Methods of making pharmaceutical compositions

Abstract

To provide an improved method for isolating carbetocin so as to produce a large amount of carbetocin having sufficient purity.SOLUTION: The invention provides a method for producing carbetocin in crystalline form, comprising crystallizing carbetocin, and a pharmaceutical composition comprising carbetocin according to the invention.SELECTED DRAWING: Figure 1

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-carbamoyl Oxytocin is a long-acting synthetic oligopeptide of oxytocin with agonist activity. Carbetocin is an analog of oxytocin. Carbetocin incorporates the following substitutions related to oxytocin: a) replacement of the amino group of cysteine ​​(position 1) by a hydrogen atom; b) thioether and c) replacement of its disulfide bond with a methyloxyl group. Replacement of the hydroxyl group (position 2) of carbetocin. RATOCIN™ is currently used to deliver infants by Caesarean section under epidural or spinal anesthesia. It is approved for the prevention of uterine atony after childbirth. The dosage used for this medical indication is The amount is relatively small, for example around 100 micrograms per dose.

[0003] Recently, there has been an increased need for oxytocin receptor agonists, especially carbetocin. For example, oxytocin receptors have recently been shown to treat Prader-Willi syndrome ( (See WO2016 / 044131.) Prader-Willi syndrome is characterized by hyperphagia, food-seeking behavior, and It is a genetic disorder characterized by hyperactivity, rapid weight gain, obsessive-compulsive behavior, and aggression in young children. As described in WO2016 / 044131, patients treated with carbetocin , bulimia, obsessive-compulsive disorder, food-seeking behavior, and Clinical Global Impression Scale measures after 15 days. The doses used are sufficient to treat uterine atony. The doses are significantly higher than those used in medical treatment, for example, on the order of tens of milligrams per day. Because treatment is longer term, relatively large amounts of peptides are produced for this indication. For such indications, it is necessary to produce relatively large quantities of highly pure carbetocin. It would be desirable to

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

[0005] After synthesis of the crude peptide, the desired peptide is typically purified by cleavage of various peptide and non-peptide amino acids. It must be separated from the pure product, this step being known as the purification step.

[0006] Many methods for purifying peptides are known to those skilled in the art. The method typically includes at least one chromatography step, such as size exclusion chromatography. hydrophobic interaction chromatography, ion exchange chromatography, Leaf-flow electrophoresis, affinity chromatography, high-performance liquid chromatography The most commonly used form of HPLC is "reversed-phase" HPLC. LC (also known as RP-HPLC), in which peptides are separated by acetonitrile depending on their hydrophobicity. The mixture is eluted with increasing amounts of organic solvent such as ethanol.

[0007] After the purification step, the peptide usually has to be separated from the volatile solvent. This step is known as the isolation step. Examples include ultrafiltration and freeze-drying.

[0008] Lyophilization (also known as freeze-drying) is a method of preserving liquids, usually in liquid form. The step of rapid freezing of the peptide-containing solution by immersing the container holding the peptide in liquid nitrogen was performed. The container is then placed in a vacuum chamber containing cooling coils. The volatile solvent is then evaporated under vacuum. The sublimation process ensures that the purified sample remains cool. Lyophilization is the most commonly used method in the art for isolating peptides from solution. This is mainly because the technique is well known, reproducible, and easy to perform. Furthermore, the stability of peptides is generally increased at low temperatures.

[0009] Methods for purifying and isolating carbetocin and related peptides are known in the art. be:

[0010] CN104592362 is a novel method for the liquid chromatography purification of carbetocin followed by The freeze-drying step is described. In most cases, liquid chromatography is used. The step is HPLC.

[0011] WO2015185584 discloses the purification and characterization of oxytocin agonists other than carbetocin. and freeze-drying.

[0012] CN102977192 is a method for analyzing the chromatographic properties of chromatographically purified ... After purification, The product then undergoes desalting and freeze-drying steps.

[0013] CN104744567 Purification of Carbetocin by Ion Exchange Chromatography and the subsequent freeze-drying process.

[0014] CN101531705 was used to purify carbetocin using reversed-phase HPLC, followed by isopropyl alcohol. This paper describes a process for converting the product to acetate using an ion exchange method. After conversion, the product is subsequently freeze-dried.

[0015] WO2009 / 122285 describes a method for preparing a HPLC step followed by a freeze-drying step. Rudko AD et al., "A method for purifying oxytocin analogs containing ..." Crystalline Salts of Oxytocin:X-ray crys tallographic data” J. Crystal Growth, vol. 10, no. 3, 1971, pp. 260-262, characterization of crystallized oxytocin salts. Bryn S et al., "Pharmaceutical Solid s:A Strategic Approach to Regulatory Control siderations” Pharmaceutical Research, vol. 12, no. 7, 1995, pp. 945-954, describes the properties of pharmaceutical solids. There are.

[0016] From the above references, the synthesis of carbetocin and other oxytocin receptor agonists There is a strong bias in the art against using lyophilization as an isolation step in It can be seen that...

[0017] However, there are some problems associated with freeze-drying, such as the need to process peptides. It requires a lot of time and the cost of refrigerants and equipment is very high.

