PHARMACEUTICAL COMPOSITION

Abstract

The present invention relates to pharmaceutical compositions having improved stability.

Claims

1-37. (canceled)

38. A stable aqueous pharmaceutical composition comprising a pharmaceutically active compound of formula (II): ##STR00006## or a pharmaceutically acceptable salt thereof; wherein the composition comprises: 2 mg/mL of the compound of formula (II) or a pharmaceutically acceptable salt thereof; a citrate-phosphate buffer; and an isotonicity agent; wherein the pH of the composition is from 5.4 to 5.65; and wherein, after storage at 40° C. for 176 days, greater than 95% of the compound of formula (II) or pharmaceutically acceptable salt thereof remains in the composition.

39. The composition of claim 38, wherein the isotonicity agent is NaCl.

40. The composition of claim 38, wherein the pH of the composition is 5.5.

41. A kit, comprising: the stable aqueous pharmaceutical composition of claim 38; and a container for the composition.

42. A method of treating one or more conditions selected from compromised lactation conditions, labor induction impairment, uterine atony conditions, excessive bleeding, inflammation, pain, abdominal pain, back pain, male and female sexual dysfunction, irritable bowel syndrome (IBS), constipation, gastrointestinal obstruction, autism, stress, anxiety, depression, anxiety disorder, surgical blood loss, post-partum haemorrhage, wound healing, infection, mastitis, placenta delivery impairment, osteoporosis, comprising administering a composition according to claim 38 to a patient in need thereof.

43. A method for diagnosis of one or more conditions selected from cancer and placental insufficiency, comprising administering a composition according to claim 38 to a patient in need thereof.

44. of treating one or more conditions selected from uterine atony and excessive bleeding following vaginal delivery comprising administering a composition according to claim 38 to a patient in need thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will now be illustrated with reference to the attached drawings in which:

[0052] FIG. 1 shows a UPLC chromatogram of an impurity mix of carbetocin and degradation products;

[0053] FIG. 1a shows the chemical formulae of carbetocin and degradation products;

[0054] FIG. 2 shows the content of degradation (hydrolysis) product [Gly.sup.9OH]carbetocin (see FIG. 1a) in the antioxidant study samples as a function of time (constant pH);

[0055] FIG. 3 shows the content of degradation (oxidation) product sulfoxide II carbetocin (see FIG. 1a) in the antioxidant study samples as a function of time (constant pH);

[0056] FIG. 4 shows individual degradation products at different pH (pH study, constant antioxidant);

[0057] FIG. 5 shows the sum of degradation products at different pH (pH study, constant antioxidant),

[0058] FIG. 6 shows the Stability of FE 202767 in selected buffers; and

[0059] FIG. 7 shows the sum of impurities of various carbetocin formulations after 12 months at 40° C. and 75% relative humidity (R.H.), as described in Experiment 3A.

ANALYTICAL METHOD

[0060] This is the analytical method for the carbetocin examples (Examples 1 to 6), below.

[0061] All solutions were analysed on a Waters Acquity UPLC (Ultra-high Pressure Liquid chromatography) system using isocratic conditions. The Mobile phase was 20% acetonitrile (JT Baker, Ultra Gradient Grade) in 5 mM unbuffered ammonium acetate (Fluka, Ultra≥99.0%). The column was a Waters Acquity UPLC BEH Shield RP18, 2.1*100 mm, 1.7 μm (Flow: 0.5 ml/min, Column Temp: 50° C.). The injection volume was 20 μl. Detection was performed by UV at 220 nm. The different impurities were evaluated as area % of total area.

[0062] FIG. 1 shows a chromatogram of an impurity mix of carbetocin and its degradation products. The solution contained, carbetocin, the hydrolysis products [Gly.sup.9OH], [Asp.sup.5] and [Glu.sup.4]carbetocin, the oxidation products sulfoxide I and sulfoxide II-carbetocin, the alkaline degradation products [βAsp.sup.5] and [D-Asn.sup.5]carbetocin and the synthesis related impurity [D-Cys.sup.6]carbetocin. The chemical formulae of carbetocin and the degradation products (hydrolysis products, oxidation products and alkaline degradation products) are shown in FIG. 1a. The “[Glu.sup.4]carbetocin” type nomenclature is well known in the art. The resolutions between all peaks were ≥2.0.

