# Patent

## Title
Stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives

## Abstract

Stabilized pharmaceutical formulations of insulin analogs and/or insulin derivatives are disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application is related to and claims the benefit of U.S. Provisional Patent Application Ser. No. 61/761,434 filed Feb. 6, 2013, and European Patent Application No. 13305126.8, filed Feb. 4, 2013, the entire contents of which are incorporated by reference herein.


INTRODUCTION
The present invention relates to a pharmaceutical formulation of at least one insulin analogue and/or insulin derivative, a process for preparing the pharmaceutical formulation of at least on insulin analogue and/or insulin derivative, and a related kit. It also relates to the pharmaceutical formulation of at least one insulin analogue and/or insulin derivative and to the related kit for use in the treatment of diabetes mellitus, hyperglycemia, and/or for use in lowering blood glucose levels. The present invention also relates to the use of a medical device for administering the pharmaceutical formulation of at least one insulin analogue and/or insulin derivative to an animal and/or human.
BACKGROUND OF THE INVENTION
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is more or less completely lost.
For decades, insulin has been used in the treatment of diabetes mellitus. Several insulin formulations have been developed, e.g. insulin zinc (Zn (II)) suspension, formulations containing protamine, etc. Further, the active pharmaceutical ingredient insulin itself has been modified by developing fast acting insulin analogues (e.g. insulin aspart, insulin lispro, insulin glulisine) and long acting insulin analogues and derivatives (e.g. insulin detemir, insulin degludec, insulin glargin). Fast acting insulin preparations are usually solutions of insulin, while long acting insulin preparations can be suspensions containing insulin in crystalline and/or amorphous form precipitated by the addition of zinc (Zn(II)) salts (e.g. zinc chloride) alone or by addition of protamine or by a combination of both.
The chemical and physical stability of insulin formulations is very important. Insulin formulations are often administered by using pen injection devices or insulin pumps in which an insulin formulation is stored in cartridges until the entire cartridge is empty. Insulin formulations may also be stored in vials, requiring a stable formulation with respect to chemical and physical stability across the shelf life of the formulation.
The chemical and/or physical stability of insulin, insulin analogues and/or insulin derivatives strongly depends on the pharmaceutical formulation, e.g. the solvent, the pH value and the excipients. Brange et al. (Acta Pharm. Nord. 4(3), pp. 149-158, 1992) disclose several aspects in connection with the chemical stability of insulin. WO 2004/080480 discloses pharmaceutical preparations comprising acid-stabilized insulin. GB 835,638 discloses insulin crystal suspensions having a protracted effect. WO 98/56406 discloses stable insulin formulations. U.S. Pat. No. 6,489,292 discloses stable aqueous insulin preparations without phenol and cresol. U.S. Pat. No. 6,211,144 discloses stable concentrated insulin preparations for pulmonary delivery. Bhatt et al. (Pharmaceutical Research, Vol. 7, No. 6, pp. 593-599, 1990) disclose chemical pathways of peptide degradation. Patel et al. (Pharmaceutical Research, Vol. 7, No. 7, pp. 703-711, 1990) disclose chemical pathways of peptide degradation. Tyler-Cross et al. (Journal of Biological Chemistry, Vol. 266, No. 33, Issue of November 25, pp. 22549-22556, 1991) disclose effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. GB 840,870 discloses improvements in or relating to insulin preparations. U.S. Pat. No. 6,852,694 discloses stabilized insulin formulations. Galloway et al. (Diabetes—The Journal of the American Diabetes Association, Vol. 21, No. Suppl. 2, pp. 637-648, 1972) disclose new forms of insulin. Jackson et al. disclose several aspects with regard to neutral regular insulin (Diabetes—The Journal of the American Diabetes Association, Vol. 21, No. 4, pp. 235-245, 1972). Lill (Pharmazie in unserer Zeit, No. 1, pp. 56-61, 2001) discloses general aspects in connection with insulin formulations. The German product specification of the medicinal product Berlinsulin® H Normal 3 mL Pen discloses a formulation containing human insulin, metacresol, glycerol, water and optionally hydrochloric acid and sodium hydroxide. The German product specification of the medicinal product Actrapid® discloses a formulation containing human insulin, zinc chloride, glycerol, metacresol, sodium hydroxide, hydrochloric acid and water.
The solubility of insulin, insulin analogues and/or insulin derivatives in aqueous media depends on the pH value. For example, the lowest solubility is shown close to the isoelectric point which for human insulin is around pH 5.3 and 5.4. Very good solubility can be observed at pH values below 4 and above 7. However, insulin suffers from degradation at strong acidic conditions and strong alkaline conditions. Therefore, most of the medicinal products containing insulin, insulin analogues and/or insulin derivatives have a pH value in the range of 7.2 to 7.4 and mostly buffering agents are used to achieve and maintain the pH within this range.
It has now surprisingly been found that an alternative aqueous pharmaceutical formulation comprising at least one insulin analogue and/or insulin derivative comprising sodium chloride, without any additional buffering agent, shows an excellent chemical and physical stability, which qualifies this aqueous pharmaceutical formulation as a medicinal product having a defined shelf life.
SUMMARY OF THE INVENTION
One embodiment of the present invention relates to a pharmaceutical formulation comprising
(a). at least one analogue and/or derivative of insulin; and
(b). Zn(II); and
(c). sodium chloride; and
(d). optionally protamine;
wherein the pharmaceutical formulation has a pH value in the range of from 6.0 to 9.0 and is free of any additional buffering agent.
In another embodiment, the pharmaceutical formulation according to the present invention is an aqueous pharmaceutical composition.
In another embodiment, the pharmaceutical formulation according to the present invention does not contain any additional buffering agent.
In another embodiment, the pharmaceutical formulation according to the present invention does not contain any additional buffering agent other than the at least one analogue and/or derivative of insulin and the optionally present protamine.
In another embodiment, the pharmaceutical formulation according to the present invention is free of any additional buffering agent selected from the group consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid or citrate salts, acetic acid and salts thereof, glycylglycine and methionin.
In another embodiment, the pharmaceutical formulation according to the present invention does not contain any additional buffering agent selected from the group consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid or citrate salts, acetic acid and salts thereof, glycylglycine and methionin.
In another embodiment, the pharmaceutical formulation according to the present invention comprises at least one analogue and/or derivative of insulin and optionally protamine, wherein the only component contributing any buffering activity is the at least one analogue and/or derivative of insulin and optionally protamine.
In another embodiment, the pharmaceutical formulation according to the present invention comprises at least one analogue and/or derivative of insulin and optionally protamine, wherein the total concentration of any buffering agent in the present invention is in the range from 3.496 mg/mL (rounded: 3.5 mg/mL) to 3.996 mg/mL (rounded: 4.0 mg/mL), from 3.496 mg/mL (rounded: 3.5 mg/mL) to 3.816 mg/mL (rounded: 3.8 mg/mL) or the total concentration of any buffering agent in the present invention is 3.496 mg/mL (rounded: 3.5 mg/mL).
In another embodiment, the pharmaceutical formulation according to the present invention consists of (a). at least one analogue and/or derivative of insulin; and (b). Zn(II); and (c). sodium chloride; and (d). optionally protamine; and (e). metacresol; (f). phenol; and (g). polysorbate 20; and (h). sodium hydroxide and/or hydrochloric acid for pH adjustment to a pH value in the range of from 6.0 to 9.0; and (i). water.
In another embodiment, the pharmaceutical formulation according to the present invention is an aqueous pharmaceutical formulation.
In another embodiment, the pharmaceutical formulation according to the present invention comprises at least one analogue and/or derivative of insulin which has or have an isoelectric point (IEP) in the range from 4.0 to 6.0, from 4.5 to 6.0, from 4.5 to 5.5, from 5.0 to 5.5, from 5.0 to 5.2 or 5.1.
In another embodiment, the pharmaceutical formulation according to the present invention has a pH value in the range from 6.5 to 8.5, in the range from 7.0 to 8.0, from 7.0 to 7.8, from 7.1 to 7.6, 7.2, 7.3, 7.4 or 7.5, or 7.4.
In another embodiment, the pharmaceutical formulation according to the present invention comprises at least one analogue of insulin which is selected from the group consisting of insulin aspart, insulin lispro and/or insulin glulisine. In one embodiment, the pharmaceutical formulation according to the present invention comprises an analogue of insulin which is insulin lispro. In one embodiment, the pharmaceutical formulation according to the present invention comprises an analogue of insulin which is insulin aspart. In one embodiment, the pharmaceutical formulation according to the present invention comprises an analogue of insulin which is insulin glulisine.
In another embodiment, the pharmaceutical formulation according to the present invention comprises a derivative of insulin which is selected from the group consisting of insulin detemir and/or insulin degludec. In one embodiment, the pharmaceutical formulation according to the present invention comprises a derivative of insulin which is insulin detemir. In one embodiment, the pharmaceutical formulation according to the present invention comprises a derivative of insulin which is insulin degludec.
In another embodiment, the pharmaceutical formulation according to the present invention comprises at least one analogue and/or derivative of insulin which is present in a concentration from 10 U/mL to 1000 U/mL, from 10 U/mL to 600 U/mL, from 10 U/mL to 300 U/mL, from 50 U/mL to 300 U/mL or 100 U/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises an analogue and/or derivative of insulin which is present in a concentration from 60 to 6000 nmol/mL, from 60 nmol/mL to 3600 nmol/mL, from 60 nmol/mL to 1800 nmol/mL, from 300 nmol/mL to 1800 nmol/mL or 600 nmol/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises Zn(II) which is present in a concentration from 0.0100 mg/mL to 0.0600 mg/mL, from 0.0150 mg/mL to 0.0500 mg/mL, from 0.0150 mg/mL to 0.0300 mg/mL, from 0.0150 mg/mL to 0.0200 mg/mL, from 0.0190 mg/mL to 0.0200 mg/mL, or 0.0196 mg/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises Zn(II) which is present in a concentration from 0.0100 mg/100 U to 0.0600 mg/100 U, from 0.0150 mg/100 U to 0.0500 mg/100 U, from 0.0150 mg/100 U to 0.0300 mg/100 U, from 0.0150 mg/100 U to 0.0200 mg/100 U, from 0.0190 mg/100 U to 0.0200 mg/100 U, or 0.0196 mg/100 U.
In another embodiment, the pharmaceutical formulation according to the present invention comprises sodium chloride which is present in a concentration from 0.01 mg/mL to 15 mg/mL, from 0.1 mg/mL to 15 mg/mL, from 0.1 mg/mL to 10 mg/mL, from 1 mg/mL to 10 mg/mL, from 2.0 mg/mL to 10 mg/mL, from 3.0 mg/mL to 9.0 mg/mL, from 4.0 mg/mL to 9.0 mg/mL, from 5.0 mg/mL to 9.0 mg/mL, from 6.0 mg/mL to 9.0 mg/mL, from 6.8 mg/mL to 8.3 mg/mL, 6.9 mg/mL, 7.0 mg/mL, 7.1 mg/mL, 7.2 mg/mL, 7.3 mg/mL, 7.4 mg/mL, 7.5 mg/mL, 7.6 mg/mL, 7.7 mg/mL, 7.8 mg/mL, 7.9 mg/mL, 8.0 mg/mL, 8.1 mg/mL, 8.2 mg/mL or 8.3 mg/mL, or 6.8 mg/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises protamine or protamine sulfate which is present in a concentration from 0.10, 0.15, 0.20, 0.25, 0.30, 0.32, 0.35, 0.40, 0.45 or 0.5 mg/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises a stabilizing agent, which is in one embodiment a surfactant, a polyoxyethylene derivative of sorbitan monolaurate (e.g. polysorbate 20), a polyethoxylethylene derivate of oleic acid (e.g. polysorbate 80), poloxamer (which is a polyoxyethylene-polyoxypropylene copolymer), or polysorbate 20 or polysorbate 80 or mixtures thereof. In another embodiment, the stabilizing agent, in one embodiment the surfactant, the polyoxyethylene derivative of sorbitan monolaurate (e.g. polysorbate 20), the polyethoxylethylene derivate of oleic acid (e.g. polysorbate 80), poloxamer (which is a polyoxyethylene-polyoxypropylene copolymer), and in another polysorbate 20 or polysorbate 80 or mixtures thereof are/is present in a concentration from 0.01 to 0.05 mg/mL, 0.010 mg/mL, 0.015 mg/mL, 0.020 mg/mL, 0.025 mg/mL, 0.03 mg/mL, or 0.02 mg/mL.
