Methods for producing pharmaceutical compositions

Abstract

The present invention relates to crystalline forms of carbetocin, methods for their preparation, and pharmaceutical compositions thereof.

Description

[Technical Field]

[0001] The present invention relates to crystalline forms of carbetocin, methods for their preparation, and pharmaceutical compositions thereof. [Background technology]

[0002] Carbetocin (also known as 1-desamino-1-monocarba-2-(O-methyl)-tyrosine)oxytocin or 1-butanoic acid-2-(O-methyl-L-tyrosine)-1-carbaoxytocin) is a long-acting synthetic oligopeptide analog of oxytocin with agonist activity. Carbetocin incorporates the following substitutions relative to oxytocin: a) a hydrogen atom replacing the amino group of cysteine ​​(position 1); b) a thioether bond replacing its disulfide bond; and c) a methyloxyl group replacing the hydroxyl group of tyrosine (position 2). Carbetocin (PABAL™, DURATOCIN™) is currently approved for the prevention of uterine atony after delivery of an infant by cesarean section under epidural or spinal anesthesia. The dosage used for this medical application is relatively low, e.g., 100 micrograms per administration.

[0003] Recently, there has been an increasing need for oxytocin receptor agonists, particularly carbetocin. For example, oxytocin receptor agonists have recently been shown to treat Prader-Willi syndrome (see WO 2016 / 044131). Prader-Willi syndrome is a genetic disorder characterized by hyperphagia, food-seeking behavior, rapid weight gain, obsessive-compulsive behavior, and aggression in young children. As described in WO 2016 / 044131, patients treated with carbetocin show statistically significant improvements in measures of hyperphagia, obsessive-compulsive disorder, food-seeking behavior, and Clinical Global Impression after 15 days compared with placebo-treated patients. Because the dosages used are significantly higher than those used to treat uterine atony, e.g., on the order of tens of milligrams per day, and treatment is longer-term, relatively large quantities of the peptide must be produced for this indication. For such an indication, it would be desirable to produce relatively large quantities of highly pure carbetocin.

[0004] Synthesis of peptides can be carried out using solid phase synthesis procedures well known in the art. Solution phase synthesis is an alternative method that may be useful for small amounts of peptide. This step of peptide production is known as "upstream processing" and results in the formation of a crude peptide product.

[0005] After synthesis of a crude peptide, it is usually necessary to separate the desired peptide from various peptidic and non-peptidic impurities, a step known as the purification step.

[0006] Many methods for purifying peptides are known to those skilled in the art. However, peptide purification methods usually include at least one chromatography step, such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, free-flow electrophoresis, affinity chromatography, high-performance liquid chromatography (HPLC), etc. The most commonly used form of HPLC is "reverse-phase" HPLC (also known as RP-HPLC), in which peptides are eluted with increasing amounts of organic solvents, such as acetonitrile, depending on their hydrophobicity.

[0007] After the purification step, the peptide must usually be separated from the volatile solvent. This step is known as the isolation step. Known methods for separating peptides from solvent include ultrafiltration and lyophilization.

[0008] Lyophilization (also known as freeze-drying) involves the rapid freezing of a peptide-containing solution, usually by immersing the container holding the solution in liquid nitrogen. The container is then placed in a vacuum chamber containing cooling coils. The volatile solvent sublimes in the vacuum. The sublimation process ensures that the purified sample remains cold. Lyophilization is the most commonly used technique in the art for isolating peptides from solution, primarily because it is well known, reproducible, and easy to perform. Furthermore, the stability of peptides is usually increased at low temperatures.

[0009] Methods for the purification and isolation of carbetocin and related peptides are known in the art:

[0010] CN104592362 describes a liquid chromatography purification step of carbetocin followed by lyophilization. In most cases, the liquid chromatography step is HPLC.

[0011] WO2015185584 describes the purification and lyophilization of oxytocin agonists other than carbetocin.

[0012] CN102977192 describes a process for purifying carbetocin by combining liquid chromatography and ion exchange chromatography. After purification, the product undergoes desalting and lyophilization steps.

[0013] CN104744567 describes the process of purifying carbetocin by ion exchange chromatography followed by lyophilization.

[0014] CN101531705 describes a process for purifying carbetocin using reverse-phase HPLC, followed by conversion of the product to the acetate salt using ion exchange. After conversion to the salt, the product is then lyophilized.

[0015] WO2009 / 122285 discloses a method for purifying oxytocin analogs, which comprises an HPLC step followed by a freeze-drying step. Rudko AD et al., "Crystalline Salts of Oxytocin: X-ray crystallographic data," J. Crystal Growth, vol. 10, no. 3, 1971, pp. 260-262, describe the characterization of crystallized oxytocin salts. Bryn S et al., "Pharmaceutical Solids: A Strategic Approach to Regulatory Considerations," Pharmaceutical Research, vol. 12, no. 7, 1995, pp. 945-954, describe the properties of pharmaceutical solids.

[0016] The above references show that there is a strong bias in the art against the use of lyophilization as an isolation step in the synthesis of carbetocin and other oxytocin receptor agonists.

[0017] However, there are several problems associated with freeze-drying, such as the time required to process the peptides and the high cost of refrigerants and equipment.

[0018] These problems may be tolerable when producing small amounts of peptide. However, when producing large amounts of peptide, lyophilization becomes a "bottleneck" in the manufacturing process. Furthermore, the proportion of the total manufacturing cost spent on lyophilization increases with the mass of peptide produced.