Example 1: Formulation Antioxidant Study (Constant pH)

[0063] 5.0 grams of D(−)-Mannitol (Ph Eur, Prolabo) was dissolved in 1000 ml of milliQ-water. This solution was adjusted with acetic acid (Ph.Eur., Merck) to pH 5.2. This solution was then divided into four 200 ml aliquots. To aliquot 1, 0.2 gram of EDTA disodium, dihydrate (Fluke) was added and dissolved. To aliquot 2, 1.0 gram of L-methionine (Sigma, non-animal source) was added and dissolved. To aliquot 3, 0.2 gram of EDTA disodium, dihydrate and 1.0 g of L-methionine was added and dissolved. Nothing was added to aliquot 4. The pH of aliquots 1-3 was adjusted with acetic acid to pH 5.2±0.1. 1 mg of carbetocin (Polypeptide Laboratories) was transferred to four 10 ml volumetric flasks. Aliquots 1-4 were used to dissolve the substance and for dilution to volume (0.1 mg/ml carbetocin). The solutions were transferred to 25 ml blue cap flasks, and placed in a cabinet at 40° C. and 75% RH. A sample of the current PABAL® formulation, pH 3.9 (measured), was placed in the same cabinet for comparison.

[0064] The solutions were analysed after 2, 6, 12, 22 and 33 weeks at 40° C. The largest impurities of this study was found to be the hydrolysis product [Gly.sup.9OH]carbetocin and the oxidation product [sulfoxide II]carbetocin. The pH in this study (pH 5.2) was not high enough to start any alkaline degradation of carbetocin. The content % (w/w) of the major impurity formed by hydrolysis, [Gly.sup.9OH]carbetocin, and by oxidation, sulfoxide II-carbetocin, are shown in FIGS. 2 and 3. The product specification allowed for each impurity is also plotted in each figure as a reference. Thus it can be seen from FIG. 2 that if the concentration of [Gly.sup.9OH] carbetocin increases above 1.5% the sample is “out of specification”, that is the sample has degraded such that is no longer suitable for administration.

[0065] As shown in FIG. 2, the antioxidant study (constant pH) showed that the formation of hydrolysis products, mainly [Gly.sup.9OH]carbetocin, was very fast in the current PABAL formulation (pH 3.9). With regard to the content of [Gly.sup.9OH]carbetocin the current formulation was quickly out-of-specification (>1.5%), after 6 weeks at 40° C. All formulations at pH 5.2 were well below the specification limit after 33 weeks at 40° C. (0.4-0.6%), indicating that these formulations are stable for at least 6 months at 40° C. and 75% RH, which is generally accepted to indicate a likely stability for at least 24 months at 25° C. and 60% RH (i.e. a stable RT formulation) The results applied for all three hydrolysis products. In FIG. 3, the addition of antioxidant was shown to be very effective in slowing down the oxidation of carbetocin, despite the increased pH of the formulations. The formulation at pH 5.2 that did not contain any additives was out-of-specification (>0.8%) with regard to the content of sulfoxide II-carbetocin after approx 20 weeks at 40° C. The formulations containing methionine or EDTA were all well below the specification limit after 33 weeks (0.2-0.4%). The formulation containing a combination of EDTA and methionine did not show any increase of oxidation products at all, compared to the levels found in the substance batch. Due to the low pH, the current formulation was not prone to degradation by oxidation (pH 3.9). The results are shown in numerical form in the following table (Table 1).

TABLE-US-00001 TABLE 1 Individual and sum of degradation products (%) after 33 weeks at 40° C. (constant pH). Sulfoxide Sulfoxide Sum of Formulation Gly.sup.9OH Asp.sup.5 Glu.sup.4 I II βAsp5 D-Asn.sup.5 impurities Current formulation 6.43 1.15 5.41 0.42 0.38 0.13 0.15 16.4 Mannitol pH 5.2 0.53 0.14 0.42 0.51 0.93 0.13 0.12 3.5 Mannitol pH 0.63 0.20 0.51 0.14 0.25 0.15 0.16 3.2 5.2 + methionine Mannitol pH 0.52 0.15 0.38 0.31 0.39 0.15 0.10 2.9 5.2 + EDTA Mannitol pH 0.44 0.13 0.35 0.10 0.16 0.19 0.17 2.4 5.2 + methionine + EDTA

[0066] Table 1 includes the sum of degradation products for all samples, and after 33 weeks the effect of EDTA is clearer. Further, the sample containing both methionine and EDTA is clearly better than the others. The evidence points to a linear degradation: assuming this is indeed the case, the mannitol pH 5.2+methionine+EDTA sample is likely to be in specification—i.e., suitable for use—for a remarkable 86 weeks at 40° C. This is based, as is well known in the art, on a linear extrapolation of the increase in impurity over time to determine when the amount of impurity would be sufficiently high for the formulation to be “out of specification”.