In another embodiment, the pharmaceutical formulation according to the present invention comprises more than one analogue and/or derivative of insulin, wherein one analogue and/or derivative of insulin is a fast acting insulin and one analogue and/or derivative of insulin is a long acting insulin. In another embodiment, the pharmaceutical formulation according to the present invention comprises a fast acting insulin selected from the group comprising insulin aspart, insulin lispro and/or insulin glulisine and a long acting insulin selected from the group comprising insulin glargin, insulin detemir and/or insulin degludec.
In another embodiment, the pharmaceutical formulation according to the present invention comprises one or more further active pharmaceutical ingredients. In one embodiment the further active pharmaceutical ingredient is an antidiabetic agent. In another embodiment, the pharmaceutical formulation according to the present invention comprises one or more antidiabetic agents as further active pharmaceutical ingredients selected from the group comprising: GLP-1 receptor agonists, dual GLP-1 receptor/glucagon receptor agonists, human FGF-21, FGF-21 analogues, FGF-21 derivatives, insulins, human insulin, analogues of insulin, and derivatives of insulin. In another embodiment, the pharmaceutical formulation according to the present invention comprises one or more further active pharmaceutical ingredients selected from the group comprising: insulin and insulin derivatives, GLP-1, GLP-1 analogues and GLP-1 receptor agonists, polymer bound GLP-1 and GLP-1 analogues, dual GLP1/GIP agonists, dual GLP1/Glucagon receptor agonists, PYY3-36 or analogues thereof, pancreatic polypeptide or analogues thereof, glucagon receptor agonists or antagonists, GIP receptor agonists or antagonists, ghrelin antagonists or inverse agonists, Xenin and analogues thereof, DDP-IV inhibitors, SGLT2 inhibitors, dual SGLT2/SGLT1 inhibitors, biguanides thiazolidinediones, dual PPAR agonists, sulfonylureas, meglitinides, alpha-glucosidase inhibitors, amylin and amylin analogues, GPR119 agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low-absorbable TGR5 agonists, Cycloset, inhibitors of 11-beta-HSD, activators of glucokinase, inhibitors of DGAT, inhibitors of protein tyrosinephosphatase 1, inhibitors of glucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrogenase kinase, alpha2-antagonists, CCR-2 antagonists, modulators of glucose transporter-4, somatostatin receptor 3 agonists, HMG-CoA-reductase inhibitors, fibrates, nicotinic acid and the derivatives thereof, nicotinic acid receptor 1 agonists, PPAR-alpha, gamma or alpha/gamma) agonists or modulators, PPAR-delta agonists, ACAT inhibitors, cholesterol absorption inhibitors, bile acid-binding substances, IBAT inhibitors, MTP inhibitors, modulators of PCSK9, LDL receptor up-regulators by liver selective thyroid hormone receptor B agonists, HDL-raising compounds, lipid metabolism modulators, PLA2 inhibitors, ApoA-I enhancers, cholesterol synthesis inhibitors, lipid metabolism modulators, omega-3 fatty acids and derivatives thereof, active substances for the treatment of obesity, such as sibutramine, tesofensine, orlistat, CB-1 receptor antagonists, MCH-1 antagonists, MC4 receptor agonists and partial agonists, NPY5 or NPY2 antagonists, NPY4 agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the 5HT2c receptor, or the combinations of bupropione/naltrexone (CONTRAVE), bupropione/zonisamide (EMPATIC), bupropione/phentermine or pramlintide/metreleptin, QNEXA (Phentermine+topiramate), lipase inhibitors, angiogenesis inhibitors, H3 antagonists, AgRP inhibitors, triple monoamine uptake inhibitors (norepinephrine and acetylcholine), MetAP2 inhibitors, nasal formulation of the calcium channel blocker diltiazem, antisense oligonucleotides against production of fibroblast growth factor receptor 4, prohibitin targeting peptide-1, drugs for influencing high blood pressure, chronic heart failure or atherosclerosis, such as angiotensin II receptor antagonists, ACE inhibitors, ECE inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, thrombocyte aggregation inhibitors.
In another embodiment, the pharmaceutical formulation according to the present invention comprises more than one analogue and/or derivative of insulin, wherein one analogue and/or derivative of insulin is a fast acting insulin and one analogue and/or derivative of insulin is a long acting insulin. In another embodiment the fast acting insulin is selected from the group comprising insulin aspart, insulin lispro and/or insulin glulisine and the long acting insulin is selected from the group comprising insulin detemir and/or insulin degludec.
In another embodiment, the pharmaceutical formulation according to the present invention consists of (a). 3.496 mg/mL insulin aspart (rounded: 3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol; and (d). 0.04087 mg/mL Zn(II); and (e). 6.8 mg/mL sodium chloride; and (f). 0.02 mg/mL polysorbate 20; and (g). sodium hydroxide and/or hydrochloric acid to adjust the pH to 7.4 and (h). water.
In another embodiment, the pharmaceutical formulation according to the present invention consists of (a). 3.496 mg/mL insulin aspart (rounded: 3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol; and (d). 0.04087 mg/mL Zn(II); and (e). from 6.8 mg/mL to 8.3 mg/mL sodium chloride; (f). 0.02 mg/mL polysorbate 20; (g) from 0.1 mg/mL to 0.5 mg/mL protamine sulfate; and (h). sodium hydroxide and/or hydrochloric acid to adjust the pH to a pH in the range from 7.1 to 7.6 and (i). water.
In another embodiment, the pharmaceutical formulation according to the present invention consists of (a). 3.496 mg/mL insulin aspart (rounded: 3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol; and (d). 0.04087 mg/mL Zn(II); and (e). 6.8 or 6.9 or 7.0 or 7.1 or 7.2 or 7.3 or 7.4 or 7.5 or 7.6 or 7.7 or 7.8 or 7.9 or 8.0 or 8.1 or 8.2 or 8.3 mg/mL sodium chloride; (f). 0.02 mg/mL polysorbate 20; (g) 0.1 or 0.15 or 0.2 or 0.25 or 0.3 or 0.32 or 0.35 or 0.4 or 0.45 or 0.5 mg/mL protamine sulfate; and (h). sodium hydroxide and/or hydrochloric acid to adjust the pH to 7.4 and (i). water.
The present invention also provides a pharmaceutical formulation for use in the treatment of diabetes mellitus, hyperglycemia and/or for use in lowering blood glucose levels.
The present invention also provides a process for preparing the pharmaceutical formulation according to the present invention, wherein the components are mixed together in the form of a solution or suspension, the desired pH is adjusted and the mixture is made up to the final volume with water.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention and a medical device. In one embodiment the medical device is selected from the group comprising: syringe, insulin injection system, insulin infusion system, insulin pump, insulin pen injection device.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device. In one embodiment the medical device is selected from the group comprising: syringe, insulin injection system, insulin infusion system, insulin pump, insulin pen injection device.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device, wherein the further active pharmaceutical ingredient is an antidiabetic agent.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device, wherein the further active pharmaceutical ingredient is an antidiabetic agent selected from the group comprising: GLP-1 receptor agonists, dual GLP-1 receptor/glucagon receptor agonists, human FGF-21, FGF-21 analogues, FGF-21 derivatives, insulins, human insulin, analogues of insulin, and derivatives of insulin. In another embodiment, the pharmaceutical formulation according to the present invention comprises one or more further active pharmaceutical ingredients selected from the group comprising: insulin and insulin derivatives, GLP-1, GLP-1 analogues and GLP-1 receptor agonists, polymer bound GLP-1 and GLP-1 analogues, dual GLP1/GIP agonists, dual GLP1/Glucagon receptor agonists, PYY3-36 or analogues thereof, pancreatic polypeptide or analogues thereof, glucagon receptor agonists or antagonists, GIP receptor agonists or antagonists, ghrelin antagonists or inverse agonists, Xenin and analogues thereof, DDP-IV inhibitors, SGLT2 inhibitors, dual SGLT2/SGLT1 inhibitors, biguanides thiazolidinediones, dual PPAR agonists, sulfonylureas, meglitinides, alpha-glucosidase inhibitors, amylin and amylin analogues, GPR119 agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low-absorbable TGR5 agonists, Cycloset, inhibitors of 11-beta-HSD, activators of glucokinase, inhibitors of DGAT, inhibitors of protein tyrosinephosphatase 1, inhibitors of glucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrogenase kinase, alpha2-antagonists, CCR-2 antagonists, modulators of glucose transporter-4, somatostatin receptor 3 agonists, HMG-CoA-reductase inhibitors, fibrates, nicotinic acid and the derivatives thereof, nicotinic acid receptor 1 agonists, PPAR-alpha, gamma or alpha/gamma) agonists or modulators, PPAR-delta agonists, ACAT inhibitors, cholesterol absorption inhibitors, bile acid-binding substances, IBAT inhibitors, MTP inhibitors, modulators of PCSK9, LDL receptor up-regulators by liver selective thyroid hormone receptor B agonists, HDL-raising compounds, lipid metabolism modulators, PLA2 inhibitors, ApoA-I enhancers, cholesterol synthesis inhibitors, lipid metabolism modulators, omega-3 fatty acids and derivatives thereof, active substances for the treatment of obesity, such as sibutramine, tesofensine, orlistat, CB-1receptor antagonists, MCH-1 antagonists, MC4 receptor agonists and partial agonists, NPY5 or NPY2 antagonists, NPY4 agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the 5HT2c receptor, or the combinations of bupropione/naltrexone (CONTRAVE), bupropione/zonisamide (EMPATIC), bupropione/phentermine or pramlintide/metreleptin, QNEXA (Phentermine+topiramate), lipase inhibitors, angiogenesis inhibitors, H3 antagonists, AgRP inhibitors, triple monoamine uptake inhibitors (norepinephrine and acetylcholine), MetAP2 inhibitors, nasal formulation of the calcium channel blocker diltiazem, antisense oligonucleotides against production of fibroblast growth factor receptor 4, prohibitin targeting peptide-1, drugs for influencing high blood pressure, chronic heart failure or atherosclerosis, such as angiotensin II receptor antagonists, ACE inhibitors, ECE inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, thrombocyte aggregation inhibitors.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device, wherein the kit comprises more than one analogue and/or derivative of insulin, wherein one analogue and/or derivative of insulin is a fast acting insulin and one analogue and/or derivative of insulin is a long acting insulin. In one embodiment the fast acting insulin is selected from the group comprising insulin aspart, insulin lispro and/or insulin glulisine and wherein the long acting insulin is selected from the group comprising insulin glargin, insulin detemir and/or insulin degludec.
The present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device for use in the treatment of diabetes mellitus, hyperglycemia and/or for use in lowering blood glucose levels.
In another embodiment, the present invention also relates to a kit or combination comprising separate packages of the pharmaceutical formulation according to the present invention, of at least one further active pharmaceutical ingredient and optionally of a medical device, wherein the pharmaceutical formulation according to the present invention and the further active pharmaceutical ingredient, in one embodiment an antidiabetic agent, are administered continuously, separately, sequentially and/or stepwise.
The present invention also relates to the use of a medical device for administering the pharmaceutical formulation to an animal and/or human. In one embodiment, the medical device is selected from the group comprising: syringe, insulin injection system, insulin infusion system, insulin pump, insulin pen injection device