[0019] Thus, there is a need in the art for improved methods of isolating carbetocin to remove the "bottleneck" of lyophilization so that large quantities of carbetocin can be produced that are sufficiently pure to meet the needs for indications such as the treatment of Prader-Willi syndrome. Summary of the Invention

[0020] In one aspect, the present invention relates to crystalline forms of carbetocin.

[0021] In another aspect, the present invention relates to a method for preparing a crystalline form of carbetocin, comprising the step of crystallizing carbetocin.

[0022] In another aspect, the present invention relates to a pharmaceutical composition comprising carbetocin according to the invention or carbetocin made according to the invention.

[0023] The drawings relevant to the present invention are described below: [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows the X-ray diffraction diagram (Cu) of solvated crystalline form I of carbetocin. [Figure 2] 1 shows the X-ray diffraction diagram (Cu) of desolvated crystalline form II of carbetocin. [Figure 3a] HPLC chromatograms of the solid isolated from Example 1 (FIG. 3a); and the solid isolated from Example 4 (FIG. 3b) are shown. [Figure 3b] HPLC chromatograms of the solid isolated from Example 1 (FIG. 3a); and the solid isolated from Example 4 (FIG. 3b) are shown. [Figure 4a] 4 shows TG / DTA data relating to solvated crystalline form I of carbetocin from Example 1 (FIG. 4a) and desolvated crystalline form II of carbetocin from Example 4 (FIG. 4b). [Figure 4b]4 shows TG / DTA data relating to solvated crystalline form I of carbetocin from Example 1 (FIG. 4a) and desolvated crystalline form II of carbetocin from Example 4 (FIG. 4b). [Figure 5] 1 shows the X-ray diffraction pattern (Cu-Kα1) of the crystalline carbetocin obtained in Example 5. [Figure 6] 1 shows an HPLC chromatogram of the solid isolated from Example 5. [Figure 7] 1 shows differential scanning calorimetry (DSC) data relating to crystalline carbetocin isolated from Example 5. [Figure 8a] Gravimetric water sorption (GVS) data from crystalline carbetocin isolated from Example 5: mass change plot (FIG. 8a) and isotherm plot (FIG. 8b). [Figure 8b] Gravimetric water sorption (GVS) data from crystalline carbetocin isolated from Example 5: mass change plot (FIG. 8a) and isotherm plot (FIG. 8b). DETAILED DESCRIPTION OF THE INVENTION

[0025] Carbetocin has not previously been known to form crystals. Applicant has surprisingly discovered, as described herein, that it is possible to form three crystalline forms of carbetocin, two of which may be designated Form I and Form II. Form I is solvated (e.g., hydrated), while Form II is desolvated. Form II has high stability (see FIG. 4B) and an acceptably low ethylene glycol content (see Example 3), allowing it to be used, for example, as a pharmaceutical. The acceptably low ethylene glycol content represents an ethylene glycol content below the ICH limit of 620 ppm, as determined by gas chromatography. Form I can be used as a synthetic intermediate in the production of Form II. A third crystalline form is also described herein (see Example 5).

[0026] According to the present invention, a first aspect provides a crystalline form of carbetocin. A second aspect provides a solvated (e.g., hydrated) crystalline form of carbetocin. A third aspect provides a desolvated crystalline form of carbetocin.

[0027] Solvated means that the crystal structure contains ordered or disordered solvent molecules. Disordered means that the positions of the solvent molecules or the positions of the atoms therein can vary within the crystal structure. The solvent molecules may be liquid or gaseous at room temperature and atmospheric pressure. The solvent molecules may consist of only one type of molecule. Alternatively, the solvent molecules may consist of two or more different types of molecules (one of which may be water). There may be at least 0.1 or more solvent molecules per carbetocin molecule, such as at least 0.2 solvent molecules per carbetocin molecule, such as at least 0.5 solvent molecules per carbetocin molecule, such as at least 1 solvent molecule per carbetocin molecule, such as at least 2 solvent molecules per carbetocin molecule, for example at least 5 solvent molecules per carbetocin molecule. Thus, the solvated crystalline form of carbetocin may be in the form of a mono-, di-, tri-, tetra-, penta-, or hexa-hydrate solvated crystalline form. Preferably, when carbetocin is in a solvated crystalline form, the solvated crystalline form is a monohydrate or a pentahydrate. Thus, in one embodiment, carbetocin is in a monohydrate or a pentahydrate crystalline form. Such carbetocin may contain ordered or disordered solvent molecules. It is believed that the number of solvent molecules does not affect whether it is ordered or disordered.

[0028] Desolvated means that the crystalline structure contains few or no ordered or disordered solvent molecules. It may contain no more than 2 solvent molecules per carbetocin molecule, such as no more than 1 solvent molecule per carbetocin molecule, for example no more than 0.5 solvent molecules per carbetocin molecule, for example no more than 0.2 solvent molecules per carbetocin molecule, for example no more than 0.1 solvent molecule per carbetocin molecule, for example no more than 0.05 solvent molecules per carbetocin molecule, for example no more than 0.02 solvent molecules per carbetocin molecule, for example no more than 0.01 solvent molecules per carbetocin molecule.

