PHARMACEUTICAL COMPOSITION COMPRISING A GLP-1 AGONIST, AN INSULIN AND METHIONINE

Abstract

A liquid composition comprising a GLP-1 agonist or/and a pharmacologically tolerable salt thereof, an insulin or/and a pharmacologically tolerable salt thereof, and, optionally, at least one pharmaceutically acceptable excipient, wherein the composition comprises methionine, as add-on therapy with metformin where appropriate.

Claims

1. An aqueous solution for subcutaneous injection comprising: (a) 0.033 mg desPro.sup.36exendin-4(1-39)-Lys.sub.6-NH.sub.2, (b) 3.64 mg Gly(A21)-Arg(B31)-Arg(B32) human insulin, (c) 30 μg zinc, (d) 2.7 mg m-cresol, (e) 3 mg L-methionine, (f) 20 mg glycerol, and (g) water for injection, to a volume of 1 mL.

2. The composition of claim 1, further comprising hydrochloric acid and sodium hydroxide as needed achieve a pH of about 4.5.

3. The composition of claim 1, wherein the amount of water is at least 10% by weight.

4. A prefilled syringe for subcutaneous injection comprising an aqueous solution comprising per 1 mL volume of the solution: (a) about 0.033 mg to about 0.1 mg desPro.sup.36exendin-4(1-39)-Lys.sub.6-NH.sub.2 or a pharmacologically tolerable salt; (b) about 3.64 mg insulin glargine or a pharmacologically tolerable salt thereof; (c) about 3 mg methionine; (d) about 2.7 mg m-cresol; (e) about 20 mg 85% glycerol; (f) about 0.06 mg zinc chloride; and (g) water for injection, to a volume of 1 mL.

5. The composition of claim 4, further comprising hydrochloric acid and sodium hydroxide as needed achieve a pH of about 4.5.

Description

EXAMPLE 1

[0200] 1. Purpose of Study

[0201] The physical and chemical stability of compositions comprising a GLP-1 agonist (AVE0010) and an insulin (insulin glargine, Lantus) was tested.

[0202] 2. Formulations Used

[0203] For the formulations tested, the substances were used in the following concentrations/amounts:

TABLE-US-00011 Amount used Substance Pharmacopeia Manufacturer Designation [mg/mL] Insulin glargine Sanofi-Aventis 3.63 7.27 10.67 AVE0010 Poly Peptide 0.1 LabTorrance CA, 0.025 USA Methionine USP MP Biomedicals 3 Zinc chloride Ph. Eur., USP, Merck 0.03 BP 0.06 0.09 Glycerol, 85% Ph. Eur., JP Hedinger, Stuttgart 20 18 m-Cresol Ph. Eur., USP Hedinger, Stuttgart 2.7 Polysorbate 20 Ph. Eur., JP Kolb Tween 20 0.02 Polysorbate 80 Ph. Eur. SEPPIC Tween 80 0.02 Poloxamer 188 BASF, Ludwigshafen Lutrol F68 0.02 Benzalkonium Ph. Eur., JP Sigma-Aldrich 0.02 chloride L-Lysine Resum, F-Ham, 1.0 Degussa 5.0 Acetate 1.75 3.5 NaOH Ph. Eur., JP Merck 0.1N, for adjusting to pH 4.0 or 4.5 HCl Ph. Eur., JP Merck 0.1N, for adjusting to pH 4.0 or 4.5 Wfl Ad 1 mL

[0204] When a factor is mentioned in conjunction with a constituent of a formulation (e.g., ½, ¼, 2×, 3×, 5×, as in ½ acetate, 5× lysine, 2× Lantus, and 3× Lantus), the concentrations of the substance concerned were used at a reduced or increased concentration depending on the factor.

[0205] 3. Test Method

[0206] 3.1 Physical Stability

[0207] 3.1.1 THT Test

[0208] Thioflavin T (THT) binds specifically to protein fibrils, which leads to a change in THT fluorescence. THT does not bind to AVE0010 or insulin. The kinetics of fibril formation can be measured in the presence of THT as the change in fluorescence. An increase in fluorescence corresponds to fibril formation. The shape of the curves allows conclusions about the tendency of a formulation to form fibrils.

