Methods of manufacturing peptide salts

Abstract

The present disclosure relates to methods for preparing peptide salts, in particular pharmaceutically acceptable peptide salts, such as salts of atosiban (e.g. atosiban pamoate). The present disclosure further extends to the preparation of particles of such salts, such as via a spray drying process.

Description

[0001] Methods of manufacturing peptide salts

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to methods for preparing peptide salts, in particular pharmaceutically acceptable peptide salts, such as salts of atosiban. The present disclosure further extends to the preparation of particles of such salts, such as via a spray drying process. The disclosure further relates to the peptide salts and particles of such salts that are made by these methods.

[0004] BACKGROUND

[0005] Atosiban is a synthetic peptide that acts as an inhibitor of oxytocin and vasopressin. Atosiban is described in the literature as having a number of therapeutic uses, including as a tocolytic agent (e.g. to delay preterm birth and / or prevent premature labour). Atosiban comprises the following structure:

[0006] Atosiban comprises a free base and may form acid addition salts. By way of example, atosiban may form an acid addition salt with acetic acid to provide atosiban acetate. Atosiban acetate is marketed under the brand name Tractocile® for use in delaying pre-term birth.

[0007] Pamoic acid is an organic acid that may be used to form acid addition salts. The chemical name of pamoic acid is 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid). Its structure is shown below:

[0008] CN114249800A (SHENZHEN SHENYIN PHARMACEUTICAL CO LTD) describes a method of preparing polypeptide pamoate salts. The method involves the use of a chromatographic column with a reversed-phase polymer filler as a stationary phase. The column must be equilibrated prior to loading of a peptide solution and subsequently, a pamoic acid inorganic salt aqueous solution and an organic solvent are loaded onto the column in a certain proportions, to allow the ion exchange to be performed on the column. CN114249800A describes that its chromatographic method provides a better control of impurities, ions and / or solvent residues in the salts produced.

[0009] SUMMARY

[0010] The present disclosure is based on the finding that an atosiban pamoate salt can be prepared via a precipitation method. Surprisingly the present inventors have identified that the atosiban pamoate produced by way of the disclosed precipitation methods provides compounds with a high degree of purity without the need for complex processing and / or purification steps. Such methods may be particularly amenable to being scaled-up for manufacture.

[0011] Thus, according to a first aspect there is provided a method for preparing an atosiban pamoate salt comprising:

[0012] (i) contacting and / or reacting together a first atosiban salt and an inorganic pamoate salt, wherein atosiban pamoate is formed as a precipitate.

[0013] The first atosiban salt may be any suitable atosiban salt that is soluble in a solvent in which atosiban pamoate is insoluble (e.g. under the temperature and pressure conditions at which the reaction takes place). The first atosiban salt is not atosiban pamoate. In some examples, the first atosiban salt may be a salt having a solubility of at least 10 mg / mL at 25 °C in aqueous solution. By way of representative example, the first atosiban salt may be selected from atosiban acetate, atosiban hydrochloride and atosiban trifluoroacetate, or the like.

[0014] By way of example, the method may comprise contacting the first atosiban salt (e.g. atosiban acetate) and an inorganic pamoate salt under such conditions so as to allow them to react. For example, the first atosiban salt (e.g. atosiban acetate) and an inorganic pamoate salt may be contacted and / or reacted together in a reaction mixture (e.g. a suspension or solution).

[0015] In particular, under the conditions as disclosed herein, when the first atosiban salt (e.g. atosiban acetate) is contacted and / or reacted together with an inorganic pamoate salt in a reaction mixture (e.g. a solution), an ion exchange reaction may take place, following which the atosiban pamoate can precipitate out. Surprisingly, the inventors have identified that this precipitation method can provide atosiban pamoate salt products with a high degree of purity. This method of providing atosiban pamoate consequently offers improved ease of manufacture whilst maintaining good levels of purity.

[0016] Suitable solvents to form the reaction mixtures (e.g. solutions) that are used in the methods described herein include aqueous solutions. In some examples, the solvent may be water, e.g. deionised water. By way of further example, suitable solvents may include aqueous buffer solutions, such as sodium phosphate buffer, phosphate- buffered saline (PBS) buffer, Tris buffer (tris(hydroxymethyl)aminomethane buffer) and HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethane sulfonic acid) buffer and the like.

[0017] In some examples, the method may comprise:

[0018] (i) providing a solution of the first atosiban salt (e.g. atosiban acetate); and

[0019] (ii) contacting the solution of the first atosiban salt (e.g. atosiban acetate) with an inorganic pamoate salt; wherein atosiban pamoate is formed as a precipitate.

[0020] In some examples, the method further comprises preparing a solution of the first atosiban salt, e.g. preparing a solution of the atosiban acetate. For example, the solution of the first atosiban salt may be prepared by adding and / or dissolving the first atosiban salt in any suitable solvent. Suitable solvents for use in preparing solutions of the first atosiban salt include any of those described herein (e.g. aqueous solutions such as water (e.g. deionized water)). By way of further example, suitable solvents may include aqueous buffer solutions, such as sodium phosphate buffer, phosphate- buffered saline (PBS) buffer, Tris buffer (tris(hydroxymethyl)aminomethane buffer) and HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethane sulfonic acid) buffer and the like.

