLC testing

[0113] A unit composition of Example 1 (insulin glargine solution), which is an ultra-long-acting insulin glargine solution, at 100 IU / ml was used as a test sample. The unit composition of Example 1 was diluted to different concentrations using the dilution solution prepared above. The dilution solution was used as a blank. A 2 ml aliquot of the diluted sample was placed into a 5 ml 30kd cutoff Vivaspin tube. The tube was centrifuged in a chilled centrifuge at 5,000 rpm for 30 minutes. The filtrate was used for HPLC analysis as described below.

[0114] <HPLC 시험 방법>

[0115] Chromatography System: Agilent HPLC

[0116] Mode - LC

[0117] Detector - 214 nm

[0118] Column - Kromasil C18, 3.0mm x 250mm, 4μ

[0119] Column temperature -35℃

[0120] Flow rate - 0.6 mL / min

[0121] Injection volume - 5 μL

[0122] Time (minutes) Solution A (%) Solution B (%) 0 96 4 20 83 17 30 63 37 33 96 4

[0123] Analysis - 94.0% to 105.0% on anhydrous basis.

[0124] <Test Analysis Results>

[0125] The analysis results are as follows, and the standard curve is shown as a graph in Figure 1.

[0126] Insulin glargine concentration-dependent AUC (Area Under Curve) at room temperature IU / mL AUC 8.000 2,569 4.000 1,300 2.500 690 1.250 358 0.625 182 0.313 79

[0127] Example 5: Setup of an in vitro model simulating the subcutaneous release of insulin glargine

[0128] Validation of an in vitro test model mimicking the blood pK profile of insulin glargine in vivo requires understanding the in vivo pK profile of drugs delivered via the subcutaneous ("Sc") route and understanding the in vitro model proposed as a test method.

[0129] Insulin glargine solution is a clear, unbuffered acidic solution with an isoelectric point of 7.4, which is the physiological pH, and forms a precipitate after injection into Sc extracellular matrix (ECM). Meanwhile, after injection into the body, insulin glargine dissociates into a hexamer; due to dilution by surrounding body fluids, this hexamer loses Zn and m-cresol and is converted into dimers and monomers. The insulin glargine monomer is then metabolized in the blood into M1 and M2, with M1 being the major metabolite. M1 interacts with insulin receptors in target body tissues to produce pharmacological effects (hyperglycemic response) and is subsequently broken down into smaller peptides by insulinases and then into amino acids.

[0130] According to the Tojeo model, 300 IU insulin glargine forms a more condensed precipitate, so the release of the hexamer is relatively slower. Consequently, the formation of insulin glargine dimers and monomers is also slower. Therefore, it was observed that the absorption of monomers and their conversion to M1 and M2 are proportionally slower, as shown in Figure 2 below.

[0131] However, no difference in plasma glargine / glargine metabolite pK profiles was observed for the same dose of the drug administered via the intravenous route.

[0132] Meanwhile, all proposed in vitro models must be able to mimic in vivo models. That is, when insulin glargine is delivered via the Sc pathway, it must be possible to make the important observation that reaching Cmax during the insulin release phase is proportionally slower when using 300 IU / ml insulin glargine than when using the 100 IU / ml version of the same dose.

[0133] To perform in vitro validation of subcutaneously administered glargine pK, we constructed an in vitro test model divided into two compartments (see Fig. 3) and intended to verify the in vivo response by testing the reaction of insulin glargine precipitates in a physiological buffered saline (PBS) dialysis tube containing Sc ECM. We established a test method in which PBS solution in the blood is dialyzed through a dialysis membrane, in which glargine precipitates are redissolved in Compartment 1, dissociating into glargine hexamers, dimers, and monomers, and in Compartment 2, monomers are absorbed and dispersed through 0.1 micron subcutaneous tissue pores (capillary pores exist in subcutaneous tissue) to form metabolites M1 and M2. In other words, Compartment 1 corresponds to the redissolution of insulin glargine precipitates within the dialysis tube, while Compartment 2 corresponds to the transport / absorption dispersion of these soluble glargine monomers and the examination of their metabolites. However, if the target tissue is absent or compartment 2 in vitro is not the same as subcutaneous tissue conditions, drug degradation may not occur.

[0134] If glargine 100 IU / ml (Example 1) or glargine 300 IU / ml (Example 2) is treated to the in vitro model of the present invention and the observed kinetics appear as those observed in the in vivo model, it means that the in vitro model of the present invention is suitable for testing the controlled release of insulin / insulin glargine formulations. Figure 3 illustrates the concept of the in vitro model proposed in the present invention.