[0018] These problems may be tolerable when producing small amounts of peptide. When mass-producing tides, freeze-drying becomes a "bottleneck" in the manufacturing process. The percentage of total manufacturing costs spent on lyophilization increases with the mass of peptide produced. do.

[0019] Therefore, there is a need in the art for treatment of indications such as Prader-Willi syndrome. Lyophilized "bottles" can be used to produce large quantities of carbetocin of sufficient purity to fill Improved methods for isolating carbetocin to remove the "neck" are needed. Summary of the Invention

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

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

[0022] In another aspect, the present invention relates to a pharmaceutical composition comprising carbetocin according to the invention, or This invention relates to carbetocin prepared according to the method disclosed herein.

[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. Interestingly, as described herein, three crystalline forms of carbetocin can be formed. Two of these forms can be designated Form I and Form II. Form I is solvated (e.g., hydrated), whereas Form II is desolvated. It has high stability (see Figure 4B) and an acceptably low ethylene glycol content (see Example 4). 3), can be used, for example, as a pharmaceutical. Acceptably low levels were found to be within the ICH limit as determined by gas chromatography. Form I represents an ethylene glycol content of less than 620 ppm in the production of Form II. A third crystalline form is also described herein and can be used as a synthetic intermediate in (See Example 5).

[0026] According to the present invention, from a first aspect, there is provided a crystalline form of carbetocin. In accordance with an embodiment, there is provided a solvated (e.g., hydrated) crystalline form of carbetocin. In accordance with an embodiment, a desolvated crystalline form of carbetocin is provided.

[0027] Solvated means that the crystal structure contains ordered or disordered solvent molecules. Disorder means that the positions of solvent molecules or the positions of atoms within them can vary within the crystal structure. Solvent molecules can be liquid or gaseous at room temperature and atmospheric pressure. Alternatively, the solvent molecules may be composed of two or more different species. One molecule of carbetocin may consist of a group of molecules, one of which may be water. at least 0.1 or more solvent molecules per molecule, e.g., at least 0. 2 solvent molecules, e.g., at least 0.5 solvent molecules per carbetocin molecule, e.g., carbe at least one solvent molecule per tocin molecule, e.g., at least one solvent molecule per carbetocin molecule There may be at least 2 solvent molecules, e.g., at least 5 solvent molecules per carbetocin molecule. Thus, the solvated crystalline forms of carbetocin include mono-, di-, tri-, tetra-, penta-, It may be in the form of a solvated crystalline form of a hydrate, a hexahydrate, or a hexahydrate. When tosine is in a solvated crystalline form, the solvated crystalline form is a monohydrate or a pentahydrate. Thus, in one embodiment, carbetocin is in the monohydrate or pentahydrate crystalline form. Such carbetocin may contain ordered or disordered solvent molecules. The number of molecules is not thought to affect whether they are ordered or disordered. .

[0028] Desolvation is the process by which a crystal structure is freed from most or all ordered or disordered solvent molecules. This means that there are no more than two solvent molecules per one carbetocin molecule, e.g., carbetocin Not more than 1 solvent molecule per synth molecule, e.g., not more than 0.5 solvent molecules per carbetocin molecule , e.g., 0.2 solvent molecules or less per carbetocin molecule, e.g., 0.5 solvent molecules or less per carbetocin molecule Not more than 0.1 solvent molecules per carbetocin molecule, e.g., not more than 0.05 solvent molecules per carbetocin molecule, e.g., Not more than 0.02 solvent molecules per carbetocin molecule, e.g., 0. It may be less than 0.1 solvent molecule.

[0029] To determine the crystalline form, X-ray powder diffraction (XRPD) analysis can be performed. In the present invention, as described in more detail in Example 1, XRPD analysis was performed using PANaly CuK radiation (α1λ = 1.54060 Å; α2 = 1.54443 Å; β = 1.39225 Å; α1:α2 ratio = 0.5) The crystalline form of carbetocin and / or the solvated crystalline form of carbetocin has a pH of about 4.83 , 7.43, 9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu) and / or can be characterized by X-ray powder diffraction peaks in FIG. The crystals can be substantially characterized by the X-ray powder diffraction (Cu) pattern shown in and / or 5 or more, 6 or more, 7 or more of the (Cu) X-ray powder diffraction peaks shown in Table 1 , 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, Thus, in one aspect, The crystalline form of carbetocin is sensitive to CuK radiation (αλ = 1.54060 Å; α = 1. 54443 Å; β = 1.39225 Å; α1:α2 ratio = 0.5), approximately 4.83, 7.43, 9.20, 17.87, 19.60, 20.43, and 21.3 It is characterized by an X-ray powder diffraction peak at 2θ of 4 degrees.