[0021] The concentration of the first atosiban salt (e.g. atosiban acetate) in the solution may be between about 0.05 %w / v and about 50%w / v, between about 0.1%w / v and about 20%w / v, between about 0.5 %w / v and about 10%w / v, or between about 1%w / v and about 5%w / v. In some examples, the concentration of the first atosiban salt (e.g. atosiban acetate) in the solution may be between about 1.5%w / v and 4%w / v. In yet further examples, the concentration of the first atosiban salt (e.g. atosiban acetate) in the solution may be about 2.5%w / v or about 3.3%w / v. References to the concentration may refer to the concentration of the first atosiban salt (e.g. atosiban acetate) in a starting solution and / or the concentration of the first atosiban salt (atosiban acetate) in the solution following addition of the metal pamoate (e.g. if the volume has increased due to the addition of the inorganic pamoate in the form of a solution).

[0022] Inorganic pamoate salts useful in the disclosed methods may include metal pamoate salts. Examples of metal pamoate salts that may be used in the methods disclosed herein include alkali and alkali earth metal pamoate salts. Representative examples include, but are not limited to, sodium pamoate and potassium pamoate. In particular examples of the present disclosure, the metal pamoate salt may be sodium pamoate.

[0023] The step of contacting the solution of the first atosiban salt (e.g. atosiban acetate) with an inorganic (e.g. metal) pamoate salt may comprise adding the inorganic pamoate salt to the solution of the first atosiban salt. The inorganic pamoate salt may be added in the form of a solution or a suspension. By way of example, a solution of the inorganic pamoate salt may be prepared by adding and / or dissolving the inorganic pamoate in any suitable solvent.

[0024] Suitable solvents for use in preparing solutions of the inorganic pamoate salt include aqueous solutions. In some examples, the solvent may be water, e.g. deionised water. By way of further example, suitable solvents may include aqueous buffer solutions, such as sodium phosphate buffer, phosphate-buffered saline (PBS) buffer, Tris buffer (tris(hydroxymethyl)aminomethane buffer) and HEPES (2-[4-(2-Hydroxyethyl)-1- piperazinyl]ethane sulfonic acid) buffer and the like.

[0025] The concentration of the inorganic pamoate salt in the solution may be between about 0.01 %w / v and about 50%w / v, between about 0.05%w / v and about 20%w / v, between about 0.1%w / v and about 10%w / v, or between about 0.25%w / v and about 5%w / v. In some examples, the concentration of the inorganic pamoate salt in the solution may be between about 0.5%w / v and 4%w / v. In yet further examples, the concentration of the inorganic pamoate salt in the solution may be about 2.3%w / v or about 0.6%w / v.

[0026] References to the concentration may refer to the concentration of inorganic pamoate in a starting solution and / or the concentration of inorganic pamoate in the final solution following addition to the solution of the first atosiban salt.

[0027] The molar ratio of the first atosiban salt (e.g. atosiban acetate) to inorganic pamoate used, contacted and / or reacted together in the reaction may be anywhere between about 10:1 and about 1 :10, such as between about 5:1 and about 1 :5, or between 3:1 and 1:3. In some examples, the molar ratio of the first atosiban salt to inorganic pamoate used, contacted and / or reacted together in the reaction may be anywhere between about 2.5:1 and about 1:1 , e.g. about 2.1 :1 and about 1.1 :1.

[0028] In some examples, a solution of the inorganic pamoate salt (e.g. an aqueous solution of sodium pamoate) may be added to the solution of the first atosiban salt in portions and / or incrementally. By way of further example, the solution of the inorganic pamoate salt may be added dropwise to the solution of the first atosiban salt.

[0029] During the addition of the inorganic pamoate salt (which may optionally be added in the form of a solution as outlined above), the resultant reaction mixture may be stirred e.g. continuously stirred to assist in facilitating and / or promoting the ion exchange reaction.

[0030] The method may proceed under any conditions suitable to facilitate and / or promote the ion exchange and / or the precipitation of atosiban pamoate from the reaction mixture (e.g. solution). The method may be conducted under ambient conditions. For example, the method may be conducted at room temperature and / or under atmospheric pressure. In some cases, the method may be carried out at temperatures above about 0 °C, above about 10 °C, or above about 15 °C. In some cases, the method may be carried out at a temperature between about 0 °C and about 100 °C. For example, the method may be carried out at a temperature between about 10 °C and about 50 °C, or between about 15 °C and about 35 °C. In some cases, the method may be carried out at about room temperature (e.g. at a temperature between about 18 °C and about 27 °C or between about 20 °C and about 25 °C).

[0031] In some cases, the method may be carried out under atmospheric pressure. For example, at a pressure in the region of 101.325 kPa or 1 atmosphere.

[0032] Suitable solvents for the freeze-drying step may be any solvent in which atosiban pamoate has a solubility of at least 20 mg / mL (e.g. at 25 °C). Preferably the solvents for the freeze-drying step may also have a melting point of at least -80°C. Suitable solvents for the freeze-drying step may comprise mixtures of acetonitrile and water. By way of representative example, the solvent for the freeze-drying step may be or comprise a mixture of acetonitrile and water at a ratio anywhere between 1:10 to 10:1 (v / v) (e.g. in acetonitrile / water (1 / 2, v / v) mixture).

[0033] According to a further aspect, there is provided an atosiban pamoate salt made in accordance with the methods described herein. In particular, using the methods disclosed herein, the present inventors have identified that a specific salt form of atosiban pamoate may be obtained. In particular, in some examples, the atosiban pamoate salts provided by the methods of the present disclosure may be a semi-salt.