[0135] Example 6: Verification of the kinetics of in vitro protease-induced release of insulin glargine

[0136] Two compartments of insulin glargine verification devices (in vitro models) were set up as described above (Example 5), and each device has a 1L Schott bottle with a wide opening and a 100 Kd cut-off dialysis tube with a 10 ml stopper inside the bottle.

[0137] Specifically, 5 ml of sterile PBS (pH 7.4) was added to a dialysis tube that had been completely pre-washed and pre-sterilized (immersed in a 70% ethanol aqueous solution and equilibrated with PBS). 1 L of sterile PBS was added to a pre-sterilized 1 L bottle, and the dialysis tube was placed inside. The dialysis tube was kept vertical in a floater position. A sterile Teflon-coated 5.08 cm x 0.95 cm magnetic rod was placed in each bottle, and the device was placed on a magnetic stirrer under a sterile hood.

[0138] In Set 1, 0.6 ml of Lantus solution, having an insulin glargine concentration of 100 IU / ml identical to Example 1, was added to the PBS solution in the dialysis tube. In Set 2, 0.2 ml of Tojeo, having an insulin glargine concentration of 300 IU / ml identical to Example 2, was added.

[0139] 20 microliters of a sterile solution of 10 milli units of recombinant rat trypsin were added to each compartment 1 (dialysis tube). Subsequently, the magnetic stirrer was turned on and the stirring speed was set to 100 rpm. 0.5 ml samples were collected from each compartment (from the dialysis tube and bottle) at different intervals. The samples were analyzed by HPLC for soluble insulin glargine, and the results are shown below.

[0140] Results of observing the kinetics of soluble insulin glargine released by in vitro proteases in two compartments Time (h) Released glargine (Milli IU / ml) Lantus (Glargine 100 IU / ml; 0.6 ml) Tojeo(300 IU / ml ; 0.20 ml) Section 1 Section 2 Section 1 Section 2 0 0 0 0 0 0.50 90 89 75 66 1.00 118 120 124 126 2.00 230 228 155 153 4.00 288 290 194 196 6.00 360 362 248 246 8.00 450 460 310 307

[0141] From the results presented in the table above, insulin glargine release after 30 minutes was found to be similar in both compartments. Subsequently, it was confirmed that the release of soluble insulin glargine was proportionally delayed in Tojeo (300 IU / ml glargine formulation) compared to Lantus (100 IU / ml glargine formulation) due to the formation of more compact insulin glargine precipitates / crystals in compartment 1. This implies that observations similar to those in in vivo human and animal pK studies were confirmed in the in vitro model experiments of the present invention.

[0142] Therefore, in the present invention, it was decided to use this in vitro method to evaluate the effect of adding rapid polymerization collagen (RPC) on the redissolution of glargine from the depot of compartment 1 and the absorption / diffusion of glargine into compartment 2.

[0143] Examples 7 and 8: Verification of the effects of different collagen concentrations on insulin glargine

[0144] 10 ml of insulin glargine at twice the concentration of 100 IU / ml was added to each of five 20 ml glass bottles and labeled from 1 to 5. Glass bottle No. 1 was for the blank test, and 10, 20, 40, and 50 mg of rapid polymerization collagen (RPC) (volumes of 0.25, 0.5, 1.0, and 1.25 ml) were added to glass bottles No. 2 through 5, respectively, in double the amount. Subsequently, each was diluted using a dilution solution to a final volume of 20 ml (final insulin glargine concentration of 100 IU / ml).

[0145] Five insulin glargine verification devices (in vitro models) were prepared. 0.5 ml of insulin glargine solution (100 IU / ml) containing 0 mg / ml RPC was added as a control to compartment 1 (dialysis tube). In the same manner, 0.5 ml of insulin glargine solutions containing 1 mg / ml RPC, 2 mg / ml RPC, 4 mg / ml RPC, and 5 mg / ml RPC were added to compartment 1 of devices 2 through 5 in the order of the indicated numbers, respectively. 0.5 ml samples were collected from compartment 2 (bottles) of each device at different time intervals, and the insulin glargine content was confirmed by RPHPLC analysis of the samples.

