RU2830121C2 - Method of producing pharmaceutical composition - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing carbetocin in a solvated crystalline form, 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), in which carbetocin in crystalline form is crystallized from a mixture containing carbetocin and one or more liquids, wherein the one or more liquids are a mixture of ethylene glycol and acetonitrile, by cooling the mixture containing carbetocin and one or more liquids, from 40 to 5 °C or by cyclic change of temperature of mixture containing carbetocin and one or more liquids, from 40 to 5 °C. Invention relates to a method of producing carbetocin in a solvated crystalline form, characterized by X-ray powder diffraction peaks at 4.34, 6.43, 8.66, 17.37, 19.03 and 19.39 degrees 2θ (Cu-K), in which carbetocin in crystalline form is crystallized from a mixture containing carbetocin and one or more liquids, wherein one or more liquids is an aqueous acetate buffer, by maintaining the mixture containing carbetocin and one or more liquids at a temperature of at least 15 °C for 3–100 days. Invention also relates to a method of producing carbetocin in a desolvated crystalline form, characterized by X-ray powder diffraction peaks at approximately 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ (Cu-K), involving the method according to claim 1, which includes an additional step of removing the solvent and drying carbetocin in crystalline form.

EFFECT: obtaining carbetocin in crystalline form.

8 cl, 11 dwg, 6 tbl, 5 ex

Description

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

State of the art

Carbetocin (also known as 1-desamino-1-monocarba-2-(O-methyltyrosine)oxytocin or 1-butanoic acid-2-(O-methyl-L-tyrosine)-1-carbaoxytocin) is a long-acting synthetic oligopeptide analogue of oxytocin with agonist activity. Carbetocin contains the following substitutions relative to oxytocin: a) the amino group of cysteine (position 1) by a hydrogen atom, b) its disulfide bond by a thioether bond, and c) the hydroxyl group of tyrosine (position 2) by a methyloxyl group. Carbetocin (PABAL ^® , DURATOCIN ^® ) is currently approved for the prevention of uterine atony following delivery of a baby by cesarean section under epidural or spinal anesthesia. The doses used for these medical indications are relatively small, for example, on the order of 100 micrograms at a time.

Recently, there has been an increased demand for oxytocin receptor agonists, particularly carbetocin. For example, oxytocin receptors have recently been indicated in the treatment of Prader-Willi syndrome (see WO 2016/044131). Prader-Willi syndrome is a genetic disorder characterized by excessive feeding, food-seeking behavior, rapid weight gain, obsessive behavior, and aggression in young children. As described in WO 2016/044131, patients treated with carbetocin show statistically significant improvements compared to placebo-treated patients after 15 days in quantitative assessments of excessive feeding, obsessive-compulsive disorder, food-seeking behavior, and clinical global impression. For this indication, it is necessary to obtain a relatively large amount of the peptide, because the doses used are significantly higher than those used in the treatment of uterine atony, for example, on the order of tens of milligrams per day, and this treatment is longer term. It would be desirable for such indications to obtain a relatively large amount of high-purity carbetocin.

Peptide synthesis can be accomplished using solid-phase synthesis techniques that are well known in the art. Liquid-phase synthesis is an alternative method that may be suitable for small amounts of peptide. This step in peptide production is known as the "preparatory process" and results in the formation of a crude peptide product.

Following the synthesis of a crude peptide, it is usually necessary to separate the peptide of interest from various peptide and non-peptide impurities. This step is known as the purification step.

There are many methods for purifying peptides known in the art. However, methods for purifying peptides usually include at least one chromatographic step, such as gel permeation chromatography, hydrophobic interaction chromatography, ion exchange chromatography, free moving boundary electrophoresis, affinity chromatography, high performance liquid chromatography (HPLC), etc. The most commonly used form of HPLC is reversed-phase HPLC (also known as RP-HPLC), in which peptides are eluted with an increasing amount of an organic solvent, such as acetonitrile, according to their hydrophobicity.

After the purification step, the peptide usually needs to be separated from the volatile solvents. This step is known as the isolation step. Known methods for separating the peptide from solvents include ultrafiltration and lyophilization.

Lyophilization (also known as freeze-drying) involves the step of rapidly freezing a peptide-containing solution, typically by immersing a container holding the solution in liquid nitrogen. The container is then placed in a vacuum chamber that contains a cooling coil. Volatile solvents are sublimated in the vacuum. The sublimation method ensures that the purified sample is kept cold.