[0034] As such, according to a further aspect of the present disclosure, there is provided an atosiban pamoate salt wherein the molar ratio of the atosiban to pamoate in the salt is about 2:1.

[0035] Atosiban pamoate salts with the stoichiometries described herein may allow higher drug loadings than a mono salt form, which can assist in reducing formulation volumes when the salts are formulated for pharmaceutical uses (and which may in turn assist in increasing patient compliance in the clinic). In some examples, it may be desirable to provide atosiban pamoate in particulate form (e.g. as particles). For example, injectable formulations generally require particles of a suitable and / or uniform size. One option to provide such particles is via the use of a spray drying technique.

[0036] As such, the methods described herein may further comprise a step of spray drying atosiban pamoate to provide atosiban pamoate particles.

[0037] Thus, according to a further aspect of the disclosure, there is provided a method for providing atosiban pamoate particles comprising:

[0038] (i) providing a loading solution of atosiban pamoate; and

[0039] (ii) spray drying the loading solution to form the atosiban pamoate particles.

[0040] As used herein, spray drying may refer to a process in which a dry powder (e.g. containing a compound) is formed from a liquid. The spray drying process typically involves the atomization of a solution (referred to herein as a “loading solution”) by spraying followed by a rapid evaporation of the sprayed droplets into solid powder by a hot gas.

[0041] In order for a spray drying process to be effective, various factors have to be balanced. One such factor is the identification of a suitable loading solution for the compound to be spray dried. The loading solution should preferably be volatile at temperatures at which the compound in question remains stable. The compound in question should show reasonable solubility in the solvent and also a good level of stability in the spray drying process. The latter point may be particularly important where compounds are to be manufactured on an industrial scale, e.g. where compounds may be sat in solution for longer periods of time ahead of a spray drying step. The need for stability becomes more significant for compounds (such as atosiban pamoate) that are intended for use in pharmaceutical formulations, where purity is highly important. Furthermore, the use of this loading solution in the spray drying process itself should preferably yield particles of suitable and / or uniform size with a good appearance and syringeability properties (e.g. where the particles are intended for use in an injectable formulation).

[0042] The present inventors encountered difficulties in identifying a suitable loading solution for use in the spray drying of atosiban pamoate, e.g. a loading solution for this particular compound that would at least provide acceptable levels of solubility whilst also balancing the need for maintaining good levels of stability in the loading solution. In other words, a loading solution that would be compatible with atosiban pamoate to provide a reproducible and reliable spray drying process that would be amenable to scaled up manufacturing.

[0043] In particular, the present inventors have identified a cohort of loading solutions in which the atosiban pamoate may show good levels of solubility and stability. The loading solutions may also show suitable volatilities for the spray drying of atosiban pamoate and / or may be used to provide particles of suitable and / or uniform size (e.g. for therapeutic use applications).

[0044] In particular, the atosiban pamoate may have a solubility in the loading solution of at least about 15 mg / mL, at least about 20 mg / mL or at least about 25 mg / mL. In some examples, the atosiban pamoate may have a solubility in the loading solution between about 15 mg / mL and 100 mg / mL, between about 20 mg / mL and 75 mg / mL, or between about 20 mg / mL and 60 mg / mL. As described herein, the solubility is measured at room temperature and under atmospheric pressure.

[0045] The atosiban pamoate may show good levels of stability in the loading solution (e.g. when stored in the loading solution prior to a spray drying step).

[0046] As used herein, the expression “good levels of stability” may mean that the atosiban pamoate present in the loading solutions as described in the present disclosure is substantially stable and / or exhibits minimal or no degradation.

[0047] In particular, the atosiban pamoate may be substantially stable and / or exhibit minimal or no degradation in the disclosed loading solutions when stored over a period of at least 1 hour, at least 2 hours, at least 3 hours or at least 4 hours. In other examples, the atosiban pamoate may be substantially stable and / or exhibit minimal or no degradation in the disclosed loading solutions when stored over a period of at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours. In further examples, the atosiban pamoate may be substantially stable and / or exhibit minimal or no degradation in the disclosed loading solutions when stored over a period of at least 1 day, at least 2 days, at least 3 days or at least 4 days. In these examples, the loading solutions may be stored at room temperature, or under refrigerated conditions. In particular, the loading solutions may be stored at room temperature (e.g. at a temperature between about 15 °C and about 30 °C, such as between about 20 °C and about 25 °C) and / or under atmospheric pressure.

[0048] As used herein, “stable” may mean that the atosiban pamoate may maintain at least about 98.9%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4% or at least about 99.5% of an original level of purity when stored in the loading solution.

[0049] The loading solutions may show suitable levels of volatility for the spray drying of atosiban pamoate. In some examples, the loading solutions may have a boiling point less than or equal to about 150 °C, less than or equal to about 125 °C or less than or equal to about 100 °C.

[0050] The step of providing a loading solution of atosiban pamoate may comprise preparing a loading solution of atosiban pamoate. For example, the loading solution of atosiban pamoate may be prepared by adding and / or dissolving atosiban pamoate in any suitable solvent as described herein.

[0051] In some examples, the loading solution may comprise one or more solvents, e.g. two, three or four solvents. In some cases, the loading solution may comprise two solvents.

[0052] The one or more solvents may be selected from alcohol, water and acetonitrile. The alcohol may be a saturated or unsaturated alcohol. Branched or straight chain alcohols may be used. Suitable alcohols include, but are not limited to, Ci-Ce alcohols or C1-C4 alcohols. Representative examples of suitable alcohols include (but are not limited to) methanol, ethanol, isopropanol, n-propanol, iso-butanol, sec-butanol, tert-butanol, pentanol and hexanol.