[0030] The crystalline form of carbetocin and / or the solvated crystalline form of carbetocin has a pH of about 4.11 , 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ( Cu) and / or as shown in FIG. can be substantially characterized by the X-ray powder diffraction (Cu) pattern shown in the example, and / or 5 or more, 6 or more, 7 or more, 8 or more of the (Cu) X-ray powder diffraction peaks shown in Table 2 or greater than, 9 or greater, 10 or greater, 11 or greater, 12 or greater, 13 or greater, 14 or greater, 15 or greater, or Thus, in one aspect, the invention can be characterized by having substantially all of: Carbetocin in crystalline form is sensitive to CuK radiation (αλ = 1.54060 Å; α = 1.54 443 Å; β = 1.39225 Å; α1:α2 ratio = 0.5), which was carried out using approximately 4. 11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 4 degrees 2 It is characterized by an X-ray powder diffraction peak at θ.

[0031] The crystalline form of carbetocin has a mean blood glucose concentration of approximately 4.34, 6.43, 8.66, 17.37, and 19.0 mg / kg. It is characterized by X-ray powder diffraction peaks at 3 and 19.39 degrees 2θ (Cu-Kα1). and / or X-ray powder diffraction (Cu-Kα1) as illustrated in FIG. can be substantially characterized by a pattern and / or as shown in Table 2 (Cu) X-ray powder diffraction peaks of 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 Thus, in one embodiment, crystalline forms of carbetocin can be characterized as Cu K performed using α1 radiation (α1λ=1.54060Å), ca. 4.34, 6.43, X-ray powder diffraction peaks at 2θ of 8.66, 17.37, 19.03, and 19.39 degrees It is characterized by a

[0032] [Table 1] JPEG2026000991000003.jpg110161

[0033] [Table 2]

[0034] [Table 3] JPEG2026000991000006.jpg146140

[0035] Table 3 shows the data obtained from Example 5. The radiation source for the values ​​reported in Table 3 was: The radiation source for the values ​​reported in Tables 1 and 2 is a Cu-K source. Note that the XPRD peak table, Table 3 (FIG. 5, Example 5) also Regardless, Examples 1 and 2 (Figure 1, Table 1) and Examples 3 and 4 (Figure 2, Table 2) , which represent different crystalline forms or polymorphs of carbetocin.

[0036] In another aspect, the present invention provides a method for preparing a crystalline form of carbetocin, The method includes the step of crystallizing carbetocin.

[0037] Crystallization is the process of forming crystalline forms of carbetocin from carbetocin dissolved in a solvent. Crystalline morphology refers to the internal arrangement of atoms and the external planes that are regularly repeated. Crystalline forms are distinguishable from amorphous forms based on X-ray powder diffraction analysis. The crystalline forms may be characterized by X-ray powder diffraction peaks as described herein. In amorphous solid forms, the XPRD pattern is essentially continuous in appearance, i.e. That is, there is no clear peak.

[0038] The crystalline form of carbetocin is obtained from a mixture containing carbetocin and one or more liquids. The one or more liquids may be water, aqueous acetate buffer, ethylene glycol, Recall, acetonitrile, ethanol, methanol, propanol, isopropanol , 1,2-propanediol, and dimethylformamide; Several liquids, e.g., a mixture of ethylene glycol and acetonitrile, e.g., ethanol, mixtures of ethylene glycol and acetonitrile, e.g., propanol, ethylene glycol mixtures of alcohol and acetone, e.g., isopropanol, ethylene glycol and acetone; Mixtures of dimethylformamide, ethylene glycol and acetonitrile a mixture of dimethylformamide and acetonitrile, e.g. a mixture of dimethylformamide and acetonitrile, e.g. a mixture of ethanol and acetonitrile, e.g. Mixtures of methanol and acetonitrile, e.g., 1,2-propanediol and acetonitrile The solvent may include a mixture of alcohols, for example a mixture of 1,2-propanediol and acetone. If one or more liquids include two or more liquids, one of the two or more liquids may be an antisolvent (as defined below). The liquids are ethylene glycol and antisolvent in a ratio of 15:85 to 25:75, e.g. For example, a ratio of 17.5:82.5 to 22.5:77.5, for example, a ratio of about 20:80, where an anti-solvent is added to bring the ratio of ethylene glycol to acetonitrile to 1: Ratios of 99 to 30:70, e.g., 2:98 to 25:75, e.g., 3:97 to 20:80 Ratios such as 5:95 to 20:80, for example, ratios of 5:95 to 15:85, for example, 7. Ratios of 5:92.5 to 12.5:87.5, for example, ratios of about 10:90, for example, 5:95 to 1 Ratios of 0:90, for example, 5:95 to 7.5:92.5, for example, a ratio of about 6.7:93.3 changes to.

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

[0040] The liquid or liquids are ethylene glycol and acetonitrile at a ratio of 1:99 to 50. :50 ratio, for example, 2:98 to 40:60 ratio, for example, 3:97 to 35:65 ratio, for example For example, the ratio is 5:95 to 35:65, for example, the ratio is 8:92 to 30:70, for example, the ratio is 10:90 to 3 Ratios of 0:70, e.g., 15:85 to 25:75, e.g., 17.5:82.5 to 22. It may be present in a ratio of 5:77.5, for example a ratio of about 20:80.

[0041] The one or more liquids may include water. The one or more liquids may include aqueous acetic acid. In certain embodiments, the crystalline form of carbetocin is and one or more liquids, wherein the one or more liquids are water and aqueous acetate buffer.