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows three impurities identified by liquid chromatography-mass spectrometry (LC/MS) in a formulation of the present invention.



DETAILED DESCRIPTION
As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a fill material containing “a carrier” includes one or more carriers, reference to “an additive” includes reference to one or more of such additives.
As used herein, the term “active pharmaceutical ingredient” (API) includes any pharmaceutically active chemical or biological compound and any pharmaceutically acceptable salt thereof and any mixture thereof, that provides some pharmacologic effect and is used for treating or preventing a condition. Exemplary pharmaceutically acceptable salts include hydrochloric, sulfuric, nitric, phosphoric, hydrobromic, maleric, malic, ascorbic, citric, tartaric, pamoic, lauric, stearic, palmitic, oleic, myristic, lauryl sulfuric, naphthalinesulfonic, linoleic, linolenic acid, and the like. As used herein, the terms “active pharmaceutical ingredient”, “drug”, “active agent”, “active ingredient”, “active substance” and “drug” are meant to be synonyms, i.e., have identical meaning.
In a one embodiment the active pharmaceutical ingredient is an antidiabetic agent. Examples of antidiabetic agents are found in the Rote Liste 2012, chapter 12. Examples of antidiabetic agents include but not limited to (a) insulin, insulin analogues and insulin derivatives, (b) glucagon-like-peptide 1 (GLP-1) and its analogues and receptor agonists, (c) dual GLP-1/GIP agonists, and (d) dual GLP-1/glucagon receptor agonists, as described in detail next.
(a). Insulin, insulin analogues, and insulin derivatives Examples of insulin, insulin analogues, and insulin derivatives include but are not limited to insulin glargine (Lantus®), insulin glulisine (Apidra®), insulin detemir (Levemir®), insulin lispro (Humalog®/Liprolog®), insulin degludec (Tresiba®), insulin aspart (NovoLog®/NovoRapid®), basal insulin and analogues (e.g. LY2605541, LY2963016), PEGylated insulin lispro, Humulin®, Linjeta®, SuliXen®, NN1045, insulin plus Symlin®, fast-acting and short-acting insulins (e.g. Linjeta®, PH20 insulin, NN1218, HinsBet®), oral, inhalable, transdermal and sublingual insulins (e.g. Exubera®, Nasulin®, Afrezza®, insulin tregopil, TPM-02/Insulin, Capsulin®, Oral-lyn®, Cobalamin® oral insulin, ORMD-0801, NN1953, VIAtab®). Additionally included are also those insulin derivatives which are bonded to albumin or another protein by a bifunctional linker.
(b). Glucagon-like-peptide 1 (GLP-1), GLP-1 analogues and GLP-1 receptor agonists Examples of GLP-1, GLP-1 analogues and GLP-1 receptor agonists include but are not limited to lixisenatide (AVE0010/ZP10/Lyxumia®), exenatide/exendin-4 (Byetta®/Bydureon®/ITCA 650, liraglutide/Victoza®), semaglutide, taspoglutide, albiglutide, dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, HM-112600, CM-3, GLP-1 Eligen, ORMD-0901, NN9924, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, MAR-701, ZP-2929, ZP-3022, CAM-2036, DA-15864, ARI-2651, ARI-2255, exenatide-XTEN and glucagon-XTEN, AMX-8089+VRS-859 and polymer bound GLP-1 and GLP-1 analogues.
(c). Dual GLP-1/glucose-dependent insulinotropic peptides (GIP) agonists
Examples of dual GLP-1/GIP agonists include but are not limited to MAR701, MAR-709, BHM081/BHM089/BHM098).
(d). Dual GLP-1/glucagon receptor agonists
Examples of dual GLP-1/glucagon receptor agonists include but are not limited to OAP-189 (PF-05212389, TKS-1225), TT-401/402, ZP2929, LAPS-HMOXM25, MOD-6030).
Other suitable active pharmaceutical ingredients which may be included in the pharmaceutical formulations of the invention include but are not limited to the following:
Further gastrointestinal peptides such as peptide YY 3-36 (PYY3-36) or analogues thereof and pancreatic polypeptide (PP) or analogues thereof.
Glucagon receptor agonists or antagonists, GIP receptor agonists or antagonists, ghrelin antagonists or inverse agonists and xenin and analogues thereof. Dipeptidyl peptidase-IV (DPP-4) inhibitors, for example: alogliptin/Nesina®, linagliptin/BI-1356/Ondero®/Trajenta®/Tradjenta®/Trayenta®, saxagliptin/Onglyza®, sitagliptin/Januvia®/Xelevia®/Tesavel®, sitagliptin+metformin/Janumet®/Velmetia®, aildagliptin, anagliptin, aemigliptin, tenegliptin, melogliptin, trelagliptin, DA-1229, MK-3102, KM-223, KRP-104 and Ari-2243.
Sodium-dependent glucose transporter 2 (SGLT2) inhibitors, for example: canagliflozin, dapagliflozin, remogliflozin, sergliflozin, empagliflozin, ipragliflozin, tofogliflozin (RO-4998452), luseogliflozin, LX-4211, ertugliflozin (PF-04971729), EGT-0001442 and DSP-3235.
Dual SGLT2/SGLT1 inhibitors.
Biguanides (e.g. metformin, buformin, phenformin), thiazolidinediones (e.g. pioglitazone, rivoglitazone, rosiglitazone, troglitazone), dual PPAR agonists (e.g. aleglitazar, muraglitazar, tesaglitazar), sulfonylureas (e.g. tolbutamide, glibenclamide, glimepiride/Amaryl®, glipizide), meglitinides (e.g. nateglinide, repaglinide, mitiglinide), alpha-glucosidase inhibitors (e.g. acarbose, miglitol, voglibose), amylin and amylin analogues (e.g. pramlintide/Symlin®). G-protein coupled receptor 119 (GPR119) agonists (e.g. GSK-1292263, PSN-821, MBX-2982,
APD-597, ARRY-981).
GPR40 agonists (e.g. TAK-875, TUG-424, P-1736, JTT-851, GW9508). GPR120 agonists and GPR142 agonists.
Systemic or low-absorbable TGR5 (GPBAR1=G-protein-coupled bile acid receptor 1) agonists (e.g. INT-777, XL-475, SB756050).
Bromocriptine/Cycloset®, inhibitors of 11-beta-hydroxysteroid dehydrogenase (11-beta-HSD) (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585), activators of glucokinase (e.g. PF-04991532, TTP-399, GK1-399, ARRY-403 (AMG-151), TAK-329, ZYGK1), inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g. pradigastat (LCQ-908), LCQ-908), inhibitors of protein tyrosinephosphatase 1 (e.g. trodusquemine), inhibitors of glucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrogenase kinase, alpha2 adrenergic receptor antagonists, C—C chemokine receptor type 2 (CCR-2) antagonists, modulators of glucose transporter-4 and somatostatin receptor 3 agonists (e.g. MK-4256).
One or more lipid lowering agents are also suitable as active pharmaceutical ingredients, such as for example: 3-hydroxy-3-methylglutaryl-coenzym-A-reductase (HMG-CoA-reductase) inhibitors (e.g. simvastatin, atorvastatin, rosuvastatin), fibrates (e.g. bezafibrate, fenofibrate), nicotinic acid and derivatives thereof (e.g. niacin, including slow release formulations of niacin), nicotinic acid receptor 1 agonists (e.g. GSK-256073), peroxisome proliferator-activated receptors (PPAR-)(alpha, gamma or alpha/gamma) agonists or modulators (e.g. aleglitazar), PPAR-delta agonists, acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g. avasimibe), cholesterol absorption inhibitors (e.g. ezetimibe), bile acid-binding substances (e.g. cholestyramine, colesevelam), ileal bile acid transport inhibitors (IBAT) (e.g. GSK-2330672), microsomal triglyceride transfer protein (MTP) inhibitors (e.g. lomitapide (AEGR-733), SLx-4090, granotapide), modulators of proprotein convertase subtilisin/kexin type 9 (PCSK9) (e.g. REGN727/SAR236553, AMG-145, LGT-209, PF-04950615, MPSK3169A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDL receptor up-regulators, for example liver selective thyroid hormone receptor beta agonists (e.g. eprotirome (KB-2115), MB07811, sobetirome (QRX-431), VIA-3196, ZYT1), HDL-raising compounds such as: CETP inhibitors (e.g. torcetrapib, anacetrapib (MK0859), dalcetrapib, evacetrapib, JTT-302, DRL-17822, TA-8995, R-1658, LY-2484595) or ABC1 regulators, lipid metabolism modulators (e.g. BMS-823778, TAP-301, DRL-21994, DRL-21995), phospholipase A2 (PLA2) inhibitors (e.g. darapladib/Tyrisa®, varespladib, rilapladib), ApoA-I enhancers (e.g. RVX-208, CER-001, MDCO-216, CSL-112, VRX-HDL, VRX-1243, VIRxSYS), cholesterol synthesis inhibitors (e.g. ETC-1002) and lipid metabolism modulators (e.g. BMS-823778, TAP-301, DRL-21994, DRL-21995) and omega-3 fatty acids and derivatives thereof (e.g. icosapent ethyl (AMR101), Epanova®, AKR-063, NKPL-66).
Other suitable active pharmaceutical ingredients which may be included in the pharmaceutical formulations include one or more active substances for the treatment of obesity, including but not limited to:
Sibutramine, tesofensine, orlistat, cannabinoid receptor 1 (CB1) antagonists (e.g. TM-38837), melanin-concentrating hormone (MCH-1) antagonists (e.g. BMS-830216, ALB-127158(a)), MC4 receptor agonists and partial agonists (e.g. AZD-2820, RM-493), neuropeptide Y5 (NPY5) or NPY2 antagonists (e.g. velneperit, S-234462), NPY4 agonists (e.g. PP-1420), beta-3-adrenergic receptor agonists, leptin or leptin mimetics, agonists of the 5-hydroxytryptamine 2c (5HT2c) receptor (e.g. lorcaserin), or the combinations of bupropione/naltrexone (Contrave®), bupropione/zonisamide (Empatic®), bupropione/phentermine or pramlintide/metreleptin, phentermine/topiramate (Qsymia®), lipase inhibitors (e.g. cetilistat/Cametor®), angiogenesis inhibitors (e.g. ALS-L1023), histamine H3 antagonists (e.g. HPP-404), AgRP (agouti related protein) inhibitors (e.g. TTP-435), triple monoamine uptake inhibitors (dopamine, norepinephrine and serotonin reuptake) (e.g. tesofensine), methionine aminopeptidase 2 (MetAP2) inhibitors (e.g. beloranib), nasal formulations of the calcium channel blocker diltiazem (e.g. CP-404) and antisense oligonucleotides against production of fibroblast growth factor receptor 4 (FGFR4) (e.g. ISIS-FGFR4Rx) or prohibitin targeting peptide-1 (e.g. Adipotide®).
Further suitable active pharmaceutical ingredients which may be included in the pharmaceutical formulations include but are not limited to:
Angiotensin II receptor antagonists (e.g. telmisartan, candesartan, valsartan, losartan, eprosartan, irbesartan, olmesartan, tasosartan, azilsartan), angiotensin converting enzyme (ACE) inhibitors, endothelin converting enzyme (ECE) inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, thrombocyte aggregation inhibitors and others or combinations thereof are suitable.
As used herein, the terms “analogue of insulin” and “insulin analogue” refer to a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and/or exchanging at least one amino acid residue occurring in the naturally occurring insulin and/or adding at least one amino acid residue. The added and/or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. Examples of analogues of insulin include, but are not limited to, the following:
(i). ‘Insulin aspart’ is created through recombinant DNA technology so that the amino acid B28 in human insulin (i.e. the amino acid no. 28 in the B chain of human insulin), which is proline, is replaced by aspartic acid;
(ii). ‘Insulin lispro’ is created through recombinant DNA technology so that the penultimate lysine and proline residues on the C-terminal end of the B-chain of human insulin are reversed (human insulin: ProB28LysB29; insulin lispro: LysB28ProB29);
(iii). “Insulin glulisine” differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid;
(iv). “Insulin glargine” differs from human insulin in that the asparagine at position A21 is replaced by glycine and the B chain is extended at the carboxy terminal by two arginines.
As used herein, the term “aqueous” refers to a solution in which the solvent is water and/or to a suspension in which the external phase is water and/or to an emulsion in which the dispersed or continuous phase is water.
As used herein, the term “buffering agent” refers to a weak acid or base used to maintain the acidity (pH) of a solution, a suspension and/or an emulsion near a chosen value after the addition of another acid or base. The function of a buffering agent is to prevent a rapid change in the pH value when acids or bases are added to the solution. In an aqueous solution, suspension and/or emulsion, a buffering agent is present in a mixture of a weak acid and its conjugate base or a in a mixture of a weak base and its conjugated acid. Examples of buffering agents include, but are not limited to, the following: sodium bicarbonate; acetic acid or acetate salts (e.g. sodium acetate, zinc acetate); boric acid or boric salts; N-cyclohexyl-2-aminoethanesulfonic acid (CHES) or salts thereof; 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid (TAPS) or salts thereof; 2-(N-morpholino)ethanesulfonic acid (MES) and salts thereof; piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES) and salts thereof; N-(2-acetamido)-2-aminoethane-sulfonic acid (ACES) and salts thereof; cholamine chloride; BES; 2-[[1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl]amino]ethanesulfonic acid (TES) and salts thereof; 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) and salts thereof; acetamidoglycine; N-(2-hydroxy-1,1-bis(hydroxyl-methyl)ethyl)glycine (tricine); glycinamide; 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine) and salts thereof; propionate salts; 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]-amino]-2-hydroxy-propane-1-sulfonic acid (TAPSO) and salts thereof; 3-morpholinopropane-1-sulfonic acid (MOPS) and salts thereof; saline-sodium citrate (SSC) buffer; 2-amino-2-hydroxymethyl-propane-1,3-diol (synonyms: TRIS, trisamine, THAM, tromethamine, trometamol, tromethane); citric acid or citrate salts (e.g. sodium citrate); trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium phosphate, monopotassium phosphate and/or any other buffering agent containing phosphate. Amino acids (having free basic or acidic functional groups, e.g. methionin, arginine) or peptides (having free basic or acidic functional groups) may also be used as buffering agent. As used herein, the term “buffering agent” also comprises amino acids, peptides and proteins. As insulin analogues and/or insulin derivatives and/or protamine are peptides or derivatives of peptides (i.e. both contain amino acids having free basic or acidic functional groups), they may also have a certain buffering capacity, i.e. are also to be considered as buffering agent.
As used herein, the term “fast acting insulin” or “short acting insulin” refers to insulin analogues and/or insulin derivatives, wherein the insulin-mediated effect begins within 5 to 15 minutes and continues to be active for 3 to 4 hours. Examples of fast acting insulins include, but are not limited to, the following: (i). insulin aspart; (ii). insulin lispro and (iii). insulin glulisine.
As used herein, the terms “free of additional buffering agent” or “buffer-free” means that there is no further buffering agent next to the analogue and/or derivative of insulin and optionally protamine. As mentioned above, analogues and/or derivatives of insulin as well as protamine contain amino acids having acidic or basic side chains and are therefore also to be considered as buffering agent. Independently from the absence of any buffering agents, the aqueous pharmaceutical formulation according to the present invention may optionally comprise protamine.
As used throughout the description and the claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises” is not intended to exclude other additives, components, integers or steps.
As used herein, the terms “derivative of insulin” and “insulin derivative” refer to a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, in which one or more organic substituents (e.g. a fatty acid) is bound to one or more of the amino acids. Optionally, one or more amino acids occurring in the naturally occurring insulin may have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or amino acids, including non-codable, have been added to the naturally occurring insulin. Examples of derivatives of insulin include, but are not limited to, the following:
(i). ‘Insulin detemir’ which differs from human insulin in that the C-terminal threonine in position B30 is removed and a fatty acid residue (myristic acid) is attached to the epsilon-amino function of the lysine in position B29.
(ii). ‘Insulin degludec’ which differs from human insulin in that the last amino acid is deleted from the B-chain and by the addition of a glutamyl link from LysB29 to a hexadecandioic acid.
As used herein, the term “FGF-21” means “fibroblast growth factor 21”. FGF-21 compounds may be human FGF-21, an analogue of FGF-21 (referred to “FGF-21 analogue”) or a derivative of FGF-21 (referred to “FGF-21 derivative”).
As used herein, the term “formulation” refers to a product comprising specified ingredients in predetermined amounts or proportions, as well as any product that results, directly or indirectly, from combining specified ingredients in specified amounts. In relation to pharmaceutical formulations, this term encompasses a product comprising one or more active ingredients, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. In general, pharmaceutical formulations are prepared by uniformly bringing the active pharmaceutical ingredient (i.e. the analogue and/or derivative of insulin) into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. The pharmaceutical formulation includes enough of the active pharmaceutical ingredient to produce the desired effect upon the progress or condition of diseases. As used herein, the term “formulation” may refer to a solution as well as to a suspension or to an emulsion. As used herein, the terms “formulation” and “composition” are meant to be synonyms, i.e., have identical meaning. The pharmaceutical compositions are made following conventional techniques of pharmaceutical technology involving mixing, filling and dissolving the ingredients, as appropriate, to give the desired oral, parenteral, rectal, transdermal, or topical products.
As used herein, the term “GLP-1 receptor agonist” refers to compounds which have an agonistic activity at the glucagon-like peptide-1 receptor. Examples of GLP-1 receptor agonists include, but are not limited to, the following: exenatide/exendin-4, liraglutide, lixisenatide, dulaglutide, albiglutide, semaglutide, taspoglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, HM-112600, CM-3, GLP-1 Eligen, ORMD-0901, NN9924, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, MAR-701, ZP-2929, ZP-3022, CAM-2036, DA-15864, ARI-2651, ARI-2255, exenatide-XTEN and glucagon-XTEN, AMX-8089+VRS-859 and polymer bound GLP-1 and GLP-1 analogues.
As used herein, the term “dual GLP-1 receptor/glucagon receptor agonist” refers to compounds which have agonistic activity at both the GLP-1 receptor and the glucacon receptor. Examples of dual GLP-1 receptor/glucagon receptor agonist include, but are not limited to, the following: oxyntomodulin, MAR701, MAR-709, and BHM081/BHM089/BHM098.
As used herein, the term “human insulin” refers to the human hormone whose structure and properties are well-known. Human insulin has two polypeptide chains (chains A and B) that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteins in position 6 and 11 of the A-chain; the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain; and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain.
As used herein, the term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
As used herein, the term “isoelectric point” (pl, IEP) refers to the pH value at which a particular molecule carries no net electrical charge. The isoelectric point can be determined by using isoelectric focusing, which is a technique for separating different molecules by differences in their isoelectric point and which is well known in the art. It can also be calculated (see e.g. Levene and Simms, “Calculation of isoelectric point” J. Biol. Chem., 1923, pp. 801-813).
As used herein, the term “kit” refers to a product (e.g. medicament, kit-of-parts) comprising one package or one or moreseparate packages of:
(i). A pharmaceutical formulation containing an active pharmaceutical ingredient and at least one further active pharmaceutical ingredient and optionally a medical device. The at least one further active pharmaceutical ingredient may be present in said pharmaceutical formulation, i.e. the kit may comprise one or more packages, wherein each package comprises one pharmaceutical formulation which comprises two or more active pharmaceutical ingredients. The further active pharmaceutical ingredient may also be present in a further pharmaceutical formulation, i.e. the kit may comprise separate packages of two or more pharmaceutical formulations, wherein each pharmaceutical formulation contain one active pharmaceutical ingredient.