[0053] In some examples, the loading solution may comprise an alcohol (e.g. methanol).

[0054] In particular, the loading solution may comprise an alcohol and water. By way of further example, the loading solution may comprise methanol and water, or ethanol and water. In other examples, the loading solution may comprise acetonitrile and water.

[0055] In particular, in some examples, the loading solution may comprise a solvent system selected from: (i) an alcohol (e.g. a C1-C4 alcohol such as methanol); (ii) an alcohol (e.g. a C1-C4 alcohol such as methanol) and water; and (iii) acetonitrile and water. In particular, the inventors have identified that such solvent systems may be used to provide effective loading solutions for atosiban pamoate, to provide good levels of both solubility and volatility, whilst maintaining good levels of stability of the atosiban pamoate. In yet further examples, the loading solution may comprise a solvent system selected from: (i) an alcohol (such as methanol) and water; and (ii) acetonitrile and water.

[0056] Where two or more solvents are present in the loading solution, they may be present in any suitable ratio that provides suitable levels of volatility, solubility and / or stability of the atosiban pamoate. In some examples, where two solvents are present, they may be present in a ratio (volume of a first solvent : volume of a second solvent) anywhere between about 100:1 to about 1 :100, between about 10:1 to about 1 :10, or between about 1 :5 and about 5:1 .

[0057] By way of further example, where the loading solution comprises an alcohol (e.g. C1-C4 alcohol such as methanol) and water, the two solvents may be present in a ratio anywhere between about 10:1 and about 1 :10 of volume of alcohol (e.g. C1-C4 alcohol such as methanol) : volume of water. In some examples, the loading solution may comprise any ratio between about 1 :1 and about 10:1 , or between about 2:1 and about 8:1 , or between about 3:1 and 7:1 of alcohol (e.g. C1-C4 alcohol such as methanol) : water (v / v). In some examples, the loading solution may comprise approximately 5:1 of alcohol (e.g. C1-C4 alcohol such as methanol) : water (v / v).

[0058] By way of a yet further representative example, where the loading solution comprises acetonitrile and water, the two solvents may be present in a ratio anywhere between about 10:1 and about 1 :10 of volume of acetonitrile : volume of water. In some examples, the loading solution may comprise any ratio between about 1 :1 and about 10:1 , or between about 1 :1 and about 5:1 , or between about 2:1 and about 4:1 of acetonitrile : water (v / v). In some examples, the loading solution may comprise approximately 3:1 of acetonitrile: water (v / v). As stated above, the method comprises a step of spray drying the disclosed loading solutions to form the atosiban pamoate particles (sometimes referred to herein as the atosiban pamoate spray-dried particles).

[0059] The spray-drying step may be carried out using any of the spray drying systems known in the art.

[0060] Spray dryers typically includes a drying chamber into which the liquid feed (e.g. the loading solution as described above) is introduced by way of an inlet. This inlet generally takes the form of an atomizer. A drying gas is also introduced to the drying chamber, which causes the loading solution to evaporate leaving behind the solid particles (e.g. the solid particles of atosiban pamoate). These solid particles may typically be separated using a particle separator (such as a cyclone) and collected in a vessel. An overview of the spray drying process in the pharmaceutical industry may be found in Gaspar et al, European Pharmaceutical Review, 28 October 2014, “Spray drying in the pharmaceutical industry”. By way of a representative example, the spray drying step may be carried out by a Mini Spray Dryer B-290 (Buchi, Switzerland).

[0061] In some examples, during the spray drying step, the inlet temperature (e.g. the temperature of the drying air at the inlet to the drying chamber) may be between about 50 °C and about 150 °C, between about 75 °C and about 125 °C, between about 90 °C and about 110 °C, or between about 95 °C and about 105 °C. In some examples, the inlet temperature may be about 100 °C.

[0062] In some examples, during the spray drying step, the feed rate (e.g. the rate the loading solution is introduced into atomizer prior to entry into the drying chamber) may be between about 0.5 mL / min and about 20 mL / min, between about 1 and 10 mL / min, or between about 2 and 7 mL / min. In some examples, the feed rate may be between about 3 mL / min and about 6 mL / min. In yet further examples, the feed rate may be about 4 mL / min.

[0063] In some examples, during the spray drying step, the atomizing gas flow rate (e.g. the rate the atomizing gas is introduced into the atomizer) may provide a height readout in the gas flowmeter between about 10 and about 100 mm, between about 25 mm and 75 mm, or between about 30 mm and 50 mm. In some examples, the atomizing gas flow rate may be about 40 mm. As used herein, “mm” is the unit of height as a readout in the gas flowmeter. The unit of height may be converted to actual atomizing gas volume flow rate under standard temperature and pressure conditions. By way of example, a height of 40 mm may match the actual gas volume flow rate of 667 L / h. Accordingly, in some examples, the atomizing gas flow rate may be between about 283 L / h and about 1744 L / h, between about 355 L / h and about 1374 L / h, or between about 439 L / h and about 1052 L / h.

[0064] In some examples, during the spray drying step, the drying gas flow rate (e.g. the rate at which the drying gas is introduced into the drying chamber) may be between about 1 m3 / h and about 100 m3 / h, between about 10 m3 / h and about 75 m3 / h, between about 20 m3 / h and 50 m3 / h. In some examples, the drying gas flow rate may be about 35 m3 / h.

