Liquid pharmaceutical composition

Abstract

The present invention relates to a liquid pharmaceutical composition comprising a peptide of SEQ ID NO.: 2 as an active pharmaceutical ingredient. The composition of the present invention is suitable for medical use in humans, for example in the treatment of disorders of a metabolic syndrome or diabetes or obesity or for reduction of excess food intake.

Claims

1. A liquid composition comprising: the peptide of SEQ ID NO: 2, a pharmaceutically acceptable salt of the peptide of SEQ ID NO: 2, or a combination thereof as an active pharmaceutical ingredient; sodium chloride; a sodium phosphate buffer; m-cresol; and water wherein the liquid composition has a pH from 6.0 to 7.0.

2. The composition of claim 1, wherein the active pharmaceutical ingredient is present in an amount of 1 μg/ml to 75 mg/ml.

3. The composition of claim 1, wherein sodium chloride is present in an amount of 0.5 mg/ml to 9 mg/ml.

4. The composition of claim 1, wherein the composition is free of any organic solvent.

5. The composition of claim 1, wherein the composition exhibits biological activity, chemical integrity, physical integrity, or any combinations of activity and integrity thereof of the active pharmaceutical ingredient after storage.

6. The composition of claim 1, wherein the composition is free of surfactants.

7. The composition of claim 1, wherein the composition is free of tonicity adjusters different from sodium chloride.

8. The composition of claim 1, wherein the composition consists of: the peptide of SEQ ID NO: 2, a pharmaceutically acceptable salt of the peptide of SEQ ID NO: 2, or a combination thereof; a sodium phosphate buffer; m-cresol; sodium chloride; and water, wherein the liquid composition has a pH of 6.2 to 6.9.

9. The composition of claim 1, wherein the composition consists of: a peptide of SEQ ID NO: 2, a pharmaceutically acceptable salt of the peptide of SEQ ID NO: 2, or a combination thereof in an amount of 0.5 mg/ml to 5 mg/ml; NaH.sub.2PO.sub.4×2H.sub.2O in an amount of 3.03 mg/ml and Na.sub.2HPO.sub.4×12H.sub.2O in an amount of 3.77 mg/ml; m-cresol in an amount of 3.15 mg/ml; sodium chloride in an amount of 6.30 mg/ml; optionally NaOH and/or HCl; and water; wherein the liquid composition has a pH of 6.6.

10. A method of treating a subject suffering from a disease or disorder, the method comprising administering to the subject a therapeutically effective amount of the liquid composition of claim 1.

11. The method of claim 10, wherein the disease or disorder is diabetes mellitus or obesity.

12. The method of claim 11, wherein the diabetic patient has a HbA1c value in the range from about 7% to about 10%.

13. The method of claim 11, wherein the composition is for administration in a patient with type II diabetes as a supplement to a diet in order to improve blood glucose control.

14. The composition of claim 5, wherein the composition exhibits biological activity of the active pharmaceutical ingredient after storage, and wherein the storage is for about one month at a temperature of about +5° C.

15. The composition of claim 5, wherein the composition exhibits chemical integrity of the active pharmaceutical ingredient after storage, and wherein the storage is for about one month at a temperature of about +5° C.

16. The composition of claim 5, wherein the composition exhibits physical integrity of the active pharmaceutical ingredient after storage, and wherein the storage is for about one month at a temperature of about +5° C.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1: Purity of the 0.5 and 5 mg/mL concentrated formulations depends on pH at 40° C. and is highest at the lowest tested pH.

(2) FIG. 2: HMWP content of the 0.5 and 5 mg/mL concentrated formulations depends on pH at 40° C.

(3) FIG. 3: Purity of the 0.5 and 5 mg/mL concentrated formulations depends on the type of tonicity adjuster and stabilizer at 40° C.

(4) FIG. 4: HMWP content of the 0.5 and 5 mg/mL concentrated formulations depends on the type of tonicity adjuster and stabilizer at 40° C.

(5) FIG. 5: Binding energy of stability samples of the formulations containing sodium chloride.

(6) FIGS. 6A-6C: Comparative ThT test of Liraglutide and SEQ ID NO.: 2. FIG. 6A: SEQ ID NO.: 2, +37° C., 500 rpm; y-axis range 0-6000 RFU (relative fluorescence units); FIG. 6B: Liraglutide, +37° C., 500 rpm; y-axis range 0-6000 RFU; FIG. 6C: SEQ ID NO.: 2, +37° C., 500 rpm; y-axis range 0-200 RFU.

(7) FIG. 7: Rheology evaluation of the formulation with a capillary rheometer.

EXAMPLES

(8) Abbreviations employed are as follows:

(9) ACN: acetonitrile

(10) API: active pharmaceutical ingredient

(11) DLS: dynamic light scattering

(12) FTIR: Fourier transform infrared spectroscopy

(13) GCG: glucagon

(14) GLP-1: glucagon-like peptide 1

(15) HMWP: high molecular weight protein

(16) HPLC: high pressure liquid chromatography

(17) HPSEC: high-performance size-exclusion chromatography

(18) LLPS: liquid liquid phase separation

(19) PEG: polyethylene glycol

(20) RH: relative humidity

(21) TFA: trifluoroacetic acid

(22) ThT: Thioflavin T

Example 1: Chemical Stability Assessment

(23) Detection and quantification of impurities and degradation products are carried out by reverse phase liquid chromatography (HPLC) using a gradient method with UV detection. In HPLC, impurities were calculated using the peak area percent method, and API content was calculated by external standardization.

(24) Column: stainless steel, 150 mm×4.6 mm i.d. (XSelect CSH C18, 2.5 μm, Waters).