[0029] To determine the crystalline form, X-ray powder diffraction (XRPD) analysis can be performed. In the present invention, as further detailed in Example 1, XRPD analysis was performed on a PANalytical X'pert pro using Cu K radiation (αλ=1.54060 Å; α=1.54443 Å; β=1.39225 Å; α:α ratio=0.5). The crystalline form of carbetocin and / or the solvated crystalline form of carbetocin can be characterized by X-ray powder diffraction peaks at about 4.83, 7.43, 9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu), and / or can be substantially characterized by the X-ray powder diffraction (Cu) pattern illustrated in FIG. 1, and / or can be characterized by having 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or substantially all of the (Cu) X-ray powder diffraction peaks shown in Table 1. Thus, in one embodiment, the crystalline form of carbetocin is characterized by X-ray powder diffraction peaks at about 4.83, 7.43, 9.20, 17.87, 19.60, 20.43, and 21.34 degrees 2θ, performed using CuK radiation (αλ=1.54060 Å; α=1.54443 Å; β=1.39225 Å; α:α ratio=0.5).

[0030] The crystalline form of carbetocin and / or the solvated crystalline form of carbetocin can be characterized by X-ray powder diffraction peaks at about 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ (Cu), and / or can be substantially characterized by the X-ray powder diffraction (Cu) pattern illustrated in FIG. 2, and / or can be characterized by having 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or substantially all of the (Cu) X-ray powder diffraction peaks shown in Table 2. Thus, in one embodiment, the crystalline form of carbetocin is characterized by X-ray powder diffraction peaks at about 4.11, 4.39, 5.60, 7.45, 17.75, 19.16, and 19.454 degrees 2θ, performed using CuK radiation (αλ=1.54060 Å; α=1.54443 Å; β=1.39225 Å; α:α ratio=0.5).

[0031] The crystalline form of carbetocin can be characterized by X-ray powder diffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39 degrees 2θ (Cu-Kα1), and / or can be substantially characterized by the X-ray powder diffraction (Cu-Kα1) pattern illustrated in Figure 5, and / or can be characterized by having 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or substantially all of the (Cu) X-ray powder diffraction peaks shown in Table 2. Thus, in one embodiment, the crystalline form of carbetocin is characterized by X-ray powder diffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39 degrees 2θ, performed using Cu Kα1 radiation (α1λ=1.54060 Å).

[0032] [Table 1] JPEG0007789482000002.jpg110161

[0033] [Table 2]

[0034] [Table 3] JPEG0007789482000005.jpg146140

[0035] Table 3 shows data obtained from Example 5. Note that the source of radiation for the values ​​reported in Table 3 is a Cu-Kα1 source, while the source of radiation for the values ​​reported in Tables 1 and 2 is a Cu-K source. The XPRD peak table, Table 3 (FIG. 5, Example 5), nevertheless shows a different crystalline form or polymorph of carbetocin when compared to Examples 1 and 2 (FIG. 1, Table 1) and Examples 3 and 4 (FIG. 2, Table 2).

[0036] According to another aspect, the present invention provides a method for preparing a crystalline form of carbetocin, the method comprising the step of crystallizing carbetocin.

[0037] Crystallization refers to the process of forming a crystalline form of carbetocin from carbetocin dissolved in a solvent. A crystalline form refers to a solid material with a regularly repeating internal arrangement of atoms and external planes. A crystalline form can be distinguished from an amorphous form based on X-ray powder diffraction analysis. A crystalline form is characterized by X-ray powder diffraction peaks as described herein. In an amorphous solid form, the XPRD pattern is essentially continuous in appearance, i.e., lacking distinct peaks.

[0038] The crystalline form of carbetocin may be crystallized from a mixture comprising carbetocin and one or more liquids, and the one or more liquids may comprise one or more liquids from the group consisting of water, aqueous acetate buffer, ethylene glycol, acetonitrile, ethanol, methanol, propanol, isopropanol, 1,2-propanediol and dimethylformamide, such as a mixture of ethylene glycol and acetonitrile, such as a mixture of ethanol, ethylene glycol and acetonitrile, such as a mixture of propanol, ethylene glycol and acetone, such as a mixture of isopropanol, ethylene glycol and acetone, such as a mixture of dimethylformamide, ethylene glycol and acetonitrile, such as a mixture of dimethylformamide and acetonitrile, such as a mixture of dimethylformamide and acetone, such as a mixture of ethanol and acetonitrile, such as a mixture of methanol and acetonitrile, such as a mixture of 1,2-propanediol and acetonitrile, such as a mixture of 1,2-propanediol and acetone. When the one or more liquids comprise two or more liquids, one liquid of the two or more liquids may be an antisolvent (as defined below). The one or more liquids may comprise ethylene glycol and an anti-solvent in a ratio of 15:85 to 25:75, such as a ratio of 17.5:82.5 to 22.5:77.5, for example a ratio of about 20:80, wherein the addition of the anti-solvent changes the ratio of ethylene glycol to acetonitrile to a ratio of 1:99 to 30:70, for example a ratio of 2:98 to 25:75, for example a ratio of 3:97 to 20:80, such as a ratio of 5:95 to 20:80, for example a ratio of 5:95 to 15:85, for example a ratio of 7.5:92.5 to 12.5:87.5, for example a ratio of about 10:90, for example a ratio of 5:95 to 10:90, for example a ratio of 5:95 to 7.5:92.5, for example a ratio of about 6.7:93.3.

[0039] The one or more liquids may include a mixture of ethylene glycol and acetonitrile.