Example 2: Formulation pH Study (Constant Antioxidant)

[0067] 1.2 grams of succinic acid (Sigma-Aldrich, 99%) and 1.0 g of L-methionine (Sigma, non-animal source) were dissolved in 1000 ml of milliQ water (10 mM). This solution was adjusted, in aliquots, with diluted NaOH (Ph.Eur., Merck) to pH 4.0, 4.5, 5.2, 5.65, 6.1, 6.5 and 7.0. 55 mg of carbetocin (Polypeptide Laboratories) was dissolved in 50 ml of milliQ water (1.1 mg/ml). 1.0 ml of the carbetocin solution (1.1 mg/ml) was mixed with 10 ml of each buffer (0.1 mg/ml carbetocin). 0.55 g of mannitol (5%) was added to each solution and dissolved. The solutions were transferred to 15 ml glass vials with screw lid and placed in a 40° C. cabinet at 75% RH.

[0068] The solutions were analysed after 12 and 52 weeks at 40° C. The content of the individual degradation products and the sum of degradation products is presented in Tables 2a and 2b and FIGS. 4 and 5.

TABLE-US-00002 TABLE 2a Individual and sum of degradation products (%) at different pH after 12 weeks at 40° C. (pH study, constant antioxidant). Oxidation products Alkaline impurities Sample Hydrolysis products Sulfoxide Sulfoxide Unknown pH [Gly.sup.9OH] [Asp.sup.5] [Glu.sup.4] I II [D-Asn.sup.5] [β Asp.sup.5] 2.0 min Sum 4.0 2.22 0.50 1.78 N.D N.D N.D. N.D. N.D. 4.50 4.5 0.84 0.19 0.67 N.D N.D N.D. N.D. N.D. 1.70 5.2 0.23 0.10 0.27 N.D N.D 0.06 0.05 0.09 0.80 5.65 0.15 0.04 0.12 N.D 0.03 0.12 0.08 0.16 0.70 6.1 0.14 0.09 0.13 0.03 0.03 0.30 0.22 0.32 1.26 6.5 0.25 0.13 0.12 0.03 0.03 0.54 0.30 0.48 1.88 7.0 0.52 0.20 0.17 0.02 0.02 1.27 0.47 0.62 3.29

TABLE-US-00003 TABLE 2b Individual and sum of degradation products (%) at different pH after 52 weeks at 40° C. (pH study, constant antioxidant). Oxidation products Alkaline impurities Sample Hydrolysis products Sulfoxide Sulfoxide Unknown pH [Gly.sup.9OH] [Asp.sup.5] [Glu.sup.4] I II [D-Asn.sup.5] [β Asp.sup.5] 2.0 min Sum 4.0 7.55 1.09 6.23 0.04 0.07 0.00 0.19 0.94 19.3 4.5 2.92 0.57 2.49 0.04 0.05 0.00 0.19 0.40 7.8 5.2 0.78 0.26 0.72 0.05 0.14 0.26 0.25 0.22 3.6 5.65 0.48 0.22 0.52 0.02 0.02 0.45 0.40 0.40 3.8 6.1 0.56 0.35 0.34 0.08 0.11 1.06 0.73 0.77 5.9 6.5 0.88 0.49 0.32 0.03 0.06 2.30 1.25 1.31 8.8 7.0 1.53 0.71 0.43 0.05 0.04 4.10 1.68 1.65 12.9

[0069] As discussed below with reference to Example 3a, the specification limit for sum of impurities (for the current PABAL® formulation) is ≤5%. As can be seen from Table 2b (“Sum”), the Samples at pH 5.2 and 5.65 (examples of the invention) are still within specification after 52 weeks (1 year) at 40° C., while all other samples are out of specification after 52 weeks (1 year) at 40° C.

[0070] The results of the pH study (FIGS. 4, 5) confirmed the observations of the antioxidant study. The formation of hydrolysis products ([Gly.sup.9OH], [Asp.sup.5] and [Glu.sup.4]carbetocin) was effectively reduced by an increase in pH from pH 4.0 to about pH 5.65. At higher pH values (pH 6.1-7.0) the content of hydrolysis products was again increased. The effectiveness of the antioxidant at about 1 mg/ml concentration was also confirmed (in the antioxidant study the concentration of antioxidant was 5 mg/ml). However, if oxidation is limited in the drug or drug solution (e.g. if the drug or drug solution is not prone to oxidation), the amount of antioxidant may be reduced, or use of antioxidant may not be necessary. Due to the antioxidant, the oxidation of carbetocin was negligible, regardless of pH. The upper pH limit of an optimised formulation was instead limited by the alkaline degradation of carbetocin. The main impurity by alkaline degradation was [D-Asn.sup.5]carbetocin, which was rapidly increased at pH values above pH 6.1. The formation of two other minor impurities at high pH was also observed, [βAsp.sup.6]carbetocin and one unknown impurity eluting early in the chromatogram (tR: 2.0 min).