(25) Mobile phase: water/acetonitrile/trifluoroacetic acid.

(26) Flow rate: 1.0 mL/min.

(27) Temperature: +45° C.

(28) Detection wavelength: UV at 210 nm

(29) Test solution: 0.5 mg/mL of test sample (undiluted for 0.5 mg/formulation, diluted with acetonitrile/water for 5.0 mg/mL formulation).

(30) The amount of high molecular weight proteins is determined using high performance size exclusion chromatography (HPSEC). In HPSEC, HMWPs were calculated using the peak area percent method.

(31) Column: stainless steel, 300 mm×7.8 mm i.d. (Insulin HMWP Column, Waters, or equivalent).

(32) Mobile phase: arginine buffer/acetonitrile/acetic acid.

(33) Flow rate: 0.45 mL/min.

(34) Temperature: room temperature

(35) Detection wavelength: UV at 283 nm

(36) Test solution: 0.5 mg/mL of test sample (undiluted for 0.5 mg/formulation, diluted with acetonitrile/water for 5.0 mg/mL formulation).

(37) A study was initiated with 20 runs of a design of experiment (DoE) study to determine the optimal type of the tonicity adjuster and/or stabilizer and to verify the pH, which ensures the highest chemical stability of peptide SEQ ID NO.: 2 as the active pharmaceutical ingredient (API). The stability study was initiated at 5° C., 25° C. and at 40° C. with 2 and 4 weeks storage conditions.

(38) In the statistical design, the effect of concentration of the API was tested at 2 levels, the effect of pH at three levels, the effect of preservatives at three levels, the effect of tonicity adjusters at three levels and the effect of additives at two levels. Table 1a below shows a summary of the controlled factors, and Table 1b shows details of the 20 runs.

(39) The proposed design is an IV-optimal response surface design which allows studying main effects and level 2 interactions with API in a minimum of experiments. The results at the beginning and after 14 and 28 days of storage are combined for analysis to allow the estimation of linear and quadratic effect of the factor time and corresponding level 2 interactions.

(40) The data for the different storage conditions is analyzed independently. A backward selection is performed to keep in the model only the terms that have a significant effect at 10%.

(41) The Box-Cox method is also used to apply a data transformation if necessary. The “Box-Cox method” refers to a systematic test of possible transformations to ensure normal distribution of the data and low variability around the model (e.g. for a linear regression, all individual measurements are as close as possible to the regression line). After analysis of each parameter a desirability function, using the relative weights defined by the scientist, is performed to obtain a global optimum.

(42) TABLE-US-00003 TABLE 1a List of controlled factors Factor (unit) Description Experimental domain API Concentration 2 fixed levels: 0.5 mg/ml, (Quantitative, 5 mg/ml mg/mL) pH 3 fixed levels: 6.5, 7.0, 7.4 (Quantitative, NA) Preservatives Phenol: 6 mg/g, 3 modalities: m-cresol, phenol, (Qualitative, NA) m-cresol: 3 mg/g m-cresol + phenol Tonicity adjusters Glycerol 85%: 5.0 mg/g, mannitol: 3 modalities: glycerol, mannitol, (Qualitative, NA) 8.7 mg/g, NaCl: 1.7 mg/g NaCl Solvent Propylene glycol: 7.5 mg/g 2 modalities: propylene (Qualitative, NA) glycol, none NA: not applicable

(43) TABLE-US-00004 TABLE 1b Proposed design with 20 runs API concentration Tonicity Run (mg/ml) pH Preservatives adjusters Additives  1 5 6.5 phenol Mannitol None  2 0.5 7.0 m-cresol NaCl propylene glycol  3 5 7.0 m-cresol + phenol Mannitol None  4 5 7.4 phenol NaCl None  5 0.5 6.5 m-cresol Glycerol None  6 5 7.4 m-cresol + phenol Glycerol None  7 0.5 7.4 m-cresol + phenol NaCl None  8 5 6.5 m-cresol + phenol NaCl propylene glycol  9 0.5 6.5 m-cresol + phenol Mannitol propylene glycol 10 5 7.4 m-cresol Mannitol propylene glycol 11 0.5 7.4 phenol Glycerol propylene glycol 12 5 7.0 phenol Glycerol propylene glycol 13 5 6.5 m-cresol Glycerol None 14 0.5 7.0 m-cresol + phenol Glycerol propylene glycol 15 0.5 7.0 phenol Mannitol None 16 5 7.0 m-cresol NaCl None 17 0.5 6.5 phenol NaCl propylene glycol 18 0.5 7.4 m-cresol Mannitol propylene glycol 19 0.5 7.0 m-cresol NaCl propylene glycol 20 5 7.0 m-cresol + phenol Mannitol None

(44) The purity of the active ingredient (API; SEQ ID NO.:2) and the content of high molecular weight protein (HMWP) in the test formulations depend on the pH value, as can be seen in the results presented in FIGS. 1 and 2.

(45) FIG. 1 is an interaction plot showing differences in purity between different pH values for low API concentration. The circle is the design point; X1-axis=API concentration; X2-axis=pH; actual factors: preservative=phenol; tonicity adjuster=NaCl; solvent=propylene glycol; time=14 d.

(46) FIG. 2 is an interaction plot showing differences in HMWP depending on the pH for low API concentration. The circle is the design point; X1-axis=API concentration; X2-axis=pH; actual factors: preservative=phenol; tonicity adjuster=NaCl; solvent=propylene glycol; time=14 d.

(47) The highest purity and the lowest content of HMWP were found at pH 6.5/6.6.