[0040] The one or more liquids may comprise ethylene glycol and acetonitrile in a ratio of 1:99 to 50:50, such as a ratio of 2:98 to 40:60, for example a ratio of 3:97 to 35:65, for example a ratio of 5:95 to 35:65, for example a ratio of 8:92 to 30:70, for example a ratio of 10:90 to 30:70, for example a ratio of 15:85 to 25:75, for example a ratio of 17.5:82.5 to 22.5:77.5, such as a ratio of about 20:80.

[0041] The one or more liquids may comprise water. The one or more liquids may comprise an aqueous acetate buffer. In certain embodiments, the crystalline form of carbetocin is crystallized from a mixture comprising carbetocin and one or more liquids, wherein the one or more liquids comprise one or both of water and an aqueous acetate buffer.

[0042] In one embodiment, the one or more liquids may be water. When the one or more liquids are water, the water may have a pH of about 2 to 6, preferably a pH of about 3 to 4, and more preferably a pH of about 3.5.

[0043] In one embodiment, the one or more liquids may be an aqueous acetate buffer. The aqueous acetate buffer may be formed from an aqueous mixture of acetic acid and an acetate salt. The acetate salt may have a suitable counterion. Suitable counterions may be, for example, alkali metal ions, alkaline earth metal ions, or organic cations. The counterion may be lithium, sodium, potassium, magnesium, calcium, or ammonium. Preferably, the counterion may be sodium or potassium, and most preferably, the counterion may be sodium. Preferably, the aqueous acetate buffer has a pH of about 4-7, preferably about 5-6, and most preferably about 5.5. Preferably, the aqueous acetate buffer has a concentration of 20-30 mM, and most preferably, a concentration of about 25 mM.

[0044] Crystalline carbetocin can be obtained by cooling a mixture containing carbetocin and one or more liquids, for example, from 40°C to 5°C, or by cycling the temperature of a mixture containing carbetocin and one or more liquids, for example, between 40°C and 5°C. The temperature can be changed at a rate of 5°C to 50°C per hour, such as 35°C per hour. Cycling the temperature of a mixture means that the temperature must be subsequently lowered and then raised, or vice versa. The temperature may be lowered and then raised, or vice versa, two or more times, for example, three or more times, for example, four or more times, for example, five or more times, for example, ten or more times. Following cooling or temperature cycling of a mixture containing carbetocin and one or more liquids, the temperature can be maintained, such as at 5°C, for a length of time appropriate for the formation of crystalline carbetocin. Typically, crystalline carbetocin can be formed and isolated within 6 to 24 hours, for example, about 12 or about 18 hours. Cooling, cycling, or maintaining the temperature can occur with or without stirring the mixture.

[0045] Alternatively, crystalline carbetocin can surprisingly be obtained by maintaining a mixture comprising carbetocin and water or a mixture comprising carbetocin and an aqueous acetate buffer at a temperature of at least 15°C, e.g., 20°C, e.g., 30°C, e.g., 40°C, for a suitable length of time to form crystalline carbetocin. Typically, crystalline carbetocin forms in 3 to 100 days, more typically 3 to 60 days, and most typically 7 to 12 days. In one alternative embodiment, crystalline carbetocin can be obtained by maintaining a mixture comprising carbetocin and water or a mixture comprising carbetocin and an aqueous acetate buffer at a temperature of 20°C for about 3 to 60 days. In another alternative embodiment, crystalline carbetocin can be obtained by maintaining a mixture comprising carbetocin and water or a mixture comprising carbetocin and an aqueous acetate buffer at a temperature of 40°C for about 7 to 12 days. In these alternative embodiments, all other steps described below can be performed, except for the step relating to the addition of an antisolvent.

[0046] Carbetocin can be crystallized from a mixture containing at least one solvent and at least one anti-solvent. The solvent refers to a liquid in which carbetocin is readily dissolved or readily soluble. The solvent may be any solvent in which carbetocin is soluble at a concentration of 0.01 mg / ml or more, for example, 0.05 mg / ml or more, for example, 0.1 mg / ml or more, for example, 0.5 mg / ml or more, for example, 1 mg / ml or more, for example, 5 mg / ml or more, for example, 10 mg / ml or more, for example, 20 mg / ml or more under standard conditions. The anti-solvent refers to a liquid in which carbetocin is less soluble or less soluble than the solvent. The antisolvent may be selected relative to the solvent and may be any solvent in which carbetocin is soluble in an amount under standard conditions of less than 20 mg / ml, for example, less than 10 mg / ml, for example, less than 5 mg / ml, for example, less than 1 mg / ml, for example, less than 0.5 mg / ml, for example, less than 0.1 mg / ml, for example, less than 0.05 mg / ml, for example, less than 0.01 mg / ml. It will be understood by those skilled in the art that if carbetocin is soluble in a solvent in an amount of, for example, 10 mg / ml or more, it will be less soluble in the antisolvent, i.e., it will be soluble in an amount of less than 10 mg / ml, for example, less than 5 mg / ml, for example, less than 1 mg / ml, for example, less than 0.5 mg / ml, for example, less than 0.1 mg / ml, for example, less than 0.05 mg / ml, for example, less than 0.01 mg / ml. Unless otherwise specified, the terms solvent and antisolvent refer to the solubility behavior of carbetocin at room temperature and atmospheric pressure.