Or

(ii). A pharmaceutical formulation containing an active pharmaceutical ingredient and medical device.

A kit may comprise one package only or may comprise one or more separate packages For example, the kit may be a product (e.g. medicament) containing two or more vials each containing a defined pharmaceutical formulation, wherein each pharmaceutical formulation contains at least one active pharmaceutical ingredient. For example, the kit may comprise (i.) a vial containing a defined pharmaceutical formulation and (ii). further a tablet, capsule, powder or any other oral dosage form which contains at least one further active pharmaceutical ingredient. The kit may further comprise a package leaflet with instructions for how to administer the pharmaceutical formulation and the at least one further active pharmaceutical ingredient.
As used herein, the term “medical device” means any instrument, apparatus, implant, in vitro reagent or similar or related article that is used to diagnose, prevent, or treat a disease of other condition, and does not achieve its purpose through pharmacological action within or on the body.
As used herein, a medical device may be a syringe, an insulin injection system, an insulin infusion system, an insulin pump or an insulin pen injection device. As used herein, a medical device may be mechanically or electromechanically driven.
As used herein, unless specifically indicated otherwise, the conjunction “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or”.
As used herein, the term “pH” and “pH value” refer to the decimal logarithm of the reciprocal of the hydrogen ion activity in a solution.
As used herein, the term “pharmaceutical” refers to the intended use in the medical diagnosis, cure, treatment and/or prevention of diseases.
As used herein, the term “pharmaceutically acceptable” refers to physiologically well tolerated by a mammal or a human.
As used herein, the term “protamine” refers to a mixture of strongly basic peptides. It was originally isolated from the sperm of salmon and other species of fish but is now produced primarily recombinant through biotechnology. It contains more than two-thirds of L-arginine. As protamine contains amino acids having free basic side chains, it has a certain buffering capacity and is therefore considered to be a buffering agent. Protamine may be used as protamine sulfate or protamine hydrochloride.
Concentrations, amounts, solubilities, particle size, wavelength, pH values, weight mass, molecular weight, percent and other numerical date may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
As used herein, the term “long acting insulin” refers to insulin analogues and/or insulin derivatives, wherein the insulin-mediated effect begins within 0.5 to 2 hours and continues to be active for about or more than 24 hours. Examples of fast acting insulins include, but are not limited to, the following: (i). insulin glargin; (ii). insuline detemir and (iii). insulin degludec.
As used herein, the term “stability” refers to the chemical and/or physical stability of active pharmaceutical ingredients, in particular of insulin analogues and/or derivatives. The purpose of stability testing is to provide evidence on how the quality of an active pharmaceutical ingredient or dosage form varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a shelf life for the active pharmaceutical ingredient or dosage form and recommended storage conditions. Stability studies can include testing of those attributes of the active pharmaceutical ingredient that are susceptible to change during storage and are likely to influence quality, safety, and/or efficacy. The testing can cover, as appropriate, the physical, chemical, biological, and microbiological attributes, preservative content (e.g., antioxidant, antimicrobial preservative), and functionality tests (e.g. for a dose delivery system). Analytical procedures can be fully validated and stability indicating. In general, significant changes for an active pharmaceutical ingredient and/or dosage form with regard to stability are defined as:

    
    
        a 5% change in assay from its initial value; or failure to meet the acceptance criteria for potency when using biological or immunological procedures;
        any degradation products exceeding its acceptance criterion;
        failure to meet the acceptance criteria for appearance, physical attributes, and functionality test (e.g., color, phase, separation, resuspendibility, caking, hardness, dose delivery per actuation); however, some changes in physical attributes (e.g. softening of suppositories, melting of creams) may be expected under accelerated conditions; and, as appropriate for the dosage form:
        failure to meet the acceptance criterion for pH; or
        failure to meet the acceptance criteria for dissolution for 12 dosage units.
    
    


The significant changes may also be evaluated against established acceptance criteria prior to starting the evaluation of the stability.
Acceptance criteriacan be derived from the monographs (e.g. monographs for the European Pharmacopeia, of the United States Pharmacopeia, of the British Pharmacopeia, or others), and from the analytical batches of the active pharmaceutical ingredient and medicinal product used in the preclinical and clinical studies. Acceptable limits should be proposed and justified, taking into account the levels observed in material used in preclinical and clinical studies. Product characteristics may be visual appearance, purity, color and clarity for solutions/suspensions, visible particulates in solutions, and pH. As a non-limiting example, suitable acceptance criteria for insulin aspart formulations are shown below:











Test item
Acceptance criteria for clinical trials







Appearance of solution (visual)



Clarity and degree of opalescence
Monitoring


Degree of coloration
Monitoring


Assay insulin aspart units (HPLC)
90.0 insulin aspart units/mL to



110.0 insulin aspart units/mL


Related impurities (HPLC)



B28isoAsp insulin aspart
equal or below to 2.5%


Total of A21Asp insulin aspart,
equal or below to 5.0%


B3Asp insulin aspart and B3isoAsp



insulin aspart



Any other unspecified, unidentified
equal or below to 2.0%


impurity



Total of other impurities
equal or below to 3.5%


High molecular weight proteins
equal or below to 1.5%


(HPSEC)



pH
7.0 to 7.8


Particulate matter (visible particles)
Practically free of visible particles


Particulate matter (subvisible
Number of particles per container:


particles)
equal or larger to 10 μm:



equal or below to 6000



equal or larger to 25 μm:



equal or below to 600


Assay m-cresol
1.55 to 1.89 [mg/mL]


Assay phenol
1.35 to 1.65 [mg/mL]


Zinc (Zn(II)) (AAS)
below 40 μg per 100 units



insulin aspart









The acceptance criteria shown above are based on monographed acceptance limits (e.g. British Pharmacopoeia, Volume III, 2012 or Pharmacopoeial Forum, Volume 36(6), November-December 2010) and/or are derived from extensive experience in the development of insulin formulations.
As used herein, the term “treatment” refers to any treatment of a mammalian, for example human condition or disease, and includes: (1) inhibiting the disease or condition, i.e., arresting its development, (2) relieving the disease or condition, i.e., causing the condition to regress, or (3) stopping the symptoms of the disease.
As used herein, the unit of measurement “U” and/or “international units” refers to the blood glucose lowering activity of insulin and is defined (according to the World Health Organization, WHO) as follows: 1 U corresponds to the amount of highly purified insulin (as defined by the WHO) which is sufficient to lower the blood glucose level of a rabbit (having a body weight of 2-2.5 kg) to 50 mg/100 mL within 1 hour and to 40 mg/100 mL within 2 hours. For human insulin, 1 U corresponds to approximately 35 kg (Lill, Pharmazie in unserer Zeit, No. 1, pp. 56-61, 2001). For insulin aspart, 100 U correspond to 3.5 mg (product information NovoRapid®). For insulin lispro, 100 U correspond to 3.47 mg (product information Humalog®). For insulin glulisine, 100 U correspond to 3.49 mg (product information Apidra® cartridges). For insulin determir, 100 U correspond to 14.2 mg (product information Levemir®). For insulin glargin, 100 U correspond to 3.64 mg (product information Lantus®).
Further embodiments of the present invention include the following:
In one aspect, the invention provides a pharmaceutical formulation comprising (a). at least one analogue and/or derivative of insulin; and (b). Zn(II); and (c). sodium chloride; and (d). optionally protamine; wherein the pharmaceutical formulation has a pH value in the range of from 6.0 to 9.0 and is free of any additional buffering agent.
In one aspect, the pharmaceutical formulation of the invention is an aqueous pharmaceutical formulation.
In one aspect, the pharmaceutical formulation of the invention has a pH value in the range from 7.0 to 7.8.
In one aspect, the pharmaceutical formulation of the invention comprises an analogue of insulin selected from the group consisting of insulin aspart, insulin lispro and insulin glulisine.
In one aspect, the pharmaceutical formulation of the invention comprises a derivative of insulin which is insulin detemir and/or insulin degludec.
In one aspect, the pharmaceutical formulation of the invention comprises an analogue and/or derivative of insulin which is present in a concentration from 10 U/mL to 1000 U/mL.
In one aspect, the pharmaceutical formulation of the invention comprises Zn(II) in a concentration from 0.0100 to 0.0600 mg/100 U of the analogue and/or derivative of insulin.
In one aspect, the pharmaceutical formulation of the invention comprises sodium chloride in a concentration from 0.01 to 15 mg/mL.
In one aspect, the pharmaceutical formulation of the invention comprises sodium chloride in a concentration from 6.8 to 8.3 mg/mL.
In one aspect, the pharmaceutical formulation of the invention comprises protamine sulfate which in a concentration from 0.1 to 0.5 mg/mL.
In one aspect, the pharmaceutical formulation of the invention is free of any additional buffering agent selected from the group consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid, citrate, acetic acid, acetate, glycylglycine and methionine.
In one aspect, the pharmaceutical formulation of the invention comprises one or more further active pharmaceutical ingredients.
In one aspect, the pharmaceutical formulation of the invention comprises a further active pharmaceutical ingredient which is an antidiabetic agent.
In one aspect, the pharmaceutical formulation of the invention comprises a further active pharmaceutical ingredient which is an antidiabetic agent selected from the group consisting of (a) a GLP-1 receptor agonist; (b) a dual GLP-1 receptor/glucagon receptor agonist; (c) human FGF-21; (d) an FGF-21 analogue; (e) an FGF-21 derivative; (f) insulin; (g) human insulin; (h) an analogue of insulin; and (i) a derivative of insulin.
In one aspect, the pharmaceutical formulation of the invention comprises more than one analogue and/or derivative of insulin, wherein one analogue and/or derivative of insulin is a fast acting insulin and one analogue and/or derivative of insulin is a long acting insulin.
In one aspect, the pharmaceutical formulation of the invention comprises a fast acting insulin is selected from the group consisting of insulin aspart, insulin lispro, and insulin glulisine, and wherein the long acting insulin is one or more insulin selected from the group consisting of insulin detemir and insulin degludec.
In one aspect, the pharmaceutical formulation of the invention consists of:

    
    
        (a). 3.5 mg/mL insulin aspart;
        (b). 1.72 mg/mL metacresol;
        (c). 1.50 mg/mL phenol;
        (d). 0.04087 mg/mL Zn(II);
        (e). 6.8 mg/mL sodium chloride;
        (f). 0.02 mg/mL polysorbate 20;
        (g). sodium hydroxide and/or hydrochloric acid to adjust the pH to 7.4, and
        (h). water.
    