Lyophilization is the most commonly used technology in the state of the art for isolating peptides from solution. In principle, the reason is that this technology is well known, reproducible and easy to perform. Additionally, the stability of peptides is usually increased at low temperatures.

Methods for the purification and isolation of carbetocin and related peptides are known in the art.

CN 104592362 describes a liquid chromatographic purification step for carbetocin followed by lyophilization. In most cases, the liquid chromatographic step is HPLC.

WO 2015/185584 describes the purification and lyophilization of oxytocin agonists other than carbetocin.

CN 102977192 describes a method for purifying carbetocin by combining liquid chromatography and ion exchange chromatography. After purification, the product is subjected to a desalting and lyophilization step.

CN 104744567 describes a method for purifying carbetocin using ion exchange chromatography followed by lyophilization.

CN 101531705 describes a method for purifying carbetocin using reversed-phase HPLC, followed by conversion of the product to the acetate salt using an ion exchange method. After conversion to the salt, the product is then lyophilized.

WO 2009/122285 describes a method for purifying oxytocin analogues comprising an HPLC step followed by a lyophilization step. Rudko A D et al. "Crystalline Salts of Oxytocin: X-ray crystallographic data" J. Crystal Growth, vol. 10, no. 3, 1971, pages 260-262 describes 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, pages 945-954 describes the characterization of pharmaceutical solids.

From the above references it can be seen that there is a strong trend in the state of the art towards the use of lyophilization as an isolation step in the synthesis of carbetocin and other oxytocin receptor agonists.

However, there are several problems associated with lyophilization, such as it takes a long time to process the peptide, and the cost of refrigerant and equipment is very high.

These problems may be acceptable when producing small amounts of peptide. However, when producing peptide in large quantities, lyophilization becomes a bottleneck in the production method. In addition, the proportion of total production costs spent on lyophilization increases with the mass of the peptide produced.

Accordingly, there is also a need in the art for an improved method for isolating carbetocin by eliminating the lyophilization bottleneck so that a larger amount of carbetocin of sufficient purity can be obtained to meet the needs of indications such as the treatment of Prader-Willi syndrome.

Brief description of the invention

In one aspect, the present invention relates to carbetocin in crystalline form.

In a further aspect, the present invention relates to a method for producing carbetocin in crystalline form, comprising a step of crystallizing the carbetocin.

In a further aspect, the present invention relates to a pharmaceutical composition comprising carbetocin of the present invention or carbetocin prepared according to the present invention.

Drawings relating to the present invention

The drawings relating to the present invention are described below:

Fig. 1 shows the X-ray diffraction pattern (Cu) of the solvated crystalline form I of carbetocin,

Fig. 2 shows the X-ray diffraction pattern (Cu) of desolvated crystalline form II of carbetocin,

Fig. 3 shows the HPLC chromatograms of the isolated solids from Example 1 (Fig. 3a) and the isolated solids from Example 4 (Fig. 3b),

Fig. 4 shows the TG/DTA (thermogravimetry/differential thermal analysis) data related to the solvated crystalline form I of carbetocin from Example 1 (Fig. 4a) and the desolvated crystalline form II of carbetocin from Example 4 (Fig. 4b),

Fig. 5 shows the X-ray diffraction pattern (Cu-Kα ₁ ) of crystalline carbetocin obtained in Example 5,

Fig. 6 shows the HPLC chromatogram of the isolated solids from Example 5,

Fig. 7 shows differential scanning calorimetry (DSC) data related to the isolated crystalline carbetocin from Example 5,

Fig. 8 shows the gravimetric vapor sorption (GVS) data of the isolated crystalline carbetocin from Example 5: the change in the mass plot (Fig. 8a) and in the isotherm plot (Fig. 8b).

Detailed description of the invention

Carbetocin was not previously known to form crystals. The inventors of the present application have surprisingly found that three crystalline forms of carbetocin can be formed as described herein, two of which can be designated as Form I and Form II. Form I is solvated (e.g., hydrated), while Form II is desolvated. Form II is highly stable (see Fig. 4B) and has an acceptably low ethylene glycol content (see Example 3) and can be used, for example, as a drug. Acceptably low ethylene glycol content is understood to mean an ethylene glycol content below the International Conference on Harmonization limit of 620 ppm, determined by gas chromatography. Form I can be used as a synthetic intermediate in the preparation of Form II. A third crystalline form is also described herein (see Example 5).

According to the present invention, in a first aspect, carbetocin is provided in crystalline form. In a second aspect, carbetocin is provided in solvated (e.g., hydrated) crystalline form. In a third aspect, carbetocin is provided in desolvated crystalline form.