(48) The purity and high molecular weight protein (HMWP) content of the test formulations further depend on the type of the tonicity adjuster and stabilizer as shown in FIGS. 3 and 4. Formulations manufactured with NaCl as tonicity adjuster have significantly higher stability, higher purity and lower HMWP formation than formulations manufactured with other type of tonicity adjusters such as mannitol and glycerol.

(49) FIG. 3 is a main effects plot showing a smaller decrease in purity when sodium chloride is used as tonicity adjuster, as compared to mannitol and glycerol. The circles in the figure are the design points, the dashed lines the Cl bands; X1-axis=time; X2-axis=tonicity; actual factors: API concentration=0.5 mg/ml; pH=6.5; preservative=phenol; solvent=propylene glycol.

(50) FIG. 4 is an Interaction plot showing the differences between isotonizers with respect to HMWP in time. The circles in the figure are the design points, the dashed lines the Cl bands; X1-axis=time; X2-axis=tonicity; actual factors: API concentration=0.5 mg/ml, pH=6.5; preservative=phenol; solvent=propylene glycol.

(51) The highest purity and the lowest content of HMWP were found for the compositions contain NaCl as a tonicity adjuster and stabilizer.

(52) As can be seen in FIG. 1-4, the strongest effects besides time and its interactions are API concentration, pH and tonicity.

(53) To summarize the data represented in FIGS. 1-4, the highest purity is obtained for a high API concentration, low pH and NaCl as a tonicity adjuster.

Example 2: Physical Stability Evaluation

(54) a) Colloidal Stability

(55) The purpose of the study was to evaluate the colloidal stability of the formulation that contains an ionic tonicity adjuster and stabilizer with Liquid Liquid Phase Separation (LLPS) method. Colloidal stability, which is a measure of interaction of colloidal molecules in the solution, can be expressed in terms of binding energy. Liquid-liquid phase separation is caused by net attraction between the colloidal molecules (e.g. proteins, peptides), thus measures the interaction of molecules in the solution in terms of binding energy. Polyethylene glycol (PEG) induced liquid-liquid phase separation is a rapid quantitative way of estimating net intermolecular attractive interaction of the colloidal (antibodies or peptide) solution and can be used to get information on the propensity of a candidate or formulations to form aggregates.

(56) Binding energy is indicative of colloidal stability was calculated using the equation of Wang et al. (Mol. Pharm. 11(5), 1391-1402, 2014):

(57) $\ln \left(\frac{v_0{N}_A}{M_1}{C}_I^1\right)=-\frac{{\mathrm{.Math.}}_B}{\mathrm{kT}}-\Delta v\frac{{\Pi }_2}{\mathrm{kT}}$ with N.sub.A=Avogadro's number M.sub.1=molecular weight of the molecule C.sup.1.sub.I=molecule mass concentration in the supernatant k=Boltzmann constant T=absolute temperature v.sub.0=volume per molecule in the condensed phase Π=osmotic pressure of PEG Δv=depletion zone per protein in the liquid condensed phase

(58) To evaluate the colloidal stability of a solution for injection comprising peptide SEQ ID NO.: 2 as active ingredient at 5.0 mg/mL and 10.0 mg/mL, a PEG induced LLPS study was conducted on nine and eighteen months stability samples.

(59) Preparation of Polyethylene glycol solution was carried out as follows. 40% (w/v) solution of Polyethylene glycol was dissolved in Placebo solution. The pH of solution was adjusted to 6.6 using 1N sodium hydroxide. PEG solution was stored at +2° C.-+8° C. for 3 days and pH was again checked for pH drift.

(60) A mixture of an API (SEQ ID NO.: 2) containing sample (5.0 mg/ml or 10.0 mg/ml API, sodium phosphate buffer, m-cresol, sodium chloride, pH for details see Table 2 below) and PEG solution was incubated at +5° C. for 2 days. After incubation sample-PEG mixture was centrifuged for two minutes at 16900 G at +5° C. After centrifugation, supernatants were transferred for RP-HPLC analysis for equilibrium peptide concentration measurements, the same HPLC method was applied as it is explained under Example 1.

(61) Binding energy of all three stability samples was negative, which suggests that inter-peptide interactions between the peptide molecules are highly repulsive (FIG. 5). Binding energy of the peptide at 5 mg/mL, stability at 5° C. for 9 and 18 months and binding energy of the peptide at 10 mg/mL, stability at 5° C. for 9 months was not significantly different. Binding energy data of the peptide at 5 mg/mL concentrated formulation incubated at 5° C. for 9 and 18 months suggests that the colloidal stability of the peptide in 5 mg/mL concentration does not change upon extended incubation at 5° C. Comparison of 5 mg/mL and 10 mg/mL samples incubated at 5° C. for 9 months suggests that the peptide at both concentrations is equally stable.

(62) b) Small Angle X-Ray Scattering (SAXS)

(63) Small angle X-ray scattering (SAXS) reveals solution structures of biological macromolecules and synthetic nanoparticles at 1-2 nm resolution.

(64) SAXS measurements were performed at a synchrotron light source (DESY Hamburg). The scattering data from all samples were collected loading 20 microliters of solutions into the capillary and flowing the whole volume across the X-ray beam. Per each sample 20 frames of 0.045 s exposure were collected and, after a statistical consistency check, averaged. The corresponding signals collected for the placebo was then subtracted. Each sample measurement was repeated at least twice to ensure reproducibility.

(65) The best fit between the experimentally observed small angle X-ray scattering curve and the calculated one is obtained using a model in which SEQ ID NO.: 2 is all hexameric in the formulations. All hexameric means that all peptide molecules are arranged in physical oligomers containing 6 molecules of SEQ ID NO.: 2.