[0042] In one embodiment, the one or more liquids may be water. When the solution is water, the water has a pH of about 2 to 6, preferably about 3 to 4, and more preferably about 5 to 8. It may be about 3.5.

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

[0044] The crystalline form of carbetocin is a mixture containing carbetocin and one or more liquids. by cooling, for example, from 40°C to 5°C, or by adding carbetocin and one or more or by cycling the temperature of a mixture containing multiple liquids between, for example, 40°C and 5°C. The temperature can be obtained by setting the temperature between 5°C and 50°C per hour, such as 35°C per hour. The temperature of the mixture can be changed at a rate of Indicates that the number must be lowered and then raised, or vice versa. For example, four or more times, for example, five or more times, for example, ten or more times, the temperature is lowered, then raised, or Cooling the mixture containing carbetocin and one or more liquids or temperature cycling followed by a period of time appropriate to form crystalline carbetocin. Crystalline carbetocin can be maintained at a temperature of 5°C or higher. The product can be formed and isolated within, for example, about 12 hours or about 18 hours. The stirring or maintaining can occur with or without stirring the mixture.

[0045] Alternatively, crystalline carbetocin can be surprisingly obtained by dissolving carbetocin in a mixture containing carbetocin and water. or a mixture containing carbetocin and an aqueous acetate buffer to form crystalline carbetocin. for a suitable length of time at a temperature of at least 15°C, e.g. 20°C, e.g. 30°C, e.g. 40°C Crystalline carbetocin can be obtained by maintaining it at a constant temperature for 3 to 100 days. Typically, the formation occurs in 3 to 60 days, most typically in 7 to 12 days. Crystalline forms of carbetocin are available in mixtures containing carbetocin and water, or carbetocin and aqueous vinegar. The mixture containing the acid buffer is maintained at a temperature of 20°C for about 3 to 60 days. In another alternative embodiment, the crystalline form of carbetocin comprises carbetocin and water. The mixture, or a mixture containing carbetocin and aqueous acetate buffer, is heated at a temperature of 40°C for approximately 7-1 In these alternative embodiments, other methods described below can be used. All steps can be carried out except for the step relating to the addition of the anti-solvent.

[0046] Carbetocin is a mixture containing at least one solvent and at least one antisolvent. Carbetocin can be crystallized from a solvent. The solvent is a liquid in which carbetocin is soluble. 5 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 The solvent may be any solvent in which the compound is soluble in an amount of 1000 mg / ml or more under standard conditions. Carbetocin is less soluble in the solvent than in the solvent, or carbetocin is more soluble in the solvent than in the solvent. The antisolvent may be selected relative to the solvent, and the carbetocin may be 20 mg or less. / ml, for example less than 10mg / ml, for example less than 5mg / ml, for example less than 1mg / ml less than, for example less than 0.5 mg / ml, for example less than 0.1 mg / ml, for example 0.05 mg Any solvent in which the compound is soluble under standard conditions in an amount of less than 0.01 mg / ml, e.g., less than 0.01 mg / ml. If carbetocin is soluble in the solvent, for example, in an amount of 10 mg / ml or more, It will be less soluble in the solvent, i.e. less than 10 mg / ml, e.g. 5 mg / ml less than 1 mg / ml, for example less than 0.5 mg / ml, for example less than 0.1 mg / ml soluble in an amount of less than 0.01 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 solvents and antisolvents will be used unless otherwise specified. The term refers to the solubility behavior of carbetocin at room temperature and atmospheric pressure.

[0047] The solvents were water, aqueous acetate buffer, ethylene glycol, ethanol, methanol, propane, one selected from the group consisting of alcohol, isopropanol, and 1,2-propanediol; The solvent may contain multiple liquids. olvents and Solvent Effects in Organic C hemistry, Wiley-VCH Publishers, 3rd edition, 2003) The relative polarity index (RPI) is greater than 0.5, e.g., greater than 0.6, as described by , such as greater than 0.7, such as greater than 0.8, such as greater than 0.9, such as greater than 1.0. The solvent may be any one or more of water, aqueous acetate buffer, or alcohol, e.g., any One or more of water (RPI=1.000), acetate buffer solution, ethylene glycol ( RPI=0.790), ethanol (RPI=0.654), and methanol (RPI=0. 762), propanol (RPI=0.803), isopropanol (RPI=0.78 7), or 1,2-propanediol (RPI=0.72) It's okay to be.

[0048] The crystalline form of carbetocin is obtained from a mixture containing carbetocin and one or more liquids. Carbetocin can be crystallized from the solution, preferably at a concentration of about 1 mg / ml to 200 mg / ml. Preferably about 10 mg / ml to 150 mg / ml, most preferably about 20 mg / ml to 100 mg / ml The solvent may be present at a concentration of 0.05 mg / ml. In one embodiment, the solvent may be water. In one embodiment, the solvent may be an aqueous acetate buffer. It may also be ethylene glycol.

[0049] The method includes adding an antisolvent to a mixture containing carbetocin and one or more liquids. The reaction may include a further step, for example adding an anti-solvent before cooling the mixture. .