[0065] The atosiban pamoate particles produced by the spray drying process may comprise a substantially spherical or near-spherical shape. The spray dried atosiban pamoate particles produced by the methods (and using the loading solutions) described herein may comprise diameters between about 0.1 and about 50 pm (microns), such as between about 0.5 pm and about 25 pm, or between about 1 pm and 10 pm. In particular, the spray dried atosiban pamoate particles produced by the methods (and using the load solutions) described herein may comprise diameters between about 1 pm and 8 pm. Spray dried atosiban pamoate particles as described herein may find particular application in injectable formulations.

[0066] Following the spray drying step, the purity of the atosiban pamoate present in the particles may be least about 90%, at least about 95%, at least about 97%, or at least about 99% (based on the total weight of the particles).

[0067] According to a further aspect of the present disclosure, there is further provided atosiban pamoate particles obtainable by the methods disclosed herein.

[0068] There is further provided a loading solution comprising atosiban pamoate in any one of the loading solutions described herein. By way of particular example, there is provided a loading solution for a spray drying process comprising atosiban pamoate and a solvent system selected from: (i) approximately 5:1 of alcohol (e.g. a C1-C4 alcohol such as methanol) : water (v / v); and (ii) approximately 3:1 of acetonitrile: water (v / v).

[0069] The methods described herein may find particular application in the preparation of atosiban pamoate for use in pharmaceutical compositions. Such pharmaceutical compositions may comprise atosiban pamoate together with one or more additional carrier ingredients such as diluents, excipients, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like.

[0070] Definitions

[0071] In the present disclosure, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds described herein, is intended to be in accordance with the rules of the International Union of Pure and Applied Chemistry (IUPAC) for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)” (see A. D. Jenkins et al., Pure & Appl. Chem., 68, 2287-2311 (1996)). For the avoidance of doubt, if an IUPAC rule is contrary to a definition provided herein, the definition herein is to prevail.

[0072] As used herein, the term “about” when qualifying a number or value, may refer to values that lie within ± 5% of the value specified. For example, where a particle is described as having a diameter of about 50 pm, a range of 47.5 pm to 52.5 pm is included.

[0073] In addition, as used herein, where any range is described as being between a lower limit and an upper limit, the defined range includes the defined endpoints (and so includes both the lower limit and upper limit values).

[0074] Isotopically-labelled compounds The disclosure also encompasses various deuterated forms of atosiban pamoate and any other compounds as described herein. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. A person of ordinary skill in the art will know how to synthesize deuterated forms of the compounds disclosed herein, including those referred to above. For example, deuterated materials, such as alkyl groups may be prepared by conventional techniques (see for example: methyl-cfe -amine available from Aldrich Chemical Co., Milwaukee, Wl, Cat. No.489, 689-2).

[0075] The disclosure also includes isotopically-labelled compounds of the present disclosure, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as3H,11C,14C,18F,123l or125l. Compounds of the present disclosure and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present disclosure. Isotopically labelled compounds of the present disclosure, for example those into which radioactive isotopes such as3H or14C have been incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.3H, and carbon- 14, i.e.14C, isotopes are particularly preferred for their ease of preparation and detectability.11C and18F isotopes are particularly useful in PET (positron emission tomography).

[0076] It should be noted that throughout this specification the term “comprising” is used to denote that embodiments of the invention “comprise” the noted features and as such, may also include other features. However, in the context of this invention, the term “comprising” may also encompass embodiments in which the invention “consists essentially of” the relevant features or “consists of’ the relevant features.

[0077] DETAILED DESCRIPTION

[0078] The present disclosure will now be further described with reference to the following, non-limiting examples. Figure 1 :1H NMR spectrum of atosiban acetate.

[0079] Figure 2:1H NMR spectrum of atosiban pamoate made according to an exemplary method of the disclosure.

[0080] Figure 3: LIPLC assay of atosiban pamoate powder produced by an exemplary method of the disclosure.

[0081] Figure 4: LIPLC assay of spray dried atosiban pamoate particles produced by an exemplary spray drying method of the disclosure.

[0082] Figure 5: Scanning Electron Microscopy (SEM) images of lyophilized atosiban pamoate (Left (500x), Right (1000x)).

[0083] Figure 6: Scanning Electron Microscopy (SEM) images of spray dried atosiban pamoate particles (Left (3000x), Right (5500x)).

[0084] PART A:

[0085] Chemistry - Materials and Methods

[0086] All chemicals, unless otherwise stated were commercially available and used without further purification.

[0087] NMR Analysis

[0088] NMR data was acquired in a Bruker Avance NEO 400 MHz NMR Spectrometer (Bruker, US). Chemical Shift is reported in ppm relative to dimethyl Sulfoxide (5 2.50) as indicated in NMR spectral data. For sample preparation, a small amount (1-5 mg) of sample (atosiban acetate or atosiban pamoate) was dissolved in DMSO-cfe(0.6ml) and transferred to the testing tube for NMR analysis. All the experiments were performed at 25 °C temperature.