[0071] The U-shape of the pH vs. sum of degradation products curve illustrates the stability plateau of carbetocin at pH 5.0-6.0. At pH 5.2 the sum of degradation products was found to be only 16% of the sum of degradation products at pH 4.0 (current formulation). The optimal pH was found to be somewhere between pH 5.1 to 6, for example between around pH 5.2 and 5.65.

[0072] Examples 1 and 2 give a very strong indication that formulations of the invention are room temperature stable for up to two years.

Example 3: Formulation Study of Isotonicity Agents, NaCl Vs. Mannitol, at 30° C., 40° C.

[0073] 4.22 grams of citric acid monohydrate (Merck, pro analysi) was dissolved in 2000 ml of milliQ water (20 mM). This solution was divided into ten 200 mi aliquots. 1.8 g of sodium chloride (Merck, pro analysi) was added to five of the flasks, to the other five flasks 10 g of mannitol (VWR, Ph Eur) was added. According to an experimental design, 0.2, 0.6 or 1.0 g of L-methionine (Sigma, non-animal source) was added and the pH was adjusted with 1% NaOH (Merck, pro analysi) to pH 5.2, 5.65 or 6.1, see Table 3a and 3b. 2 mg of Carbetocin (Polypeptide Laboratories) was transferred to twelve 20 ml volumetric flasks and the substance was dissolved in each buffer (0.1 mg/ml carbetocin). The samples containing 3 mg/ml methionine were prepared in duplicate, see Table 3a and 3b.

[0074] Two mL of each solution was transferred to LC-vials and placed in a 30° C./75% RH cabinet. The remaining solutions were transferred to 25 ml blue cap flasks and placed in a 40° C./75% R.H. cabinet. The level of impurities after 25 weeks in 30′C/75% R.H are shown in the following Tables 3a and 3b.

TABLE-US-00004 TABLE 3a Sulfoxide Sulfoxide Unknown Formulation Gly.sup.9OH Asp.sup.5 Glu.sup.4 I II 2 min βAsp.sup.5 D-Asn.sup.5 D-Cys.sup.6 Sum ** Mannitol, pH 0.18 0.04 0.18 0.04 0.03 N.D. N.D. N.D. 0.09 0.80 5.2, 1 mg/mL methionine Mannitol, pH 0.07 0.03 0.07 0.03 N.D. 0.17 0.15 0.01 0.12 0.95 6.1, 1 mg/mL methionine Mannitol, pH 0.10 0.03 0.08 0.04 0.05 0.09 0.07 0.05 0.12 0.78 5.65, 3 mg/mL methionine, sample 1 Mannitol, pH 0.09 0.02 0.11 0.03 N.D. 0.08 0.04 N.D. 0.11 0.63 5.65, 3 mg/mL methionine, sample 2 Mannitol, pH 0.17 0.03 0.20 0.02 N.D. N.D. 0.04 N.D. 0.11 0.75 5.2, 5 mg/mL methionine Mannitol, pH 0.10 0.05 0.05 0.04 N.D. 0.23 0.18 0.11 0.12 1.11 6.1, 5 mg/mL methionine Years to OOS* 8 12 7 “infinity” “infinity” 6 12 “infinity” N/A 4.2 for 3 mg/mL, pH 5.65 sample 2. *Out of specification ** of degradation products

TABLE-US-00005 TABLE 3b (NaCl) Sulfoxide Sulfoxide Unknown Beta Formulation Gly.sup.9OH Asp.sup.5 Glu.sup.4 I II 2 min Asp.sup.5 D-Asn.sup.5 D-Cys.sup.8 Sum ** NaCl, pH 5.2, 0.21 0.06 0.15 0.02 0.03 ND 0.02 0.12 0.82 1 mg/mL methionine NaCl, pH 6.1, 0.08 0.05 0.04 0.04 0.04 0.2  0.12 0.12 0.11 0.99 1 mg/mL methionine NaCl, pH 5.65, 0.09 0.03 0.07 0.01 ND 0.12 0.04 0.05 0.11 0.74 3 mg/mL methionine sample 1 NaCl, pH 5.65, 0.09 0.04 0.08 0.02 0.03 0.10 0.05 0.14 0.11 0.78 3 mg/mL methionine sample 2 NaCl, pH 5.2, 0.17 0.04 0.15 0.02 0.04 ND 0.05 0.14 0.86 5 mg/mL methionine NaCl, pH 6.1, 0.09 0.04 0.06 0.03 ND 0.23 0.17 0.14 0.11 1.11 5 mg/mL methionine ** of degradation products

[0075] Tables 3a and Tables 3b show that there is very little degradation in all samples. This level of degradation corresponds to that seen after 6 weeks at 40° C.