[0050] The antisolvent was prepared by Christian Reichardt (Solvents and olvent Effects in Organic Chemistry, Wile (VCH Publishers, 3rd edition, 2003) , the relative polarity index (RPI) is less than 1, e.g., less than 0.9, e.g., less than 0.8, e.g. It may be less than 0.75, such as less than 0.7, such as less than 0.6, such as less than 0.5 The anti-solvent may be any one or more of an ester, a ketone, a nitrile, or an ether, For example, any one or more of acetonitrile (RPI=0.460), ethyl acetate ( RPI=0.228), acetone (RPI=0.355), or methyl tert-butyl It may be or contain methyl ether (RPI=0.124).

[0051] Thus, in one embodiment of the method, the carbetocin is carbetocin and one or Crystallization can be performed from a mixture containing multiple liquids, one or more of which may be ethylene. Carbetocin contains carbetocin and one or more or a mixture of liquids, at a concentration of 10 mg / ml to 150 mg / ml, most preferably about It may be present at a concentration of 100 mg / ml. Ethylene glycol and acetonitrile The crystalline forms of carbetocin may be present in a ratio of 5:95 to 35:65. A mixture containing rubetosin and one or more liquids is heated from 40°C to 35°C per hour. Cool to 5°C and allow to stand for a suitable length of time to form crystalline carbetocin, e.g., up to about 12 hours. This can be achieved by maintaining the temperature at 5°C for about 18 hours.

[0052] The method comprises adding crystals, e.g., carbetocin, to a mixture comprising carbetocin and one or more liquids. Further seeding with crystals of carbetocin, e.g., solvated crystalline Form I of carbetocin. It may also include a step.

[0053] Seeding is the process of growing crystals by adding seed crystals to the mixture, either uniform or non-uniform. This means nucleating and / or growing further carbetocin in the form of a homogeneous Crystalline means crystalline carbetocin in any of its forms. Heterogeneous crystals mean different crystals. It means the crystals of the material.

[0054] The method further comprises inducing crystallization in a mixture comprising carbetocin and one or more liquids. The crystallization may include a step of: Carbetocin and one or more liquids may be mixed by any suitable means, for example, For example, carbetocin and 1 Pipetting liquid up and down from the surface of a mixture containing a species or multiple liquids The surface of the mixture containing carbetocin and one or more liquids is It can be induced by scratching the surface of the container where it comes into contact.

[0055] The method includes desolvating (and optionally drying) the crystalline form of carbetocin, It may include steps.

[0056] Desolvation is the process of purifying a crystal structure containing few or no ordered or disordered solvent molecules. To prevent this, some or substantially all solvated molecules are removed from the crystalline structure of carbetocin. In a preferred embodiment, desolvation means converting carbetocin into the pentahydrate crystalline form. This means converting the crystalline form from the monohydrate form to the monohydrate form.

[0057] Desolvation of crystalline carbetocin involves heating the crystalline carbetocin at a temperature below 20°C. For example, the temperature may be from -30°C to 20°C, for example, from -20°C to 20°C. The temperature may be, for example, -10°C to 20°C, for example, -5°C to 15°C, for example At a temperature which may be, for example, 0°C to 10°C, for example, about 5°C, an anti-solvent, for example Washing in acetonitrile followed by drying, e.g., vacuum drying. Drying can be carried out for a suitable length of time to effect desolvation, for example, more than 1 hour, for example, about 2 hours. This may occur under vacuum for 4 hours. Preferably, the crystalline form of carbetocin is added to acetonitrile. The solvent was removed by washing in trityl at a temperature of about 5°C and drying under vacuum at a temperature of about 20°C for about 24 hours. can be carried out.

[0058] Desolvation also involves desolvating crystalline carbetocin at temperatures of at least 40°C and at most 190°C. or by heating the crystalline form of carbetocin at low relative humidity, e.g. This can be done by exposing it to an environment with a humidity of 40% or less.

[0059] Thus, in one embodiment of the method, the carbetocin is carbetocin and one or Crystallization can be performed from a mixture containing multiple liquids, one or more of which may be ethylene. Carbetocin contains 10mg / ml to 150mg Carbetocin and one or more of the above at a concentration of about 100 mg / ml, most preferably about 100 mg / ml It may be present in a mixture with several liquids, such as ethylene glycol and acetonitrile. The solvent may be present in a ratio of 5:95 to 35:65. A solvent may be added. The crystalline form of carbetocin is prepared by mixing carbetocin and one or more The mixture containing the liquid was cooled from 40°C to 5°C at a rate of 35°C per hour to form crystalline carbetocin. Maintain the temperature at 5°C for a length of time appropriate for isolation, e.g., about 12 hours or about 18 hours. Carbetocin in crystalline form can be obtained by dissolving it in acetonitrile at a temperature of about 5°C. The solvent may be removed by washing with 50° C. and drying under vacuum at a temperature of about 20° C. for about 24 hours.

[0060] Crystallization may be preceded by a filtration step, which is preferably carried out by centrifugation. Thus, in one aspect, the method for producing carbetocin in crystalline form comprises: (1) filtration, preferably by centrifugation; and (2) crystallization steps.

