Preparation of a raloxifene hydrate for cancer and osteoporosis therapy

Abstract

The invention relates to a method of producing a hydrate of Raloxifene (RLX) with increased solubility and to a hydrate of RLX produced according to the method. Also provided are pharmaceutical compositions comprising a hydrate of RLX prepared according to the method. The invention further relates to a hydrate of RLX or pharmaceutical compositions comprising the hydrate of RLX for use in methods of preventing and / or treating osteoporosis and / or cancer. In particular, the hydrate of RLX is prepared by combining a co-former mixture of RLX, or a salt thereof, and Arginine.

Description

[0001] PREPARATION OF A RALOXIFENE HYDRATE FOR CANCER AND OSTEOPOROSIS THERAPY

[0002] BACKGROUND OF THE INVENTION

[0003] The present invention relates to a method of producing a hydrate of Raloxifene (RLX) with increased solubility and to a hydrate of RLX produced according to the method. The invention also relates to pharmaceutical compositions comprising a hydrate of RLX prepared according to the method. The invention further relates to a hydrate of RLX or pharmaceutical compositions comprising the hydrate of RLX for use in methods of preventing and / or treating osteoporosis and / or cancer. In particular, the hydrate of RLX produced by combining a coformer mixture of RLX-HCI and Arginine .

[0004] Raloxifene (RLX) is a nonsteroidal selective oestrogen receptor modulator (SERM) that has been approved by the FDA for the treatment of osteoporosis and to lower the risk of invasive breast cancer in postmenopausal women. Raloxifene was specifically developed in order to maintain the beneficial estrogenic activity on bone and lipids and to exert anti- oestrogenic activity on endometrial and breast tissue. Raloxifene works by inducing conformational changes in the oestrogen receptor, which in turn, enables the expression of certain oestrogen- regulated genes in different tissues. For instance, the agonist properties of raloxifene on bone tissues were recently attributed to the activation of the human transforming growth factor-b3 gene, which is generally regarded to be essential in bone remodelling.

[0005] RLX is a class II Biopharmaceutical Classification System (BCS) active pharmaceutical ingredient (API), which has previously been shown to exhibit poor solubility and high permeability. Raloxifene HCI has a molecular weight of 510.05 g-mol"1. It is off-white to pale yellow non-volatile solid. Its solubility in water is 627.4±132.0 pg mL-1and it is classified as very slightly soluble in water. Raloxifene HCI is absorbed rapidly after oral administration and has an absolute bioavailability of about 2%. The drug has a half-life of about 28 h and is eliminated primarily in the faeces after hepatic glucuronidation.

[0006] The main problem associated with preparations containing raloxifene, particularly those with raloxifene hydrochloride, is that they display a great hydrophobicity and extremely low solubility of the compounds in water and consequent very low bioavailability, most especially in the gastro-intestinal tract, which has negative implications on patient therapy. Therefore, in order to achieve a good therapeutic effect, it is necessary to use a high dose of the active compound, which is unfortunately connected with heavier side effects.

[0007] Raloxifene hydrochloride salts are usually used orally, therefore the solubility in gastric juice, where the main constituent is diluted hydrochloric acid, is particularly important. Specifically, due to the low pH and the presence of chloride ions, the solubility of any salt used decreases close to the solubility level of raloxifene hydrochloride. The present invention relates to a Raloxifene hydrate having improved solubility and bioavailability, and to uses thereof.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to a hydrate of Raloxifene (RLX), with increased solubility and bioavailability and also relates to a method of producing the hydrate of RLX. Also described herein are pharmaceutical compositions comprising a hydrate of RLX. The invention further relates to methods of preventing and / or treating osteoporosis and / or cancer using the hydrate of RLX, as well as to compositions for use in methods of preventing and / or treating osteoporosis and / or cancer.

[0010] According to a first aspect of the present invention there is provided for a method for producing a hydrate of Raloxifene (RLX), the method comprising: a) combining RLX, or a salt thereof, with an amino acid to form a co-former mixture; b) dissolving the co-former mixture in a solvent to form a solution; and c) evaporating the solvent from the solution to form a hydrate of RLX.

[0011] In a first embodiment of the method for producing a hydrate of RLX, the salt of RLX in step (a) that is combined with the amino acid may be a hydrochloride salt of RLX (RLX-HCI). In some embodiments, the HCI is removed from the solution after producing the hydrate of RLX, for example by mixing the solution of step (b) with a base, such as NaOH, preferably before step (c).

[0012] In a second embodiment of the method for producing a hydrate of RLX, the amino acid may be L-tyrosine, phenylalanine, L-arginine, L-tryptophan, proline and / or glycine. In one embodiment the amino acid is phenylalanine, tyrosine, tryptophan, or arginine. In a preferred embodiment, the amino acid is arginine.

[0013] According to a third embodiment of the method for producing a hydrate of RLX of the invention, the molar weight ratio of the RLX, or salt thereof, to amino acid is 2:1 , 1 :1 or 1 :2 (w / w). In one embodiment, the molar weight ratio of the RLX: amino acid is 1 :1 (w / w).

[0014] In a fourth embodiment of the composition of the method for producing a hydrate of RLX, the solvent may be an organic solvent, such as ethanol or methanol.

[0015] According to a fifth embodiment of the method for producing a hydrate of RLX, the hydrate of RLX has increased solubility compared to RLX or a salt thereof, such as RLX-HCI.

[0016] According to a second aspect of the present invention there is provided for a hydrate of RLX produced according to the method as described herein.

[0017] According to a third aspect of the present invention there is provided for a pharmaceutical composition comprising a hydrate of RLX prepared according to the method as described herein.

[0018] In a first embodiment of the pharmaceutical composition of the invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. According to a fourth aspect of the present invention there is provided for a method of treating osteoporosis in a subject, wherein the method comprises administering a hydrate of RLX or pharmaceutical composition as described herein to the subject. In some embodiments, the hydrate of RLX or the pharmaceutical composition has increased solubility and / or bioavailability in the subject compared to RLX, a salt thereof, such as RLX-HCI, or a pharmaceutical composition comprising RLX, or a salt thereof, such as RLX-HCI.

[0019] According to a fifth aspect of the present invention there is provided for a method of treating and / or preventing breast cancer in a subject, wherein the method comprises administering a hydrate of RLX or pharmaceutical composition as described herein to the subject.

[0020] According to a sixth aspect of the present invention there is provided for a hydrate of RLX or a pharmaceutical composition as described herein for use in a method of treating osteoporosis in a subject, wherein the method comprises administering the hydrate of RLX or the pharmaceutical composition to the subject.

[0021] According to a seventh aspect of the present invention there is provided for a hydrate of RLX or a pharmaceutical composition as described herein for use in a method of treating and / or preventing breast cancer in a subject, wherein the method comprises administering the hydrate of RLX or the pharmaceutical composition to the subject. In an embodiment of the invention, the hydrate of RLX or the pharmaceutical composition has increased solubility and / or bioavailability in the subject compared to RLX, a salt thereof, such as RLX-HCI, or a pharmaceutical composition comprising RLX, or a salt thereof, such as RLX-HCI.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0024] Figure 1 : Overlay of FTIR spectra for RLX and Arg compared with the RLX-Arg crystals in molar ratios of 2:1 , 1 :1 , and 1 :2 prepared through solvent evaporation with corresponding physical mixtures.

[0025] Figure 2: Overlay of DSC thermograms obtained for RLX-HCI, Arg and the prepared hydrate of RLX-HCl-Arg in molar ratios 2:1 , 1 :1 and 1 :2 through solvent evaporation.

[0026] Figure 3: PXRD diffractograms acquired for RLX-HCI, Arg, and the synthesized hydrate of RLX-HCl-Arg in molar ratios of 2:1 , 1 :1 , and 1 :2 through solvent evaporation with corresponding physical mixtures.

[0027] Figure 4: HSM micrographs obtained for the prepared (a) RLX-HCI, (b) ARG, (c)

[0028] RLX-ARG (1 :1 ) and (d) RLX-ARG (1 :2) and (e) RLX-ARG (2:1 ) during heating at 10 °C / min.

[0029] Figure 5: SEM images for RLX (150X), Arg (60X), RLX-Arg PM (1 :1 ), (106X) and RLX-Arg (1 :1 ) (100X). Figure 6: Perspective view showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

[0030] Figure 7: Solubility profile for RLX-HCI and the hydrate of RLX-Arg (1 :1 ).

[0031] Figure 8: Dissolution profile of RLX-HCI and the hydrate of RLX-Arg (1 :1 ).

[0032] Figure 9: FTIR Spectra of the hydrate of RLX-Arg (1 :1 ).

[0033] Figure 10: DSC Thermogram of the hydrate of RLX-Arg (1 :1 ).

[0034] Figure 11 : PXRD Diffractogram of the hydrate of RLX-Arg (1 :1 ).

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0037] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0038] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0039] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of such an embodiment “comprising” is to be understood as having the meaning of “consisting of’.