    


In one aspect, the pharmaceutical formulation of the invention consists of:

    
    
        (a). 3.5 mg/mL insulin aspart;
        (b). 1.72 mg/mL metacresol;
        (c). 1.50 mg/mL phenol;
        (d). 0.04087 mg/mL Zn(II);
        (e). from 6.8 mg/mL to 8.3 mg/mL sodium chloride;
        (f). 0.02 mg/mL polysorbate 20;
        (g). from 0.1 mg/mL to 0.5 mg/mL protamine sulfate;
        (h). sodium hydroxide and/or hydrochloric acid to adjust the pH to a pH in the range from 7.1 to 7.6; and
        (i). water.
    
    


In one aspect, the invention provides a process for preparing the pharmaceutical formulation of the invention, wherein the components are mixed together in the form of a solution or suspension, the pH is adjusted to reach the desired pH, and water is added to reach the final volume.
In one aspect, the invention provides a kit comprising one or more separate packages of:

    
    
        (a). a pharmaceutical formulation of the invention; and
        (b). a medical device.
    
    


In one aspect, the invention provides a kit comprising one or more separate packages of:

    
    
        (a). a pharmaceutical formulation of the invention; and
        (b). at least one further active pharmaceutical ingredient;
        (c). and optionally a medical device.
    
    


In one aspect, the kit of the invention comprises a further active pharmaceutical ingredient which is an antidiabetic agent.
In one aspect, the kit of the invention comprises a further active pharmaceutical ingredient which is an antidiabetic agent selected from the group consisting of:

    
    
        (a). a GLP-1 receptor agonist;
        (b). a dual GLP-1 receptor/glucagon receptor agonist;
        (c). human FGF-21;
        (d). an FGF-21 analogue;
        (e). an FGF-21 derivative;
        (f). insulin;
        (g). human insulin;
        (h). an analogue of insulin; and
        (i). a derivative of insulin.
    
    


In one aspect, the kit of the invention comprises more than one analogue and/or derivative of insulin, wherein one analogue and/or derivative of insulin is a fast acting insulin and one analogue and/or derivative of insulin is a long acting insulin.
In one aspect, the kit of the invention comprises a fast acting insulin selected from the group consisting of insulin aspart, insulin lispro and nsulin glulisine, and a long acting insulin selected from the group consisting of insulin glargin, insulin detemir and insulin degludec.
In one aspect, the invention provides a pharmaceutical formulation or kit for use in the treatment of diabetes mellitus.
In one aspect, the invention provides a pharmaceutical formulation or kit for use in the treatment of hyperglycemia.
In one aspect, the invention provides a pharmaceutical formulation or kit for use in lowering blood glucose level.
In one aspect, the invention provides a method of treating diabetes mellitus in a subject in need thereof comprising administering a pharmaceutical formulation of the invention.
In one aspect, the invention provides a method of treating hyperglycemia in a subject in need thereof comprising administering a pharmaceutical formulation of the invention.
In one aspect, the invention provides a method of lowering blood glucose levels in a subject in need thereof comprising administering a pharmaceutical formulation of the invention.
In one aspect, the invention provides a medical device for administering a pharmaceutical formulation of the invention to an animal and/or human.
The present invention is illustrated by the following Examples. However, it should be understood that the present invention is not limited to the specific details of these examples.
EXAMPLES
Example 1
Manufacturing Process
(a) Polysorbate Solution
Polysorbate Solution was prepared by dissolving 1.00 g polysorbate 20 in water for injection (according to Ph. Eur.) and by filling up with water for injection to final volume of 1000 mL.
(b) Zinc Chloride Solution
Zinc Chloride Solution (containing Zn(II)) was prepared by dissolving 2.00 g zinc chloride in water for injection and by filling up with water for injection to final volume of 1000 mL.
(c) Solution A
The final composition of Solution A is given in Table 1:







TABLE 1







Composition of Solution A













Composition
Composition
Composition



Excipient
per 200 mL
per 400 mL
per 1000 mL





1.
Sodium chloride
6.8 g
13.60 g 
34.00 g 


2.
Phenol
1.5 g
 3.0 g
7.5 g


3.
m-Cresol
1.72 g 
3.44 g
8.6 g


4.
Sodium hydroxide
ad pH 8.65
ad pH 8.65
ad pH 8.65



solution
(rounded:
(rounded:
(rounded:




pH 9.0)
pH 9.0)
pH 9.0)


5.
Hydrochloric acid
ad pH 8.65
ad pH 8.65
ad pH 8.65




(rounded:
(rounded:
(rounded:




pH 9.0)
pH 9.0)
pH 9.0)


6.
Water for Injection
ad 200 mL =
ad 400 mL =
ad 1000 mL =




205.2 g
410.4 g
1026 g









Solution A was prepared as described in the following:

    
    
        1. It was started with approximately 500 mL water for injection.
        2. 34.00 g sodium chloride, 7.5 g phenol and 8.6 g m-cresol were dissolved while stirring constantly.
        3. Solution was filled up to approximately 900 g with water for injection.
        4. Solution was stirred for approximately 15 min using a magnetic stirrer.
        5. pH was checked (pH should be 8.65, rounded: pH 9.0). If pH value is not 8.65, the pH was adjusted to said range using hydrochloric acid 1 N or sodium hydroxide solution 1 N.
        6. Solution was filled up to 1026 g with water for injection.

(d) Final Solution

    
    


The final composition of Final Solution is given in Table 2:







TABLE 2







Composition of Final Solution













Composition
Composition
Composition



Excipient
per mL
per 1000 mL
per 2000 mL





1.
Insulin aspart
3.496 mg
3.496 g
6.992 g




(rounded:
(rounded:
(rounded:




3.5 mg)
3.5 g)
7.0 g)


2.
Zn(II)
40.87 μg 
0.04087 g  
0.08174 g  


3.
Sodium chloride
6.80 mg
6.80 g
13.60 g 


4.
Phenol
1.50 mg
1.50 g
3.00 g


5.
m-Cresol
1.72 mg
1.72 g
3.44 g


6.
Polysorbat 20
0.02 mg
0.02 g
0.04 g


7.
Sodium hydroxide
ad pH 7.4
ad pH 7.4
ad pH 7.4



solution





8.
Hydrochloric acid
ad pH 7.4
ad pH 7.4
ad pH 7.4


9.
Water for Injection
ad 1 mL =
ad 1000 mL =
ad 2000 mL =




1.005 g
1005 g
2010 g









In the following, preparation of the composition per 2000 mL is described. Other volumes (e.g. composition per 1000 mL) can be prepared in the same way (using the corresponding amount of insulin aspart and excipients).
Final Solution was prepared as described in the following:

    
    
        1. It was started with approximately 300 mL water for injection (according to Ph. Eur.).
        2. 6.992 g (rounded 7.0 g) insulin aspart were added to the 300 mL water for injection while stirring constantly (a suspension of insulin aspart in water for injection is formed).
        3. pH value was checked.
        4. pH value was changed to approximately 3.1 to 3.2 by adding hydrochloric acid 0.1 N or sodium hydroxide solution 0.02 N to dissolve the insulin aspart.
        5. Solution was stirred for approximately 15 min using a magnetic stirrer.
        6. 40.866 g Zinc Chloride Solution was added to the solution while stirring constantly.
        7. 40 g Polysorbate Solution was added while stirring constantly.
        8. Solution was filled up to 600 g with water for injection.
        9. 410.4 g Solution A was added slowly and carefully while stirring constantly.
        10. pH was adjusted to 7.4 (range 7.2 to 7.6) using hydrochloric acid 0.1 N or sodium hydroxide solution 0.02 N.
        11. Solution was filled up to 2010 g (corresponds to 100% of the Final Solution).
    
    


Quality control: Final solution was a clear and uncolored solution, showed a pH value of 7.4 (plus/minus 0.2; at 20-25° C.).
The Final Solution was applied to sterile filtration using “Sartopore Minisart high flow” filter (filter material: polyethersulfone; pore size: 0.2 km; supplier: Sartorius).
The Final Solution after sterile filtration was a clear and uncolored solution and showed an osmolality of 260 mOsmol/kg (plus/minus 30).
The Final Solution after sterile filtration was filled into appropriate vials (volume: 5 and 10 mL; 13 mm; clear glas; glas type 1).
The vials—containing the Final Solution after sterile filtration—was stored between +2° C. and +8° C. and protected from light.
Example 2
Control of the Formulation
(a) Analytical Procedures
Tests were carried out using compendial analytical test methods, where applicable. The quality control concept has been established taking into account the cGMP requirements as well as the current status of the ICH process.
The non-compendial and chromatographic analytical procedures used to control the formulation are summarized in the following:
Description
Visually examined a number of containers for conformance to the acceptance criteria.
Identification (HPLC)
The identity of the active ingredient was ensured by comparing the retention time of the drug formulation sample with the retention time of the reference standard using a reverse phase HPLC method. The method was also used for the determination of assay of the active ingredient, for the determination of the related compounds and impurities, and for quantifying the preservatives m-cresol and phenol.
Assay (HPLC)
The test was carried out by reverse phase liquid chromatography (HPLC). The method was also used for the identification, the determination of assay of the active ingredient, for the determination of the related compounds and impurities, and for quantifying the preservatives m-cresol and phenol. Column: Lichrosorb RP18, particle size 5 μm, pore size 100 {acute over (Å)} (250 mm×4.0 mm), thermostated at +35° C. Autosampler: Thermostated at ° C. Mobile phase A: Sodium sulfate solved in water, 14 g/mL, adjusted with phosphoric acid and sodium hydroxide to a pH of 3.4.
Mobile phase B: Water/acetonitrile (50:50 v/v). Gradient is shown in Table 3.







TABLE 3







HPLC gradient









Time [min]
Mobile phase A [%]
Mobile phase B [%]












0 to 42
57.7
42.3


42 to 47 linear to
20
80


47 to 52
20
80


52 to 53 linear to
57.7
42.3


53 to 60 equilibration
57.7
42.5









Flow rate: 1.0 mL/min. Injection volume: 10 μL. Detection: 214 nm (for the active ingredient) and
260 nm (for m-cresol and phenol). Typical run time: 60 min.
Assay of the active ingredient, m-cresol and phenol were calculated by external standardization.
Impurities were calculated using the peak area percent method.
Test solution: The formulation was used without any dilution or further treatment.
Related Compounds and Impurities (HPLC)
The same chromatographic conditions as for “Assay (HPLC)” were used for the determination of related compounds and impurities. Related compounds and Impurities were calculated using the peak area percent method.
High Molecular Weight Proteins (HMWPs)
The high molecular weight proteins were determined using high pressure size exclusion chromatography (HPSEC). Column: Waters Insulin HMWP, particle size 5-10 μm, pore size 12-12.5 nm (300 mm×7.8 mm), thermostated at room temperature. Autosampler: thermostated at ≦+8° C. Mobile phase: 650 mL of arginine solution (1 g/L) was mixed with 200 mL of acetonitrile and 150 mL of glacial acetic acid. Isocratic elution Flow rate: 1.0 mL/min. Injection volume: 100 μL.
Detection: 276 nm. Typical run time: 35 min.
HMWPs were calculated using the peak area percent method. Test solution: The formulation was used without any dilution or further treatment.
Antimicrobial Preservative Assay
The same chromatographic conditions as for “Assay (HPLC)” were used for the determination of assay of m-Cresol and of phenol m-cresol and phenol were calculated by external standardization.
(b) Validation of Analytical Procedures
The HPLC analytical procedure for the formulation for the determination of identification, assay, and related compounds and impurities was validated to demonstrate specificity, linearity, limit of detection and limit of quantification, accuracy, precision and range.
(c) Justification of the Acceptance Criteria Tests and acceptance criteria, as previously presented, were selected based on ICH Q6B and on published monographs, analytical results obtained, precision of procedures used, Pharmacopoeial and/or regulatory guidelines, and were in agreement with the standard limits at this stage of development.
Example 3
Stability of the Formulation
(a) Stability of the Formulation
Stability studies for the formulation were initiated according to the stability protocol summary described in the following table. The composition and manufacturing method of the stability batches were representative of the material. The stability profile was assessed for storage under long term, accelerated, and stress testing conditions according to ICH guidelines. Samples were packed and stored in glass vials with flanged cap with inserted disc and flip-off lid. The stability data obtained using this packaging material were representative for the preliminary shelf life and storage direction for both packaging configurations (10 mL glass vials and 3 mL cartridges). Up to now, 12 months stability data are available from a batch filled into 10 mL vials and 12 months of a batch filled into 3 mL cartridges ongoing stability studies of the formulation.