[0209] Fluorescence measurements were carried out on a Tecan Infinite 200 fluorescence measurement instrument. For analysis of fibrillation kinetics, a Photomed FluoDia 770 high-temperature fluorescence microplate reader was used. The thioflavin T fluorescence spectra were carried out with a Tecan Infinite 200 fluorescence measurement instrument at 23° C. Insulin (900 μl) was mixed with 10 μl of thioflavin T (1 mM in H.sub.2O). The mixture was then distributed into a black V-shaped 96-well plate from Biozym (100 μl per well). The emission of fluorescence was measured between 470 and 600 nm (in increments of 1 nm) after excitation at 450 nm with a gain of 100, an integration time of 200 μs, and 25 readings at room temperature.

[0210] The binding kinetics of thioflavin T were measured on a Photomed FluoDia 770 high-temperature fluorescence microplate reader. The instrument consists essentially of a 50 W quartz halogen lamp for excitation, filter wheels for excitation and emission which can each contain up to 4 filter sets, and a PMT detector. The heating plate for 96-well plates allows very high precision with regard to temperature (better than ±0.3° C.).

[0211] A solution (10 μl) of thioflavin T (10.1 mM in ultrapure water) was added to 1 ml of the formulations and gently mixed by inverting the small tubes several times. The mixture was then distributed into a black V-shaped 96-well plate from Biozym (100 μl per well, 8 wells per sample). All measurements were carried out with the following parameters:

[0212] Number of cycles: 181

[0213] Excitation filter: 450 nm

[0214] Interval: 1 min

[0215] Emission filter: 486 nm

[0216] Integration time: 20 ms

[0217] Temperature control: Standard temperature-control mode

[0218] Number of averagings: 4

[0219] Target temperature: 70° C.

[0220] Attenuation: 4

[0221] Fluorescence mean values were determined from 8 parallel measurements.

[0222] 3.2 Chemical Stability

[0223] The formulations were tested for chemical stability after preparation (t0) or after storage for 1 month at 4° C., 25° C. (60% relative humidity), and 40° C. (75% relative humidity). The measurements were carried out on an HPLC instrument (model: alliance) from Water Systems, using the 100% peak area method. For separation, a gradient of 0.1% TFA and acetonitrile as the mobile phase and a C18 reversed-phase column (Jupiter) as the stationary phase were used. For analysis, the formulation was treated with a zinc acetate solution, which led to precipitation of insulin glargine. The precipitates were centrifuged down, and only the supernatant was analyzed.

[0224] Impurities of insulin glargine: the amount of impurities was determined with an HPLC (Water Systems), using the 100% peak area method. For separation, a sodium phosphate-buffered solution (pH 2.5) with NaCI and acetonitrile gradients was used as the mobile phase. A C18 reversed-phase column (Supersher) was used as the stationary phase.