[0061] A washing step may be carried out before crystallization. For example, carbetocin, e.g. crude carbetocin, may be washed with water. Vetocin can be slurried, for example, in acetonitrile. for 2 hours to 1 week, or for about 18 hours in acetonitrile with continuous stirring. The crude carbetocin can be washed to improve the purity before crystallization. Approximately 1-2% increase, significantly increasing assay value from approximately 44% to approximately 70% (in acetonitrile) Thus, in one aspect, a method for preparing a crystalline form of carbetocin includes: (1) adding carbetocin; washing the rubetocin, e.g., crude carbetocin, in acetonitrile; and (2) a crystallization step. In another aspect, the method for preparing crystalline forms of carbetocin comprises: (1) Carbetocin, e.g., crude carbetocin, is washed in acetonitrile. (2) a filtration step, preferably by centrifugation; and (3) a crystallization step. include.

[0062] Applicant has advantageously and surprisingly found that it is possible to crystallize carbetocin, e.g., from solution. Isolating carbetocin by crystallizing it without the need for lyophilization I discovered that it is possible.

[0063] The method also produces highly pure carbetocin in acceptable yield without the need for a lyophilization step. Drop.

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

[0065] As used herein, the term "crude" as in "crude carbetocin" refers to a pharmaceutical The crude peptide was purified by UV-HPLC. Purity less than 95%, e.g., less than 92.5%, e.g., 90% to 93%, as measured by C The crude peptide may contain one or more impurities, for example, 91% to 93%. Multiple inorganics, residual solvents (e.g., DMF), peptide-related impurities, and residual peptide chains The antibody may contain a pulling reagent.

[0066] (Product) Carbetocin in crystalline form / Carbetocin in solvated (e.g., hydrated) crystalline form / Desolvated crystalline forms of carbetocin may be greater than 95% pure.

[0067] Crude carbetocin can be prepared by methods well known to those skilled in the art, e.g., Ferring BV WO2009 / 122285 (International Patent Application No. PCT / IB2009 / 005351 No. 4,239,493.

[0068] According to the invention, in another aspect, a carbetocin according to the invention or a method according to the invention is Accordingly, there is provided a pharmaceutical composition comprising carbetocin. The pharmaceutical composition of the present invention can be used as a pharmaceutical. for use in treating reproductive disorders, e.g., for use in treating Prader-Willi syndrome (Ferring BV, International Patent Application No. WO2016 / 044131 (PCT / U or for example, vaginal delivery of an infant, cesarean section, For use in the treatment or prevention of, for example, uterine atony after delivery of an infant by incision; or Used to treat or prevent 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 (F WO2009 / 122285 (International Patent Application No. PCT / IB2 009 / 005351).

[0069] The present invention is illustrated below. The examples may also illustrate preferred embodiments of the invention. But that doesn't mean it's limiting. [Example]

[0070] Preparation of solvated crystalline form I of carbetocin Step i: Synthesis WO2009 / 122285 (International Patent Application No. PCT / 1999 / 122285) of Ferring BV It was synthesized in a purity of approximately 1.5g by a synthesis similar to that described in B2009 / 005351. 91% crude carbetocin was obtained.

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

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

[0073] The precipitated material was isolated.

[0074] XRPD analysis was performed on a PANalytical X'pert pro. The material was gently crushed to break up any agglomerates and to support the sample. Place in a multiwell plate with a Kapton or Mylar polymer film for The multiwell plate was then placed in the diffractometer and the 40 kV / 40 mA generator settings were used. CuK radiation (α) was measured in transmission mode (step size 0.0130° 2θ). 1λ=1.54060Å;α2=1.54443Å;β=1.39225Å;α1:α2 The analysis was performed using a ratio of 0.5.

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

[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 determined by UV-HP according to the method outlined in Table 4. Calculated at 96.2% by LC (Fig. 3a).

[0078] [Table 4] [Example]

[0079] Preparation of solvated crystalline form I of carbetocin Step i: Synthesis WO2009 / 122285 (International Patent Application No. PCT / 1999 / 122285) of Ferring BV It was synthesized in a purity of approximately 1.5g by a synthesis similar to that described in B2009 / 005351. 91% crude carbetocin was obtained.

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

[0081] Step iii: Addition of antisolvent Add enough acetonitrile to achieve an ethylene glycol:acetonitrile ratio of 6.7: The concentration was adjusted to 93.3 (v / v).

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

[0083] The precipitated material was isolated.

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

[0085] Carbetocin having an X-ray diffraction pattern substantially as shown in Table 1 and Figure 1 was obtained from the solution. It crystallized from the solution. [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 To do this, the crystallized material of Example 1 or Example 2 was desolvated and dried.

[0087] Form I crystalline material was washed in acetonitrile at 5°C followed by drying under vacuum to give the solvated form The desolvation of Form I results in a desolvation of Form I having a diffraction pattern substantially as shown in Table 2 and FIG. Solvated Form II crystals were produced. Form II crystals were determined by gas chromatography. have ethylene glycol levels below the ICH limit of 620 ppm The parameters were 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 a 30% ethanol solution prepared in advance. Add 3 mL of ethylene glycol:70% acetonitrile (v / v) solvent mixture and The mixture was heated to 40° C. with stirring for 30 minutes.