[0047] The solvent may comprise one or more liquids selected from the group consisting of water, aqueous acetate buffer, ethylene glycol, ethanol, methanol, propanol, isopropanol, and 1,2-propanediol. The solvent may have a relative polarity index (RPI) greater than 0.5, such as greater than 0.6, such as greater than 0.7, such as greater than 0.8, such as greater than 0.9, for example greater than 1.0, as described by Christian Reichardt (Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, 3rd Edition, 2003). The solvent may be or include any one or more of water, aqueous acetate buffer, or alcohol, for example, any one or more of water (RPI=1.000), aqueous acetate buffer, ethylene glycol (RPI=0.790), ethanol (RPI=0.654), methanol (RPI=0.762), propanol (RPI=0.803), isopropanol (RPI=0.787), or 1,2-propanediol (RPI=0.72).

[0048] The crystalline form of carbetocin can be crystallized from a mixture comprising carbetocin and one or more liquids, wherein the carbetocin is present in a solvent at a concentration of about 1 mg / ml to 200 mg / ml, preferably about 10 mg / ml to 150 mg / ml, and most preferably about 20 mg / ml to 100 mg / ml. In one embodiment, the solvent can be water. In one embodiment, the solvent can be an aqueous acetate buffer. In one embodiment, the solvent can be ethylene glycol.

[0049] The method may comprise the further step of adding an anti-solvent to the mixture comprising carbetocin and one or more liquids, for example adding the anti-solvent before cooling the mixture.

[0050] The anti-solvent may have a relative polarity index (RPI) of less than 1, such as less than 0.9, for example less than 0.8, such as less than 0.75, for example less than 0.7, for example less than 0.6, for example less than 0.5, as described by Christian Reichardt (Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, 3rd Edition, 2003). The anti-solvent may be or comprise any one or more esters, ketones, nitriles, or ethers, for example any one or more of acetonitrile (RPI=0.460), ethyl acetate (RPI=0.228), acetone (RPI=0.355), or methyl tert-butyl ether (RPI=0.124).

[0051] Thus, in one embodiment of the method, carbetocin can be crystallized from a mixture containing carbetocin and one or more liquids, the one or more liquids including ethylene glycol and acetonitrile. Carbetocin may be present in the mixture containing carbetocin and one or more liquids at a concentration of 10 mg / ml to 150 mg / ml, most preferably about 100 mg / ml. Ethylene glycol and acetonitrile may be present in a ratio of 5:95 to 35:65. Crystalline carbetocin can be obtained by cooling the mixture containing carbetocin and one or more liquids from 40°C to 5°C at a rate of 35°C per hour and maintaining the temperature at 5°C for a suitable length of time to form crystalline carbetocin, e.g., about 12 hours or about 18 hours.

[0052] The method may include the further step of seeding the mixture comprising carbetocin and one or more liquids with crystals, eg, carbetocin crystals, eg, crystals of solvated crystalline Form I of carbetocin.

[0053] Seeding refers to the addition of homogeneous or heterogeneous crystals, i.e., seed crystals, to a mixture to nucleate and / or grow additional carbetocin in crystalline form. Homogeneous crystals refer to crystalline carbetocin in any of its forms. Heterogeneous crystals refer to crystals of another material.

[0054] The method may include the further step of inducing crystallization in the mixture comprising carbetocin and one or more liquids. Crystallization may be induced by any suitable means to promote crystal nucleation and growth, such as by disturbing the surface of the mixture comprising carbetocin and one or more liquids to create seed crystals, for example by pipetting liquid up and down from the surface of the mixture comprising carbetocin and one or more liquids, or by scratching the surface of the mixture comprising carbetocin and one or more liquids where it contacts the surface of the container in which the mixture is held.

[0055] The method may include the further step of desolvating (and optionally drying) the crystalline form of carbetocin.

[0056] Desolvation refers to the removal of some or substantially all solvate molecules from the crystalline structure of carbetocin such that the crystalline structure contains few or no ordered or disordered solvent molecules. In a preferred embodiment, desolvation refers to the conversion of carbetocin from the pentahydrate crystalline form to the monohydrate crystalline form.

[0057] Desolvation of crystalline carbetocin can be carried out by washing the crystalline carbetocin in an anti-solvent, for example, acetonitrile, at a temperature which may be 20°C or lower, for example, between -30°C and 20°C, for example, between -20°C and 20°C, for example, between -10°C and 20°C, for example, between -5°C and 15°C, for example, between 0°C and 10°C, for example, about 5°C, and then drying, for example, under vacuum. Drying may occur under vacuum for a suitable length of time to effect desolvation, for example, more than 1 hour, for example, about 24 hours. Preferably, the crystalline carbetocin can be desolvated by washing in acetonitrile at a temperature of about 5°C and drying under vacuum at a temperature of about 20°C for about 24 hours.

[0058] Desolvation can also be carried out by heating the crystalline form of carbetocin to a temperature of at least 40°C and at most 190°C, or by exposing the crystalline form of carbetocin to an environment with low relative humidity, for example, a relative humidity of 40% or less.