TABLE 4







Storage Conditions









Storage Condition
Sampling Intervals
Container





Long Term




+5° C. ± 3° C.
1, 2, 3, 6, 9, and
10 mL vials



12 months



+5° C. ± 3° C.
1, 2, 3, 6, 9, 12,
 3 mL vials



18, 24 and 36 months



Accelerated




+25° C. ±
1, 2, 3, and 6 months
10 mL vials and


2° C./60% ± 5% RH

3 mL cartridges


Stress




+40° C. ±
 1 month
10 mL vials and


5° C./75% ± 5% RH

3 mL cartridges


Photostability




Sun test according to ICH
 1 day
3 mL cartridges


guidelines*




Indoor light**
14 days
3 mL cartridges





*Overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours/m2. A dark control sample is stored under the same conditions to eliminate any effects due to local temperature changes


**Variolux, Heraeus, standard fluorescent tubes, GE-Lightening, Type F40/33, irradiance approximately 8 W/m2, 2000 Lux. A dark control sample is stored under the same conditions to evaluate any effects due to local temperature changes






The following tests were performed during stability testing: appearance, assay, related impurities, high molecular weight proteins, pH, particulate matter (visible and subvisible particles), assay of antimicrobial preservatives (m-cresol and phenol), content of zinc. The investigations on physical and chemical properties after 12 months of storage at the long term storage condition of +5° C. confirm the stability of the formulation when stored at the recommended storage condition. Only very slight changes of the related impurities could be observed.
When stored at accelerated conditions for 3 months at +25° C./60% RH the related impurities and high molecular weight proteins increased, however stayed within the current acceptance limit. When stored at stress conditions (1 month at +40° C./75% RH) one of the related impurities increased above the acceptance criterion. The content of the active ingredient, m-cresol and phenol remained basically unchanged under accelerated conditions.
Due to the present results of the stability studies of the formulation, the chemical and physical stability of the formulation can be confirmed.
Tables 5-12 show the long term stability results, wherein batch no. “_0105” is referring to a formulation according to the present invention filled into 10 mL vials and wherein batch no. “_318” refers to a formulation according to the present invention filled into 3 mL cartridges.
(b) Comparison of Stability of the Buffer-Free Formulation Against Buffer Formulations
A buffered formulation containing a phosphate buffer showed physical instability by formation of anorganic particles (formation of sodium zinc phosphate hydrate, Na6(ZnPO4)6.8 H2O, Sodalite-type crystal structure). Another buffer formulation containing citrate buffer showed physical instability as well, by turning turbid after exposing to slight physical stress. A buffered formulation containing 2-amino-2-hydroxymethyl-propane-1,3-diol (synonym: TRIS) showed an increase of the related impurities when stored under accelerated conditions. Additionally, the buffered formulation containing 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) showed three additional impurites (identified by LC/MS) when stored under stress conditions (+40° C./75% RH), i.e. a TRIS-adduct +103 Da; a corresponding Desamido-adduct +104 Da; and a Formaldehyde-adduct +12 Da (see FIG. 1). The formulation according to the invention does not show these impurities.
The relevant items tested and the analytical results after one month stability, at long term, accelerated and stress conditions regarding the physico-chemical stability in comparison to the formulation according to the invention are listed in Table 13.
After storage of the formulations at long-term storage conditions (1 month at +5° C.), the formulations were applied to mechanical stress (shaking) and thermal stress (+37° C.). The turbidity was measured (nephelometric investigation).
The stability of the buffer-free formulation (i.e., the alternative aqueous pharmaceutical formulation comprising at least one insulin analogue and/or insulin derivative comprising sodium chloride and without any additional buffering agent) shows an excellent chemical and physical stability which qualifies said aqueous pharmaceutical formulation as medicinal product having a defined shelf life.







TABLE 5







Long term stability +5° C. - batch _0105










Acceptance
Time [months]















Test item
criteria
Initial
1
2
3
6
9
12










Appearance of solution (visual)















Clarity
Monitoring
<I (water
<I (water
<I (water
<I (water
<I (water
<I (water
<I (water




clear)
clear)
clear)
clear)
clear)
clear)
clear)


Color
Monitoring
B9
B9
B9
B9
B9
B9
<B9


Assay in insulin aspart
90.0 to 110.0
104.7
104.5
104.3
104.9
104.9
103.1
103.0


units (HPLC)
insulin
insulin
insulin
insulin
insulin
insulin
insulin
insulin



aspart
aspart
aspart
aspart
aspart
aspart
aspart
aspart



units/mL
units/mL
units/mL
units/mL
units/mL
units/mL
units/mL
units/mL







Related Compounds(HPLC)















B28isoAsp insulin aspart
≦2.5%
0.20%
0.29%
0.36%
0.42%
0.53%
0.63%
0.79%


A21 Asp insulin aspart
Monitoring
1.29%
1.14%
1.09%
1.14%
1.19%
1.23%
0.44%


B3 Asp insulin aspart
Monitoring
<RT
<RT
<RT
<RT
<RT
<RT
0.24%


B3 iso Asp insulin aspart
Monitoring
0.08%
0.09%
0.10%
0.13%
0.14%
0.30%
0.14%


Total of A21 Asp insulin
≦5.0%
1.37%
1.23%
1.19%
1.27%
1.32%
2.16%
1.61%


aspart, B3Asp insulin aspart










and B3isoAsp insulin aspart










Any other impurity
≦2.0%
0.69%
0.73%
0.59%
0.65%
0.83%
0.58%
0.46%


Total of other impurities
≦3.5%
0.87%
0.92%
0.75%
0.84%
0.91%
0.81%
0.62%


High molecular weight
≦1.5%
0.24%
0.24%
0.26%
0.28%
0.31%
0.33%
0.32%


proteins (HPSEC)










pH
Between
7.33
7.33
7.35
7.34
7.31
7.38
7.49



7.0 to 7.8









Particulate matter
Practically
complies
complies
complies
complies
complies
complies
Complies


(visible particles)
free from










visible










particles









Assay m-cresol
1.55 to 1.89
1.72 mg/mL
1.72 mg/mL
1.71 mg/mL
1.69 mg/mL
1.69 mg/mL
1.68 mg/mL
1.71 mg/mL



mg/mL (90.0%
(100.0%)
(100.0%)
(99.2%)
(98.3%)
(98.3%)
(97.7%)
(99.2%)



to 110.0% of










label claim)









Assay phenol
1.35 to 1.65
1.49 mg/mL
1.48 mg/mL
1.49 mg/mL
1.47 mg/mL
1.47 mg/mL
1.48 mg/mL
1.49 mg/mL



mg/mL (90.0%
(99.5%)
(98.6%)
(99.3%)
(98.0%)
(98.0%)
(98.6%)
(99.5%)



to 110.0% of










label claim)









Zinc (AAS)
<40 μg per
19.7 μg per
Not tested
Not tested
Not tested
Not tested
Not tested
19.5 μg per



100 units
100 units





100 units



insulin aspart
insulin aspart





insulin aspart




(20.6 μg/mL)





(20.1 μg/mL)


Nephelometry
Monitoring

0.77
Not tested
0.77
Not tested
<I (water
Turbid after









clear)
3 days
















TABLE 6







Accelerated stability +25° C./60%RH - batch _0105










Acceptance criteria
Time













Test item
for clinical trials
Initial results
1 month
2 months
3 months
6 months










Appearance of solution (visual)













Clarity and degree of
Monitoring
<I (water
<I (water
<I (water
<I (water
<I (water


opalescence

clear)
clear)
clear)
clear)
clear)


Degree of coloration
Monitoring
B9
B9
B9
B9
B9


Assay insulin aspart
90.0 insulin
104.6
104.6
104.3
103.7
101.8


units (HPLC)
aspart units/mL
units/mL
units/mL
units/mL
units/mL
units/mL



to 110.0 insulin








aspart units/mL












Related impurities (HPLC)













B28isoAsp insulin aspart
≦2.5%
0.20%
0.84%
1.49%
2.24%
4.11%


Total of A21Asp insulin aspart,
≦5.0%
1.37%
1.50%
1.74%
2.14%
2.90%


B3Asp insulin aspart and








B3isoAsp insulin aspart








Any other unspecified,
≦2.0%
0.69%
0.88%
0.93%
1.26%
2.16%


unidentified impurity








Total of other impurities
≦3.5%
0.87%
1.08%
1.11%
1.50%
2.40%


High molecular weight
≦1.5%
0.24%
0.35%
0.48%
0.67%
1.07%


proteins (HPSEC)








pH
7.0 to 7.8
7.3
7.3
7.4
7.3
7.2


Particulate matter
Practically free
conforms
conforms
conforms
conforms
conforms


(visible particles)
of visible particles







Particulate matter
Number of particles







(subvisible particles)
per container








≧10 μm: ≦6000
33  
not tested
not tested
not tested
3 



≧25 μm: ≦600 
1 



0 


Assay m-cresol
1.55 to 1.89
1.72 mg/mL
1.72 mg/mL
1.71 mg/mL
1.70 mg/mL
1.69 mg/mL



[mg/mL]







Assay phenol
1.35 to 1.65
1.49 mg/mL
1.48 mg/mL
1.49 mg/mL
1.47 mg/mL
1.47 mg/mL



[mg/mL]







Zinc (MS)
<40 μg per 100
19.7 μg/
not tested
not tested
not tested
testing



units insulin
100 U



ongoing



aspart
















TABLE 7







Stress stability +40° C./75%RH - batch _0105










Acceptance criteria
Time










Test item
for clinical trials
Initial result
1 months










Appearance of solution (visual)










Clarity and degree of
Monitoring
<I (water clear)
<I (water clear)


opalescence





Degree of coloration
Monitoring
B9
B9


Assay insulin aspart
90.0 insulin aspart
104.6 units/mL
97.1 units/mL


units (HPLC)
units/mL to 110.0





insulin aspart units/mL









Related impurities (HPLC)










B28isoAsp insulin aspart
≦2.5%
0.20%
4.44%


Total of A21Asp insulin
≦5.0%
1.37%
2.81%


aspart, B3Asp insulin





aspart and B3isoAsp





insulin aspart





Any other unspecified,
≦2.0%
0.69%
2.62%


unidentified impurity





Total of other impurities
≦3.5%
0.87%
2.97%


High molecular weight
≦1.5%
0.24%
1.28%


proteins (HPSEC)





pH
7.0 to 7.8
7.3
 7.3


Particulate matter
Practically free
conforms
conforms


(visible particles)
of visible particles




Particulate matter (subvisible
Number of particles per container




particles)
≧10 μm: ≦6000
33  
8



≧25 μm: ≦600 
1 
1


Assay m-cresol
1.55 to 1.89 [mg/mL]
1.72 mg/mL
1.70 mg/mL


Assay phenol
1.35 to 1.65 [mg/mL]
1.49 mg/mL
1.47 mg/mL


Zinc (Zn(II)) (AAS)
<40 μg per 100 units insulin aspart
19.7 μg/100 U
20.9 μg/100 U
















TABLE 8







Long term stability +5° C. - batch _318 - up to 12 months


Container/closure: 3 mL cartridges


Storage condition: +5° C. ± 3° C.


Storage orientation: Horizontal










Acceptance
Time














Test item
criteria
Initial results
1 month
3 months
6 months
9 months
12 months










Appearance of solution (visual)














Clarity
Monitoring
<I (water
<I (water
<I (water
<I (water
<I (water
<I (water




clear)
clear)
clear)
clear)
clear)
clear)


Color
Monitoring
B9
B9
B9
B9
B9
B9


Assay in insulin aspart
90.0 to 110.0
100.9
100.5
99.9
102.6
97.9
100.2


units (HPLC) a
insulin aspart
insulin
insulin
insulin
insulin
insulin
insulin



units/mL
aspart
aspart
aspart
aspart
aspart
aspart




units/mL
units/mL
units/mL
units/mL
units/mL
units/mL







Related compounds(HPLC)














B28isoAsp insulin aspart
≦2.5%
0.22%
0.40%
0.45%
0.59%
0.61%
0.74%


Total of A21Asp insulin
≦5.0%
0.68%
1.01%
1.04%
1.11%
0.62%
0.80%


aspart, B3Asp insulin aspart









and B3isoAsp insulin aspart









Any other unspecified,
≦2.0%
0.45%
0.62%
0.65%
0.67%
0.87%
0.34%


unidentified impurity









Total of other impurities
≦3.5%
0.53%
0.72%
0.74%
0.84%
0.99%
0.53%


High molecular weight
≦1.5%
0.19%
0.24%
0.24%
0.23%
0.22%
0.26%


proteins (HPSEC)









pH
7.0 to 7.8
7.31
7.37
7.41
7.20
7.39
7.35


Sterility
Complies
complies




complies


Particulate matter
Practically free
complies
complies
complies
complies
complies
complies


(visible particles)
from visible









particles













Particulate matter (subvisible particles)














Number of particles per
≦6000
60  
not tested
not tested
not tested
not tested
213  


container ≧10 μm:









Number of particles per
 ≦600
0 




0 


container ≧25 μm:














Antimicrobial preservative assay














m-cresol
1.55 mg/mL to
1.69 mg/mL
1.70 mg/mL
1.69 mg/mL
1.70 mg/mL
1.67 mg/mL
1.69 mg/mL



1.89 mg/mL
(98.3%)
(98.8%)
(98.3%)
(101.2%)
(97.2%)
(98.4%)



(90.0% to 110.0%









of label claim)








phenol
1.35 mg/mL to
1.48 mg/mL
1.49 mg/mL
1.49 mg/mL
1.48 mg/mL
1.46 mg/mL
1.49 mg/mL



1.65 mg/mL
(98.7%)
(99.3%)
(99.3%)
(98.7%)
(97.2%)
(99.4%)



(90.0% to 110.0%









of label claim)








Zinc (AAS)
<40 μg/100
19.4 μg/
not tested
not tested
not tested
not tested
not tested



units insulin
100 units








aspart
insulin aspart









(19.6 μg/mL)







Preservative efficacy
Complies with
Complies




Complies



Ph. Eur./USP
with Ph. Eur




with Ph. Eur




criteria A




criteria A




and with USP




and with USP





a The content of insulin aspart is calculated together with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulin aspart. 1 unit is equivalent to 0.0350 mg of insulin aspart













TABLE 9







Long term stability +5° C. - batch _318 - 18 to 36 months


Container/closure: 3 mL cartridges


Storage condition: +5° C. ± 3° C.