[0040] In it broadest sense, the present invention pertains to novel hydrates of Raloxifene and their methods of synthesis, wherein protection is extended to encompass a broad range of synthesis conditions, reactants, and methodologies.

[0041] The synthesis of the Raloxifene hydrate described herein involves the use of amino acids, including but not limited to Arginine (Arg), Phenylalanine (Phe), Tyrosine (Tyr), and Tryptophan (Trp). It is understood that any amino acid or combination thereof may be employed in the synthesis process to yield the hydrate form. The scope of this invention, therefore, extends to the use of any naturally occurring, semi-synthetic, or synthetic amino acid as a co-former in the formation of the Raloxifene hydrate.

[0042] The Raloxifene hydrate can be synthesized using varying molar ratios of RLX-HCI to amino acids, including but not limited to 2:1 , 1 :1 , and 1 :2. The present invention encompasses any ratio of RLX-HCI to amino acid that results in the formation of the Raloxifene hydrate, including ratios that may deviate from those explicitly disclosed herein. It is a particular aim of the present invention to provide for the preparation of Raloxifene hydrate with improved solubility and bioavailability. The problem of poor solubility and consequent low bioavailability is solved by the introduction of hydrate into the raloxifene hydrochloride molecule. In the present invention, the solvent evaporation technique was used to develop a hydrate of the Raloxifene with amino acids as co-formers. The inventors of the present invention show herein that RLX-HCI and amino acid arginine (Arg) in ethanol as the solvent exhibited the best combination for crystal formation after characterisation and led to improved solubility and bioavailability of Raloxifene HCI. To synthesise the hydrate of RLX, the inventors used RLX-HCI as the starting material, combining it with an amino acid, preferably Arg. After the synthesis process, the final product did not include HCI and Arginine, although the inventors determined that HCI and Arginine played a significant role in forming the hydrate of Raloxifene. The synthesis of the hydrate of Raloxifene was not successful when using the RLX-HCI alone without the amino acid co-former. Similarly, based on preliminary results, the desired hydrate was not obtained when the pure RLX was used alone, although the inventors are of the view that it could be used to obtain a hydrate of RLX under certain conditions.

[0043] The term “raloxifene” as used herein means the selective oestrogen receptor modulator molecule known as raloxifene having the IUPAC name: [6-hydroxy-2-(4-hydroxyphenyl)- benzothiophen-3-yl]-[4-[2-(1 -piperidyl)ethoxy]phenyl]-methanone, and includes any other name therefor, such as Keoxifene, Pharoxifene, LY-139481 , LY-156758, CCRIS-7129. In some embodiments, the term may encompass known salts and derivatives thereof. While the present invention has primarily employed the hydrochloride salt form of Raloxifene (RLX-HCI) in hydrate synthesis, it is further contemplated that the neutral (free base) form of Raloxifene (RLX) may also be used to obtain the hydrate of RLX. Alternative salts of RLX may also be employed in the method of producing the hydrate of RLX, including but not limited to salts of RLX obtained by reacting RLX with hydrobromic acid, acetic acid, tartaric acid, or methanesulfonic acid. The scope of this invention therefore extends to the use of any pharmaceutically acceptable form of RLX in the RLX hydrate synthesis method.

[0044] There are different methodologies for hydrate formulation, including (i) solvent evaporation without heat; (ii) solvent evaporation with heat; (iii) solvent evaporation with heat and cooling; and (iv) recrystallization. A formulated sample of RLX-HCI with Arginine using the solvent evaporation technique may not form the crystals but a large particle-size powder. This powder can be further re-crystalized by placing it in methanol or ethanol and leaving for at least 3 to 7 days without stirring or agitation and without adding any heat, to allow for the growth of crystals. Another method that can be used to grow crystals is to place samples of RLX and Arg in a solvent (ethanol or methanol) without agitation or heat and leave the samples for at least 3 to 7 days, allowing the precipitate to grow clear crystals. Additionally, another method comprises placing a sample of RLX-HCl-Arginine into a test tube with 10ml ethanol, heating it at 78 °C while stirring at 200rpm until the ethanol boils, allowing it to cool-down for one minute then extracting the top clear solution, transferring it into another test tube and then placing the test tube in a beaker with ice or placing it in a fridge. Visible crystals will form if the sample is refrigerated for at least 3 to 7 days after the solvent has evaporated.

[0045] The synthesis of the hydrate of the present invention has been demonstrated using solvents such as ethanol and methanol. However, the invention is not limited to these solvents alone and extends to any pharmaceutically acceptable solvent that facilitates hydrate formation, including but not limited to isopropanol, dimethyl sulfoxide (DMSO), ethyl acetate, water, or any other related solvent.

[0046] The present invention also encompasses any isomeric or polymorphic forms of the hydrate that may arise during synthesis. This includes but is not limited to stereoisomers, tautomers, and conformational isomers of the hydrate structure. Any hydrate exhibiting substantially similar physicochemical properties and therapeutic efficacy is considered within the scope of this invention. Additionally, any crystalline or amorphous forms resulting from the disclosed methods are included within the scope of the invention.

[0047] As used herein the term “subject” includes mammals, preferably human or animal subjects, but most preferably the subjects are human subjects. The terms “subject” and “patient” are used interchangeably herein.

[0048] The invention also includes pharmaceutical compositions comprising an effective amount of a fatty acid raloxifene derivative and a pharmaceutically acceptable carrier. The invention includes a fatty acid raloxifene derivative provided as a pharmaceutically acceptable prodrug, hydrate, salt, such as a pharmaceutically acceptable salt, enantiomers, stereoisomers, or mixtures thereof.

[0049] Representative “pharmaceutically acceptable salts” include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2- naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2-hydroxy-3-naph-thoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p- toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethio-dide, and valerate salts.

[0050] The term “preventing”, when used in relation to a medical disease or condition, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition.

[0051] As used herein, the terms “improve”, “ameliorate”, and “heal” are used interchangeably and refer to when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency.

[0052] The term “therapeutic treatment” is well known to those of skill in the art and includes administration to a subject of one or more of the hydrates or pharmaceutical compositions of the invention. If the composition is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilise the existing unwanted condition or side effects thereof).

[0053] The term “disorder” as used herein means, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0054] The term “administer”, “administering”, or “administration” as used in this disclosure refers to either directly administering a compound or pharmaceutically acceptable salt of the compound or a composition comprising the compound to a subject or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject’s body.

[0055] The term “prodrug,” as used in this disclosure, means a compound which is convertible in vivo by metabolic means (e.g., by hydrolysis) to a fatty acid raloxifene derivative.

[0056] Suitable formulations or compositions to administer the pharmaceutical compositions of the present invention to subjects fall within the scope of the invention. Any appropriate route of administration may be employed, such as, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, or oral administration.

[0057] An “effective amount” of a hydrate or pharmaceutical composition according to the invention includes a therapeutically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of osteoporosis and / or breast cancer. The outcome of the treatment may for example be the prevention of osteoporosis, cancer metastasis, preventing recurrence of the cancer, a decrease in cancer markers, a decrease in tumour size, inhibition of target metabolic pathways, delay in development of a pathology associated with cancer, or any other method of determining a therapeutic benefit. A therapeutically effective amount of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, the ability of the composition to elicit a desired response in the individual, previous therapeutic treatments, the nature and severity of the cancer or osteoporosis to be treated, the route of administration, and the form of the composition. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0058] The hydrate of RLX or pharmaceutical compositions of the invention can be provided either alone or in combination with other compounds, in the presence of a liposome, an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans.

[0059] As used herein a “pharmaceutically acceptable carrier” or “excipient” includes any and all coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible. A “pharmaceutically acceptable carrier” may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the hydrates or pharmaceutical compositions to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions. The use of media and agents for the preparation of pharmaceutically active substances is well known in the art. Where any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is not contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0060] The invention also relates in part to a method of preventing breast cancer, or treating breast cancer, in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount, of the hydrates or compositions or formulations thereof of the present invention, in order to treat breast cancer in the subject.

[0061] Further, the invention also relates in part to a method of providing osteoporosis therapy, for treating osteoporosis in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount, of the hydrates or compositions or formulations thereof of the present invention, in order to treat osteoporosis in the subject.

[0062] Typically, an effective amount of the hydrates, or compositions of the invention will be administered to a subject. For pharmaceutical compositions, an effective amount of the hydrate of the present invention can be provided, either alone or in combination with other compounds, or they may be linked with suitable carriers and / or other molecules.

[0063] Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD5o and the ED5o. Data obtained from the cell cultures and / or animal studies may be used to formulate a dosage range for use in a subject. The dosage of any composition of the invention lies preferably within a range of circulating concentrations that include the ED5o but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. For compounds and compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.

[0064] Dosage values may vary and be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the hydrates or pharmaceutical compositions or compounds of the invention. It may be advantageous to formulate the hydrates or compositions in dosage unit forms for ease of administration and uniformity of dosage.

[0065] In some embodiments, the hydrates or pharmaceutical compositions according to the invention may be provided in a kit, optionally with a carrier, together with instructions for use.