[0089] Ultra Performance Liquid Chromatography (UPLC) Analysis

[0090] UPLC was performed on a Waters Acquity UPLC H-class system (Waters, US) equipped with a Waters Acquity UPLC ®BEH Phenyl 1.7 pm, 2.1 x 100 mm column. The UPLC parameters were listed below: Description Parameter

[0091] Column Waters Acquity UPLC ®BEH Phenyl 1.7 pm, 2.1 x 100 mm

[0092] Mobile Phase A 0.1% TFA (trifluoroacetic acid) in H2O

[0093] Mobile Phase B 0.1% TFA in ACN (acetonitrile)

[0094] Detection 210 nm (bandwidth 4 nm); spectra collection 190-400 nm

[0095] Flow rate 0.3 ml / min

[0096] Injection Volume 3 pL

[0097] Column Temp 30 °C

[0098] Autosampler Temp 5 °C

[0099] Running Time 10 min

[0100] Needle Wash 0.1% TFA in water / ACN (90:10)

[0101] The UPLC mobile phase gradient was as follows:

[0102] Time (min) %Mobile Phase B

[0103] 7.01 30

[0104] 10 30

[0105] Scanning Electron Microscopy (SEM) Analysis:

[0106] SEM was performed on a Phenom™ Pure transmission electron microscope (Thermo Fisher Scientific, US). For the sample preparation, spray dried powder was dispersed directly onto the carbon adhesive and excess powder was removed by blowing a jet of particle-free compressed gas across each, and then coated with gold platinum under a high vacuum. SEM images were recorded digitally at higher magnification. The particle sizes were determined by examining the microphotographs using the self-contained measuring tool.

[0107] A.1 Preparation of atosiban pamoate

[0108] Atosiban pamoate salt was prepared via a precipitation method. 5g atosiban acetate (94.5% free base basis, MW=994.5 Da) was dissolved in 150mL deionized (DI) water. Separately, 1.15g sodium pamoate (Na2Pamoate; MW=432.34 Da, 1.1 mole ratio of acid to base) was dissolved in 50mL deionized (DI) water. Mixing was performed by adding atosiban acetate solution to the sodium pamoate solution dropwise under continuous stirring by an overhead stirrer in a 500mL beaker at room temperature. Precipitates formed during the continuous stirring and the stirring proceeded for additional 4 hrs at room temperature. The jelly-like precipitate was quickly rinsed twice with deionized (DI) water, and re-dissolved in acetonitrile / water (1 / 2, v / v) mixture. Finally, the re-dissolved atosiban pamoate solution was pre-freezed in -40 °C for 24 hrs, and then placed into lyophilizer for 72 hrs to obtain atosiban pamoate in a powder form.

[0109] To confirm the successful synthesis of atosiban pamoate,1H-NMR and LIPLC were utilized to analyze the synthetized product. The equilibrium solubility study was performed to verify the change of atosiban solubility in aqueous medium after hydrophobic counter ion exchange.

[0110] The stoichiometry of the starting material atosiban : acetate was determined to be approximately 0.93:1 by1H NMR, (DMSO-de), indicating that the atosiban acetate was a mono-acetate salt (see Figure 1).

[0111] The stoichiometry of atosiban : pamoate was determined to be 1 :0.5 by1H NMR (DMSO-de,) (see Figure 2). The signal of acetate was not found, suggesting a pamoate salt of atosiban as semi-pamoate was formed.

[0112] Figure 3 shows LIPLC graphs of atosiban acetate (top curve) and atosiban pamoate (bottom curve). The LIPLC analysis was carried out in accordance with the method outlined in the “Materials and Methods” section and indicates that the purity of atosiban pamoate is nearly 100%. (Noting that in the LIPLC curve of atosiban pamoate, the peak at ~2.6 min is for atosiban free base, while the peak at ~6.6 min is for pamoic acid. By contrast, there is only atosiban free base peak at ~2.6 min in the curve of atosiban acetate, as acetic acid has no peak under the experimental condition).

[0113] A.2 Equilibrium solubility studies:

[0114] The results of equilibrium solubility studies of atosiban acetate and atosiban pamoate in phosphate buffered saline (PBS) solution are shown in Table 1 below.

[0115] Table 1 showing equilibrium solubility results of atosiban acetate and atosiban pamoate in phosphate buffered saline (PBS) solution. S: solubility (mg / mL), the solubility was calculated with freebase.

[0116] The approximate solubility of atosiban acetate in PBS solution (pH = 7.4) at room temperature (~25 °C) was above 197 mg / mL. Atosiban acetate showed high solubility (> 60 mg / mL), which solution was clear after shaking at 37 °C for 8 hours and kept clear within 48 hours. In comparison, pamoate salt showed solubility (~ 7.8 mg / mL, average value) in PBS solution at 37 °C within 48 hours. In addition, gel was observed at the bottom after shaking for 8 hours. The result indicated that the pamoate salt showed a significant lower solubility than that of the atosiban acetate in PBS.

[0117] PART B: Spray drying of atosiban pamoate

[0118] Gelling of atosiban pamoate had been observed in certain solvent systems typically used in a spray drying process indicating that they would be unsuitable for use in the spray drying of atosiban pamoate. The inventors conducted further investigations to identify a suitable solvent to provide a loading solution for the spray drying step.

[0119] B1: Initial solubility studies

[0120] The approximate solubility of atosiban pamoate was performed at room temperature and under atmospheric pressure.