[0225] 4. Summary of Experimental Data on Physical Stability

TABLE-US-00012 THT 3 h, 70° C. relative Formulation fluorescence No. Batch Composition pH intensity at 486 nm 1 630 AVE0010 standard, 4.5 536 industrial scale 2 567 AVE0010 standard, 4 518 fresh 3 631 Lantus standard, 4.0 2952 industrial scale 4 560 Lantus standard, fresh 4 1566 5 568 Lantus form., AVE0010 4 2037 6 569 Lantus form., AVE0010, 4 11763 1/2 acetate buffer 7 570 Lantus form., AVE0010, 4 69184 acetate buffer 8 582 Lantus form., AVE0010, 4 2053 methionine 9 583 Lantus form., AVE0010, 4 18814 1/2 acetate buffer, methionine 10 584 Lantus form., AVE0010, 4 8183 polysorbate 20 11 585 Lantus form., AVE0010, 4 6731 polysorbate 20, methionine 12 586 Lantus form., AVE0010, 4 13897 polysorbate 20, 1/2 acetate buffer 13 587 Lantus form., AVE0010, 4 22200 polysorbate 20, 1/2 acetate buffer, methionine 14 588 Lantus form., AVE0010, 4 134093 polysorbate 20, acetate buffer, methionine 15 590 Lantus form., AVE0010, 4 3362 lysine 16 591 Lantus form., AVE0010, 4 19677 lysine, 1/2 acetate buffer 17 592 Lantus form., AVE0010, 4 30176 lysine, 1/2 acetate buffer, polysorbate 20 18 593 Lantus form., 4 3107 1/4 AVE0010 19 594 Lantus form., 4 74662 1/4 AVE0010, 5x lysine 20 595 2x Lantus AVE0010 4 4504 21 596 3x Lantus AVE0010 4 30251 22 604 Lantus form., AVE0010 4.5 4357 23 605 Lantus form., AVE0010, 4.5 36338 1/2 acetate buffer 24 606 Lantus form., AVE0010, 4.5 72370 acetate buffer 25 607 Lantus form., AVE0010, 4.5 5429 methionine 26 608 Lantus form., AVE0010, 4.5 34714 1/2 acetate buffer, methionine 27 609 Lantus form., AVE0010, 4.5 1166 polysorbate 20 28 610 Lantus form., AVE0010, 4.5 5564 polysorbate 20, methionine 29 611 Lantus form., AVE0010, 4.5 12115 polysorbate 20, 1/2 acetate buffer 30 612 Lantus form., AVE0010, 4.5 16397 polysorbate 20, 1/2 acetate buffer, methionine 31 613 Lantus form., AVE0010, 4.5 779 polysorbate 20, acetate buffer, methionine 32 614 Lantus form., AVE0010, 4.5 9726 lysine 33 615 Lantus form., AVE0010, 4.5 74027 lysine, 1/2 acetate buffer 34 616 Lantus form., AVE0010, 4.5 9520 lysine, 1/2 acetate buffer, polysorbate 20 35 617 Lantus form., 4.5 3713 1/4 x AVE0010 36 618 Lantus form., 4.5 83384 1/4 x AVE0010, 5x lysine 37 619 2x Lantus AVE0010 4.5 13120 38 620 3x Lantus AVE0010 4.5 41684 39 657 Lantus form., AVE0010, 4 9309 polysorbate 80, methionine 40 658 Lantus form., AVE0010, 4 767 poloxamer 188, methionine 41 659 Lantus form., AVE0010, 4 1040 benzalkonium chloride, methionine 42 660 Lantus form., AVE0010, 4.5 16803 polysorbate 80, methionine 43 661 Lantus form., AVE0010, 4.5 689 poloxame 188, methionine 44 662 Lantus form., AVE0010, 4.5 942 benzalkonium chloride, methionine

[0226] 5. THT Test

[0227] Methionine has no influence on the tendency to form fibrils. The formulations

TABLE-US-00013 Fluorescence intensity No. Composition at 486 nm 2 AVE0010 standard 518 4 Lantus standard 1566 8 Lantus form., AVE0010, methionine, 2053 pH 4 25 Lantus form., AVE0010, methionine, 5429 pH 4.5
have fluorescence values like the reference formulations (no. 2 and 4). With values below approximately 6000, no tendency to form fibrils is present.

[0228] When AVE0010, Lantus, and methionine are combined with acetate buffer with or without polysorbate 20 at pH 4, there is a greater tendency to form fibrils:

TABLE-US-00014 Fluorescence No. Composition intensity at 486 nm 2 AVE0010 standard 518 4 Lantus standard 1566 9 Lantus form., AVE0010, ½ acetate, Met, pH 4 18814 13 Lantus form., AVE0010, polysorbate 20, 1/2 22200 acetate, Met, pH 4 14 Lantus form, AVE0010, polysorbate 20, acetate, 134093 Met, pH 4

[0229] The values for formulations 13 and 14 lie clearly above the threshold for a tendency to form fibrils.

[0230] 6.1 Summary

[0231] Polysorbate 20 and polysorbate 80 can lead to turbidity, which is detectable in the double refraction test. Hence, both of these substances can lead to physical instability of a formulation of AVE0010 and insulin.