[0066] The following examples are offered by way of illustration and not by way of limitation.

[0067] In the following examples, RLX-HCI with purity of 99.85% was purchased from Iffect chemphar Ltd. (Jiangsu, China). The amino acids arginine, tryptophan, phenylalanine, and tyrosine were purchased from Glentham Lifesciences (Corsham, United Kingdom). Analytical grade ethanol was purchased from Rochelle Chemicals (Johannesburg, South Africa). HPLC grade of acetonitrile was purchased from Fisher Scientific (Loughborough, United Kingdom). Potassium di-hydrogen ortho phosphate monohydrate and Ortho-phosphoric acid from Merck Chemicals (Pty) Ltd (Gauteng, South Africa). For the preparation of buffers for dissolution and solubility studies, the analytical reagents sodium hydroxide, di-sodium hydrogen phosphate anhydrous were purchased from Rochelle Chemicals (Johannesburg, South Africa). Hydrochloric acid was purchased from Sigma-Aldrich (Saint Louis, United States of America), acetic acid glacial was purchased from Associated Chemical Enterprises (Pty) Ltd (Johannesburg, South Africa).

[0068] EXAMPLE 1

[0069] Preparation of RLX-HCl-Amino acid crystals

[0070] Preparation of R LX- HCI- L-tyrosine crystals through slow solvent evaporation

[0071] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX-HCI to Tyrosine in a 51 .004 mg of RLX-HCI and 18.119 mg of L-Tyrosine mixture. In the 2:1 mixture 102.008 mg of RLX-HCI and 18.1 19 mg of Tyrosine were mixed and in the 1 :2 mixture 51.004 mg of RLX-HCI and 36.238 mg of Tyrosine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0072] Preparation of R LX- HCI -Phenylalanine crystals through slow solvent evaporation

[0073] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX-HCI to Phenylalanine in a 51.004 mg of RLX-HCI and 16.519 mg of Phenylalanine mixture. In the 2:1 mixture 102.008 mg of RLX-HCI and 15.519 mg of Phenylalanine were mixed and in the 1 :2 mixture 51 .004 mg of RLX-HCI and 33.038 mg of Phenylalanine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0074] Preparation of RLX- HCI- L-Arginine crystals through slow solvent evaporation

[0075] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX-HCI to Arginine in a 51.004 mg of RLX-HCI and 17.02 mg of Arginine mixture. In the 2:1 mixture 102.008 mg of RLX-HCI and 17.02 mg of Arginine were mixed and in the 1 :2 mixture 51 .004 of RLX-HCI and 34.04 mg of Arginine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0076] Preparation of R LX- HCI - L-tryptophan crystals through slow solvent evaporation

[0077] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX-HCI to Tryptophan in a 51.004 mg of RLX-HCI and 20.423 mg of Tryptophan mixture. In the 2:1 mixture 102.008 mg of RLX-HCI and 20.423 mg of Tryptophan were mixed and, in the 1 :2 mixture, 51 .004 mg of RLX-HCI and 40.846 mg of Tryptophan were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0078] EXAMPLE 2

[0079] Extraction of RLX from RLX-HCI

[0080] Pure RLX was extracted from RLX-HCI for possible use in the method of obtaining a hydrate of RLX, however it was decided to conduct the further experiments using RLX-HCI.

[0081] To calculate mass of RLX-HCI and Sodium Hydroxide needed in molar ratios of 1 :1 , a decided amount of 2.9 g of RLX-HCI was needed, the number of moles for RLX-HCI were calculated to be 0.0056858286 and mass of NaOH was 227.42 mg. During extracting, the reaction to be used was RLX-HCI (dissolved in ethanol) + NaOH (dissolved in water) = R + NaCI (Salt) + water. Thereafter, 2.9 g of RLX-HCI was dissolved in 50 ml of Ethanol and 227.42 mg of NaOH was dissolved in 50 ml of distilled water. The two mixtures were separately placed on a digital heating magnetic stirrer and mixed without heating. The mixture of water and NaOH was poured into the mixture of RLX-HCI and ethanol as they stirred. An instant colour change to reddish was observed. The mixture was then placed in a heating oven to dry overnight. The resultant dried solid mixture was mixed with water, and the mixture remained solid. Filtration was conducted to filter out the water and the NaCI salt, remaining with the pure RLX-HCI. The Molecular weight of Raloxifene is 473.58 g / mol. Therefore, with a ratio of 0.1 mmol: 0.1 mmol, the mass of Raloxifene was calculated to be 47.358 mg. Adopted formula is 0.0001 x MW x 1000.

[0082] EXAMPLE 3

[0083] Synthesis of pure RLX-L-Amino acid crystals through slow solvent evaporation

[0084] Synthesis of pure RLX-L-tyrosine crystals through slow solvent evaporation

[0085] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX to Tyrosine in a 47.358 mg of RLX and 18.119 mg of Tyrosine mixture. In the 2:1 mixture 94.716 mg of RLX and 18.1 19 mg of Tyrosine were mixed and in the 1 :2 mixture 47.358 mg of RLX and 36.238 mg of Tyrosine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0086] Synthesis of pure RLX-Phenylalanine crystals through slow solvent evaporation

[0087] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX to Phenylalanine in a 47.358 mg of RLX and 16.519 mg of Phenylalanine mixture. In the 2:1 mixture 94.716 mg of RLX and 16.519 mg of Phenylalanine were mixed and in the 1 :2 mixture 47.358 mg of RLX and 33.038 mg of Phenylalanine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0088] Synthesis of pure RLX-L-Arginine crystals through slow solvent evaporation

[0089] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX to Arginine in a 47.358 mg of RLX and 17.02 mg of Arginine mixture. In the 2:1 mixture 94.716 mg of RLX and 17.02 mg of Arginine were mixed and in the 1 :2 mixture 47.358 mg of RLX and 34.04 mg of Arginine were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization.

[0090] Synthesis of pure RLX- L-tryptophan crystals through slow solvent evaporation

[0091] Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve 1 :1 molar ratio of RLX to Tryptophan in a 47.358 mg of RLX and 20.423 mg of Tryptophan mixture. In the 2:1 mixture 94.716 mg of RLX and 20.423 mg of Tryptophan were mixed and in the 1 :2 mixture 47.358 mg of RLX and 40.846 mg of Tryptophan were mixed. The components were mixed with a Z83 vortex mixer for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterization. EXAMPLE 4

[0092] Optimization of RLX-HCl-Arginine crystals

[0093] Preparation and optimization of RLX-HCl-Arginine crystals through Solvent evaporation with application of Heat and Stirring

[0094] This process was aimed at re-crystallizing the newly formulated Raloxifene Hydrate for purposes of optimization. Furthermore, to grow crystals from the newly formulated powdered hydrate as well as grow new crystals using a different method. Materials used were polytope containers, magnetic stirrers, heater and solvents such as methanol and ethanol. Raloxifene HCI with arginine in molar ratios of 1 :1 was weighed and placed in different test tubes. Amounts varied with increase in weight by x1 (51.004 mg: 17.02 mg), X2 (102.008 mg: 34.04), X5 (255.02 mg: 85.1 mg), X8 (408.032 mg: 136.16 mg), X10 (510.04 mg: 170.2 mg) and X20 (1 .02008 g: 340.4 mg). The mixture was heated at 78 °C and stirring at 200 rpm.

[0095] Preparation and optimization of RLX-HCl-Arginine crystals through Solvent evaporation without application of Heat and Stirring

[0096] This process was aimed at re-crystallizing the newly formulated Raloxifene Hydrate for purposes of optimization. Furthermore, to grow crystals from the newly formulated powdered hydrate as well as grow new crystals using a different method. Materials used were polytope containers, magnetic stirrers, heater and solvents such as methanol and ethanol. Raloxifene HCI with arginine in molar ratios of 1 :1 was weighed and placed in different test tubes. Amounts varied with increase in weight by x1 (51.004 mg: 17.02 mg), X2 (102.008 mg: 34.04), X5 (255.02 mg: 85.1 mg), X8 (408.032 mg: 136.16 mg), X10 (510.04 mg: 170.2 mg) and X20 (1 .02008 g: 340.4 mg). The mixture was not heated or stirred; however, the sample was left for 3 days until a precipitate formed at the bottom of the beaker. A crystal looking cake formed after 3 days. The sample was filtered using filter paper and the remaining solvent was left to evaporate.

[0097] Recrystallization of an already formulated sample of RLX-HCI-ARG powder through Solvent evaporation

[0098] A sample of the powder was placed into a test tube and 5mls of methanol was added. No stirring occurred and the sample was placed in an enclosed cupboard for 4 days to allow for the methanol to completely evaporate.

[0099] Preparation of RLX-HCI -ARG crystals by recrystallization through applying heat and cooling of the solvent to facilitate evaporation

[0100] Raloxifene HCI (51 Omg) with Arginine (170mg) in equi-molar ratios of 1 :1 was weighed and placed in a test tube. 10ml of ethanol was added into the test tube which was then placed on a magnetic stirrer. Stirring was set at 200rpm for 30 minutes and temperature was set at 78°C until the ethanol reached its boiling point. The test tube was allowed to cool down for at least 1 minute and was then placed into a beaker with ice. The mixture was then left for an hour until the ice melted. The beaker with the test tube was then placed into the fridge for 3-7 days. The resultant crystals were characterized on the FTIR, HSM, and DSC.