[0059] Thus, in one embodiment of the method, carbetocin can be crystallized from a mixture containing carbetocin and one or more liquids, the one or more liquids comprising ethylene glycol and acetonitrile. Carbetocin may be present in the mixture containing carbetocin and one or more liquids at a concentration of 10 mg / ml to 150 mg / ml, most preferably about 100 mg / ml. Ethylene glycol and acetonitrile may be present in a ratio of 5:95 to 35:65. An additional antisolvent, such as acetonitrile, may be added. Crystalline carbetocin can be obtained by cooling the mixture containing carbetocin and one or more liquids from 40°C to 5°C at a rate of 35°C per hour and maintaining the temperature at 5°C for a suitable length of time to isolate crystalline carbetocin, e.g., about 12 hours or about 18 hours. Crystalline carbetocin may be desolvated by washing in acetonitrile at about 5°C and drying under vacuum at about 20°C for about 24 hours.

[0060] A filtration step may be carried out before crystallization. The filtration step preferably comprises filtration by centrifugation. Thus, in one aspect, a method for producing crystalline carbetocin comprises the steps of: (1) filtration, preferably by centrifugation; and (2) crystallization.

[0061] A washing step may be performed before crystallization. For example, carbetocin, e.g., crude carbetocin, can be slurried, e.g., in acetonitrile, e.g., for 2 hours to 1 week, e.g., for about 18 hours with continuous stirring. Washing the crude carbetocin increases its purity before crystallization by about 1-2%, significantly increasing the assay value from about 44% to about 70% (in acetonitrile). Thus, in one embodiment, a method for producing crystalline carbetocin includes: (1) washing carbetocin, e.g., crude carbetocin, in acetonitrile; and (2) crystallization. In another embodiment, a method for producing crystalline carbetocin includes: (1) washing carbetocin, e.g., crude carbetocin, in acetonitrile; (2) filtration, preferably by centrifugation; and (3) crystallization.

[0062] Applicants have advantageously and surprisingly discovered that it is possible to isolate carbetocin without the need for lyophilization by crystallizing carbetocin, for example by crystallizing carbetocin from a solution.

[0063] The process results in highly pure carbetocin in acceptable yield without the need for a lyophilization step.

[0064] The carbetocin in the mixture comprising carbetocin and one or more liquids can be substantially pure carbetocin or crude carbetocin.

[0065] As used herein, the term "crude," as in "crude carbetocin," refers to carbetocin that is insufficiently pure for use as a pharmaceutical. The crude peptide may be less than 95% pure, e.g., less than 92.5%, e.g., 90%-93%, e.g., 91%-93%, as measured by UV-HPLC. Impurities found in the crude peptide may include one or more inorganic substances, residual solvents (e.g., DMF), peptide-related impurities, and residual peptide coupling reagents.

[0066] The (Product) crystalline form of carbetocin / solvated (eg hydrated) crystalline form of carbetocin / desolvated crystalline form of carbetocin may be 95% or more pure.

[0067] Crude carbetocin can be synthesized by methods well known to those skilled in the art, for example by methods similar to those described in WO2009 / 122285 to Ferring BV (International Patent Application No. PCT / IB2009 / 005351).

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising carbetocin according to the present invention or carbetocin produced according to the method of the present invention. The pharmaceutical composition of the present invention can be for use as a medicament. The pharmaceutical composition of the present invention can be for use in the treatment of neurological or reproductive disorders, for example, for the treatment of Prader-Willi syndrome (as described in WO 2016 / 044131 (International Patent Application No. PCT / US2015 / 04911) to Ferring BV); or for use in the treatment or prevention of uterine atony, for example, after vaginal delivery of an infant, delivery of an infant by cesarean section; or for use in the treatment or prevention of uterine atony in patients at risk of developing postpartum hemorrhage (PPH); and / or for use in the treatment or prevention of excessive bleeding after vaginal delivery (as described in WO 2009 / 122285 (International Patent Application No. PCT / IB2009 / 005351) to Ferring BV).

[0069] The present invention is illustrated below. The examples may illustrate preferred embodiments of the invention, but are not meant to be limiting in any way. [Example]

[0070] Preparation of solvated crystalline form I of carbetocin Step i: Synthesis Crude carbetocin was obtained with a purity of about 91% by a synthesis similar to that described in WO2009 / 122285 (International Patent Application No. PCT / IB2009 / 005351) of Ferring BV.

[0071] Step ii: Preparation of solutions 60 mg of crude carbetocin obtained in step i) was dissolved in 0.6 mL of a 30:70 (v / v) mixture of ethylene glycol (first liquid):acetonitrile (second liquid) at 40° C. The vessel was then charged with Form I (solvated) carbetocin crystals. It is understood that seeding is not required but may facilitate crystallization.

[0072] Step iii: Crystallization The solution obtained in step ii) was heated to 40°C and held at this temperature for 30 minutes. The mixture was then filtered by centrifugation to remove insoluble impurities. The mixture was then stirred at 40°C for 30 minutes, cooled to 5°C over 1 hour, and then held at 5°C overnight with continuous stirring.

[0073] The precipitated material was isolated.

[0074] XRPD analysis was performed on a PANalytical X'pert pro. Samples were scanned from 3 to 35°2θ. The material was gently ground to release aggregates and placed in a multiwell plate with a Kapton or Mylar polymer film to support the sample. The multiwell plate was then placed in the diffractometer and analyzed using CuK radiation (αλ = 1.54060 Å; α = 1.54443 Å; β = 1.39225 Å; α:α ratio = 0.5) running in transmission mode (step size 0.0130°2θ) with a 40 kV / 40 mA generator setting.

[0075] Carbetocin crystallized from the solution having an X-ray diffraction pattern substantially as shown in Table 1 and Figure 1 (Form I).

[0076] The solid was analyzed by TG / DTA to simplify the mass loss / thermal event (Figure 4a).