(66) c) Dynamic Light Scattering (DLS)

(67) The light-scattering intensity of particles is proportional to the diameter and therefore extremely sensitive to aggregation. For the analysis of incretin peptides, DLS is used as a standard method to measure aggregation. If high molecular weight particles are detected, this may be an artifact of the fact that the buffer has not been sufficiently filtered. On the other hand, if no high molecular weight intensity is measured; this means that no aggregates were present in the sample.

(68) DLS experiments were performed with a Malvern Zetasizer Nano ZS. The formulation was pipetted into the measuring cell, the temperature was equilibrated to +25° C. and measured in NIBS (Non-Invasive Back-Scatter) mode.

(69) Dynamic light scattering was performed with a composition of the invention (0.5 mg/ml and 5.0 mg/ml, respectively, phosphate buffer, NaCl and m-cresol, pH of about 6.6; for details see Table 2 below) to monitor the hydrodynamic diameter. For both API concentrations, a hydrodynamic diameter of about 6 nanometers was found. The value of 6 nm indicates that the API is present in the form of a hexamer (see above). These results further support the absence of undesired larger aggregates, for which larger hydrodynamic diameters (such as 100 nm) would have been measured. These results are in line with the SAXS results.

Example 3: Fibrillation Tendency

(70) Investigations were carried out to determine fibrillation tendencies under stress conditions using a ThT assay. ThT is a dye that, after binding to beta-sheet-rich structures displays an enhanced fluorescence and a characteristic red shift of the emission spectrum. Amyloid-like structures show a beta-sheet rich structure. Fibrillation of peptide solutions is induced by thermic and mechanical stress. Samples are stressed under defined conditions using Fluoroskan Ascent FL.

(71) For the tests in Fluoroskan Ascent FL 200 μL sample were placed into 96 well microtiterplate PS, flat bottom, Greiner Fluotrac No. 655076. Plates were sealed with Scotch Tape (Quiagen). Samples were stressed by continuous cycles of 10 s shaking at 960 rpm and 50 s rest period at +37° C. The kinetic was monitored by measuring fluorescence intensity every 20 minutes.

(72) Solutions were transferred in 2 mL Eppendorf vials and 20 μL of a 10.1 mM ThT solution in H.sub.2O were added to 2 mL of peptide solution to receive a final concentration of 100 μM ThT. For each sample eight replicates were tested.

(73) It was found that the peptide of SEQ ID NO.: 2 (at a concentration of 1 mg/ml in PBS) is more resistant to mechanical and thermal stress than Liraglutide (SEQ ID NO.: 1; 1 mg/ml in PBS), see FIGS. 6A-6C. The figure illustrates that the peptide of SEQ ID NO.:2, in contrast to Liraglutide, shows negligible fibrillation even after 100 h incubation at +37° C. and 500 rpm. In a further experiment, no fibrillation was observed after 100 hours at 37° C. shaking (500 rpm) in a solution containing 5 mg/mL SEQ ID NO.:2, phosphate buffer, NaCl and m-cresol, pH≈6.6, see Table 2 below).

Example 4: Chemical and Biophysical Evaluation

(74) The purpose of the study was to evaluate the chemical and biophysical stability of formulations containing SEQ ID NO.: 2 with different peptide and natural salt ratios, which are 0.08 (0.5 mg: 6.30 mg) for the 0.5 mg/mL concentrated formulation, 0.8 (5 mg: 6.30 mg) for the 5.0 mg/mL concentrated formulation and 0.24 (1.5 mg: 6.30 mg) for the 1.5 mg/mL concentrated formulation.

(75) The following composition was tested:

(76) TABLE-US-00005 TABLE 2 tested composition Specification according to Substance pharmacopeia Amount per unit Peptide of SEQ ID NO.: 2 Sanofi-Aventis 0.50 mg (Batch A) or 5.0 mg (Batch B) or 1.5 mg (Batch C) Sodium dihydrogen Ph. Eur./USP 3.03 mg phosphate dihydrate Di-sodium hydrogen Ph. Eur./USP 3.77 mg phosphate dodecahydrate m-cresol Ph. Eur./USP 3.15 mg Sodium chloride Ph. Eur./USP 6.30 mg 0.1 N Hydrochloric acid Ph. Eur./USP ad pH 6.6 0.1 N NaOH solution Ph. Eur./USP ad pH 6.6 Water for injection (WfI) Ph. Eur./USP ad 1.0 ml

(77) The formulations are stored in units which are intended for clinical studies and for sales and distribution.

(78) Storage times, storage conditions, time points are summarized in the following table.

(79) TABLE-US-00006 TABLE 3 summary conditions Test interval (months) Condition 0 1 3 6 Long-term storage +5 ± 3° C. x x x x Accelerated conditions (temperature, humidity) +25 ± 2° C./60 ± 5% RH x x x x +37 ± 2° C./75 ± 5% RH x x — —

(80) The formulations storage orientation was inverted. RH means relative humidity. Time point 0 is the start of storage. The measurements at time point 0 are used as a reference for all conditions tested.

(81) The physical and chemical stability of the stored formulations is determined with the help of the following tests: Dynamic light scattering (DLS) method was applied to access the subvisible particle size distribution Fourier transform infrared spectroscopy (FTIR) to analyze the ratio of alpha and beta sheets Chemical stability (purity and impurities), determined by HPLC Proteins of high molecular weight, determined by HPSEC

(82) All values were determined by HPLC with the so-called 100% method. Here, in particular, it involves reversed-phase HPLC (C 18 column), in which a gradient method was used for the mobile phase: a) 0.1% TFA, 15% ACN and b) 0.1% TFA, 75% ACN.

(83) Detection at 210 nm (UV).

(84) The high-molecular-weight proteins (HMWP) were detected by HPSEC, described in European Pharmacopeia 6.0 for injectable insulin preparations.