[0101] EXAMPLE 5

[0102] Initial Screening of Crystal Systems

[0103] Crystal Screening

[0104] In the initial stages, the study utilized a fast-screening process to assess the co-former feasibility in developing crystalline systems. These included citric acid, maleic acid, tartaric acid, malonic acid, gallic acid, L-tyrosine, phenylalanine, L-arginine, L-tryptophan, proline, glycine, and stearic acid which are all GRAS (generally regarded as safe) elements.

[0105] The preliminary screening procedure was employed to identify the suitable co-formers in the preparation of RLX-HCI hydrate. During the screening process, 4 different co-formers which included phenylalanine, tyrosine, tryptophan, and arginine were evaluated using the Hansen solubility parameter (HSR) and the pKa based models.

[0106] The Hansen Solubility Parameter (HSP) Studies

[0107] The Hansen solubility parameter (HSP) was used on RLX-HCI to predict the co-crystal formation between RLX-HCI and the co-formers. The HSP values of the co-formers were compared with those of RLX-HCI to determine whether the two materials were likely to be compatible and form crystalline systems as part of the theoretical prediction of potential cocrystal formation. The HSP can provide a first approximation of the likelihood of co-crystal formation. Co-formers with a smaller Delta value are typically more likely to form hydrate with the drug substance, as their similar solubility characteristics may lead to enhanced molecular interactions and stability in the solid state.

[0108] The HSP for RLX-HCI, HCI, and various co-formers are provided in Table 1. The HSP values are divided into three components which include dispersion (5D), polar (bP), and hydrogen bonding (bH), along with the total HSP (bT) and a delta value (ST of RLX-HCI - ST of co-former), which is the difference in solubility parameter between the drug and the coformer. The delta values represent the radius of the Hansen sphere or the distance in the Hansen space between the drug and each co-former. A smaller delta value indicates a closer match in the Hansen space and suggests better compatibility between the drug and the coformer for co-crystal formation. It has previously been suggested that, if the difference in the solubility parameter (Abt) value of the API and co-former is < 5MPa1 / 2, then potential hydrate might be formed (Mohammad et al, 201 1 ). Other studies on the prediction of miscibility between the API and co-former suggested the formation of potential hydrate if the difference is < 7 MPa1 / 2(Greenhalgh et al, 1999), or suggest a cut-off value of 8.18 MPa1 / 2(Nagy et al, 2019).

[0109] For Raloxifene, the dispersion and polar forces suggested its hydrophobic nature. Hydrophobic molecules tend to have higher dispersion forces relative to their polar forces. Besides hydrogen bonding parameter (bH), RLX-HCI can also engage in hydrogen bonding due to the presence of hydroxyl groups. The results obtained for the HSP, in particular the solubility difference (Abt) indicates that all the screened co-formers are within the suggested limit, indicating the potential to form hydrate with RLX-HCI (Table 1 ). Among the co-formers screened for, L-Arginine and phenylalanine, have the smallest delta values when paired with Raloxifene, with L-arginine having the Abt value of 3.2 and phenylalanine having 2.9.

[0110] This is consistent with the principle that a good solvent for a co-crystal formation is one that solubilizes both components equally well, leading to a uniform solution from which hydrate can precipitate upon solvent removal or evaporation. The HSP data suggests that although many of the co-formers presented show the potential to be promising co-formers. Among the qualified co-formers, the L-arginine was found to be more favourable.

[0111] Table 1. Hansen solubility parameter (HSP) for RLX-HCI with other co-formers

[0112] Drug 3D 3P 3H 8T

[0113] RLX-HCI 21.3 6.0 8.6 23.8

[0114] HCI 20.0 0.1 19.8 28.1

[0115] 25.95

[0116] Co-formers Delta

[0117] Citric acid 17.6 1 1.4 26.0 33.4 -7.5

[0118] Maleic Acid 18.2 10.8 23.0 31.2 -5.3

[0119] Tartaric Acid 17.8 14.0 29.2 37.0 -1 1.1

[0120] Malonic Acid 17.5 1 1.9 24.2 34.2 -8.3

[0121] Gallic acid 21.0 9.8 18.8 29.8 -3.9

[0122] L-Tyrosine 19.5 7.9 17.1 27.1 -1.2

[0123] Phenylalanine 18.5 6.0 12.5 23.1 2.9

[0124] L-Arginine 16.8 9.6 12.1 22.8 3.2

[0125] L-tryptophan 19.6 8.3 13.0 24.9 1.1 pKa Based Model

[0126] The pKa based model was used as a pharmacokinetic model to predict the absorption, distribution, metabolism, and elimination of RLX-HCL Hydrochloride (HCI) based on its acid dissociation constant (pKa) values in relation to the pKa values of the co-formers. In this study, 2 pKa values, including the strongest acidic as well as the strongest basic were used for each co-former in relation to the API. The difference between the pKa of the strongest acidic and the strongest basic was then determined for the API as well as the other co-formers. In this study, the pKa (acid dissociation constant) based model was used for the prediction of the acid dissociation constants of the co-formers. This is crucial in understanding their protonation and deprotonation during co-crystal formation. This model allows for the prediction of ionization states, hydrogen bonding patterns, and overall molecular reactivity of the API and co-formers. The transfer of proton can be observed if the difference in the pKa value is more than 3. According to previous studies, if the ApKa value is less than zero, then hydrate might be formed but if the ApKa value is greater than 3, it could result in the formation of salts. However, the ApKa is in between 0-3, then either a co-crystal or salt can be expected to be formed (Thayyil et al, 2020). As indicated in Table 2, considering that RLX-HCI is the base in the formula, ApKa = [pKa (base) - pKa (acid)], the four amino acids (tyrosine, phenylalanine, arginine, and tryptophan) showed a predicted formation of hydrate as compared to other coformers. These amino acids presented negative delta pKa values (below zero). According to the pKa based model, the four amino acids tyrosine, phenylalanine, arginine, and tryptophan with pKa values of -0.8, -1.1 , -0.6 and -0.9 respectively have higher potential of forming hydrate as compared to the other co-formers. Studies have highlighted the role of amino acids in cocrystal formation. For the carboxylic acids, maleic acid, and malonic acid, the ApKa values were 2.2 and 2.724 respectively. According to the guideline provided, a ApKa between 0-3 indicates that either a co-crystal or salt may form. Furthermore, a ApKa greater than 3 indicates the formation of salts as is the case with citric acid, tartaric acid and gallic acid with ApKa of 3.72, 4.12 and 3.52 respectively.

[0127] Table 2: pKa based model results for RLX-HCI with different co-formers.

[0128] EXAMPLE 6

[0129] Preparation and Characterisation of the Crystalline Systems

[0130] Solvent evaporation

[0131] The present study explored the possibility of preparing RLX-HCl-hydrate using amino acids Rhe, Tyr, Trp and Arg as co-formers. The hydrate were further investigated with the aid of DSC, HSM, FTIR and XRD. The binary physical mixtures (PMs) of RLX-HCl-Phe, RLX-HCI- Tyr, RLX-HCl-Trp and RLX-HCl-Arg prepared by gently grinding the accurately weighed (Radwag analytical balance, Radom, Poland) quantities of RLX-HCI (API) and Phe, Tyr, Trp, and Arg (co-formers) at different drug to co-former molar weight ratios of 2:1 , 1 :1 and 1 :2 (w / w). The components were then placed in a test tube and mixed using a mortar and pestle. Approximately 5-10 ml of analytical grade ethanol or methanol was added to dissolve the RLX- HCL-co-former mixture, and the components were mixed with a Z83 vortex mixer (FisherbrandTM, Milan, Italy) for at least 30 seconds. The test tube was placed under fume cupboards at room temperature to allow the solvent to evaporate. The resultant mixtures were further left to completely dry in a desiccator prior to characterisation. For optimization of the RLX-HCl-Arg hydrate, a re-crystallization technique was used. Where, approximately 8 ml of analytical grade ethanol was added to the weighed ingredients and the components were placed in a test tube and placed on a hot-plate magnetic stirrer. The mixture was heated at 78 °C until the ethanol reached boiling point. This was done whilst the magnetic stirrers kept stirring for about 30 minutes. The sample was then removed and left to cool down for 2 minutes. The sample was then placed in the fridge for 3-7 days. To harvest the crystals, ethanol was removed while filtering the precipitate. The extracted crystals were dried for further characterization.

[0132] Fourier transform infrared spectroscopy

[0133] Fourier Transform Infrared Spectroscopy (FTIR) absorption spectra of the samples was collected using an Agilent Cary 630 Fourier Transform spectrophotometer (Agilent Technologies, California, United States of America) and was analysed using OriginPro® 9 (Northampton, Massachusetts, United States of America) Software. Approximately 8 mg of powder was placed onto a diamond crystal and analysed at a wavenumber range of 400-4000 cm'1at a rate of 4 cm-1(Figure 1 ).