[0090] After 30 minutes, the mixture was filtered by centrifugation to remove insoluble impurities. To the 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 trinitrile.

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

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

[0093] The next day, the solid was analyzed by HPLC, mass loss / thermal activity, and HPLC for purity and assay (Figure 3b). TG / DTA for simplification of the analysis (Fig. 4b), and biased analysis for morphology and crystalline content. It was analyzed by photoluminescence microscope (PLM) and XRPD.

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

[0095] The TG / DTA data for the crystals formed by the method of Example 4 and shown in Figure 4b show a 110 This indicates that there was only about a 0.8% total mass loss up to 100°C. This indicates that the first mass loss was about A two-stage process with a mass loss of about 0.5% up to 60°C and a second mass loss of about 0.3% up to about 110°C. These mass losses were found to be due to the weakly bound acetonitrile and water on the surface. This loss of solvent is therefore not indicative of solvation of the crystalline form itself. The crystallinity of the solvated crystalline form II is not altered.

[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 WO2009 / 122285 (International Patent Application No. PCT / 1999 / 122285) of Ferring BV It was synthesized in a purity of approximately 1.5g by a synthesis similar to that described in B2009 / 005351. 93.5% crude carbetocin was obtained.

[0098] Step ii: Preparation of solutions Acetate buffer, 25 mM, pH 5.5, 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 the A 500 μL portion was dispensed into a vial, which was then sealed. The pH of the carbetocin solution was , 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, remove the vial and gently dissolve the solution using an Eppendorf glass pipette. Aspirate and return to the vial, thereby creating some seeds for crystallization. After mixing, the vial was resealed and kept at 40°C. After 9 days, a crystalline-like Particles formed and the precipitated material was isolated.

[0100] XRPD analysis was performed using a Cu-Kα1 monochromator (α1λ=1.54060 Å). The analysis was carried out on a PANalytical X'pert pro. The sample was analyzed at 2 to 35° 2θ. The material was gently crushed and smeared onto a Si zero-background wafer. , then it was scanned in transmission mode (scan rate 0.01) using a 45 kV / 40 mA generator setting. Slow rotation in the diffractometer performed at 0.01° / sec, step size 0.017°2θ The sample was placed in a sample holder that allows for measurement with a programmable entrance divergence slit. Ta.

[0101] The X-ray diffraction pattern of the obtained carbetocin crystals is shown in Table 3 and Figure 5. Compared with Examples 1 and 2 (Fig. 1, Table 1) and Examples 3 and 4 (Fig. 2, Table 2), 1 shows carbetocin in various crystalline forms or polymorphs.

[0102] Differential scanning calorimetry (DSC) analysis was performed on a Netzsch DSC 204F1. Milligrams of crystals were isolated from the mother liquor and stored in a fume hood at approximately 20% relative humidity (RH) for several hours. The crystals were gently crushed to a powder material, and 1.2 mg of this material was taken. 25 μL was placed in an Al pan. Before drilling a pinhole (0.25 mm diameter), The sample was analyzed from 20 to 250 °C using a heating rate of 5 K / min. did.

[0103] The DSC data for the crystals formed by the method of Example 5 and shown in Figure 7 indicate that the crystals are weakly bound to the surface. There was a loss of volatiles in the range of 40–120 °C, corresponding to the loss of dissolved and solvated water. The 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 used. was added to an Al pan and exposed to stepwise relative humidity (RH) changes during two successive cycles: 0-30-40-50-60-70-80-70-60-50-40-30-20-10 -0-10-20-30-40-50-60-70-80-90-80-70-60-5 Open loop mode of 0-40-30-20-10-0%RH. Temperature is maintained at 25°C. A nitrogen flow rate of 200 mL / min was used. The dm / dt criterion applied was 0.001 for 5 min. All steps were performed except for the step at 0% RH, which was set to 6 hours in weight percent per minute. The maximum time was 150 minutes.

[0105] The GVS data are shown in Figure 8. The GVS isothermal plot is shown in Figure 8b. The plateau at 80°C corresponds to the monohydrate and some loosely bound surface water. The second plateau in (w / w) corresponds to the pentahydrate and some loosely bound surface water. The pentahydrate exists above approximately 60% RH (sorption) and at approximately 40-90% RH (desorption). .

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

[0107] [Table 6]

Claims

1. Carbetocin in crystalline form.

2. Solvated crystalline forms of carbetocin.

3. Desolvated crystalline form of carbetocin.

4. Approximately 4.83, 7.43, 9.20, 17.87, 19.60, 20.43 and 21. characterized by an X-ray powder diffraction peak at 34 degrees 2θ (Cu-K). Carbetocin according to claim 1 or 2.