By solvated is meant that the crystal structure contains either ordered or disordered solvent molecules. By disordered is meant that the positions of the solvent molecules or the positions of the atoms therein may vary within the crystal structure. The solvent molecules may be in a liquid or gaseous state 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 in some cases 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, such as at least 5 solvent molecules per carbetocin molecule. Accordingly, carbetocin in solvated crystalline form may be in the form of a mono-, di-, tri-, tetra-, penta- or hexa-hydrate of the solvated crystalline form. Preferably, when carbetocin is in solvated crystalline form, the solvated crystalline form is a monohydrate or pentahydrate. Accordingly, in one aspect, carbetocin is in a crystalline form of a monohydrate or pentahydrate. Such carbetocin may contain either ordered or disordered solvent molecules. It is believed that the number of solvent molecules does not affect whether they are ordered or disordered.

By desolvated is meant that the crystal structure contains few or no ordered or disordered solvent molecules. There may be no more than 2 solvent molecules per carbetocin molecule, e.g., no more than 1 solvent molecule per carbetocin molecule, e.g., no more than 0.5 solvent molecule per carbetocin molecule, e.g., no more than 0.2 solvent molecule per carbetocin molecule, e.g., no more than 0.1 solvent molecule per carbetocin molecule, e.g., no more than 0.05 solvent molecule per carbetocin molecule, e.g., no more than 0.02 solvent molecule per carbetocin molecule, e.g., no more than 0.01 solvent molecule per carbetocin molecule.

In order to determine the crystalline form, powder X-ray diffraction analysis (PSXDA) can be performed. In the present invention, PSXDA was performed using Cu K radiation (α ₁ λ=1.54060 , α ₂ = 1.54443 , β=1.39225 , ratio _α1 : _α2 =0.5) on PANalytical X'pert pro, as further detailed in Example 1. Carbetocin in crystalline form and/or carbetocin in solvated crystalline form may be characterized by powder X-ray diffraction peaks at about 4.83, 7.43, 9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu), and/or may be characterized by an X-ray powder diffraction pattern (Cu) substantially as shown in Fig. 1, and/or may 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 powder X-ray diffraction peaks (Cu) shown in Table 1. Accordingly, in one aspect, carbetocin in crystalline form is characterized by powder X-ray diffraction peaks at about 4.83, 7.43, 9.20, 17.87, 19.60, 20.43, and 21.34 degrees 2θ, performed using Cu K radiation (α ₁ λ=1.54060 α ₂ = 1.54443 , β=1.39225 ratio α ₁ : α ₂ = 0.5).

Carbetocin in crystalline form and/or carbetocin in desolvated crystalline form may be characterized by powder X-ray diffraction peaks at about 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ (Cu), and/or may be characterized by a powder X-ray diffraction pattern (Cu) substantially as shown in Fig. 2, and/or may 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) powder X-ray diffraction peaks shown in Table 2. Accordingly, in one aspect, carbetocin in crystalline form is characterized by powder X-ray diffraction peaks at about 4.11, 4.39, 5.60, 7.45, 17.75, 19.16, and 19.454 degrees 2θ, performed using Cu K radiation (α ₁ λ=1.54060 α ₂ = 1.54443 β=1.39225 ratio α ₁ : α ₂ = 0.5).

Carbetocin in crystalline form may be characterized by powder X-ray diffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03 and 19.39 degrees 2θ (Cu-Kα ₁ ), and/or may be characterized by a powder X-ray diffraction pattern (Cu-Kα ₁ ) substantially as shown in Fig. 5, and/or may 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 powder X-ray diffraction (Cu-Kα ₁ ) peaks shown in Table 3. Accordingly, in one aspect, carbetocin in crystalline form is characterized by powder X-ray diffraction peaks at about 4.34, 6.43, 8.66, 17.37, 19.03, and 19.39 degrees 2θ, performed using Cu Kα ₁ radiation (α1 λ=1.54060 ).

(FWHM = full line width at half height)

Table 3 shows the data obtained from Example 5. Note that the radiation source for the values presented in Table 3 is a Cu-Kα ₁ source, while the radiation source for the values presented in Tables 1 and 2 is a Cu-K source. The above table of PRSA peaks, Table 3 (Fig. 5, Example 5), however, shows carbetocin in a different crystalline form or polymorph compared to Examples 1 and 2 (Fig. 1, Table 1) and Examples 3 and 4 (Fig. 2, Table 2).

According to the present invention, in a further aspect, there is provided a method for producing carbetocin in crystalline form, comprising the step of crystallizing the carbetocin.