[0134] The FTIR spectra of RLX-HCI characteristic absorptions are evident at specific wavenumbers corresponding to functional group vibrations. The N-H stretching vibrations manifest at 3200 cm'1, while the O-H bond stretching is observed at 3140 cm'1. Carbonyl (C=O) stretching vibrations are present at 1600 cm'1, with the C=C stretching vibrations occurring in the range of 1470 cm-1to 1600 cm'1. Additionally, C-N stretching vibrations are detected at 1260 cm'1.

[0135] Arginine (Arg), in contrast, demonstrates distinct peaks at 3302 cm-1and 3357 cm'1, attributable to the guanidine and primary amine groups, respectively. In the RLX-Arg (2:1 ) crystals, a new absorption peak at 3530 cm'1is observed, alongside an alteration at 3350 cm-1. The peak at 3530 cm-1may denote the formation of a new hydrogen-bonded species, suggesting an interaction between the N-H groups of RLX-HCI and the guanidine or amine groups of Arg. The 3350 cm'1alteration could be attributed to a shift in the hydrogen bonding pattern of Arg’s guanidine group. The RLX-Arg (1 :1 ) crystals displays similar spectral features, with the 3530 cm'1peak indicating a strong interaction between the components, potentially due to the equimolar presence facilitating optimal hydrogen bond formation. The peak at 3350 cm'1in this ratio could reflect the balanced interaction between the guanidine group of Arg and the functional groups of RLX-HCI. Conversely, the RLX-Arg (1 :2) crystals, where Arg is in excess, still shows the new peak at 3530 cm'1, but the intensity and shape of this peak may differ, reflecting the altered stoichiometry and the possible saturation of hydrogen bonding sites on RLX-HCI.

[0136] The broadening of the peaks in the 1140-1228 cm'1region across all mixtures is indicative of an interaction affecting the C-N bond, potentially due to an overlap of the vibrations from both RLX-HCI and Arg. This broadening becomes more pronounced with increasing Arg content. Additionally, the elongation of the spectra in the 3100-3300 cm-1region for all mixtures denotes a deviation from the absorption patterns of both RLX-HCI and Arg, which is indicative of a complex interaction between the N-H and O-H stretching vibrations, potentially altering the hydrogen bonding network. The FTIR spectra of the RLX-Arg crystals exhibit significant deviations from the spectra of the individual components, RLX-HCI and Arg.

[0137] The appearance of new peaks and the alteration of existing ones across the different ratios signify molecular interactions, hydrogen bonding, between RLX-HCI and Arg. These interactions vary with the stoichiometry of the mixtures, which may influence the physical and chemical properties of the resultant compounds, carrying potential implications for their pharmaceutical applications. The FTIR spectra of the physical mixtures (PM) of RLX-HCI and Arg in different ratios provides insights into the potential interactions or lack thereof between the two compounds. In the context of physical mixtures, the inventors would anticipate observing superimposed spectra of the individual components, unless there are interactions that alter the chemical environment of the functional groups involved.

[0138] For the RLX-Arg (2:1 ) PM, the spectra within the 2500-3500 cm-1range do not show new peaks or significant shifts, which aligns with the characteristic peaks of both RLX-HCI and Arg. This suggests that within this ratio, the compounds are not interacting strongly enough to alter the O-H or N-H stretching vibration regions typically observed around 3200 cm-1for N-H stretching, 3140 cm'1for O-H bonds, and the 3302-3357 cm-1range for Arg's guanidine and amine groups. The absence of new peaks or shifts in this region indicates that there is no hydrogen bonding or other strong intermolecular forces affecting the hydroxyl or amine groups’ vibrational frequencies.

[0139] Similarly, the RLX-Arg (1 :1 ) PM spectrum indicates that the mixing of RLX-HCI and Arg in equal proportions does not result in significant changes in the O-H and N-H regions. The preservation of the original peaks from both components implies that the physical mixture does not exhibit new intermolecular interactions that are detectable by FTIR spectroscopy.

[0140] The RLX-Arg (1 :2) PM, where Arg is in excess, the spectrum closely resembles that of pure Arg, suggesting that the characteristics of Arg dominate the spectral features. This is especially true if the Arg peaks are more intense or broad, which would overshadow the contributions from RLX-HCI. The lack of notable change in the spectrum implies that the excess Arg does not engage in new interactions with RLX-HCI that would alter its spectral signature, particularly in the regions where Arg has distinctive absorptions. The physical mixtures can still have weak interactions that may not be detected by FTIR due to the limitation of the technique in distinguishing subtle changes when the individual components' peaks overlap significantly.

[0141] The RLX-HCI and Arg physical mixtures, in the molar ratios, largely retain their individual spectral characteristics, indicating minimal interaction, particularly in the form of hydrogen bonding, within the limitations of FTIR detection. The FTIR spectra of RLX-Arg physical mixtures at different ratios (2:1 , 1 :1 , 1 :2) suggest that the mixing of RLX-HCI and Arg does not lead to significant molecular interactions that alter the vibrational frequencies. This is evidenced by the preservation of characteristic peaks of both components in the mixtures, with the spectrum of the 1 :2 mixture closely resembling that of Arg due to its predominance in the mixture.

[0142] Differential scanning calorimetry (DSC)

[0143] Melting characteristics of the samples were performed using a DSC 3 (Mettler Toledo, Ohio, United States of America) and analysed using Starre software V17.00 (Mettler Toledo, Ohio, United States of America). Approximately 3-5 mg of the sample was weighed using an analytical balance into aluminium crucible, which was sealed with a pin-holed aluminium lid. Samples were heated against blank crimped pans from room temperature to 300 °C at the rate of 10 °C / min under nitrogen flow of 10 ml / min.

[0144] The DSC thermograms provided in Figure 2 offer insight into the thermal behaviour of RLX-HCI, Arg, and their hydrate at different molar ratios (2;1 , 1 :1 and 1 :2). For RLX-HCI, the thermogram exhibits a distinct and sharp endothermic peak at 268.8 °C, which is indicative of the melting point of the substance. This confirms the purity and identity of the RLX-HCI sample. The sharpness of the peak suggests a pure crystalline phase with a well-defined melting transition. In the case of Arg, the thermogram shows a complex thermal behaviour. The initial small endothermic peak at 100.1 °C can be attributed to a dehydration effect, likely due to the loss of water molecules from the arginine sample. Following this, the second and third peaks at 234.3 °C and 239.6 °C respectively, correspond to the melting process, with the potential for overlapping melting and decomposition processes. The reported melting points in the literature are 220.7°C and 241 .7 °C and the differences could be due to variations in the sample purity or experimental conditions.

[0145] The crystals at a 2:1 molar ratio showed a shift in the thermal events compared to the individual components. The peak observed at a slightly lower temperature, 98 °C can be ascribed to dehydration. The subsequent melting peaks at 127 °C and 184 °C do not align with the pure substances' melting points. This suggests the formation of a solid solution or a complex between RLX-HCI and Arg, altering the thermal behavior. Furthermore, these melting peaks could indicate a eutectic formation, where the mixture melts at a lower temperature than either of the pure components, or it could be indicative of a solid dispersion where one drug is molecularly dispersed within the crystal lattice of the other, leading to depression of the melting point. For the RLX-Arg (1 :1 ) molar ratio, the singular endothermic peak at 129.3 °C suggests a significant alteration in phase behavior, likely due to the formation of a new compound or a eutectic mixture or formation of a hydrate. Eutectic mixtures occur when two components exhibit complete miscibility in the liquid phase but limited solubility in the solid phase, leading to a combined melting point that is lower than either component alone. The presence of only one peak implies that the RLX-HCI and Arg have interacted to form a new phase with a unique thermal signature, distinct from either pure compound.

[0146] The 1 :2 crystals presents a dehydration peak similar to the 2:1 crystals but follows with endothermic peaks at 126 °C and 181 °C. Again, these temperatures do not match the melting points of the pure compounds, suggesting the formation of a new phase, possibly a eutectic or a compound with a depressed melting point due to the solid solution formation. In all mixtures, the absence of endothermic peaks corresponding to the pure components' melting points suggests that the RLX-HCI and Arg are not merely physically mixed but chemically or physically interacting. These interactions could be hydrogen bonding, van der Waals forces, or ionic interactions, potentially leading to hydrates or eutectic mixtures especially in the RLX- Arg (1 :1 ) ratio.

[0147] Powder x-ray diffraction (PXRD)

[0148] The PXRD diffractograms of the pure and prepared samples were recorded using a D2 phaser XE-T Edition (Brucker, Massachusetts, United States of America). The operations used a Cu Ka radiation with a voltage of 40 kV, and 40 Ma current. The divergent slit of the machine was set at 0.2 mm and monochromator scanning was performed at a scan speed of 20 per minute with step size of 0.025°.