[0076] The results indicate that the best samples are likely to be stable for 5 years at 30° C. As seen in Table 3a, rows 5 and 8, results for the methionine 3 mg/mL, pH 5.65 sample 2 indicate that this sample would remain in specification for more than 4 years at 30° C. and 75% RH. This is based, as is well known in the art, on a linear extrapolation of the increase in impurity over time to determine when the amount of impurity would be sufficiently high for the formulation to be “out of specification” (OOS). It was also found that the optimum pH at 30° C. is higher than at 40° C. (results not shown). The differences are small but pH 5.65 is slightly superior to pH 5.2 at 30° C. (vice versa at 40° C.). These results indicate there is a good margin for obtaining a climate zone III/IV stable formulation.

[0077] The applicants found that increase in methionine leads to more degradation, mainly by increase of [BetaAsp5]carbetocin. A concentration of about 1 mg/ml appears to be sufficient to provide effective stabilisation without significant degradation.

[0078] Experiment 3a—the Stability of Carbetocin at Different pH and Using Different Antioxidants

[0079] This study was designed to give a broader picture of the stability of carbetocin at different pH and using different antioxidants.

[0080] 1.2 grams of succinic acid (Sigma-Aldrich, ≥99%) was dissolved in 1000 ml of milliQ water (10 mM). This solution was adjusted, in aliquots, with diluted NaOH (Ph.Eur., Merck) to pH 4.0, 4.5, 5.2, 5.65, 6.1, 6.5 and 7.0. 55 mg of carbetocin (Polypeptide Laboratories, Strasbourg) was dissolved in 50 ml of milliQ water (1.1 mg/ml). 1.0 ml of the carbetocin solution (1.1 mg/ml) was mixed with 10 ml of each buffer (0.1 mg/ml carbetocin). 0.55 g of mannitol (5%) was added to each solution and dissolved. The solutions were transferred to 15 ml glass vials with screw lid and placed in the 40° C./75% R.H. cabinet.

[0081] The same procedure was repeated; with the exception that 1.0 g of L-methionine (Sigma, non-animal source) was added to the 1000 ml of milliQ water, giving duplicate samples containing 1 mg/ml methionine at all pH-levels. The solutions were transferred to 15 ml glass vials with screw lid and placed in the 40° C./75% R.H. cabinet.

[0082] The buffers at pH 5.65, 6.1 and 6.5 were also divided into aliquots to which EDTA disodium, dihydrate (Fluka) was added. These samples were stored at 40° C./75% for 12 months before analysis.

[0083] The sum of impurities after 12 months at 40° C./75% R.H are shown in FIG. 7. The Figure also shows the “spec limit”, above which the sum of impurities is such that the formulation is out of specification.

[0084] All formulations at pH 5.2 and pH 5.65 were within specification after 12 months at 40° C./75% R.H.

[0085] The positive effect of methionine was visible also in this study. All samples containing methionine showed very low amounts of oxidation products, regardless of composition and pH. This points to inclusion of methionine in a robust formulation, where (for example) metal ion content of the active ingredient carbetocin, which can vary with production batch and which, if high, may lead to increased oxidation, will not be a controlled parameter.

[0086] The most stable formulation was the formulation at pH 5.2 containing 1 mg/ml of methionine (results not shown). The parameter that was closest to the specification limit after 12 months at 40° C./75% R.H. was the sum of impurities (FIG. 7). The specification limit for sum of impurities (for the current PABAL® formulation) is ≤5% (i.e. 5.5%). It can be assumed that the degradation is linear over time, and it can therefore be calculated that the formulation at pH 5.2 containing 1 mg/ml of methionine would be out-of-specification after approximately 80 weeks at 40° C./75% R.H, based on the specification for the current PABAL® formulation.

[0087] A commonly used guide, supported by the Arrhenius equation, is that the rate of most chemical reactions doubles for every 10° C. increase of temperature. If we apply this relationship to the formulation at pH 5.2 containing 1 mg/ml of methionine, the estimated shelf-life of a new formulation will be 160 weeks at 30° C., i.e. slightly more than 3 years, again based on the specification for the current PABAL® formulation. This is likely to be an underestimation, since the reported “sum of impurities” in this experiment included every peak on the baseline, including synthesis related impurities and peaks below the reporting limit (<0.05%). The synthesis related impurities consist mainly of [DCys.sup.6] and [desGln.sup.4]carbetocin, which do not increase during storage. The substance batch contained 0.9% impurities according to the supplier. Thus, it is likely that a shelf-life of more than 3 years at 30° C./75% R.H. would be achieved for this formulation.