[0121] A solubility screening method by visual inspection was utilized for the measurement of approximate solubilities in accordance with the following procedure:

[0122] 1. ~2mg atosiban-pamoate salt powder was weighed out in each of the 2mL vials;

[0123] 2. For each group, the tested solvent was added sequentially according to the following steps:

[0124] Step 1 : 50pl solvent was added to the vial. If the salt dissolved, the data for Solubility was calculated as (S)>40mg / mL (The actual S was calculated case by case via equation: S=actual weight / 0.05); If the salt did not dissolve, the tester continued to Step 2;

[0125] Step 2: 50pl solvent was added to the vial. If the salt dissolved, the data for Solublility was calculated as 40>S>20mg / mL (The actual S was calculated case by case); If the salt did not dissolve, the tester continued to Step 3;

[0126] Step 3: 10OpI solvent was added to the vial. If the salt dissolved, the data for Solubility was calculated as 20>S>10mg / mL (The actual S was calculated case by case); If the salt did not dissolve, the tester continued to Step 4;

[0127] Step 4: 200pl solvent was added to the vial. If the salt dissolved, the data for Solubility was calculated as 10>S>5mg / mL (The actual S was calculated case by case); If the salt did not dissolve, the tester continued to Step 5;

[0128] Step 5: 200pl solvent was added to the vial. If the salt dissolved, the data for Solubility was calculated as 5>S>3.33mg / mL (The actual S was calculated case by case); If the salt did not dissolve, the tester continued to Step 6;

[0129] Step 6: 400pl solvent was added to the vial. If the salt dissolved, the data for Solubility was calculated as 3.33>S>2mg / mL (The actual S was calculated case by case); If the salt did not dissolve, S<2mg / mL (The actual S should be calculated case by case).

[0130] The results of initial solubility studies in pure volatile solvents are outlined in Table 2 below.

[0131] Table 2 shows the results of initial solubility studies of atosiban pamoate in pure volatile solvents.

[0132] Limited solubility of atosiban pamoate was observed in nearly all the tested pure volatile solvents (with the exception of methanol). Consequently, the inventors conducted some further investigations into the solubility of atosiban pamoate in various mixed solvents of water and organic solvents. The results are shown in Table 3 below.

[0133] Table 3 shows the results of initial solubility studies of atosiban pamoate in a number of mixed solvent systems.

[0134] These initial solubility investigations highlighted a number of possible solvent systems that provided good and / or acceptable levels of solubility for atosiban pamoate.

[0135] B2: Short-term stability studies

[0136] The inventors then conducted further investigations into the short-term stability of atosiban pamoate to confirm the compatibility of atosiban pamoate with the different solvent systems. The stability studies were performed at 25 °C and under atmospheric pressure. LIPLC was utilized to determine the purity of the atosiban pamoate at various time points after atosiban pamoate was added to the solvent system (0 minutes, 4 hours, 24 hours, and 4 days). The peak area proportion change of atosiban free base (atosiban purity) at various time points were summarized to evaluate the short-term stability of the atosiban pamoate. (The LIPLC measurement methods used in this study are summarised in the “Materials and Methods” section).

[0137] The results are shown in Table 4.

[0138] Tab e 4 shows the results of the short-term stability studies carried out on atosiban pamoate in different solvent systems at Oh, 4 h, 24 hours and 4 days after addition of atosiban pamoate to the solvent system.

[0139] The short-term stability data revealed that THF and acetone may lead to the degradation of atosiban peptide under certain conditions, while acetonitrile, MeOH and EtOH had good compatibility with atosiban-pamoate and were appropriate for use in spray drying process.

[0140] B3: Development of spray drying process:

[0141] Following the initial investigations into suitable solvents for use in the loading solution, further parameters of the spray drying process were investigated including solvent, inlet temperature and feed rate. The atomizing gas flow rate (40mm) and drying gas flow rate (35m3 / h) were kept constant during the process development. With regards to the atomizing gas flow rate, it is noted that 40 mm represents the height readout in the gas flowmeter. This number may be converted to actual atomizing gas volume flow rate under standard temperature and pressure conditions. For example, a height of 40 mm equates to an actual gas volume flow rate of 667 L / h.

[0142] Several attributes of the spray dried products were then analysed, including purity, assay and syringeability.

[0143] In particular, six batches of 300mg scale spray drying manufacturing process were conducted. The results are shown in Table 5 below.

[0144] Table 5 showing the results of investigations into the spray drying process of atosiban pamoate.

[0145] In Table 5, the purity is based on the peak area proportion change of atosiban free base (as determined by LIPLC analysis), indicating the degree of degradation of the atosiban free base in the atosiban pamoate salt, e.g. -100% purity indicates there is substantially no degradation for the atosiban free base in the atosiban pamoate salt, indicating a good stability during processing. In Table 5, %assay data indicates the content of atosiban free base in the atosiban pamoate powder. The %assay of the final atosiban pamoate particles are around 80% due to the presence of pamoic acid in the atosiban pamoate.

[0146] B4: Spray dried atosiban pamoate particles

[0147] B4.1 : Method:

[0148] A spray drying method was utilized to manufacture atosiban pamoate particles. Lyophilized atosiban pamoate powder (1.2 g) was dissolved in MeOH / FW (40 mL, 5:1, v / v) mixed solvent. The solution was filtered by a 0.22pm filter and spray-dried using a Mini Spray Dryer B-290 (Buchi, Switzerland) at an inlet temperature of 100 °C and feed rate of 4.0mL / min. The atomizing gas flow rate and drying gas flow rate were maintained at 40mm and 35m3 / h respectively.

[0149] B4.2: Analysis:

[0150] The spray dried atosiban pamoate particles produced in accordance with the method outlined in B4:1 were characterized by Ultra Performance Liquid Chromatography (UPLC) and Scanning Electron Microscopy (SEM) (in accordance the methods outlined in the “Materials and Methods” section). The results are shown in Figures 4, 5 and 6.