[0149] The PXRD pattern (Figure 3) of RLX-HCI presents sharp and distinct peaks at angles 20 = 8.7°, 11 °, 14.1 °, 14.9°, 15.9°, 18.7°, 20.3°, 21.4°, 22.4°, 24.2°, 25.6°, 27.0°, 27.8°, and 31 .5°. These peaks correspond to the crystalline planes of RLX-HCI, indicating a high degree of crystallinity. The characteristic peaks reported in the literature at 20 = 13.64°, 14.74°, 15.97°, 19.37°, 21.20°, 21.57°, 22.90°, and 24.32° are comparable to those observed, which confirms the phase purity and the crystalline nature of the RLX-HCI sample.

[0150] The PXRD pattern for Arg shows peaks at 20 = 11.5°, 17.2°, 18.1°, 18.5°, 20.2°, 25.2°, 31 °, and 33.5°. The unique set of peaks indicates that Arg has a crystalline structure that is distinct from RLX-HCI. The absence of shared peaks implies no polymorphic similarities between the two compounds in their pristine forms. The crystal derived from RLX-Arg in a 2:1 molar ratio displays peaks at 20.3°, 21.9°, 23.8°, and 25.1 °. The disappearance of most peaks from the individual compounds indicates a significant interaction that alters the original crystalline structures, possibly leading to the formation of a new phase. The visible peaks being more reflective of RLX-HCI suggest that at this ratio, the RLX-HCI crystal lattice dominates, possibly encapsulating Arg within its structure. The RLX-Arg (1 :1 ) ratio reveals new peaks at 20 = 8.7°, 10.9°, 13.3°, and 28.2°, which are absent in the individual diffractograms of RLX- HCI and Arg. The appearance of new, distinct, and sharp peaks, especially at high intensities (10.9° and 28.2°), indicates the formation of a new crystalline phase entirely. This could be due to the formation of a crystals, a eutectic or a new solid dispersion where the molecules of RLX-HCI and Arg arrange themselves into a novel crystalline network. The additional peaks at 14.4°, 15.3°, 18.4°, 20.7°, 22.9°, and 25.4° that align with RLX-HCI, and those at 18° and 22.3° that align with Arg, suggest a partial retention of the parent crystalline structures alongside the new phase. For the RLX-Arg (1 :2) ratio, the diffractogram shows a peak at 20 = 9.5°, which appears to be more related to RLX-HCI, and others at 16°, 19.74°, 20.1 °, and 24.1 °, which are more aligned with Arg. The presence of these peaks indicates a less complete interaction compared to the 1 :1 ratio, with the Arg structure seemingly more prominent in the mixture. This suggests that the Arg crystalline network has a stronger influence on the resulting structure, with RLX-HCI possibly integrating into the Arg lattice.

[0151] Furthermore, physical mixtures were also analysed (Figure 3). The RLX-Arg PM (2:1 ) PXRD pattern for this ratio shows peaks with low intensity at 20 = 15.5°, 16.5°, 20.3°, 22.1°, 23.8°, and 28.5°. The low intensity and fewer number of peaks, compared to the individual compounds, could be indicative of a dilution effect where the presence of one compound diminishes the crystalline signature of the other without significantly altering the crystal structure. This might also suggest partial amorphization or a decrease in crystallinity due to the physical mixing process. The RLX-Arg PM (1 :1 ) diffractogram for the 1 :1 physical mixture exhibits peaks at 20 = 11.2°, 13.3°, 15.6°, 16.8°, 18.7°, 19.4°, 20.6°, 23.2°, 24.9°, 26.1°, and 29.9°. The greater number of peaks and their alignment with those of pure RLX-HCI and Arg suggest that the crystalline structures of both components are preserved in the mixture. This indicates that there is no significant interaction at the molecular level that would lead to the formation of a new crystalline phase. Instead, the mixture represents an additive combination of the two crystalline patterns. The RLX-Arg PM (1 :2) physical mixture, peaks are observed at 20 = 16°, 20.8°, 22.3°, 23.8°, and 28.7°. Similar to the 2:1 mixture, the fewer peaks with low intensity could be due to the presence of Arg in a higher ratio, which may lead to a less pronounced crystalline signature of RLX-HCI. The preserved peaks from both components, however, indicate that the compounds are co-existing without forming a new phase.

[0152] Hot stage microscope (HSM)

[0153] The thermal melting behavior of the samples was investigated using a Linkam u-p03 hot stage microscope (Olympus, Tokyo, Japan), employing objective lenses with 4X and 10X magnification capabilities. The samples were subjected to a controlled temperature program with a ramp of 10 °C / min, spanning a broad thermal spectrum from an initial temperature of 25 °C to an upper limit of 300 °C. This methodological approach facilitates the precise determination of melting and other thermal events pertinent to the samples under investigation.

[0154] Hot-stage microscopy (HSM) was utilized to scrutinize the thermal dynamics and phase transitions of RLX-HCI, Arginine, and their crystals across varying ratios. The experiments were conducted at a heating rate of 10 °C / min, enabling detailed observation of the samples' behavior during thermal excursions. RLX-HCI exhibited a first melting event at 260 °C, with complete melting occurring at 269.2 °C, consistent with the DSC findings. Similarly, Arginine displayed its initial melting at 249.3 °C, with complete melting observed at 254.3 °C, aligning well with the DSC observations.

[0155] For RLX-ARG (1 :1 ) crystals, the HSM analysis revealed a notable first melting event at 127.2 °C, with complete melting observed at 150.5 °C. This was closely related to that observed in the DSC. During heating, the micrographs depicted the gradual disappearance of crystalline structures, signifying the transition from solid to liquid phase. This behavior aligns with the fundamental principles of phase transitions, where increased temperature induces molecular motion, disrupting the ordered lattice arrangement characteristic of the solid state. In the case of RLX-ARG (1 :2), the HSM observations indicated a distinct initial melting point at 129.7 °C, with complete melting occurring at 290.3 °C. As the temperature rose, the micrographs exhibited the gradual dissolution of crystalline entities, suggesting the progressive transition towards a molten state. This process reflects the thermodynamic principle of melting, where the energy supplied overcomes intermolecular forces, leading to the breakdown of the solid structure into a more disordered liquid phase. Similarly, RLX-ARG (2:1 ) crystals demonstrated a first melting event at 128.5 °C, with complete melting observed at 265.3 °C.

[0156] The HSM micrographs illustrated in Figure 4 showed the onset of melting at the designated temperature, followed by the disappearance of solid structures, indicative of the transition to a liquid phase. This behavior is consistent with the principles of thermal analysis, where the observed melting temperatures correspond to the energy required to overcome the intermolecular forces holding the crystalline lattice together. The observed melting temperatures and thermal behavior during the HSM analysis were in agreement with the results obtained from differential scanning calorimetry (DSC), providing corroborative evidence of the samples' thermal characteristics. In summary, the HSM analysis facilitated a comprehensive understanding of the thermal properties and phase transitions of RLX-HCI, Arginine, and their crystals across different ratios. These insights contribute to the elucidation of the materials' behavior under thermal stress, which is crucial for various pharmaceutical and materials science applications.

[0157] Scanning Electron Microscopy (SEM) - Energy-dispersive X-ray Spectroscopy (EDX)

[0158] Scanning electron microscopy was used for evaluation of the morphological properties of the samples whereas the energy-dispersive X-ray spectroscopy EDX, also referred to as EDS, was based on the generation of X-rays interaction of an electron beam with sample atoms which releases continuous spectrum of X-rays. These sharp X-ray signals are produced at wavelengths that are specific for a given element which is crucial for elemental mapping by SEM. The carbon coated samples were placed on a holder, vacuum dried, and examined at different magnifications. The shape and surface morphology of the formulated samples were viewed using the SEM SUPRA 55 VP, Carl Zeiss, Oberkochen (Baden-Wurttemberg, Germany) at a 2 kV accelerating voltage. The samples will be lightly sprinkled, mounted on aluminum stubs using double-sided adhesive carbon tape, and then sputter coated with approximately 15 nm gold using a Quorum T150 ES coater (East Sussex, United Kingdom) before imaging. In addition, the appearance of the samples was photographed using a camera. These prepared samples were further analysed under varying degrees of magnification to ascertain their elemental composition and distribution. The examination was conducted using an Oxford Instruments X-Max 50mm2EDX detector (Abingdon, Oxfordshire, United Kingdom). This was coupled with a 30 kV accelerating voltage for optimal resolution. The elemental analysis and mapping were done through UNION Instruments GmbH INCA analyser software (Baden-Wurttemberg, Germany) which facilitated the precise detection and quantification of the elemental constituents present within the samples.