Crystallization is the process by which a crystalline form of carbetocin is formed from carbetocin dissolved in a solvent. A crystalline form is a solid with a regularly repeating internal arrangement of atoms and external planar faces. Crystalline forms can be distinguished from amorphous forms by powder X-ray diffraction analysis. Crystalline forms are characterized by the powder X-ray diffraction peaks described in this document. In amorphous solid forms, the X-ray powder diffraction pattern is essentially continuous, i.e., without clearly defined peaks.

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

One or more liquids may include a mixture of ethylene glycol and acetonitrile.

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

One or more liquids may comprise water. One or more liquids may comprise an aqueous acetate buffer. In a particular aspect, carbetocin in crystalline form is crystallized from a mixture comprising carbetocin and one or more liquids comprising water and/or an aqueous acetate buffer.

In one embodiment, one or more liquids may be water. If 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, more preferably a pH of about 3.5.

In one embodiment, one or more liquids can be an aqueous acetate buffer. The aqueous acetate buffer can be formed from an aqueous mixture of acetic acid and an acetate salt. The acetate salt can be any acetate salt with a suitable counterion. A suitable counterion can be, for example, an alkali metal ion, an alkaline earth metal ion, or an organic cation. The counterion can be lithium, sodium, potassium, magnesium, calcium, or ammonium. Preferably, the counterion can be sodium or potassium, most preferably, the counterion can be sodium. Preferably, the aqueous acetate buffer has a pH of about 4 to 7, preferably a pH of about 5 to 6, most preferably a pH of about 5.5. Preferably, the aqueous acetate buffer has a concentration of 20 to 30 mM, most preferably a concentration of about 25 mM.

Carbetocin in crystalline form 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 from 5°C to 50°C per hour, for example 35°C per hour. By cycling the temperature of the mixture is meant that the temperature is successively lowered and then increased, or vice versa. The temperature can be lowered and then increased, 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. After cooling or cycling the temperature of the mixture containing carbetocin and one or more liquids, the temperature can be maintained, for example at 5°C, for a period of time suitable for the formation of crystalline carbetocin. Typically, crystalline carbetocin may form and be recovered within 6-24 hours, such as within about 12 hours or within about 18 hours. Cooling, cycling or maintaining the temperature may be done with or without stirring the mixture.

Alternatively, it appears that carbetocin in crystalline form 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 at least 15°C, such as 20°C, such as 30°C, such as 40°C, for a period of time suitable for the formation of crystalline carbetocin. Typically, crystalline carbetocin is formed within 3-100 days, more typically within 3-60 days, most typically within 7-12 days. In one alternative embodiment, carbetocin in crystalline form 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 3-60 days. In another alternative embodiment, carbetocin in crystalline form can be obtained by maintaining a mixture containing carbetocin and water, or a mixture containing carbetocin and an aqueous acetate buffer, at a temperature of 40°C for about 7-12 days. In these alternative embodiments, all other steps described below can be performed, except for the step involving the addition of an antisolvent.

Carbetocin can be crystallized from a mixture comprising at least one solvent and at least one antisolvent. The solvent is a liquid in which carbetocin is readily soluble or readily soluble. The solvent may be any solvent in which carbetocin is soluble in an amount under standard conditions of 0.01 mg/ml or more, such as 0.05 mg/ml or more, such as 0.1 mg/ml or more, such as 0.5 mg/ml or more, such as 1 mg/ml or more, such as 5 mg/ml or more, such as 10 mg/ml or more, such as 20 mg/ml or more. The antisolvent is a liquid in which carbetocin dissolves less readily relative to the solvent, or in which carbetocin is less soluble relative to 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, such as less than 10 mg/ml, such as less than 5 mg/ml, such as less than 1 mg/ml, such as less than 0.5 mg/ml, such as less than 0.1 mg/ml, such as less than 0.05 mg/ml, such as less than 0.01 mg/ml. It is clear to the skilled person that when carbetocin is soluble in an amount of, for example, 10 mg/mL or more in a solvent, it is less soluble in an antisolvent, i.e. it dissolves 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.

The solvent may comprise one or more liquids 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) as described by Christian Reichardt (Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, ^3rd ed., 2003) of 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, such as greater than 1.0. The solvent may be or comprise any one or more of water, an aqueous acetate buffer, or an alcohol, such as any one or more of water (ODI = 1.000), an aqueous acetate buffer solution, ethylene glycol (ODI = 0.790), ethanol (ODI = 0.654), methanol (ODI = 0.762), propanol (ODI = 0.803), isopropanol (ODI = 0.787), or 1,2-propanediol (ODI = 0.72).