[0077] The purity of the (solvated) crystalline form of carbetocin was calculated at 96.2% by UV-HPLC (Figure 3a) according to the method outlined in Table 4.

[0078] [Table 4] [Example]

[0079] Preparation of solvated crystalline form I of carbetocin Step i: Synthesis Crude carbetocin was obtained with a purity of about 91% by a synthesis similar to that described in WO2009 / 122285 (International Patent Application No. PCT / IB2009 / 005351) of Ferring BV.

[0080] Step ii: Preparation of solutions 60 mg of crude carbetocin obtained in step i) was dissolved in 0.6 mL of a 30:70 (v / v) mixture of ethylene glycol (first liquid):acetonitrile (second liquid) at 40° C. The vessel was then charged with Form I (solvated) carbetocin crystals. It is understood that seeding is not required but may facilitate crystallization.

[0081] Step iii: Addition of antisolvent Sufficient acetonitrile was added to adjust the ethylene glycol:acetonitrile ratio to 6.7:93.3 (v / v).

[0082] Step iv: Crystallization The solution obtained in step iii) was heated to 40°C and held at this temperature for 30 minutes. The mixture was then filtered by centrifugation to remove any insoluble impurities. The mixture was then stirred at 40°C for 30 minutes, cooled to 5°C over 1 hour, and then held at 5°C overnight with continuous stirring.

[0083] The precipitated material was isolated.

[0084] XRPD analysis was performed as described above for Example 1.

[0085] Carbetocin crystallized from the solution having an X-ray diffraction pattern substantially as shown in Table 1 and Figure 1. [Example]

[0086] Preparation of desolvated crystalline form II carbetocin. To remove the ethylene glycol present in the crystallized material produced by Examples 1 and 2, the crystallized material of Example 1 or Example 2 was desolvated and dried.

[0087] Washing of the Form I crystalline material in acetonitrile at 5°C followed by drying under vacuum resulted in desolvation of the solvated Form I to produce desolvated Form II crystals having a diffractogram substantially as shown in Table 2 and Figure 2. The Form II crystals were found to have ethylene glycol levels below the ICH limit of 620 ppm as determined by gas chromatography, with parameters as shown in Table 5 below.

[0088] [Table 5] [Example]

[0089] Preparation of desolvated crystalline form II carbetocin. Approximately 300 mg of crude carbetocin (purity approximately 91.3%) was added to 3 mL of a previously prepared 30% ethylene glycol:70% acetonitrile (v / v) solvent mixture, and the mixture was heated to 40°C for 30 min with continuous stirring.

[0090] After 30 minutes, the mixture was filtered by centrifugation to remove insoluble impurities. To this mixture (still at 40°C) was added 1.5 mL of acetonitrile in 0.5 mL aliquots. No precipitation was observed at 40°C, even after complete addition of acetonitrile.

[0091] The mixture was then stirred at 40° C. for 1 hour, cooled to 5° C. over 1 hour, and then held at 5° C. with continuous stirring for 18 hours.

[0092] After 18 hours, the precipitated material was isolated, washed with approximately 5 mL of acetonitrile, and then dried under vacuum at ambient temperature for 24 hours.

[0093] The next day, the solid was analyzed by HPLC for purity and assay (Fig. 3b), TG / DTA for mass loss / simplification of thermal events (Fig. 4b), and polarized light microscopy (PLM) and XRPD for morphology and crystalline content.

[0094] PLM analysis showed that the final isolated solid contained a mixture of aggregates (50–100 μm) that readily dispersed into very small needle-like crystals (length <10 μm).

[0095] TG / DTA data for crystals formed by the method of Example 4 and shown in Figure 4b indicate a total mass loss of only about 0.8% up to 110°C. This was found to be a two-step process, with a first mass loss of about 0.5% up to about 60°C and a second mass loss of about 0.3% up to about 110°C. These mass losses correspond to acetonitrile and water weakly bound to the surface and do not indicate solvation of the crystalline form itself. Therefore, this solvent loss does not alter the crystallinity of the desolvated crystalline Form II.

[0096] These results indicate that desolvated crystalline Form II carbetocin is highly stable. [Example]

[0097] Preparation of solvated crystalline form I of carbetocin Step i: Synthesis Crude carbetocin was obtained with a purity of about 93.5% by a synthesis similar to that described in WO2009 / 122285 (International Patent Application No. PCT / IB2009 / 005351) of Ferring BV.

[0098] Step ii: Preparation of solutions Acetate buffer, 25 mM, pH 5.5, was prepared from sodium acetate trihydrate, glacial acetic acid, and ultrapure water. 354 mg of crude carbetocin obtained in step i) was dissolved in 16.6 mL of acetate buffer. The solution was filtered through a 0.22 μM PVDF syringe filter, and 500 μL portions of the solution were dispensed into vials, which were then sealed. The pH of the carbetocin solution was 5.3.

[0099] Step iii: Crystallization The solution obtained in step ii) was heated to 40°C and kept at this temperature in a sealed vial. After 3 days, the vial was removed and the solution was gently aspirated back into the vial using an Eppendorf glass pipette, thereby creating some seeds for crystallization. After pipetting, the vial was resealed and kept at 40°C. After 9 days, particles with a crystalline appearance had formed. The precipitated material was isolated.