(85) The percentage values are the content values (percentage values of impurities) of total impurities, and of high-molecular-weight proteins (HMWP).

(86) Results

(87) The formulations were studied separately for parallel batches A (0.5 mg/ml API), B (5.0 mg/ml API) and C (1.5 mg/ml) with regard to the following parameters:

(88) Biophysical stability with the evaluation of DLS and FTIR results. Physical instability was not observed even for a high sodium chloride containing formulation (0.5 mg/mL peptide with 6.3 mg/mL sodium chloride concentration). The DLS and FTIR data were stable during the stability study and an increase in the beta sheet was not analysed.

(89) Total impurities. An increase in the sum of impurities/degradation products was observed. This increase is not due to an increase of one single impurity, but a slight increase of about 0.1 to 0.2% is observed for various impurities. Stability results support the minimum 24 months shelf life of the drug product independently from the concentration of the active pharmaceutical ingredient within the formulation.

(90) High molecular weight proteins (HMWPs). There is only a slight increase in high molecular weight proteins observed at 5° C. and 25° C. At 37° C. after 1 month storage the increase is between 0.3 and 0.7% and results are within the specification 2.0%.

(91) The data are summarized in the following table:

(92) TABLE-US-00007 TABLE 4 Summary Results Batch A: 0.5 mg/mL +5° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. 9 Mon. 12 Mon. 18 Mon. 24 Mon. SEQ ID NO.: 2 0.50 0.50 0.49 0.50 0.50 0.51 0.51 0.50 assay in mg/mL Total impurities 3.1 3.1 3.7 3.9 4.3 4.7 4.9 6.0 in % Proteins of high 0.4 0.5 0.5 0.5 0.7 0.9 1.0 1.0 molecular weight in % FTIR: alpha/ 50.3/ — — 47.5/ — 53.2/ 48.1/ 50.2/ beta sheets in % 16.7 17.5 22.6 11.7 12.8 DLS in nm — — — — — — — — +25° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. Activity assay in 0.50 0.49 0.47 0.46 mg/mL Total impurities 3.1 4.1 7.1 10.0 in % Proteins of high 0.4 0.7 0.9 1.5 molecular weight in % FTIR: alpha/ 50.3/ — — 46.7/ beta sheets in % 16.7 20.8 DLS in nm — — — — +37° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. SEQ ID NO.: 2 0.50 0.46 — — assay in mg/mL Total impurities 3.1 8.1 — — in % Proteins of high 0.4 1.1 — — molecular weight in % FTIR: alpha/ 50.3/ — — — beta sheets in % 16.7 DLS in nm — — — — Batch B: 5.0 mg/mL +5° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. 9 Mon. 12 Mon. 18 Mon. 24 Mon. SEQ ID NO.: 2 5.13 5.20 5.08 4.96 5.12 5.26 5.25 5.18 assay in mg/mL Total impurities in 3.1 3.2 3.9 3.8 4.2 4.7 4.9 6.0 % Proteins of high 0.4 0.3 0.3 0.4 0.4 0.6 0.6 0.7 molecular weight in % FTIR: alpha/beta 46.9/ — — 48.0/ — 52.0/ 47.6/ 49.5/ sheets in % 13.9 15.5 12.3 12.8 13.9 DLS in nm 5.93 — — 6.15 — — 6.02 5.60 +25° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. Activity assay in 5.13 5.06 4.78 4.56 mg/mL Total impurities in 3.1 4.3 7.5 9.9 % Proteins of high 0.4 0.4 0.5 0.7 molecular weight in % FTIR: alpha/beta 46.9/ — — 48.1/ sheets in % 13.9 16.1 DLS in nm 5.93 — — 6.13 +37° C. t.sub.0 1 Mon. 3 Mon. 6 Mon. SEQ ID NO.: 2 5.13 4.79 — — assay in mg/mL Total impurities in 3.1 8.8 — — % Proteins of high 0.4 0.7 — — molecular weight in % FTIR: alpha/beta 46.9/ — — — sheets in % 13.9 DLS in nm 5.93 — — — Batch C: 1.5 mg/mL +5° C. t.sub.0 3 Mon. 6 Mon. 12 Mon. 18 Mon. 24 Mon. SEQ ID NO.: 2 1.47 1.46 1.39 1.43 1.43 1.39 assay in mg/mL Total impurities 3.9 4.4 5.1 5.3 6.1 6.0 in % Proteins of high 0.6 0.6 0.7 0.7 0.7 0.8 molecular weight in % +25° C. t.sub.0 3 Mon. 6 Mon. Activity assay in 1.47 1.38 1.26 mg/mL Total impurities 3.9 8.0 12.3 in % Proteins of high 0.6 1.0 1.4 molecular weight in %

Conclusion

(93) The results of the accelerated and long term stability study verified the observations of the initial short term experiments (storage for 2 and 4 weeks). Sodium chloride ensured the suitable chemical and biophysical stability of the active pharmaceutical ingredient in 0.08, 0.24 and 0.8 peptide to neutral salt ratio.

Example 5: Ratio of Alpha and Beta Sheets

(94) FTIR spectroscopy is a suitable method for the determination of a protein's secondary structure. The vibration giving the most pronounced signal in FTIR spectroscopy is the C═O stretching vibration of amide groups resulting in a signal in the so-called amide I-region (1700-1600 cm.sup.−1). This vibration is sensitive to the hydrogen-bonding pattern, dipole-dipole interactions and the geometry of the polypeptide backbone. The signals obtained in this IR-region give typical bands for the different secondary structural elements of a protein. For most proteins, different secondary structural elements influence the IR-spectrum in the amide I-region.