Example 4—Formulation in Succinate Buffer

[0088] The following preparation and decanting was performed in a pharmaceutical room under germ free conditions. 47 grams of mannitol, 1.2 grams of succinic acid buffering agent and 1.0 g of L-methionine was dissolved in about 900 ml of milliQ water (10 mM). The pH of the solution was adjusted with 5M NaOH to pH 5.4. The solution was transferred to a 1000 ml volumetric flask and diluted to volume with WFI.

[0089] 50 mg of carbetocin (Polypeptide Laboratories) was transferred to a 500 ml volumetric flask and dissolved and diluted to volume with the mannitol/succinic acid/methionine buffer pH 5.4. The solution was filtered through a 0.22 μm filter and filled in glass vials with rubber stoppers (1.1 ml per vial). Each vial included an aqueous composition comprising carbetocin (0.1 mg/mL), and the pH of the composition was 5.4 (i.e. from 5.0 to 6.0). The aqueous composition also included succinate buffer (succinic acid buffering agent), methionine (anti-oxidant) and mannitol (isotonic agent). In a further Example (Example 4A, not shown) a solution was made up exactly as Example 4 and EDTA (0.1% w/v) added. The osmolality of the solutions in Example 4 and 4A was found to be 300±20 mOsmol/kg.

[0090] The formulation of Example 4 (and that of Example 4A) is suitable for injection to a patient with uterine atony.

Example 5—Formulation in Succinate Buffer

[0091] The following preparation and decanting was performed in a pharmaceutical room under germ free conditions. 1.2 grams of succinic acid buffering agent (Sigma-Aldrich, 99%) and 1.0 g of L-methionine (Sigma, non-animal source) were dissolved in 1000 ml of milliQ water (10 mM) to provide a succinate buffer of pH 5.4, the pH being adjusted to this value with NaOH solution.

[0092] 0.55 g of mannitol (5%) was dissolved in 10 ml of succinate buffer. Methionine 0.5% (w/v) was added to the solution and dissolved. Carbetocin (Polypeptide Laboratories) was dissolved in the solution so the concentration of carbetocin was 0.1 mg/mL, and the pH adjusted to 5.4 using NaOH solution. The solution was divided into 1 mL quantities and sealed in ampoules. Each ampoule included an aqueous composition comprising carbetocin (0.1 mg/mL), and the pH of the composition was 5.4 (i.e. from 5.0 to 6.0). The aqueous composition also included succinate buffer (succinic acid buffering agent), methionine (anti-oxidant) and mannitol (isotonic agent). It will be appreciated that the composition may be made with water for injection (WFI). The formulation of Example 5 is suitable for injection to a patient with uterine atony.

Example 6—Formulation with Citrate/Phosphate Buffer

[0093] The formulation set out in the following table was made up by similar methods to those set out in Examples 4 and 5 above.

TABLE-US-00006 TABLE 4 Component Amount per mL Function Carbetocin 10 mg Active ingredient Sodium phosphate dibasic 3.24 mg Buffering agent dihdrate Citric acid monohydrate 1.43 mg Buffering agent NaCl 7.5 mg Isotonicity agent HCl q.s. adjust to pH 5.5 pH adjustment NaOH q.s. adjust to pH 5.5 pH adjustment Water for Injection Adjust to 1 mL Solvent
The composition is suitable for nasal administration.
Optionally, an antioxidant (e.g. methionine at a concentration of 1.0 mg/mL may be included in the formulation). The anti-oxidant may be any anti-oxidant commonly used in the art.
Optionally, the composition may include an enhancer. The enhancer may be any enhancer commonly used in the art, for example any enhancer approved for use as a pharmaceutical excipient. The enhancer may be, for example, methyl-β-cyclodextrin, Polysorbate 80, carboxymethylcellulose or hydroxypropylmethylcellulose.

Example 7—the Stability of FE 202767 in Citrate and Citrate-Phosphate Buffers (pH

5.0, 5.5, and 6.0) at 40° C. for a Six Month Period.