[0159] SEM images shown in Figure 5 indicate RLX-HCI at 150 times magnification. The image shows elongated, rod-like structures typical of crystalline forms. Arg is shown at 60 times magnification showing relatively large, irregularly shaped particles with a rough surface, indicating a different crystal habit. Furthermore, RLX-Arg PM (1 :1 ) is a physical mixture of RLX- HCI and Arg in a 1 :1 ratio, observed at 106 times magnification. The image shows a more heterogeneous mixture of particles, where both the elongated structures of RLX-HCI and the irregular shapes of Arg are visible. This suggests that in the physical mixture, the two substances retain their individual characteristics without forming a new compound. Finally, RLX-Arg (1 :1 ) is a sample of RLX-HCI and Arg in a 1 :1 ratio, imaged at 100 times magnification. Without additional context, it's unclear whether this represents a crystal, a solid solution, or another form of compound such as a hydrate or eutectic. However, the image shows a more homogeneous morphology compared to the physical mixture, suggesting some form of interaction between RLX and Arg, suggesting a crystallization or a more intimate mixing at the molecular level.

[0160] Single crystal XRD

[0161] Intensity data was determined on a Bruker D8 Venture Microfocus with Photon III CCD area detector diffractometer with graphite-monochromated MoK1 (0.71073 A) radiation at 173 K using an Oxford Cryostream 600 cooler. Data reduction was carried out using the program SAINT+, version 6.02 and empirical absorption corrections were made using SADABS. Space group assignments was made using XPREP. The structure was solved in the WinGX Suite of programs, using intrinsic phasing through SHELXT and refined using full-matrix least- squares / difference Fourier techniques on F using SHELXL-2019 / 3. All C-bound hydrogen atoms were placed at idealized positions and refined as riding atoms with isotropic parameters 1 .2 or 1 .5 times those of their parent atoms. All O-bound H atoms were in the difference Fourier map and their fractional coordinates and isotropic displacement parameters refined freely. The residual electron density was removed using Squeeze and could correspond to the methanol / ethanol crystallization solvent used. Diagrams were generated using ORTEP-3 and PLATON.

[0162] The SCXRD analysis revealed that the RLX-Arg (1 :1 ) compound had an empirical formula C28H29NO5S as shown in Table 3. The SCXRD was crucial in gaining significant insights into its molecular and crystal structure of this sample. The compound, having a molecular weight of 491.58, was studied at a low temperature of 173 K (-100.15 °C). This low temperature is crucial in minimizing thermal vibrations within the crystal lattice, allowing for more precise and less distorted diffraction patterns. Such clarity is essential for accurate structural determination. The use of a 0.71073 A wavelength, typical for copper K-alpha radiation, further assisted in achieving high-resolution data. The crystal system was monoclinic, and the space group C 2 / c indicates a specific kind of symmetry and molecular packing within the crystal. This information is critical for understanding the intermolecular interactions and the overall stability of the crystal. The unit cell dimensions (a = 29.394 A, b = 11 .5056 A, c = 16.3953 A) with the non-standard angle of 97.806° suggest a unique packing arrangement, which might influence both the physical and chemical properties of the compound. A large unit cell volume of 5493.5 A3, in combination with the crystal system and space group, suggests a complex arrangement of molecules within the crystal. With a calculated density of 1.189 Mg / m3, the material's interactions with light and X-rays can be inferred, affecting its absorption properties. The absorption coefficient of 0.154 mm-1also provides information on how X-rays are absorbed by the crystal, which is crucial for adjusting the parameters of the diffraction experiment. The crystal size, although relatively small (0.398 x 0.206 x 0.165 mm3), was sufficient for the collection of a large number of reflections (103245), indicating a good quality crystal. This high number of reflections, along with a completeness of 99.9% up to a theta of 25.242°, demonstrates the high quality of the data collected, ensuring reliability in the resulting structural model. The refinement details, including the final R indices (R1 = 0.0351 , wR2 = 0.0974 for I > 2sigma (I)), indicate a high degree of accuracy in the model fitting to the observed data. A goodness-of-fit on F2 of 1 .037 is another testament to the reliability of the structural data obtained.

[0163] Table 3: Crystal data and structure refinement for 23ab_yc1_A

[0164]

[0165] Figure 6 represents the molecular structure of RLX-Arg (1 :1 ) as determined by the SCXRD. This is referred to as an ORTEP diagram, which provides a visual representation of the atomic positions and their thermal vibration. In the figure, the atoms are represented by ellipsoids, which show the thermal motion at a particular probability level. Hydrogen atoms are usually depicted as small spheres or circles attached to their respective carbons. The bonds between atoms are depicted as solid lines, while dashed lines indicate hydrogen bonding or other non-covalent interactions. The structure suggests a complex organic molecule with multiple rings, likely aromatic given the delocalized pi-electron systems implied by the ring structures. The presence of oxygen and nitrogen atoms suggests functional groups that may include ketones, ethers, or amines, which are common in pharmaceutical compounds or natural organic products.

[0166] One notable feature is the hydrogen bond (dashed line) between the nitrogen atom labelled N1 and a water molecule (indicated as 01 W), suggesting the presence of a hydrate form or solvent inclusion within the crystal structure. This kind of interaction is crucial as it often influences the packing and stability of the crystal lattice. The thermal ellipsoids for some atoms appear elongated, indicating increased thermal motion or disorder in these positions. This is important for understanding the dynamic aspects of the molecular structure. The significance of Figure 6 is multifaceted. It confirms the molecular structure and stereochemistry of the compound, which is essential for understanding its reactivity and interaction with biological targets. The precise arrangement of atoms and the identification of non-covalent interactions, such as hydrogen bonds, provides insight into the compound's potential binding modes with other molecules, which is particularly relevant in drug design.

[0167] The absence of arginine in the Single Crystal X-ray Diffraction (SCXRD) structure of the compound synthesized from RLX-HCI and Arg may be due to several factors. One possibility is that during the solvent evaporation process, arginine did not co-crystallize with RLX-HCI. This could occur if the conditions favored the crystallization of RLX-HCI alone, possibly due to its higher concentration or a more favorable crystal packing arrangement. Arginine, being more soluble, might have remained in the solvent phase. Solubility differences are a common reason for the lack of co-crystallization in multi-component systems. Another reason could be related to the stoichiometry of the reaction used to synthesize the compound. If the reaction conditions or the starting material ratios did not favor the formation of a 1 :1 stoichiometric complex between RLX-HCI and arginine, the resulting crystal may not have included arginine in its lattice. It's also possible that arginine was present initially but decomposed or reacted during the crystallization process. Conditions such as pH, temperature, or the presence of other reactive species could lead to the degradation or transformation of arginine into different species that did not incorporate into the crystal lattice. Furthermore, the interaction between RLX-HCI and Arg might be non-stoichiometric or too weak to survive the crystallization process. In some cases, the intended non-covalent interactions (e.g., hydrogen bonding, ionic interactions) between components may not be strong enough to withstand the conditions during solvent evaporation, leading to the formation of a crystal lattice that excludes one of the components.

[0168] The crystal structure presented in Figure 6 confirms that this is Raloxifene hydrate. There is evidence of some solvent incorporated in the crystal lattice, but the atom position could not be resolved. The structure has not been determined previously. There is no evidence that the HCI used in the crystallization has caused a chloride salt to form.

[0169] The absence of arginine in the Single Crystal X-ray Diffraction (SCXRD) structure of the compound synthesized from RLX-HCI and Arg may be due to several scientific factors. One possibility is that during the solvent evaporation process, arginine did not co-crystallise with RLX-HCI. This could occur if the conditions favoured the crystallization of raloxifene HCI alone, possibly due to its higher concentration or a more favourable crystal packing arrangement. Arginine, being more soluble, might have remained in the solvent phase. Solubility differences are a common reason for the lack of co-crystallisation in multi-component systems. Another reason could be related to the stoichiometry of the reaction used to synthesize the compound. If the reaction conditions or the starting material ratios did not favour the formation of a 1 :1 stoichiometric complex between RLX-HCI and Arg, the resulting crystal may not have included Arg in its lattice. It's also possible that Arg was present initially but decomposed or reacted during the crystallization process. Conditions such as pH, temperature, or the presence of other reactive species could lead to the degradation or transformation of Arg into different species that did not incorporate into the crystal lattice. Furthermore, the interaction between RLX-HCI and Arg might be non-stoichiometric or too weak to survive the crystallization process. In some cases, the intended non-covalent interactions (e.g., hydrogen bonding, ionic interactions) between components may not be strong enough to withstand the conditions during solvent evaporation, leading to the formation of a crystal lattice that excludes one of the components.