[0151] Referring to Figure 4, UPLC analysis of atosiban acetate (top curve) and spray dried atosiban pamoate particles (bottom curve). The UPLC analysis indicated the purity of spray dried atosiban pamoate powders to be nearly 100%. (Again, noting that in the UPLC curve of atosiban pamoate, the peak at ~2.6 min is for atosiban free base, while the peak at ~6.6 min is for pamoic acid. By contrast, there is only atosiban free base peak at ~2.6 min in the curve of atosiban acetate, as acetic acid has no peak under the experimental condition).

[0152] SEM analysis of the spray dried atosiban pamoate particles (made in accordance with procedure outlined in “B4.1 : Method” above) showed these particles to have a spherical or near-spherical shape with diameters approximately between 1 and 8pm (see SEM images in Figure 6). Such particles are considered suitable for use in injectable formulations. In contrast, SEM analysis of the lyophilized atosiban pamoate powder showed the particles to have an irregular sheet shape with generally larger particle sizes (ranging between 20 pm and 80 pm diameter) (see SEM images in Figure 5). These were considered to be less suitable for use in injectable formulations.

[0153] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.

Claims

CLAIMS:

1. A method for preparing atosiban pamoate comprising:(i) contacting and / or reacting together a first atosiban salt and an inorganic pamoate salt in a reaction mixture, wherein the atosiban pamoate is formed as a precipitate.

2. A method according to claim 1, wherein the first atosiban salt is an atosiban salt having a solubility of at least 10 mg / mL at 25 °C in aqueous solution, and / or wherein the first atosiban salt is selected from atosiban acetate, atosiban hydrochloride and atosiban trifluoroacetate.

3. A method according to claim 1 or 2, wherein the inorganic pamoate salt is a metal pamoate salt, optionally selected from sodium pamoate and potassium pamoate.

4. A method according to any one of the preceding claims, wherein the reaction mixture is a solution, such as an aqueous solution, optionally water or an aqueous buffered solution.

5. A method according to any one of the preceding claims, further comprising:(i) providing a solution of the first atosiban salt; and(ii) contacting the solution of the first atosiban salt with the inorganic pamoate salt; and optionally further comprising:(iii) adding the inorganic pamoate salt to the solution of the first atosiban salt, optionally further comprising adding a solution of the inorganic pamoate salt to the solution of atosiban salt.

6. A method according to any one of the preceding claims, wherein the molar ratio of the atosiban salt to inorganic pamoate used, contacted and / or reacted together is any molar ratio between about 10:1 and about 1 :10, between about 5:1 and about 1 :5, or between about 3: 1 and about 1 :3.

7. A method according to any one of the preceding claims, further comprising separating and / or removing the atosiban pamoate precipitate; and optionally(i) freeze-drying the atosiban pamoate precipitate to form freeze-dried atosiban pamoate); and / or(ii) spray drying atosiban pamoate to provide spray-dried atosiban pamoate particles.

8. A method for providing atosiban pamoate particles comprising:(i) providing a loading solution of atosiban pamoate; and(ii) spray drying the loading solution to form the atosiban pamoate particles; optionally wherein the atosiban pamoate has a solubility in the loading solution of at least about 15 mg / mL, at least about 20 mg / mL or at least about 25 mg / mL.

9. A method according to claim 8, wherein the atosiban pamoate is substantially stable and / or exhibits minimal or no degradation in the loading solution when stored over a period of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 12 hours, at least 24 hours, at least 36 hours or at least 48 hours; optionally wherein the atosiban pamoate maintains at least about 98.9%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4% or at least about 99.5% of an original level of purity when stored in the loading solution.

10. A method according to claim 8 or 9, wherein the loading solution:(a) has a boiling point less than or equal to about 150 °C, less than or equal to about 125 °C or less than or equal to about 100 °C; and / or(b) comprises one or more solvents, optionally wherein the one or more solvents are selected from alcohol, water and acetonitrile; and / or(c) comprises a solvent system selected from: (i) an alcohol (e.g. a C1-C4 alcohol such as methanol); (ii) an alcohol (e.g. a C1-C4 alcohol such as methanol) and water; and (iii) acetonitrile and water.

11. A method according to any one of claims 8 to 10, wherein the loading solution comprises a solvent system selected from:(i) approximately 5:1 of alcohol (e.g. a C1-C4 alcohol such as methanol) : water (v / v); and(ii) approximately 3:1 of acetonitrile: water (v / v).

12. A loading solution for a spray drying process comprising atosiban pamoate and a solvent system selected from:(i) approximately 5:1 of alcohol (e.g. a C1-C4 alcohol such as methanol) : water (v / v); and (ii) approximately 3:1 of acetonitrile: water (v / v).

13. A spray-dried atosiban pamoate particle obtained or obtainable by a method as defined in any one of claims 8 to 11 , optionally wherein the particles comprise a diameter of between about 0.1 pm and about 50 pm (microns), between about 0.5 pm and about 25 pm, or between about 1 pm and about 10 pm.

14. An atosiban pamoate salt wherein:(i) the molar ratio of the atosiban to pamoate in the salt is approximately 2:1 ; and / or(ii) the salt is obtained or obtainable by a method as defined in any one of claims 1 to 7.

15. A pharmaceutical composition comprising atosiban pamoate obtained or obtainable by a method as defined in any one of claims 1 to 7, a spray-dried atosiban pamoate particle according to claim 13, or the atosiban pamoate salt of claim 14.

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