[0100] XRPD analysis was performed on a PANalytical X'pert pro using a Cu-Kα1 monochromator (α1λ = 1.54060 Å). Samples were scanned from 2 to 35° 2θ. Material was gently crushed and smeared onto a Si zero-background wafer, which was then placed in a slowly rotating sample holder in the diffractometer, which was run in transmission mode (scan rate 0.01° / sec, step size 0.017° 2θ) using a 45 kV / 40 mA generator setting. Measurements were performed using a programmable entrance and divergence slit.

[0101] The X-ray diffraction patterns of the resulting carbetocin crystals are shown in Table 3 and Figure 5. The X-ray diffraction patterns show carbetocin with different crystalline forms or polymorphs when compared to Examples 1 and 2 (Figure 1, Table 1) and Examples 3 and 4 (Figure 2, Table 2).

[0102] Differential scanning calorimetry (DSC) analysis was performed on a Netzsch DSC 204F1. A few milligrams of crystals were isolated from the mother liquor and left to air-dry for several hours at approximately 20% relative humidity (RH) in a fume hood. The crystals were gently crushed to a powder material, and 1.2 mg of this material was placed in a 25 μL Al pan. A lid was crimped onto the pan before a pinhole (0.25 mm diameter) was drilled. Samples were analyzed from 20 to 250 °C using a heating rate of 5 K / min.

[0103] DSC data for crystals formed by the method of Example 5 and shown in Figure 7 indicate a loss of volatiles in the range of 40-120°C, corresponding to the loss of weakly surface-bound water and solvated water. A melting endotherm with an onset of 192°C corresponds to the melting of anhydrous carbetocin.

[0104] Gravimetric vapor sorption (GVS) was performed on an SMS DVS-1. 1.4 mg of crystals and powder were added to an Al pan and exposed to stepwise relative humidity (RH) changes during two consecutive cycles: 20-30-40-50-60-70-80-70-60-50-40-30-20-10-0-10-20-30-40-50-60-70-80-90-80-70-60-50-40-30-20-10-0% RH in open-loop mode. The temperature was held at 25 °C, and a pure nitrogen flow rate of 200 mL / min was used. The applied dm / dt criterion was 0.001 wt% / min for 5 min, with a maximum time of 150 min for all steps, except for the step at 0% RH, which was set at 6 h.

[0105] The GVS data are shown in Figure 8. The GVS isotherm plot is shown in Figure 8b. The plateau at approximately 2% (w / w) corresponds to the monohydrate and some loosely bound surface water. A second plateau at an additional approximately 8% (w / w) corresponds to the pentahydrate and some loosely bound surface water. The pentahydrate exists above approximately 60% RH (sorption) and between approximately 40 and 90% RH (desorption).

[0106] The purity of the (solvated) crystalline form of carbetocin was calculated to be 98.7% by UV-HPLC according to the method outlined in Table 6.

[0107] [Table 6]

Claims

1. A crystalline form of carbetocin characterized by X-ray powder diffraction peaks at 4.83, 7.43, 9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu-K).

2. A crystalline form of carbetocin characterized by X-ray powder diffraction peaks at 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ (Cu-K).

3. A crystalline form of carbetocin characterized by X-ray powder diffraction peaks at 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39 degrees 2θ (Cu-Kα1).

4. 1. A method for preparing carbetocin in crystalline form, comprising: (a) the crystalline form of carbetocin is crystallized from a mixture comprising carbetocin and one or more liquids, wherein the one or more liquids consist of ethylene glycol and acetonitrile; the ethylene glycol and acetonitrile are in a ratio of 1:99 to 50:50; The process wherein the crystalline form of carbetocin is obtained by cooling a mixture comprising carbetocin and one or more liquids from 40°C to 5°C.

5. A method for producing carbetocin in crystalline form, comprising: (b) the crystalline form of carbetocin is crystallized from a mixture comprising carbetocin and one or more liquids, wherein the one or more liquids consist of water and an aqueous acetate buffer; The method, wherein the crystalline form of carbetocin is obtained by maintaining a mixture comprising carbetocin and one or more liquids at a constant temperature of 15°C to 40°C for 3 to 100 days to crystallize carbetocin.

6. a further step of seeding the mixture comprising carbetocin and one or more liquids with crystals, which may be carbetocin crystals; It may include the further step of desolvating the crystalline form of carbetocin, and drying the carbetocin; 5. The method of claim 4, wherein the desolvation of the crystalline form of carbetocin may be carried out by washing the crystalline form of carbetocin with an anti-solvent, which may be acetonitrile, and wherein the desolvation may be carried out at a temperature of 20° C. or less.

7. The method of claim 1, further comprising the step of seeding the mixture comprising carbetocin and one or more liquids with crystals, which may be carbetocin crystals; 6. The method of claim 5, which may comprise the further step of desolvating the crystalline form of carbetocin, and drying the carbetocin.

8. A pharmaceutical composition comprising carbetocin according to any one of claims 1 to 3.

9. 9. A pharmaceutical composition according to claim 8 for use as a medicament.

10. 9. A pharmaceutical composition according to claim 8 for use in the treatment of neurological or reproductive disorders, for the treatment of Prader-Willi syndrome; or for the treatment or prevention of uterine atony after vaginal delivery of an infant, after delivery of an infant by Caesarean section; or for the treatment or prevention of uterine atony in patients at risk of developing postpartum hemorrhage (PPH); and / or for the treatment or prevention of excessive bleeding after vaginal delivery.

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