Carbetocin in crystalline form can be crystallized from a mixture comprising carbetocin and one or more liquids, wherein carbetocin is present in the solvent 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. 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.

The method may include the additional step of adding an antisolvent to the mixture containing carbetocin and one or more liquids, for example adding the antisolvent before cooling the mixture.

The antisolvent may have a relative polarity index (RPI) as described by Christian Reichardt (Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, ^3rd ed., 2003) of less than 1, such as less than 0.9, such as less than 0.8, such as less than 0.75, such as less than 0.7, such as less than 0.6, such as less than 0.5. The antisolvent may be or comprise any one or more of an ester, ketone, nitrile, or ether, such as 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).

Thus, in one embodiment of the method, carbetocin can be crystallized from a mixture comprising carbetocin and one or more liquids comprising ethylene glycol and acetonitrile. Carbetocin may be present in the mixture comprising carbetocin and one or more liquids in a concentration of from 10 mg/mL to 150 mg/mL, most preferably about 100 mg/mL. Ethylene glycol and acetonitrile may be present in a ratio of from 5:95 to 35:65. Carbetocin in crystalline form can be obtained by cooling the mixture comprising 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 period of time suitable for the formation of crystalline carbetocin, such as for about 12 hours or for about 18 hours.

The method may include the additional step of seeding a mixture comprising carbetocin and one or more liquids with a crystal, such as a crystal of carbetocin, such as a crystal of solvated crystalline form I of carbetocin.

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 a different material.

The method may include the additional step of inducing crystallization in the mixture containing carbetocin and one or more liquids. Crystallization may be induced by any suitable means to promote nucleation and crystal growth, for example by agitating the surface of the mixture containing carbetocin and one or more liquids to create seed crystals, for example by pipetting liquid from the surface and pipetting the liquid onto the surface of the mixture containing carbetocin and one or more liquids, or by scratching the location where the surface of the mixture containing carbetocin and one or more liquids contacts the surface of a container in which the mixture is held.

The method may include an additional step of removing the solvent and possibly drying the carbetocin in crystalline form.

By solvent removal is meant the removal of a portion or substantially all of the solvated molecules from the crystal structure of carbetocin such that the crystal structure includes little or no ordered or disordered solvent molecules. In a preferred embodiment, solvent removal means converting carbetocin from the pentahydrate crystal form to the monohydrate crystal form.

The removal of the solvent from the carbetocin in crystalline form can be accomplished by washing the carbetocin in crystalline form with an antisolvent, such as acetonitrile, optionally at a temperature of 20°C or lower, such as from -30°C to 20°C, such as from -20°C to 20°C, such as from -10°C to 20°C, such as from -5°C to 15°C, such as from 0°C to 10°C, such as about 5°C, and then drying, such as drying under vacuum. Drying can take place under vacuum for a period of time suitable to effect the removal of the solvent, such as more than 1 hour, such as for about 24 hours. Preferably, the carbetocin in crystalline form can be washed with acetonitrile at a temperature of about 5°C and dried under vacuum at a temperature of about 20°C for about 24 hours to effect the removal of the solvent.

Solvent removal can also be accomplished by heating the crystalline carbetocin to a temperature of at least 40°C to no more than 190°C or by exposing the crystalline carbetocin to an environment of low relative humidity, such as a relative humidity of 40% or less.

Thus, in one embodiment of the method, carbetocin can be crystallized from a mixture comprising carbetocin and one or more liquids comprising ethylene glycol and acetonitrile. Carbetocin may be present in the mixture comprising carbetocin and one or more liquids at a concentration of from 10 mg/mL to 150 mg/mL, most preferably about 100 mg/mL. Ethylene glycol and acetonitrile may be present in a ratio of from 5:95 to 35:65. An additional antisolvent such as acetonitrile may be added. Carbetocin in crystalline form can be obtained by cooling the mixture comprising 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 period of time suitable for the isolation of crystalline carbetocin, such as for about 12 hours or for about 18 hours. Carbetocin in crystalline form can be washed with acetonitrile at a temperature of about 5°C and dried under vacuum at a temperature of about 20°C for about 24 hours to remove the solvent.

If necessary, a filtration step may be performed prior to crystallization. The filtration step preferably comprises filtration by centrifugation. Accordingly, in one aspect, a method for producing carbetocin in crystalline form comprises the steps of (1) filtration, preferably by centrifugation, and (2) crystallization.