(95) As a consequence, the individual component bands that represent the different structural elements, such as α-helices, β-sheets, turns and irregular structures are often not resolved and difficult to identify in the broad amide I-band contours of the experimentally obtained spectra. The Opus software includes a chemometric method analysing certain regions of the amide I and amide II bands from a library of 43 proteins of known α-helix and β-sheet content. The proteins used for the calibration of the method covered a region of 0 to 75% for α-helix content and 0 to 48% for β-sheet content. The average error in predicted α-helix for the calibration proteins was 5.5%, the error in β-sheet content 4.4%, reasonable numbers for an empirical method. However, it should be kept in mind that the results for some proteins deviate far more (up to 15%) from their true values. The absolute values for protein conformation determined by the chemometric method can therefore be systematically biased, however, the repeatability of results for samples of the same protein is usually in the range of +/−2%.

(96) Investigations were carried out to determine the ratio of alpha and beta sheets at 5.0 mg/ml and 75 mg/mL concentration, respectively, of the biopharmaceutical protein (SEQ ID NO.: 2). The tested formulation contained a phosphate buffer, m-cresol and sodium chloride (as detailed in Table 2 above).

(97) Samples were investigated using a Bruker Tensor 27 Fourier-transform infrared (FTIR) spectrometer equipped with an AquaSpec sampling cuvette with an optical path length of 7 μm (“Confocheck” setup). Spectrometer control and data evaluation was done by the Opus software package, version 6.5. Measurements were performed as described below according to the manufacturer's instructions: Daily system check by the automated “Performance Qualification” tool of the Opus software. Flushing of the AquaSpec cell with water until a clean background line is achieved. In case residual peaks cannot be eliminated by purging with water alone, a 5% Hellmanex solution is used. Two samples are subsequently measured, first the placebo (buffer) solution followed by the protein formulation. About 50 μL are injected into the cell by means of a Hamilton syringe. The difference of the two spectra is automatically generated and represents the spectrum of the pure protein. The procedure was repeated for each formulation in order to obtain two data sets for duplicate analysis. After normalization, the spectra are evaluated by the Quant2 module of the Opus software.

(98) The measured values of alpha and beta sheets were 48.9 and 13.1%, which are in the trend of previous FTIR results of formulations containing 0.5 and 5.0 mg/mL active pharmaceutical ingredient.

Example 6: Effect of Surfactants

(99) Formulations were manufactured to evaluate the effect of surfactants such as Polysorbate 20, Polysorbate 80 and Poloxamer 188 on the biophysical stability of the active pharmaceutical ingredient in a solution formulation. Formulations were manufactured at pH=7.4 with 5.0 mg/mL active pharmaceutical ingredient concentration. Interestingly, the particle size measured with the DLS method (general method description: see above) increased for the formulations containing especially Polysorbates up to 7.8 nm, which phenomena was explained as a micelle formation.

Example 7: Adsorption to Surfaces

(100) Investigations were carried out to study the adsorption phenomena of the active pharmaceutical ingredient to glass and plastic surfaces of the primary containers in 0.05 mg/mL peptide concentration with a formulation containing a neutral salt such as sodium chloride. Dilution of the 0.5 mg/mL concentrated formulation described above (see Table 2) was done with 0.9% NaCl containing solution to reach the 0.05 mg/mL concentration. The diluted formulation was stored in glass and in plastic containers. As explained above, the formulation contains a buffer and a microbiological preservative. Assay results showed the best recovery as compared to the assay results in 10 times higher (0.5 mg/mL) concentration, close to 100%, of the theoretical assay from glass containers.

(101) Detection of the assay values at the end of the adsorption time were carried out by reverse phase liquid chromatography (HPLC) using a gradient method with UV detection (see above).

Example 8: Rheological Properties of the Formulations

(102) Investigations were carried out to evaluate the fluid property of the formulation to determine whether it is a Newtonian fluid or not. To this end, a composition as described above in Table 2 containing 5.0 mg/ml SEQ ID NO.: 2 was added to a rheometer and subjected to measurement. The experiments were performed with a MCR rotation rheometer (Anton Paar) and with capillary rheometer Kinexus (Malvern). No significant changes were observed in shear stress or viscosity at shear rates>1/s* (Newtonian fluid). High shear rates have no influence on the shear viscosity of the formulation (see also FIG. 7).

(103) The invention is further characterized by the following items:

(104) Item 1. A liquid composition comprising a peptide of SEQ ID NO.: 2 and/or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient, sodium chloride, and a liquid carrier.

(105) Item 2. The composition of item 1, wherein the active pharmaceutical ingredient is present in an amount of 1 μg/ml to 75 mg/ml.

(106) Item 3. The composition of item 1 or 2, wherein the active pharmaceutical ingredient is present in an amount of 1 μg/ml to 10 mg/ml.

(107) Item 4. The composition of any one of items 1-3, wherein the active pharmaceutical ingredient is present in an amount of 0.5 mg/ml to 5 mg/ml.

(108) Item 5. The composition of any one of items 1-4, wherein sodium chloride is present in an amount of 0.5 mg/ml to 9 mg/ml.

(109) Item 6. The composition of any one of items 1-5, wherein sodium chloride is present in an amount of 5 mg/ml to 9 mg/ml.

(110) Item 7. The composition of any one of items 1-6, wherein the composition further comprises a pharmaceutically acceptable preservative.

(111) Item 8. The composition of item 7, wherein the pharmaceutically acceptable preservative is selected from phenol and m-cresol.

(112) Item 9. The composition of item 7 or 8, wherein the pharmaceutically acceptable preservative is m-cresol.

(113) Item 10. The composition of any one of items 1-9, wherein the composition further comprises a pharmaceutically acceptable buffer.