[0146] RPC concentration-dependent delay of soluble insulin glargine (100 IU / ml) release Time (h) Released Glargine (Milli IU / ml) RPC Concentration (mg / ml) 0 1 2 4 5 0 0 0 0 0 0 0.5 80 78 65 58 60 1.0 175 170 80 98 120 2.0 230 220 160 145 160 4.0 288 260 250 195 190 6.0 360 348 376 247 255 8.0 455 440 400 305 310

[0147] Meanwhile, 10 ml of insulin glargine at double the concentration of 300 IU / ml was added to each of five 20 ml glass bottles and labeled from 1 to 5. Glass bottle No. 1 was left empty, and 10, 20, 40, and 50 mg of rapid polymerization collagen (RPC) (volumes of 0.25, 0.5, 1.0, and 1.25 ml) were added to glass bottles No. 2 through 5, respectively, in double the amount. Subsequently, each was diluted using a dilution solution to a final volume of 20 ml (final insulin glargine concentration of 300 IU / ml).

[0148] RPC concentration-dependent delay of soluble insulin glargine (300 IU / ml) release Time (h) Released Glargine (Milli IU / ml) RPC Concentration (mg / ml) 0 1 2 4 5 0 0 0 0 0 0 0.5 58 55 50 45 50 1.0 90 88 75 50 55 2.0 148 150 115 60 65 4.0 230 230 166 126 136 6.0 315 318 210 190 205 8.0 397 400 263 255 279

[0149] Example 9: In vitro release of the Example 1 unit composition (100 IU / ml)

[0150] In vitro release of insulin glargine-RPC (100 IU / ml) composition Time (h) Glargine release from Example 1 (Milli IU / ml) 2 148 4 200 6 258 8 318

[0151] Example 10: In vitro release of the Example 2 unit composition (300 IU / ml)

[0152] In vitro release of insulin glargine-RPC (300 IU / ml) composition Time (h) Glargine release from Example 2 (Milli IU / ml) 2 50 4 95 6 142 8 196

Claims

Claim 1 A stable, long-acting injectable pharmaceutical composition comprising insulin glargine having an isoelectric point of 5 to 8.5, collagen, zinc, cresol, glycerol, and polysorbate, wherein the collagen is a rapid-polymerized human collagen solution prepared by removing salts from an acidic human collagen solution using acetic acid and EDTA and adding acidic water for injection, and forms fibrils at a physiological pH, wherein the pharmaceutical composition has a pH of 2 to 5, is a solution at administration, and forms a depot at a physiological pH immediately after administration, and wherein the pharmaceutical composition exhibits increased bioavailability and a longer duration compared to an injectable pharmaceutical composition comprising 'insulin glargine having an isoelectric point of 5 to 8.5' having the same dose, and is a stable, long-acting injectable pharmaceutical composition. Claim 2 delete Claim 3 A stable, long-acting injectable pharmaceutical composition according to claim 1, characterized in that the insulin glargine is present in a concentration range of 100 IU to 1,000 IU / ml. Claim 4 delete Claim 5 In claim 1, the pharmaceutical composition is a stable, long-acting injectable pharmaceutical composition characterized by a duration of action of 24 hours or more. Claim 6 A stable, long-acting injectable pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition is administered two or three times a week. Claim 7 A stable, long-acting injectable pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition has lower immunogenicity compared to a commercially available injectable pharmaceutical composition containing an equal dose of 'insulin glargine with an isoelectric point of 5 to 8.5'. Claim 8 delete Claim 9 delete Claim 10 A stable, long-acting injectable pharmaceutical composition according to claim 1, characterized in that it is used for the treatment of one or more metabolic disorders selected from the group consisting of insulin resistance syndrome, diabetes mellitus, hyperlipidemia, fatty liver disease, cachexia, obesity, atherosclerosis, and arteriosclerosis. Claim 11 delete Claim 12 A method for preparing a stable, long-acting injectable solution comprising insulin glargine, collagen, zinc, cresol, glycerol, and polysorbate having an isoelectric point of 5 to 8.5, comprising the steps of: preparing an insulin glargine solution by solubilizing insulin glargine through treatment with a hydrochloric acid (HCl) solution and water for injection; adding zinc, cresol, glycerol, polysorbate, and collagen to the insulin glargine solution; and adjusting the final pH of the solution to 2 to 5, wherein the collagen is a rapidly polymerized human collagen solution prepared by removing salts from an acidic human collagen solution using acetic acid and EDTA and adding acidic water for injection, and is characterized in that it forms fibrils at a physiological pH. Claim 13 delete Claim 14 delete

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