[0232] The addition of methionine does not lead to physical instability.

[0233] 7. Chemical Stability

[0234] 7.1 Stability at Time Point t0

[0235] The formulations which comprise methionine (with and without sodium acetate) have the lowest amounts of impurities (overall, approximately 1.2 to 1.5%). The following formulations have low amounts of impurities:

TABLE-US-00015  8 Lantus form., AVE0010, methionine, pH 4  9 Lantus form., AVE0010, 1/2 acetate buffer, methionine, pH 4 11 Lantus form., AVE0010, polysorbate 20, methionine, pH 4 13 Lantus form., AVE0010, 1/2 acetate buffer, polysorbate 20, methionine, pH 4 14 Lantus form., AVE0010, acetate buffer, polysorbate 20, methionine, pH 4 25 Lantus form., AVE0010, methionine, pH 4.5 26 Lantus form., AVE0010, 1/2 acetate buffer, methionine, pH 4.5 28 Lantus form., AVE0010, polysorbate 20, methionine, pH 4.5 30 Lantus form., AVE0010, 1/2 acetate buffer, polysorbate 20, methionine, pH 4.5 31 Lantus form., AVE0010, acetate buffer, polysorbate 20, methionine, pH 4.5

[0236] Formulations which did not comprise methionine showed a higher fraction of impurities.

[0237] Polysorbate 20 has no negative influence on the chemical stability of the formulations.

[0238] Acetate buffer has no negative influence on chemical stability when it is combined with methionine and polysorbate 20.

[0239] When lysine is present in the formulations, the sum of impurities is greater. The same is true for formulations which comprise polysorbate 80, poloxamer 188, and benzalkonium chloride.

[0240] Determining the impurities of insulin glargine revealed that all formulations had comparable amounts of impurities (0.3 to 0.4%).

[0241] 7.2 Stability After 1 month

[0242] 7.2.1 Impurities of AVE0010

[0243] The content of oxidized methionine in the formulations was analyzed. The sequence of AVE0010 has one methionine residue at position 14. The sequence of insulin glargine has no methionine residues. Therefore, the content of oxidized methionine is indicative of oxidation of AVE0010 at the methionine residue. The data are summarized in FIG. 1. Overall, the data show that, without methionine at a pH of 4.5, the fraction of Met(ox) is higher than at pH 4.0. Without methionine as a constituent of the formulations, the fractions of Met(ox) are greatest when the content of insulin glargine is increased or the content of AVE0010 is reduced.

[0244] Generally, the greatest fractions of Met(ox) were measured after storage at 40° C./75% relative humidity. Here, the lowest fractions of Met(ox)-AVE0010 (<1%) are to be found in the formulations 8, 9, 11, 13, 14, 25, 26, 28, 30, and 31. The values of these formulations are in the range of the values for the AVE0010 reference formulations no. 1 and 2 (frame in FIG. 1).

[0245] The impurities of AVE0010 after 1 month without Met(ox) are represented in FIG. 2. The frames show the values of the AVE0010 reference formulations at 25° C. and at 40° C. Formulations which have the same or better impurity values than the AVE0010 reference formulations are within or below the frames. This is true for the formulations 24, 25, 26, 28, 29, 30, 31, 33, and 34 (40° C.). Impurity values which are above the impurity values of the AVE0010 reference formulations indicate impurities of insulin glargine. Generally, formulations having a pH of 4.5 have fewer impurities than at a pH of 4.0.

[0246] The following formulations have, after storage for one month at 40° C., the lowest content of Met(ox) and, simultaneously, the lowest content of other impurities (comparison of FIGS. 1 and 2). They are better than or the same as the AVE0010 reference formulations:

TABLE-US-00016 25 Lantus form., AVE0010, methionine, pH 4.5 26 Lantus form., AVE0010, 1/2 acetate buffer, methionine, pH 4.5 28 Lantus form., AVE0010, polysorbate 20, methionine, pH 4.5 30 Lantus form., AVE0010, 1/2 acetate buffer, polysorbate 20, methionine, pH 4.5

[0247] These formulations also belonged to those formulations which have at time point t0 the lowest amounts of AVE0010 impurities. All formulations comprise methionine. Polysorbate 20 has no negative effects on the impurities.