(114) Item 11. The composition of any one of items 1-10, wherein the pharmaceutically acceptable buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, and a phosphate-citrate buffer.

(115) Item 12. The composition of any one of items 1-11, wherein the pharmaceutically acceptable buffer is a phosphate buffer.

(116) Item 13. The composition of item 12, wherein the phosphate buffer is a sodium phosphate buffer.

(117) Item 14. The composition of any one of items 1-11, wherein the pharmaceutically acceptable buffer is a citrate buffer.

(118) Item 15. The composition of item 14, wherein the citrate buffer is a sodium citrate buffer.

(119) Item 16. The composition of any one of items 1-15, wherein the composition has a pH from 6.0 to 7.0.

(120) Item 17. The composition of any one of items 1-16, wherein the composition has a pH from 6.2 to 6.9.

(121) Item 18. The composition of any one of items 1-17, wherein the composition has a pH of about 6.6, particularly 6.55-6.64.

(122) Item 19. The composition of any one of items 1-18, wherein the liquid carrier is an aqueous liquid.

(123) Item 20. The composition of any one of items 1-19, wherein the liquid carrier is water.

(124) Item 21. The composition of any one of items 1-20, wherein the liquid carrier is free of any organic solvent.

(125) Item 22. The composition of any one of items 1-20, which is free of any organic solvent.

(126) Item 23. The composition of any one of items 1-20, wherein the liquid carrier is free of glycerol.

(127) Item 24. The composition of any one of items 1-20, which is free of glycerol.

(128) Item 25. The composition of any one of items 1-20, 23, wherein the liquid carrier is free of propylene glycol.

(129) Item 26. The composition of any one of items 1-20, 24, which is free of propylene glycol.

(130) Item 27. The composition of any one of items 1-26, which exhibits biological activity of the active pharmaceutical ingredient(s) (API(s)) after storage.

(131) Item 28. The composition of item 27, wherein the composition is stored for 1 month, 3 months, 6 months, 9 months, 12 months, 18 months or 24 months.

(132) Item 29. The composition of item 27 or 28, wherein the composition is stored at a temperature of +5° C., +25° C., or +37° C.

(133) Item 30. The composition of any one of items 27-29, wherein the biological activity of the API(s) after storage for at least one month at a temperature of +5° C. or +25° C., is at least 80%, at least 90%, at least 95% or at least 98% of the activity at the start of storage.

(134) Item 31. The composition of any one of items 27-30, wherein the biological activity of the API(s) after storage for twelve months or twenty-four months at a temperature of +5° C., is at least 80% of the activity at the start of storage.

(135) Item 32. The composition of any one of items 27-30, wherein the biological activity of the API(s) after storage for one month or three months or six months at a temperature of +25° C., is at least 80% of the activity at the start of storage.

(136) Item 33. The composition of any one of items 27-31, wherein the biological activity of the API(s) after storage for twenty-four months at a temperature of +5° C. is at least 90%, at least 95% or at least 98% of the activity at the start of storage.

(137) Item 34. The composition of any one of items 1-33, which exhibits chemical integrity of the API(s) after storage.

(138) Item 35. The composition of item 34, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage.

(139) Item 36. The composition of item 34 or 35, wherein the composition is stored for 1 month, 3 months, 6 months, 9 months, 12 months, 18 months or 24 months.

(140) Item 37. The composition of any one of items 34-36, wherein the composition is stored at a temperature of +5° C., +25° C., or +37° C.

(141) Item 38. The composition of any one of items 34-37, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 18 months at +5° C.

(142) Item 39. The composition of any one of items 34-37, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 6 months at +25° C.

(143) Item 40. The composition of any one of items 34-37, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 1 month at +37° C.

(144) Item 41. The composition of any one of items 34-37, wherein the composition is defined as exhibiting chemical integrity if the proportion of proteins of high molecular weight (HMWP) with respect to the entire mass of the peptide of SEQ ID NO.: 2 present in the composition after storage is below 2 wt-%.

(145) Item 42. The composition of item 41, wherein the proportion of proteins of high molecular weight (HMWP) with respect to the entire mass of the peptide of SEQ ID NO.: 2 present in the composition after storage is below 2 wt-% after storage for one month, 6 months, 12 months or 18 months at +5° C.

(146) Item 43. The composition of item 41, wherein the proportion of proteins of high molecular weight (HMWP) with respect to the entire mass of the peptide of SEQ ID NO.: 2 present in the composition after storage is below 2 wt-% after storage for one month, 6 months, 12 months or 18 months at +25° C.

(147) Item 44. The composition of item 41, wherein the proportion of proteins of high molecular weight (HMWP) with respect to the entire mass of the peptide of SEQ ID NO.: 2 present in the composition after storage is below 2 wt-% after storage for one month, 6 months, 12 months or 18 months at +37° C.

(148) Item 45. The composition of any one of items 41-44, wherein the proportion of proteins of high molecular weight (HMWP) with respect to the entire mass of the peptide of SEQ ID NO.: 2 present in the composition after storage is below 2 wt-% after storage for 18 months at +5° C., 6 months at +25° C. and/or one month at +37° C.

(149) Item 46. The composition of any one of items 1-45, which exhibits physical integrity of the API(s) after storage.

(150) Item 47. The composition of item 46, wherein at least 80%, at least 90%, at least 95%, or at least 98% of the API(s) are present in a substantially physically unchanged form, compared with the start of storage.

(151) Item 48. The composition of item 46 or 47, wherein the composition is stored for 1 month, 3 months, 6 months, 9 months, 12 months, 18 months or 24 months.