5. Claim 1 characterized substantially by the X-ray powder diffraction pattern illustrated in Figure 1.

10. Carbetocin according to any one of claims 1, 2 or 4.

6. Approximately 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.4 1. The method of claim 1, characterized by an X-ray powder diffraction peak at 5 degrees 2θ (Cu-K). Or carbetocin according to claim 3.

7. Claim 1 characterized substantially by the X-ray powder diffraction pattern illustrated in Figure 2.

7. Carbetocin according to any one of claims 1, 3 and 6.

8. Approximately 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39 degrees θ(Cu-Kα １ ) characterized by an X-ray powder diffraction peak at or 3. Carbetocin according to any one of claims 1 to 3.

9. A method for producing carbetocin in crystalline form, comprising the steps of crystallizing carbetocin. A method including:

10. The crystalline form of carbetocin is obtained from a mixture comprising carbetocin and one or more liquids. The one or more liquids are optionally ethylene glycol, acetonitrile, ethanol, methanol, propanol, isopropanol, 1,2-propanediol one or more of the group consisting of ethylene glycol and dimethylformamide, mixtures of ethanol and acetonitrile, e.g., ethanol, ethylene glycol and acetonitrile; 10. The method of claim 9, comprising a mixture of methyltrimethylsilyl and methyltrimethylsilyl.

11. The crystalline form of carbetocin is obtained from a mixture comprising carbetocin and one or more liquids. The one or more liquids may include one or both of water and an aqueous acetate buffer. The method of claim 9 .

12. The crystalline form of carbetocin is a mixture comprising carbetocin and one or more liquids. by cooling, for example, to 40°C to 5°C, or by adding carbetocin and one or more The temperature of a mixture containing multiple liquids can be changed, for example, from 40°C to 5°C and from 5°C to 40°C.

11. The method of claim 10, obtained by cycling.

13. The one or more liquids are ethylene glycol and acetonitrile in a ratio of 1:99 to 50. :50 ratio, for example 2:98 to 40:60 ratio, for example 3:97 to 35:65 ratio, for example 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 3 Ratios of 0:70, for example 15:85 to 25:75, for example 17.5:82.5 to 22.

13. The composition of claim 10 or claim 12, comprising a ratio of 5:77.5, for example a ratio of about 20:

80. How to do it.

14. A further step of adding an antisolvent to the mixture containing carbetocin and one or more liquids.

14. Any one of claims 10, 12 and 13, wherein the anti-solvent is optionally acetonitrile. The method according to any one of claims 1 to 4.

15. The crystalline form of carbetocin is prepared by adding a mixture containing carbetocin and one or more liquids. , for crystallization of carbetocin, 3 to 100 days, for example 3 to 60 days, for example 7 to 1 Maintain a temperature of at least 15°C, for example 20°C, for example 30°C, for example 40°C for 2 days. The method according to claim 11, wherein the method is obtained by

16. The one or more liquids include a mixture of ethylene glycol and acetonitrile, 15. The method of claim 14, wherein the solvent comprises acetonitrile.

17. The liquid or liquids are ethylene glycol and acetonitrile in a ratio of 15:85 to 2: 5:75 ratio, for example 17.5:82.5 to 22.5:77.5 ratio, for example about 20:8 0 ratio, and when acetonitrile antisolvent is added, the ratio of ethylene glycol to acetonitrile The ratio of tolyl is 1:99 to 30:70, for example, 2:98 to 25:75, for example, 3 :97 to 20:80, for example 5:95 to 20:80, for example 5:95 to 15:8 5 ratio, for example, 7.5:92.5 to 12.5:87.5 ratio, for example, about 10:90 ratio The method of claim 16 wherein

18. A mixture containing carbetocin and one or more liquids may be added to form crystals, e.g., carbetocin crystals.

18. The method of claim 9, further comprising the step of seeding the crystals. method.

19. The method includes the further step of desolvating the crystalline form of carbetocin, and drying the carbetocin.

19. The method of any one of claims 9 to 18, wherein

20. Desolvation of crystalline carbetocin involves the desolvation of crystalline carbetocin in an antisolvent, e.g., acetonitrile. The solvent removal is carried out by washing in nitrile, and the solvent removal is carried out at a temperature of 20° C. or lower, for example, −30° C. °C to 20°C, for example, -20°C to 20°C, for example, -10°C to 20°C, for example, -5°C to 15°C 20. The method of claim 19, which may be carried out at a temperature of, for example, 0°C to 10°C, for example, about 5°C. Law.

21. Carbetocin according to any one of claims 1 to 8 or any one of claims 9 to 20 A pharmaceutical composition comprising carbetocin made according to the described method.

22. 22. The pharmaceutical composition of claim 21 for use as a medicament.

23. For use in the treatment of neurological or reproductive disorders, e.g., for the treatment of Prader-Willi syndrome or for use in the treatment of infants, such as after vaginal delivery or delivery of an infant by Caesarean section. for use in the treatment or prevention of uterine atony; or for the treatment of women who develop postpartum hemorrhage (PPH). For use in the treatment or prevention of uterine atony in patients at risk of developing it; and / or for use by vaginal administration 23. A pharmaceutical composition according to claim 22 for use in the treatment or prevention of excessive postpartum bleeding. 。

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