If desired, a washing step can be performed prior to crystallization. For example, carbetocin, for example crude carbetocin, can be suspended, for example suspended in acetonitrile, for example suspended in acetonitrile for 2 hours to a week, for example suspended in acetonitrile for about 18 hours with constant stirring. Washing crude carbetocin increases the purity before crystallization by about 1 2% and significantly increases the assay content from about 44% to about 70% (in acetonitrile). Accordingly, in one aspect, a method for producing carbetocin in crystalline form includes the steps of (1) washing carbetocin, for example crude carbetocin, with acetonitrile and (2) crystallizing. In another aspect, a method for producing carbetocin in crystalline form comprises the steps of (1) washing carbetocin, such as crude carbetocin, with acetonitrile, (2) filtration, preferably by centrifugation, and (3) crystallization.

The present inventors have advantageously and unexpectedly discovered that it is possible to isolate carbetocin without any lyophilization by crystallization of carbetocin, for example, crystallization of carbetocin from solution.

The method provides high purity carbetocin with acceptable yield without the need for a lyophilization step.

The carbetocin in the mixture comprising carbetocin and one or more liquids may be substantially pure carbetocin or, alternatively, may be crude carbetocin.

As used herein, the term "crude", such as in "crude carbetocin", means carbetocin that is insufficiently pure for use as a pharmaceutical product. The crude peptide may have a purity of less than 95%, such as less than 92.5%, such as from 90% to 93%, such as from 91% to 93%, as measured by HPLC with UV detection. Impurities found in the crude peptide may include one or more inorganic substances, residual solvent (e.g., dimethylformamide (DMF)), peptide-related impurities, and residual peptide binding reagents.

(The resulting) carbetocin in crystalline form/carbetocin in solvated (e.g. hydrated) crystalline form/carbetocin in desolvated crystalline form may have a purity of greater than or equal to 95%.

Crude carbetocin can be synthesized using methods known in the art, for example methods similar to those described in WO 2009/122285 (International Patent Application No. PCT/IB2009/005351) by Ferring B.V.

According to the invention, in a further aspect, there is provided a pharmaceutical composition comprising carbetocin according to the invention, or carbetocin obtained according to the method according to the invention. The pharmaceutical composition according to the invention can be used as a medicine. The pharmaceutical composition of the invention can be used in the treatment of a neurological disorder or a reproductive disorder, for example used in the treatment of Prader-Willi syndrome (as described in WO 2016/044131 (International Patent Application No. PCT/US 2015/04911) by Ferring B.V.), or, for example, used in the treatment or prevention of uterine atony, for example after vaginal childbirth, childbirth by caesarean section, or used in the treatment or prevention of uterine atony in a patient who is at risk of developing postpartum hemorrhage, and/or used in the treatment or prevention of excessive bleeding after vaginal delivery (as described in WO 2009/122285 (International Patent Application No. PCT/IB 2009/005351) by Ferring B.V.).

The present invention is explained below using examples. The examples may describe preferred embodiments of the invention, but they are not considered to be limiting in any way.

Example 1. Preparation of solvated crystalline form I of carbetocin

Stage 1. Synthesis

Crude carbetocin with a purity of approximately 91% was obtained using synthetic methods similar to those described in WO 2009/122285 (International Patent Application No. PCT/IB 2009/005351) by Ferring B.V.

Stage 2. Obtaining a solution

60 mg of crude carbetocin obtained in step 1 were 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 seeded with crystals of (solvated) form I of carbetocin. Clearly, seeding is not essential, but it can accelerate crystallization.

Stage 3. Crystallization

The solution obtained in step 2 was heated to 40°C and maintained at that 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 thirty minutes, cooled to 5°C over one hour and then maintained at 5°C overnight with constant stirring.

The precipitated material was isolated.

XRD was performed on a PANalytical X'pert pro. Samples were scanned from 3 to 35° 2θ. The material was gently ground to release any agglomerates and loaded onto a multiwell plate with a Kapton or Mylar polymer film to support the sample. The multiwell plate was then placed in a diffractometer and analyzed using Cu K radiation (α ₁ λ=1.54060 , α ₂ = 1.54443 , β=1.39225 , ratio α ₁ : α ₂ = 0.5), operating in transmission mode (step size 0.0130° 2θ) using generator settings of 40 kV/40 mA.

Carbetocin crystallized from the solution, having an X-ray diffraction pattern essentially as shown in Table 1 and Fig. 1 (Form I).

Solids were analyzed by TG/DTA for ease of determination of mass loss/thermal effects (Fig. 4a).