Storage orientation: Horizontal









Time












Test item
Acceptance criteria
Initial results
18 months
24 months
36 months










Appearance of solution (visual)












Clarity
Monitoring
<I (water clear)
not yet available
not yet available
not yet available


Color
Monitoring
B9
not yet available
not yet available
not yet available


Assay in insulin aspart
90.0 to 110.0 insulin
100.9 insulin
not yet available
not yet available
not yet available


units (HPLC) a
aspart units/mL
aspart units/mL










Related compounds(HPLC)












B28isoAsp insulin aspart
≦2.5%
0.22%
not yet available
not yet available
not yet available


Total of A21Asp insulin
≦5.0%
0.68%
not yet available
not yet available
not yet available


aspart, B3Asp insulin aspart







and B3isoAsp insulin aspart







Any other unspecified,
≦2.0%
0.45%
not yet available
not yet available
not yet available


unidentified impurity







Total of other impurities
≦3.5%
0.53%
not yet available
not yet available
not yet available


High molecular weight
≦1.5%
0.19%
not yet available
not yet available
not yet available


proteins (HPSEC)







pH
7.0 to 7.8
7.31
not yet available
not yet available
not yet available


Sterility
Complies


not yet available
not yet available


Particulate matter
Practically free from
complies
not yet available
not yet available
not yet available


(visible particles)
visible particles











Particulate matter (subvisible particles)












Number of particles per
≦6000
60  
not yet available
not yet available
not yet available


container ≧10 μm:







Number of particles per
 ≦600
0  





container ≧25 μm:












Antimicrobial preservative assay












m-cresol
1.55 mg/mL to 1.89 mg/mL
1.69 mg/mL
not yet available
not yet available
not yet available



(90.0% to 110.0%
(98.3%)






of label claim)






phenol
1.35 mg/mL to 1.65 mg/mL
1.48 mg/mL
not yet available
not yet available
not yet available



(90.0% to 110.0%
(98.7%)






of label claim)






Zinc (AAS)
<40 μg/100 units
19.4 μg/100 units


not yet available



insulin aspart
insulin aspart







(19.6 μg/mL)





Preservative efficacy
Complies with


not yet available
not yet available



Ph. Eur./USP





a The content of insulin aspart is calculated together with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulin aspart. 1 unit is equivalent to 0.0350 mg of insulin aspart













TABLE 10







Accelerated stability +25° C./60% RH - batch _318


Container/closure: 3 mL cartridges


Storage condition: +25° C. ± 2° C./60% ± 5% RH


Storage orientation: Horizontal









Time












Test item
Acceptance criteria
Initial results
1 month
3 months
6 months










Appearance of solution (visual)












Clarity
Monitoring
<I (water clear)
<I (water clear)
<I (water clear)
<I (water clear)


Color
Monitoring
B9
B9
B9
B9


Assay in insulin aspart
90.0 to 110.0 insulin
100.9 insulin
100.5 insulin
99.2 insulin
99.8 insulin


units (HPLC) a
aspart units/mL
aspart units/mL
aspart units/mL
aspart units/mL
aspart units/mL







Related compounds(HPLC)












B28isoAsp insulin aspart
≦2.5%
0.22%
1.05%
2.34%
 4.06% b


Total of A21Asp insulin
≦5.0%
0.68%
1.35%
1.97%
2.91%


aspart, B3Asp insulin aspart







and B3isoAsp insulin aspart







Any other unspecified,
≦2.0%
0.45%
0.82%
1.15%
1.64%


unidentified impurity







Total of other impurities
≦3.5%
0.53%
0.95%
1.36%
2.12%


High molecular weight
≦1.5%
0.19%
0.34%
0.51%
0.62%


proteins (HPSEC)







pH
7.0 to 7.8
7.31
7.35
7.39
7.22


Particulate matter
Practically free from
complies
complies
complies
Complies


(visible particles)
visible particles











Particulate matter (subvisible particles)












Number of particles per
≦6000
60  
not tested
not tested
164   


container ≧10 μm:







Number of particles per
 ≦600
0  


0  


container ≧25 μm:












Antimicrobial preservative assay












m-cresol
1.55 mg/mL to 1.89 mg/mL
1.69 mg/mL
1.69 mg/mL
1.68 mg/mL
1.67 mg/mL



(90.0% to 110.0%
(98.3%)
(98.3%)
(97.6%)
(97.1%)



of label claim)






phenol
1.35 mg/mL to 1.65 mg/mL
1.48 mg/mL
1.49 mg/mL
1.49 mg/mL
1.47 mg/mL



(90.0% to 110.0%
(98.7%)
(99.3%)
(99.3%)
(98.0%)



of label claim)






Zinc (MS)
<40 μg/100 units
19.4 μg/100 units
not tested
not tested
20.5 μg/100 units



insulin aspart
insulin aspart


insulin aspart




(19.6 μg/mL)


(20.5 μg/mL)





a The content of insulin aspart is calculated together with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulin aspart. 1 unit is equivalent to 0.0350 mg of insulin aspart


b OOS result













TABLE 11







Stress stability +40° C./75% RH - batch _318


Container/closure: 3 mL cartridges


Storage condition: +40° C. ± 2° C./75% ± 5% RH


Storage orientation: Horizontal


 










Acceptance
Time










Test item
criteria
Initial result
1 month










Appearance of solution (visual)










Clarity
Monitoring
<I (water clear)
<I (water clear)


Color
Monitoring
B9
B9


Assay in insulin
90.0 to 110.0 insulin
100.9 insulin
98.2 insulin


aspart units (HPLC) a
aspart units/mL
aspart units/mL
aspart units/mL







Related compounds(HPLC)










B28isoAsp insulin aspart
≦2.5%
0.22%
 5.05% b


Total of A21Asp insulin
≦5.0%
0.68%
2.64%


aspart, B3Asp insulin





aspart and B3isoAsp





insulin aspart





Any other unspecified,
≦2.0%
0.45%
 2.38% b


unidentified impurity





Total of other impurities
≦3.5%
0.53%
2.79%


High molecular weight
≦1.5%
0.19%
1.16%


proteins (HPSEC)





pH
7.0 to 7.8
7.31
7.24


Particulate matter
Practically free from
complies
complies


(visible particles)
visible particles









Particulate matter (subvisible particles)










Number of particles per
≦6000
60  
132   


container ≧10 μm:





Number of particles per
 ≦600
0  
0  


container ≧25 μm:










Antimicrobial preservative assay










m-cresol
1.55 mg/mL to 1.89 mg/mL
1.69 mg/mL
1.68 mg/mL



(90.0% to 110.0%
(98.3%)
(97.7%)



of label claim)




phenol
1.35 mg/mL to 1.65 mg/mL
1.48 mg/mL
1.48 mg/mL



(90.0% to 110.0%
(98.7%)
(98.7%)



of label claim)




Zinc (AAS)
<40 μg/100 units
19.4 μg/100 units
20.6 μg/100 units



insulin aspart
insulin aspart
insulin aspart




(19.6 μg/mL)
(20.2 μg/mL)





a The content of insulin aspart is calculated together with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulin aspart. 1 unit is equivalent to 0.0350 mg of insulin aspart


b OOS result













TABLE 12







Photostability (Sun-test) - batch _318


Container/closure: 3 mL cartridges


Storage condition: Photostability according to ICH Q1B guideline (Sun-test) ICH option 1


Storage orientation: Horizontal









Results















Exposure 1 day






1.2 million lux


Test item
Acceptance criteria
Initial
Dark control
hours 200 W-h/m2










Appearance of solution (visual)











Clarity
Monitoring
<I (water clear)
<I (water clear)
<I (water clear)


Color
Monitoring
B9
B9
B8


Assay in insulin aspart
90.0 to 110.0 insulin
100.9 insulin
100.3 insulin
94.2 insulin


units (HPLC)a
aspart units/mL
aspart units/mL
aspart units/mL
aspart units/mL







Related compounds(HPLC)











B28isoAsp insulin aspart
≦2.5%
0.22%
0.35%
0.30% 


Total of A21Asp insulin
≦5.0%
0.68%
0.97%
0.91% 


aspart, B3Asp insulin aspart






and B3isoAsp insulin aspart






Any other unspecified,
≦2.0%
0.45%
0.70%
5.05% b


unidentified impurity






Total of other impurities
≦3.5%
0.53%
0.77%
5.54% b


High molecular weight
≦1.5%
0.19%
0.22%
3.48% b


proteins (HPSEC)






pH
7.0 to 7.8
7.31
7.30
7.24


Particulate matter
Practically free from
complies
complies
complies


(visible particles)
visible particles










Particulate matter (subvisible particles)











Number of particles per
≦6000
60  
123   
132   


container ≧10 μm:






Number of particles per
 ≦600
0  
0  
0  


container ≧25 μm:











Antimicrobial preservative assay











m-cresol
1.55 mg/mL to 1.89 mg/mL
1.69 mg/mL
1.70 mg/mL
1.69 mg/mL



(90.0% to 110.0% of label claim)
(98.3%)
(98.8%)
(98.3%)


phenol
1.35 mg/mL to 1.65 mg/mL
1.48 mg/mL
1.48 mg/mL
1.48 mg/mL



(90.0% to 110.0% of label claim)
(98.7%)
(98.7%)
(98.7%)










Photostability (indoor light) - batch _318


Container/closure: 3 mL cartridges


Storage condition: Photostability indoorlight


Storage orientation: Horizontal









Results















Exposure 14 days






2000 lux hours


Test item
Acceptance criteria
Initial
Dark control
8 W-h/m2











Appearance of solution (visual)












Clarity
Monitoring
<I (water clear)
<I (water clear)
<I (water clear)


Color
Monitoring
B9
B9
B9


Assay in insulin aspart
90.0 to 110.0 insulin
100.9 insulin
100.3 insulin
97.6 insulin


units (HPLC)a
aspart units/mL
aspart units/mL
aspart units/mL
aspart units/mL








Related compounds(HPLC)












B28isoAsp insulin aspart
≦2.5%
0.22%
0.45%
0.41%


Total of A21Asp insulin
≦5.0%
0.68%
1.06%
1.06%


aspart, B3Asp insulin aspart






and B3isoAsp insulin aspart






Any other unspecified,
≦2.0%
0.45%
0.68%
 2.51% b


unidentified impurity






Total of other impurities
≦3.5%
0.53%
0.78%
2.88%


High molecular weight
≦1.5%
0.19%
0.23%
0.23%


proteins (HPSEC)






pH
7.0 to 7.8
7.31
7.27
7.28


Particulate matter
Practically free from
complies
complies
complies


(visible particles)
visible particles











Particulate matter (subvisible particles)












Number of particles per
≦6000
60  
69  
410   


container ≧10 μm:






Number of particles per
 ≦600
0  
0  
0  


container ≧25 μm:












Antimicrobial preservative assay












m-cresol
1.55 mg/mL to 1.89 mg/mL
1.69 mg/mL
1.69 mg/mL
1.69 mg/mL



(90.0% to 110.0% of label claim)
(98.3%)
(98.3%)
(98.3%)


phenol
1.35 mg/mL to 1.65 mg/mL
1.48 mg/mL
1.48 mg/mL
1.48 mg/mL



(90.0% to 110.0% of label claim)
(98.7%)
(98.7%)
(98.7%)





aThe content of insulin aspart is calculated together with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulin aspart.


b OOS result













TABLE 13





Comparison of stability of the formulation according to the invention (_0105) against other formulations

















Composition














Formu-
Storage
Methionin
Polysorbate
m-Cresol
Phenol
ZnCl
NaCl


lation
condition
[mg/mL]
20 [mg/ml]
[mg/mL]
[mg/mL]
[μg/mL]
[mg/mL]





_0062
+5° C.
—
0.02
2.1
1.5
40.87
6.8



+25° C./60% RH
—
0.02
2.1
1.5
40.87
6.8



+40° C./75% RH
—
0.02
2.1
1.5
40.87
6.8


_0105
+5° C.
—
0.02
1.72
1.5
40.87
6.8



+25° C./60% RH
—
0.02
1.72
1.5
40.87
6.8



+40° C./75% RH
—
0.02
1.72
1.5
40.87
6.8


_0063
+5° C.
—
—
2.1
1.5
40.87
6.8



+25° C./60% RH
—
—
2.1
1.5
40.87
6.8



+40° C./75% RH
—
—
2.1
1.5
40.87
6.8


_0106
+5° C.
—
—
1.72
1.5
40.87
6.8



+25° C./60% RH
—
—
1.72
1.5
40.87
6.8



+40° C./75% RH
—
—
1.72
1.5
40.87
6.8


_0071
+5° C.
3
0.02
2.1
1.5
40.87
6.2



+25° C./60% RH
3
0.02
2.1
1.5
40.87
6.2



+40° C./75% RH
3
0.02
2.1
1.5
40.87
6.2


_0072
+5° C.
3
—
2.1
1.5
40.87
6.2



+25° C./60% RH
3
—
2.1
1.5
40.87
6.2



+40° C./75% RH
3
—
2.1
1.5
40.87
6.2













Related impurities
















Total of
Total of







A21Asp,
other





Formu-
B28isoAsp
B3Asp and
impurities
HMWPs
Visual
Nephelometry


lation
[%]
B3isoAsp [%]
[%]
[%]
inspection
(+37° C/120 rpm)