[0248] The impurities of insulin glargine are represented in FIG. 3. Formulations 3 and 4 are the reference formulations for insulin glargine. The values of these formulations are indicated as narrow frames. All formulations which were identified with regard to AVE0010 impurities as the best formulations (broad frames, more particularly formulations 25, 26, 28, and 30) are, with regard to insulin glargine impurities, better than the insulin glargine reference formulations (approximately 1.5 to 2.4% at 40° C.).

[0249] Hence, it can be deduced from this experiment that methionine engenders an increased storage stability of a composition comprising an insulin (e.g., Lantus) and a GLP-1 agonist AVE0010). The addition of methionine engenders chemical integrity of this composition.

[0250] 8. Conclusions

[0251] The data described herein lead to the following conclusions: [0252] Methionine leads to an increased chemical stability and has no negative effects on the physical stability of formulations of a combination of a GLP-1 agonist, more particularly AVE0010, and an insulin, more particularly Lantus. Therefore, methionine is advantageous as a constituent of these compositions. [0253] Acetate can lead to physical instability. This instability is greater with increasing acetate concentration. Therefore, formulations of a combination of a GLP-1 agonist, more particularly AVE0010, and an insulin, more particularly Lantus, which are free of acetate are advantageous compared with corresponding compositions which comprise acetate. [0254] Polysorbate 20 has no negative influence on the physical and the chemical stability of formulations of a combination of a GLP-1 agonist, more particularly AVE0010, and an insulin, more particularly Lantus. By combining acetate at lower concentrations (½ acetate) with polysorbate 20, the negative effects of acetate can be partially compensated. In acetate-free compositions, the addition of polysorbate 20 does not lead to any advantages. Therefore, formulations of a combination of a GLP-1 agonist, more particularly AVE0010, and an insulin, more particularly Lantus, should be prepared which are free of polysorbate 20. [0255] Lysine (at normal and higher concentrations), benzalkonium chloride, polysorbate 80, and poloxamer 188 already showed chemical instability at the beginning of the studies (t0). For lysine, this is also true for the results of the THT test.

EXAMPLE 2

[0256] The “3 pens cover all” concept (FIG. 4) [0257] 3 premix pens having 3 different predetermined proportions: [0258] (a) Mix A: 100 U Lantus+66.66 μg AVE0010 per mL [0259] (b) Mix B: 100 U Lantus+40 μg AVE0010 per mL [0260] (c) Mix C: 100 U Lantus+25 μg AVE0010 per mL [0261] Use of the 3 premix pens: The exemplary table in FIG. 4 proceeds from a therapeutic range of 15 to 80 U per dose of Lantus and 10 to 20 μg AVE0010. For a particular patient, a dose of Lantus to be administered is set or predetermined. The predetermined dose is looked up in the left-hand column. When a corresponding AVE0010 dose in the range from 10 to 20 μg is mentioned in the columns MIX A-MIX C, the corresponding MIX is selected, metered, and administered. The ranges are overlapping: for example, when 26 to 30 U Lantus is required, Mix A or MIX B (having a higher dose of AVE0010) could be selected. Accordingly, this is true for MIX B and C. If, for example, a dose of 50 U of insulin is determined, then 0.5 ml of MIX B or MIX C is to be metered. This dose contains 20 μg (MIX B) or 12.5 μg (MIX C) of AVE0010. [0262] Conclusion: Assuming that a probable AVE0010 effect in the range from 10 to 15 pg and a therapeutic effect in the range from 15 to 22 μg is achieved, almost all patients who take Lantus doses of 15-80 U can likewise receive therapeutic doses of AVE0010 in the range from 10 to 20 μg when they use one of the three premix pens, which contain three different Lantus:AVE0010 ratios (Mix A, B, or C). Due to the broad range of possible ratios of Lantus to AVE0010, the ratios in the pens can be fine-tuned such that a desired dose of AVE0010 is included for every dose of Lantus in at least one pen.