The purity of carbetocin in the (solvated) crystalline form was 96.2%, calculated by UV-HPLC (Fig. 3a) according to the method described in Table 4.

Example 2. Preparation of solvated crystalline form I of carbetocin

Stage 1. Synthesis

Crude carbetocin with a purity of approximately 91% was obtained using synthetic methods similar to those described in WO 2009/122285 (International Patent Application No. PCT/IB 2009/005351) by Ferring B.V.

Stage 2. Obtaining a solution

60 mg of crude carbetocin obtained in step 1 were 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 seeded with crystals of (solvated) form I of carbetocin. Clearly, seeding is not essential, but it can accelerate crystallization.

Step 3: Adding Antisolvent

Enough acetonitrile was added to change the ethylene glycol:acetonitrile ratio to 6.7:93.3 (v/v).

Stage 4. Crystallization

The solution obtained in step 3 was heated to 40°C and maintained at that 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 thirty minutes, cooled to 5°C over one hour and then maintained at 5°C overnight with constant stirring.

The precipitated material was isolated.

PRSA was performed as described above in connection with Example 1.

Carbetocin crystallized from the solution and had an X-ray diffraction pattern essentially the same as that shown in Table 1 and Fig. 1.

Example 3. Preparation of desolvated crystalline form II of carbetocin

To remove ethylene glycol present in the crystallized material obtained in Examples 1 and 2, the crystallized material of Example 1 or Example 2 was subjected to solvent removal and dried.

Washing of the crystallized Form I material with acetonitrile at 5°C was followed by drying under vacuum, resulting in removal of the solvent from the solvated Form I to yield crystals of desolvated Form II, which had a diffraction pattern substantially as shown in Table 2 and Fig. 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 the parameters as in Table 5 below.

Example 4. Preparation of desolvated crystalline form II of carbetocin To approximately 300 mg of crude carbetocin (purity approximately 91.3%) were added 3 ml of a pre-prepared solvent mixture of 30% ethylene glycol: 70% acetonitrile (v/v) and the mixture was heated to 40°C for 30 minutes with constant stirring.

After 30 minutes, the mixture was filtered by centrifugation to remove any 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 all the acetonitrile had been added.

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

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.

The following day, the solids were analyzed by HPLC for purity and quantification (Fig. 3b), TG/DTA for easy determination of mass loss/thermal effects (Fig. 4b), and by polarizing microscopy (PM) and PCR to determine morphology and crystalline content.

PM analysis showed that the final isolated solid contained a mixture of agglomerates (50-100 µm) that readily disintegrated into very small needle-like crystals (length <10 µm).

The TG/DTA data for the crystals formed by the method of Example 4 and shown in Fig. 4b indicate that there was an overall mass loss of only about 0.8% up to 110°C. This process was found to occur in two stages, with a first 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 correspond to weakly surface bound acetonitrile and water and are not indicative of solvation of the crystalline form itself. Accordingly, the loss of this solvent does not change the degree of crystallinity of the desolvated crystalline Form II.

These results indicate that desolvated crystalline form II of carbetocin is highly stable.

Example 5. Preparation of solvated crystalline form I of carbetocin

Stage 1. Synthesis

Crude carbetocin with a purity of approximately 93.5% was obtained using synthetic methods similar to those described in WO 2009/122285 (International Patent Application No. PCT/IB 2009/005351) by Ferring B.V.

Stage 2. Obtaining a solution

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 1 was dissolved in 16.6 mL of acetate buffer. The solution was filtered using a 0.22 μm PVDF syringe filter and 500 μL portions of the solution were aliquoted into vials, which were then sealed. The pH of the carbetocin solution was 5.3.

Stage 3. Crystallization

The solution obtained in step 2 was heated to 40°C and kept in sealed vials at this temperature. After 3 days, the vials were removed and a glass Eppendorf pipette was used to gently suck the solution upwards and back into the vials to thereby create some nuclei for crystallization. After pipetting, the vials were resealed and kept at 40°C. After 9 days, particles with a crystalline appearance had formed. The precipitated material was isolated.

PRSA was performed on a PANalytical X'pert pro with a Cu-Kα ₁ monochromator (α ₁ λ=1.54060 ). The samples were scanned from 2 to 35° 2θ. The material was carefully ground and spread onto a zero-background Si wafer, which was then placed in a slowly rotating sample holder in the diffractometer operated in transmission mode (scan rate 0.01°c, step size 0.017° 2θ) using a generator setting of 45 kV/40 mA. The measurements were performed using a programmable slit angle deflection.