_0062
0.36
1.32
0.78
0.28
conforms
clear after 10 days



1.05
1.63
0.94
0.41
conforms
not performed



4.62
2.91
2.90
1.2
conforms
not performed


_0105
0.29
1.23
0.92
0.24
conforms
clear after 10 days



0.84
1.50
1.08
0.35
conforms
not performed



4.44
2.81
2.97
1.3
conforms
not performed


_0063
0.34
1.26
0.84
0.26
conforms
clear after 10 days



1.02
1.60
0.97
0.38
conforms
not performed



4.61
2.86
2.78
1.3
conforms
not performed


_0106
0.30
1.17
0.85
0.21
conforms
clear after 10 days



0.85
1.52
1.07
0.32
conforms
not performed



4.47
2.79
2.81
1.1
conforms
not performed


_0071
0.33
1.18
0.95
0.24
conforms
clear after 10 days



0.90
1.56
1.35
0.33
does not
not performed







conform




4.62
2.81
3.59
1.1
conforms
not performed


_0072
0.33
1.18
1.02
0.24
conforms
clear after 10 days



0.90
1.53
1.46
0.33
conforms
not performed



4.61
3.00
3.68
1.1
conforms
not performed












Composition















Formu-
Storage
Methionin
Polysorbate
m-Cresol
Phenol
TRIS
ZnCl
NaCl


lation
condition
[mg/mL]
20 [mg/ml]
[mg/mL]
[mg/mL]
[mg/mL]
[μg/mL]
[mg/mL]





_0060
+5° C.
—
0.02
2.1
1.5
6
40.87
4.5



+25° C./60% RH
—
0.02
2.1
1.5
6
40.87
4.5



+40° C./75% RH
—
0.02
2.1
1.5
6
40.87
4.5


_0066
+5° C.
—
0.02
1.72
1.5
6
40.87
4.5



+25° C./60% RH
—
0.02
1.72
1.5
6
40.87
4.5



+40° C./75% RH
—
0.02
1.72
1.5
6
40.87
4.5


_0061
+5° C.
—
—
2.1
1.5
6
40.87
4.5



+25° C./60% RH
—
—
2.1
1.5
6
40.87
4.5



+40° C./75% RH
—
—
2.1
1.5
6
40.87
4.5


_0104
+5° C.
—
—
1.72
1.5
6
40.87
4.5



+25° C./60% RH
—
—
1.72
1.5
6
40.87
4.5



+40° C./75% RH
—
—
1.72
1.5
6
40.87
4.5


_0069
+5° C.
3
0.02
2.1
1.5
6
40.87
3.3



+25° C./60% RH
3
0.02
2.1
1.5
6
40.87
3.3



+40° C./75% RH
3
0.02
2.1
1.5
6
40.87
3.3


_0070
+5° C.
3
—
2.1
1.5
6
40.87
3.3



+25° C./60% RH
3
—
2.1
1.5
6
40.87
3.3



+40° C./75% RH
3
—
2.1
1.5
6
40.87
3.3













Related impurities
















Total of
Total of







A21Asp,
other





Formu-
B28isoAsp
B3Asp and
impurities
HMWPs
Visual
Nephelometry


lation
[%]
B3isoAsp [%]
[%]
[%]
inspection
(+37 °C/120 rpm)





_0060
0.36
1.27
0.77
0.24
conforms
clear after 10 days



1.08
1.62
1.02
0.34
conforms
not performed



4.70
3.14
3.68
1.1
conforms
not performed


_0066
0.34
1.27
0.80
0.22
conforms
clear after 10 days



1.03
1.57
1.01
0.30
conforms
not performed



4.76
3.09
3.46
1.2
conforms
not performed


_0061
0.37
1.25
0.80
0.22
conforms
clear after 10 days



1.08
1.47
1.10
0.31
conforms
not performed



4.73
3.10
3.68
1.1
conforms
not performed


_0104
0.33
1.18
0.76
0.20
conforms
clear after 10 days



0.87
1.55
1.08
0.27
conforms
not performed



4.57
3.05
3.45
1.1
conforms
not performed


_0069
0.33
1.25
0.89
0.22
conforms
clear after 10 days



0.90
1.64
1.26
0.28
conforms
not performed



4.64
3.27
3.41
1.0
conforms
not performed


_0070
0.33
1.24
0.97
0.21
conforms
clear after 10 days



0.89
1.63
1.28
0.27
conforms
not performed



4.61
3.29
3.40
1.0
conforms
not performed












Composition
















Formu-
Storage
Glycerol 85%
Methionin
Polysorbate
m-Cesol
Phenol
Na2HPO4*
ZnCl
NaCl


lation
condition
[mg/mL]
[mg/mL]
20 [mg/ml]
[mg/mL]
[mg/mL]
7H2O [mg/mL]
[μg/mL]
[mg/mL]





_0067
+5° C.
—
—
0.02
2.1
1.5
1.88
40.87
6.2



+25° C./60% RH
—
—
0.02
2.1
1.5
1.88
40.87
6.2



+40° C./75% RH
—
—
0.02
2.1
1.5
1.88
40.87
6.2


_0068
+5° C.
—
—
—
2.1
1.5
1.88
40.87
6.2



+25° C./60% RH
—
—
—
2.1
1.5
1.88
40.87
6.2



+40° C./75% RH
—
—
—
2.1
1.5
1.88
40.87
6.2


_0075
+5° C.
—
3
0.02
2.1
1.5
1.88
40.87
5



+25° C./60% RH
—
3
0.02
2.1
1.5
1.88
40.87
5



+40° C./75% RH
—
3
0.02
2.1
1.5
1.88
40.87
5


_0076
+5° C.
—
3
—
2.1
1.5
1.88
40.87
5



+25° C./60% RH
—
3
—
2.1
1.5
1.88
40.87
5



+40° C./75% RH
—
3
—
2.1
1.5
1.88
40.87
5


_0151
+5° C.
18.82
—
—
1.72
1.5
1.88
40.87
0.58



+25° C./60% RH
18.82
—
—
1.72
1.5
1.88
40.87
0.58



+40° C./75% RH
18.82
—
—
1.72
1.5
1.88
40.87
0.58













Related impurities
















Total of
Total of







A21 Asp,
other





Formu-
B28isoAsp
B3Asp and
impurities
HMWPs
Visual
Nephelometry


lation
[%]
B3isoAsp [%]
[%]
[%]
inspection
(+37° C./120 rpm)





_0067
0.22
1.36
0.84
0.25
does not
suspended







conform
particles at t0



0.81
1.72
1.06
0.36
does not
not performed







conform




4.46
3.22
2.73
1.3
does not
not performed







conform



_0068
0.23
1.41
0.84
0.23
conforms
suspended








particles at t0



0.81
1.68
0.97
0.35
conforms
not performed



4.49
3.18
2.68
1.2
conforms
not performed


_0075
0.24
1.36
0.82
0.23
conforms
clear after 10 days



0.85
1.69
1.05
0.31
conforms
not performed



4.47
3.17
3.06
1.0
conforms
not performed


_0076
0.25
1.33
0.84
0.21
conforms
clear after 10 days



0.83
1.66
1.06
0.30
conforms
not performed



4.41
3.20
2.94
1.0
conforms
not performed


_0151
0.30
1.10
0.98
0.38
conforms
precipitated after








7 days *



0.91
1.70
1.32
0.55
conforms
not performed



5.07
5.05
4.95
2.0
conforms
not performed












Composition















Formu-
Storage
Methionin
Polysorbate
m-Cresol
Phenol
Na-citrate
ZnCl
NaCl


lation
condition
[mg/mL]
20 [mg/ml]
[mg/mL]
[mg/mL]
[mg/mL]
[μg/ml]
[mg/ml]





_0064
+5° C.
—
0.02
2.1
1.5
4
40.87
5.6



+25° C./60% RH
—
0.02
2.1
1.5
4
40.87
5.6



+40° C./75% RH
—
0.02
2.1
1.5
4
40.87
5.6


_0107
+5° C.
—
0.02
1.72
1.5
4
40.87
5.6



+25° C./60% RH
—
0.02
1.72
1.5
4
40.87
5.6



+40° C./75% RH
—
0.02
1.72
1.5
4
40.87
5.6


_0065
+5° C.
—
—
2.1
1.5
4
40.87
5.6



+25° C./60% RH
—
—
2.1
1.5
4
40.87
5.6



+40° C./75% RH
—
—
2.1
1.5
4
40.87
5.6


_0108
+5° C.
—
—
1.72
1.5
4
40.87
5.6



+25° C./60% RH
—
—
1.72
1.5
4
40.87
5.6



+40° C./75% RH
—
—
1.72
1.5
4
40.87
5.6


_0073
+5° C.
3
0.02
2.1
1.5
4
40.87
4.4



+25° C./60% RH
3
0.02
2.1
1.5
4
40.87
4.4



+40° C./75% RH
3
0.02
2.1
1.5
4
40.87
4.4


_0074
+5° C.
3
—
2.1
1.5
4
40.87
4.4



+25° C./60% RH
3
—
2.1
1.5
4
40.87
4.4



+40° C./75% RH
3
—
2.1
1.5
4
40.87
4.4













Related impurities


















Total of
Total of








A21Asp,
other






Formu-
B28isoAsp
B3Asp and
impurities
HMWPs
Visual
Nephelometry



lation
[%]
B3isoAsp [%]
[%]
[%]
inspection
(+37° C./120 rpm)






_0064
0.28
1.34
0.84
0.23
conforms
suspended particles









after 7 days




0.98
1.67
0.98
0.33
conforms
not performed




4.63
3.48
2.93
1.1
conforms
not performed



_0107
0.27
1.28
0.89
0.24
conforms
clear after 10 days




0.83
1.60
1.03
0.31
conforms
not performed




4.31
3.30
2.55
1.0
conforms
not performed



_0065
0.29
1.36
0.90
0.22
conforms
turbid after 3 days




0.99
1.66
0.99
0.31
conforms
not performed




4.66
3.45
2.92
1.1
conforms
not performed



_0108
0.25
1.31
0.82
0.22
conforms
turbid after 3 days




0.81
1.62
1.00
0.30
conforms
not performed




4.29
3.26
2.59
0.91
does not
not performed








conform




_0073
0.33
1.24
0.84
0.24
conforms
clear after 10 days




0.90
1.64
1.12
0.31
conforms
not performed




4.43
3.72
3.57
1.2
conforms
not performed



_0074
0.33
1.19
0.84
0.22
does not
turbid after 3 days








conform





0.92
1.62
1.12
0.29
conforms
not performed




4.43
3.72
3.657
1.2
does not
not performed








conform





* data taken after 3 months storage under long term conditions (+5° C.)

## Claims

1. A pharmaceutical formulation consisting of:
(a) 3.5 mg/mL insulin aspart;
(b) 1.72 mg/mL metacresol;
(c) 1.50 mg/mL phenol;
(d) 0.04087 mg/mL zinc chloride;
(e) 6.8 mg/mL sodium chloride;
(f) 0.02 mg/mL polysorbate 20;
(g) sodium hydroxide and/or hydrochloric acid to adjust the pH to 7.4, and
(h) water.

2. The pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation.

3. The pharmaceutical formulation as claimed in claim 1, wherein the pharmaceutical formulation is formulated for injection.

4. A kit comprising one or more separate packages of
(a) the pharmaceutical formulation as claimed in claim 1; and
(b) a medical device.

5. A kit comprising one or more separate packages of
(a) the pharmaceutical formulation as claimed in claim 1; and
(b) at least one further active pharmaceutical ingredient;
(c) and optionally a medical device.

6. The kit according to claim 5, wherein the further active pharmaceutical ingredient is an antidiabetic agent.

7. The kit as claimed in claim 5, wherein the further active pharmaceutical ingredient is an antidiabetic agent selected from the group consisting of:
(a) a GLP-1 receptor agonist;
(b) a dual GLP-1 receptor/glucagon receptor agonist;
(c) human FGF-21;
(d) an FGF-21 analogue;
(e) an FGF-21 derivative;
(f) insulin;
(g) human insulin;
(h) an analogue of insulin; and
(i) a derivative of insulin.

8. A method of treating diabetes mellitus in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the pharmaceutical formulation of claim 1.

9. A method of treating hyperglycemia in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the pharmaceutical formulation of claim 1.

10. A method of lowering glucose levels in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the pharmaceutical formulation of claim 1.

11. A medical device comprising the pharmaceutical formulation as claimed in claim 1, for administering the pharmaceutical formulation to an animal and/or human.

12. A process for preparing the pharmaceutical formulation according to claim 1, wherein the components are mixed together in the form of a solution or suspension, the pH is adjusted to reach pH 7.4, and water is added to reach the final volume.