Materials and Methods

[0094] FE 202767 (Ferring) was synthesised by the method set out in WO2009/122285. FE 202767 was dissolved at a concentration of 0.2 mg/ml in either 25 mM citrate buffer (isotonic to saline) or 25 mM citrate-phosphate buffer (isotonic with saline) at varying pH (pH 5.0, 5.5, 6.0), by methods known in the art. The solutions were incubated at 40° C. for 176 days, with samples taken at day 0, 15, 30, 84, and 176.
Samples were evaluated by HPLC to determine the amount of intact peptide remaining at the various time points, by methods well known in the art, comparing the % Area. of the intact peptide peak on the sampling day vs. % Area on Day 0.
The HPLC method used an Agilent 1200 instrument. The mobile phases where HPLC Buffers A (A=0.01% TFA in water) and B (B=0.01% TFA in [70% v/v acetonitrile and 30% v/v water]) with the gradient 15% B for 1 min, then 15 to 95% B in 30 min, then 95 to 100% B in 3 min, then 100% B for 5 min and 100% B to 15% B in 1 min at flow rate 0.3 mL/min. The Phenomenex MAX-RP C18, 2.0×150 mm, 4 μm, 80 Å column was at temperature 40 with UV detection at 210 nm. The injection volume was 10 μL.
The results are shown in the following Table 5, and on the attached FIG. 6. In Table 5 and FIG. 6, CP50 is citrate phosphate buffer at pH 5.0; CP55 is citrate phosphate buffer at pH 5.5; and CP60 is citrate phosphate buffer at pH 6.0; CT50 is citrate phosphate buffer at pH 5.0; CT55 is citrate phosphate buffer at pH 5.5; and CT60 is citrate phosphate buffer at pH 6.0.

TABLE-US-00007 TABLE 5 % Intact Peptide Remaining (normalised to day 0) Day CP50 CP55 CP60 CT50 CT55 CT60 0 100.00 100.00 100.00 100.00 100.00  100.00 6 99.97 99.83 99.65 99.66 99.75 100.00 15 99.66 99.31 99.54 99.51 99.71 99.40 30 99.47 99.30 99.26 99.29 n.a. 99.30 84 98.38 97.44 97.78 98.05 98.61 97.94 176 96.98 95.60 95.48 95.19 97.34 73.36 Notes: % Intact Peptide Remaining expressed relative to % Area on Day 0. n.a. = data point excluded due to aberrant peak in HPLC chromatogram. CP = citrate-phosphate buffer; CT = citrate buffer.

Conclusion

[0095] FE 202767 showed good stability in citrate-phosphate buffers in the pH range tested (pH 5.0, 5.5, and 6.0), with >95% remaining after 176 days in each condition. It was also very stable (>95% remaining) in citrate buffer at pH 5.0 and 5.5; however, there was significant degradation after 176 days in pH 6.0 citrate buffer.
In general, a formulation suitable for nasal administration is expected to be of pH between 5.0 and 6.0, include the minimum number of reagents (e.g. no anti-oxidant). It is also preferred that the formulation is room temperature stable. Example 7 demonstrates that formulations along the lines above may be suitable for nasal administration, because they have appropriate pH and are room temperature stable without requirement for anti-oxidant or other additives that might adversely affect the nasal mucosa.

Example 8—the Stability of FE 202767 in Various Buffers at 40° C. for One and Three Months

Materials and Methods

[0096] The method was similar to Example 7. FE 202767 (Ferring) was synthesised by the method set out in WO2009/122285. The FE 202767 was dissolved at a concentration of 0.2 mg/ml in either 25 mM citrate buffer (citric acid/Na citrate), 10 mM acetate buffer (acetic acetate/Na acetate) or 10 mM succinate buffer (1 mM succinic acid+NaOH to relevant pH) at varying pH (pH 5.0, 5.2, 5.5, 5.65, 5.8, 6.0), by methods known in the art. As set out in the table below, the various samples also included isotonicity agent (NaCl, 7 mg/mL or mannitol 47 mg/mL) to achieve isotonicity. Some of the samples included oxidant (methionine 1 mg/mL, EDTA 1 mg/mL, or combination of EDTA 1 mg/mL and methionine 1 mg/mL). Each formulation (see Table below) was filled in a 10R glass vial sealed with a rubber stopper and an aluminium cap.
The solutions were incubated at 40° C. at 75% RH, with samples taken at day 30 (1 month), and day 90 (3 months).
Samples were evaluated by HPLC to determine the amount of intact peptide remaining at the various time points, by methods well known in the art, comparing the % Area of the intact peptide peak on the sampling day vs. % Area on Day 0.
The HPLC method used an Agilent 1100 instrument. The mobile phases where HPLC Buffers A (A=0.1% TFA in water) and B (B=0.1% TFA in acetonitrile) with the gradient 20 to 30% B in 40 min, then 30 to 60% B in 15 min, then 60 to 20% B in 1 min and then 20% B for 10 min at flow rate 0.5 mL/min. The Zorbax 300SB C18, 3.0×150 mm, 3.5 μm, 300 Å column was at temperature 25 with UV detection at 214 nm. The injection volume was 15 μL
The results are shown in the following Table.