The X-ray diffraction pattern of the obtained carbetocin crystals is shown in Table 3 and Fig. 5. The X-ray diffraction pattern shows carbetocin with a different crystalline form or polymorphic modification compared to Examples 1 and 2 (Fig. 1, Table 1) and Examples 3 and 4 (Fig. 2, Table 2).

Differential scanning calorimetry (DSC) analysis was performed on a Netzsch DSC 204F1. A few milligrams of crystals were isolated from the mother liquor and allowed to air dry in a fume hood for several hours at a relative humidity (RH) of approximately 20%. The crystals were carefully ground to a powder and 1.2 mg of this material was loaded into a 25 µl aluminum cuvette. A lid previously pierced with a pinhole (0.25 mm in diameter) was fitted and crimped onto the cuvette. The sample was analyzed from 20 to 250°C using a heating rate of 5 K/min.

DSC data for crystals formed by the method of Example 5 and shown in Fig. 7 indicate that there is a loss of volatile material in the region from 40 to 120°C, which corresponds to a loss of weakly surface bound water and solvated water. The melting endothermicity with an onset at 192°C corresponds to the melting of anhydrous carbetocin.

Gravimetric vapor sorption (GVS) was performed on a SMS DVS-1. 1.4 mg of crystals and powder were added to an aluminum cuvette and subjected to stepwise changes in relative humidity (RH) during two successive 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 the open-loop control mode. The temperature was maintained at 25 °C and pure nitrogen was used at a flow rate of 200 ml/min. The applied dm/dt criterion was 0.001 mass%. %/min over a 5 minute window, with a maximum time of 150 minutes for all stages except for stages at 0% RH, when it was set to 6 hours.

The GSP data are shown in Fig. 8. The GSP isotherm plot is shown in Fig. 8b. The plateau at about 2% (w/w) corresponds to the monohydrate plus some loosely bound surface water. The second plateau at about 8% (w/w) corresponds to the pentahydrate with some loosely bound surface water. The pentahydrate exists at RH of about 60% and above (sorption) and RH from about 40 to 90% (desorption).

The purity of carbetocin in the (solvated) crystalline form was 98.7%, calculated by UV-HPLC according to the method given in Table 6.

Claims (8)

1. A method for producing carbetocin in a solvated crystalline form characterized by powder X-ray diffraction peaks at 4.83, 7.43, 9.20, 17.87, 19.60, 20.43 and 21.34 degrees 2θ (Cu-K), wherein carbetocin in crystalline form is crystallized from a mixture containing carbetocin and one or more liquids, wherein the one or more liquids are a mixture of ethylene glycol and acetonitrile, by cooling the mixture containing carbetocin and one or more liquids from 40 to 5°C or by cycling the temperature of the mixture containing carbetocin and one or more liquids from 40 to 5°C. 2. A method for producing carbetocin in a solvated crystalline form characterized by powder X-ray diffraction peaks at 4.34, 6.43, 8.66, 17.37, 19.03 and 19.39 degrees 2θ (Cu-K), wherein carbetocin in crystalline form is crystallized from a mixture containing carbetocin and one or more liquids, wherein one or more liquids is an aqueous acetate buffer, by maintaining the mixture containing carbetocin and one or more liquids at a temperature of at least 15°C for 3-100 days. 3. The method according to claim 1, wherein one or more liquids contain ethylene glycol and acetonitrile in a ratio of 1:99 to 50:50. 4. The method according to any one of claims 1 or 3, comprising the additional step of adding an antisolvent to the mixture comprising carbetocin and one or more liquids, wherein the antisolvent is optionally acetonitrile. 5. The method of claim 4, wherein one or more liquids comprise ethylene glycol and acetonitrile in a ratio of 15:85 to 25:75 and wherein the addition of acetonitrile as an antisolvent changes the ratio of ethylene glycol to acetonitrile to a ratio of 1:99 to 30:70. 6. The method according to any one of claims 1 to 5, comprising the additional step of seeding the mixture containing carbetocin and one or more liquids with a crystal. 7. A method for producing carbetocin in a desolvated crystalline form characterized by powder X-ray diffraction peaks at 4.11, 4.39, 5.60, 7.45, 17.75, 19.16 and 19.45 degrees 2θ (Cu-K), comprising the method of claim 1, which includes the additional step of removing the solvent and drying the carbetocin in crystalline form. 8. The method according to claim 7, wherein the removal of the solvent from the carbetocin in crystalline form is carried out by washing the carbetocin in crystalline form with an antisolvent, optionally at a temperature of 20°C or below.