(152) Item 49. The composition of any one of items 46-48, wherein the composition is stored at a temperature of +5° C., +25° C., or +37° C.

(153) Item 50. The composition of any one of items 46-49, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 18 months at +5° C.

(154) Item 51. The composition of any one of items 46-49, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 6 months at +25° C.

(155) Item 52. The composition of any one of items 46-49, wherein at least 80%, at least 90%, at least 95% or at least 98% of the API(s) are present in a substantially chemically unchanged form after storage for 1 month at +37° C.

(156) Item 53. The composition of any one of items 46-52, which is free of aggregates, particularly covalent aggregates.

(157) Item 54. The composition of any one of items 46-53, wherein the composition is defined as exhibiting physical integrity if it is free of covalent aggregates as determined by Dynamic Light Scattering (DLS).

(158) Item 55. The composition of any one of items 27-54, wherein the peptide of SEQ ID NO.: 2 is the sole active pharmaceutical ingredient (API).

(159) Item 56. The composition of any one of items 1-55, which is free of surfactants.

(160) Item 57. The composition of any one of items 1-55, which is free of polyhydric alcohols or esters or ethers thereof, particularly free of fatty acid esters and ethers of glycerol and sorbitol.

(161) Item 58. The composition of any one of items 1-57, which is free of polyols, particularly polyols selected from the group consisting of polypropylene glycols, polyethylene glycols, polysorbates, poloxamers, Pluronics, and Tetronics.

(162) Item 59. The composition of any one of items 1-58, which is free of polysorbates.

(163) Item 60. The composition of any one of items 1-59, which is free of poloxamers.

(164) Item 61. The composition of any one of items 1-58, which is free of polysorbates and poloxamers.

(165) Item 62. The composition of any one of items 1-61, which is free of tonicity adjusters different from sodium chloride.

(166) Item 63. The composition of any one of items 1-61, which is free of glycerol, dextrose, lactose, sorbitol, and mannitol.

(167) Item 64. The composition of any one of items 1-61, which is free of mannitol and propylene glycol.

(168) Item 65. The composition of any one of items 1-64, which consists of

(169) (a) a peptide of SEQ ID NO.: 2 and/or a pharmaceutically acceptable salt thereof,

(170) (b) a sodium phosphate buffer of pH 6.2 to 6.9, particularly a sodium phosphate buffer of about pH 6.5 to 6.7,

(171) (c) m-cresol,

(172) (d) sodium chloride, and

(173) (e) water.

(174) Item 66. The composition of any one of items 1-65, which consists of

(175) (a) a peptide of SEQ ID NO.: 2,

(176) (b) a sodium phosphate buffer of pH 6.2 to 6.9, particularly a sodium phosphate buffer of about pH 6.5 to 6.7,

(177) (c) m-cresol,

(178) (d) sodium chloride, and

(179) (e) water.

(180) Item 67. The composition of any one of items 1-66, which consists of

(181) (a) a peptide of SEQ ID NO.: 2,

(182) (b) a sodium phosphate buffer of about pH 6.5 to 6.7,

(183) (c) m-cresol,

(184) (d) sodium chloride, and

(185) (e) water.

(186) Item 68. The composition of any one of items 1-64, which consists of

(187) (a) a peptide of SEQ ID NO.: 2 and/or a pharmaceutically acceptable salt thereof in an amount of 0.5 mg/ml to 5 mg/ml,

(188) (b) NaH.sub.2PO.sub.4×2H.sub.2O in an amount of 3.03 mg/ml and Na.sub.2HPO.sub.4×12H.sub.2O in an amount of 3.77 mg/ml,

(189) (c) m-cresol in an amount of 3.15 mg/ml,

(190) (d) sodium chloride in an amount of 6.30 mg/ml,

(191) (e) optionally NaOH and/or HCl at pH 6.6, and

(192) (f) water, particularly water for injection.

(193) Item 69. The composition of any one of items 1-64, which consists of

(194) (a) a peptide of SEQ ID NO.: 2 in an amount of 0.5 mg/ml to 5 mg/ml,

(195) (b) NaH.sub.2PO.sub.4×2H.sub.2O in an amount of 3.03 mg/ml and Na.sub.2HPO.sub.4×12H.sub.2O in an amount of 3.77 mg/ml,

(196) (c) m-cresol in an amount of 3.15 mg/ml,

(197) (d) sodium chloride in an amount of 6.30 mg/ml,

(198) (e) optionally NaOH and/or HCl at pH 6.6, and (f) water for injection.

(199) Item 70. The composition of any one of items 1-69 for use in medicine, particularly for use in human medicine.

(200) Item 71. The composition of item 70 for injection.

(201) Item 72. The composition of any one of items 1-69 for use in a method of treating diabetes mellitus, particularly for use in a method of treating type II diabetes mellitus.

(202) Item 73. The composition of any one of items 1-69 for use in a method of treating obesity.

(203) Item 74. The composition of any one of items 1-69 for use in a method of treating diabetes mellitus, particularly type II diabetes mellitus, and obesity.

(204) Item 75. The composition of any one of items 1-69 for use according to item 72 or 74, wherein the diabetic patients have a HbA1c value in the range from 7% to 10%.

(205) Item 76. The composition of any one of items 1-69 for use according to any one of items 69-74, which is for administration in patients with type II diabetes as a supplement to a diet in order to improve blood glucose control.

(206) Item 77. A method for treating a disorder comprising administering to a patient in need thereof a therapeutically effective amount of a composition of any one of items 1-69.

(207) Item 78. The method of item 77, wherein the patient is a human patient.

(208) Item 79. The method of item 77 or 78, wherein said disorder is selected from the group consisting of diabetes mellitus, particularly type II diabetes mellitus, and obesity.