TABLE-US-00008 TABLE 6 Initial Peptide Peptide peptide conc. conc. Sample Isotonicity conc. (mg/mL) (mg/mL) Number Buffer pH agent Antioxidant (mg/mL) at 30 days at 90 days 1 Citrate 6 NaCl No 0.186 0.187 0.182 2 Citrate 5.65 NaCl No 0.187 0.187 0.182 3 Citrate 5.8 NaCl No 0.187 0.187 0.182 4 Citrate 5 NaCl Methionine 0.187 0.183 0.162 5 Citrate 5.5 NaCl Methionine 0.187 0.187 0.171 6 Citrate 6 NaCl Methionine 0.186 0.187 0.181 7 Citrate 6 NaCl EDTA 0.187 0.188 0.183 8 Citrate 6 NaCl Methionine 0.187 0.187 0.182 and EDTA 8 placebo Citrate 6 NaCl Methionine 0.000 0.000 0.000 and EDTA 9 Citrate 5 Mannitol No 0.187 0.186 0.173 10 Succinate 6 Mannitol No 0.188 0.186 0.180 11 Succinate 5 NaCl No 0.186 0.187 0.183 12 Succinate 5.2 NaCl No 0.187 0.188 0.184 13 Succinate 5.65 NaCl No 0.187 0.188 0.183 14 Succinate 6 NaCl No 0.187 0.187 0.182 15 Succinate 5 NaCl Methionine 0.187 0.186 0.182 16 Succinate 5.2 NaCl Methionine 0.187 0.185 0.182 17 Succinate 5.65 NaCl Methionine 0.187 0.186 0.180 18 Succinate 6 NaCl Methionine 0.187 0.188 0.181 19 Succinate 5 Mannitol No 0.188 0.186 0.176 20 Succinate 6 Mannitol No 0.187 0.099 0.124 21 Succinate 5 Mannitol Methionine 0.187 0.180 0.012 21 placebo Succinate 5 Mannitol Methionine 0.000 0.000 0.000 22 Succinate 6 Mannitol Methionine 0.188 0.023 0.174 23 Acetate 5.2 NaCl No 0.186 0.186 0.183 24 Acetate 5.65 NaCl No 0.187 0.187 0.184 24 placebo Acetate 5.65 NaCl No 0.000 0.000 0.000

Conclusion

[0097] FE 202767 showed good stability in citrate and acetate buffers in the pH range tested after 30 days in each condition. It was also very stable in succinate buffer at pH 5.0 to 5.65; however, there was significant degradation after 30 days in some pH 6.0 succinate samples (sample 20, 22). The presence or absence of antioxidant seemed unimportant on a 30 day timescale.
FE 202767 also showed good stability in citrate and acetate buffers in the pH range tested after 90 days in each condition, with the best results being shown at the upper end of the pH range (e.g. between pH 5.5 and 6, see samples 1 to 6). It was also stable in succinate buffer at pH 5.0 to 5.65 after 90 days. The 30 and 90 day results for samples 21 and 22 suggest a mix up in analysis.
Again, the presence or absence of antioxidant seemed unimportant on a 90 day timescale.
The results indicate that NaCl is a better isotonicity agent than mannitol.
As indicated above, a formulation suitable for nasal administration is expected to be of pH between 5.0 and 6.0, include the minimum number of reagents (e.g. no anti-oxidant). It is also preferred that the formulation is room temperature stable. Example 8 demonstrates that formulations along the lines above may be suitable for nasal administration, because they have appropriate pH and are room temperature stable without requirement for anti-oxidant or other additives that might adversely affect the nasal mucosa.

Example 9—Formulation of FE 202767 with Citrate/Phosphate Buffer

[0098] FE 202767 (Ferring) was synthesised by the method set out in WO2009/122285. The formulation set out in the following table was made up by similar methods to those set out in Examples 4 and 5 above.

TABLE-US-00009 TABLE 7 Component Amount per mL Function carba-1-[4-FBzlGly7]dOT 0.7 mg Active ingredient (FE 202767) Sodium phosphate dibasic 3.24 mg Buffering agent dihdrate Citric acid monohydrate 1.43 mg Buffering agent NaCl 7.5 mg Isotonicity agent HCl q.s. adjust to pH 5.5 pH adjustment NaOH q.s. adjust to pH 5.5 pH adjustment Water for Injection Adjust to 1 mL Solvent
The composition is suitable for nasal administration.

[0099] Optionally, an antioxidant (e.g. methionine at a concentration of 1.0 mg/mL may be included in the formulation).