[0170] EXAMPLE 7

[0171] Solubility studies

[0172] Assay Method

[0173] An amount equivalent to 100 mg RLX-HCI was weighed and dispersed in acetonitrile and sonicated for 10 min to achieve a clear solution. To prepare the stock solution, each solution was diluted to 100 mL with distilled water. One millilitre (1 ml) from the stock solution was further diluted to 100 ml with distilled water. A similar procedure was employed to prepare an RLX-HCI reference standard solution to achieve a concentration of 100 pg / mL. Subsequently, samples were analysed using high-performance liquid chromatography (HPLC). A Shimadzu (Kyoto, Japan) HPLC system consisting of a pump (LC-20AD), an autosampler (SIL-20AHT), UV detector (SPD-M20A), and Lab solutions software were utilised. Prior to injection of the drug solution, the column was equilibrated for at least 25 min with the mobile phase flowing through the system. The API, RLX-HCI was analysed at 287 nm using a Shimadzu Shim-pack GIST column, 150 x 4.6 mm; 5pm particle size (Tokyo, Japan) at ambient temperature. A mobile phase consisting of a buffer and acetonitrile in the ratio 64:36 (v / v), at a flow rate of 1.0 mL / min and an injection volume of 50 pL injection volume was used. The buffer consisted of 50 mM potassium di-hydrogen ortho phosphate monohydrate, with pH adjusted to 3.0 using ortho-phosphoric acid. They were filtered through 0.45p membrane filter before use, degassed and were pumped from the solvent reservoir. Drug concentration was calculated by using the calibration curve in the range of 2.5 - 15 pg / mL, with a correlation coefficient (r2) of 0.9991 . The RLX-HCI concentration was measured in triplicate and the mean ± SD were reported in Table 4.

[0174] Table 4: RLX-HCI linearity data

[0175] The equilibrium solubility of RLX-HCI and RLX-Arg (1 :1 ) hydrate crystals were determined in distilled water, Hydrochloric Acid Buffer pH 1.2 HCI buffer, acetate buffer (pH 4.5) and phosphate buffer (pH 6.8). An excess amount of the RLX-HCI and RLX-Arg (1 :1 ) hydrate were added to 10 mL of the solvents mentioned above, in glass poly-top vials. The samples were kept at equilibrium for a period of 72 hrs on orbital shaking incubator at 37 ± 0.5°C and 50 rpm. The contents of vials were filtered through 0.45-micron filter and analysed by HPLC at 287 nm. With the aid of the calibration curve, the concentrations were determined. Table 5 shows the samples run on the HPLC in triplicate with the mean area under the curve calculated as well as the concentrations.

[0176] Table 5: Samples run on the HPLC in triplicate with the mean area under the curve calculated as well as the concentrations.

[0177] Further, the data shows in Figure 7 that the concentration of RLX-HCI and the formulated hydrate, RLX-Arg (1 :1 ) in solution varies with pH, which is a common observation for compounds with ionizable compounds. When dissolved in distilled water (DH2O), RLX-HCI shows a high concentration, suggesting that it has a better solubility profile in neutral conditions without the influence of pH. The RLX-Arg (1 :1 ) hydrate, however, exhibits a lower concentration (0.19 pg / ml), which could be attributed to the nature of the hydrate affecting its solubility. Moreover, at pH 1.2, the concentration is relatively low for both RLX-HCI and RLX- Arg (1 :1 ), indicating that the drug and its hydrate may not be fully dissolved or ionized at this acidic pH. The acidic environment may not be favourable for the solubility of RLX-HCI or newly formulated its hydrate, possibly due to protonation of the RLX-HCI, which reduces their solubility. When the pH is increased to 4.5, a substantial increase in the concentration of RLX- HCI to 1.8 pg / ml is observed, indicating improved solubility at this pH level. This could be due to the drug approaching its pKa value, leading to an optimal balance between ionized and nonionized forms for solubility. However, the concentration of the RLX-Arg (1 :1 ) hydrate does not increase to the same extent, which could be due to the different solubility profiles of the hydrate compared to RLX-HCI, possibly because of interactions between the RLX-HCI and Arg in the hydrate or the removal of Arg as observed in the SCXRD data. The concentration of RLX-Arg (1 :1) only increases to 0.41 pg / ml, suggesting that the hydrate may have different solubility characteristics possibly due to the formation of a less soluble complex when the solvent was incorporated into the crystal lattice and the loss of the HCI salt as observed in the SCXRD data. Furthermore, at pH 6.8, the concentration of both RLX-HCI and RLX-Arg (1 :1 ) decreases, with RLX-HCI showing a more significant reduction. This trend may be due to the ionization state at a higher pH where the drug is less ionized, reducing its solubility. It could also be indicative of the formation of less soluble aggregates at this pH. It is at this pH however where the solubility of the hydrate was greater than that of the drug.

[0178] In the context of RLX-HCI absorption, the environment of the small intestine, with a pH of around 6.8, plays a crucial role. At this pH, one would not typically expect high solubility for RLX-HCI given its Log P value (5.69), which indicates a preference for lipophilic environments. This characteristic is essential for the drug to effectively cross the lipid bilayer of intestinal cells. However, the inherent lipophilicity of RLX-HCI at pH 6.8, as suggested by its high Log P value, aligns with the requirement for efficient absorption through the lipid-rich cellular membranes. The hydrate form RLX-Arg (1 :1 ), with a significantly lower Log P (1.11 ), represents a noteworthy alteration in this balance. By enhancing the drug's hydrophilicity at this critical pH, the hydrate formulation could potentially improve RLX-HCI solubility in the intestinal environment. This increased solubility at pH 6.8, although resulting in a less lipophilic compound, could paradoxically enhance the bioavailability of RLX-HCI. Improved solubility often facilitates better dissolution rates and higher concentrations of the drug in the gastrointestinal tract, which are key factors in enhancing oral absorption. Thus, while the hydrate form of RLX-HCI exhibits decreased lipophilicity, this alteration appears to be strategically advantageous for increasing the drug's bioavailability in the intestinal environment, thereby potentially improving its overall absorption profile.

[0179] EXAMPLE 8

[0180] In vitro dissolution studies

[0181] In vitro dissolution studies RLX-HCI were determined in 900 mL of pH 6.8 (phosphate buffer) at 37 ± 0.5°C with a stirrer rotation speed of 75 rpm using the USP dissolution test apparatus type II (paddle type) ERWEKA DT 128 light series (Langen, Germany). The RLX- HCI-co-former systems were taken equivalent to 100 mg of RLX-HCI with an aliquot of 5 mL of sample withdrawn at 5, 10, 15, 25, 30, 60,120, and 720 min, filtered through 0.45-micron filter, suitably diluted and transferred into HPLC vials. Samples were analysed at wavelength of 287 nm in triplicate. As a model independent approach, dissolution efficiency (DE) was utilized to evaluate the dissolution rate of RLX-HCL. DE is defined as the area under the dissolution curve up to the time tt, expressed as a percentage of the area of the rectangle described by 100% dissolution at the same time. The DE values were calculated from the dissolution data and used for comparison using the formula: where, y is the percentage of dissolved product. DE is the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period.

[0182] Table 6 shows the cumulative drug release over time, while Figure 8 is a representation of in vitro dissolution profiles of RLX-HCI and RLX-Arg (1 :1 ) potential crystal. The graph in Figure 8 shows the cumulative drug release percentage of RLX-HCI, and its hydrate with Arg, RLX-Arg (1 :1 ) over time, measured in minutes. Initial observations reveal that both RLX-HCI and RLX-Arg (1 :1 ) demonstrate a rapid release within the first 50 minutes, achieving approximately 80% dissolution. This initial burst is typical for drugs with high solubility or those that have been processed to enhance dissolution, such as through co-crystallization. Subsequently, the dissolution rate for both RLX-HCI and RLX-Arg (1 :1 ) plateaus, reaching close to 100% release by approximately 450 minutes. This plateau suggests that the dissolution process is controlled by the dissolution rate. The RLX-Arg (1 :1 ) hydrate (A) shows a marginally faster dissolution rate than pure RLX-HCI (B), particularly noticeable between 50 to 250 minutes where the RLX-Arg (1 :1 ) curve is consistently above the RLX-HCI curve. By the end of the observed period, both have nearly identical release profiles, suggesting that while the co-crystallization with Arg may have enhanced the dissolution rate initially, the overall solubility of RLX-HCI was not significantly altered by the formation of the hydrate. The slight improvement in dissolution rate for RLX-Arg (1 :1 ) could be attributed to the improved wettability, reduced particle size, altered crystal habit, or increased porosity of the crystals compared to pure RLX-HCI. The increased dissolution rate of the RLX-Arg (1 :1 ) hydrate could lead to faster onset of action, which can be crucial for the therapeutic application of the drug.

[0183] Table 6: Cumulative drug release over time in minutes

[0184] Determination of partition coefficient (LoG P Experiment)

[0185] The partition coefficient or Log P is a critical parameter in pharmacokinetics, describing how a compound distributes itself between a hydrophobic (organic) phase and a hydrophilic (aqueous) phase. For the Log P experiment, 30mg of hydrate (RLX-Arg (1 :1 ) sample was dissolved in a water-octanol (10ml) solvent system. Magnetic stirrers were used to stir the mixture for a period of 36 hours. The solution was then centrifuged in a Hettich Rotofix 32A centrifuge (Andreas Hettich GmbH & Co., Tuttingen, Germany) for 45 minutes to allow them to separate the two phases of solution, octanol, and water. From the mixed solution, 100pg was withdrawn from each of the solvents using a micropipette into separate 10ml volumetric flasks. The solutions were diluted and made up to the volume using the mobile phase. The solutions were then injected into the Shimadzu (Kyoto, Japan) HPLC.

[0186] The results of the Log P experiment are presented in Table 7. For the RLX-Arg (1 :1 ) hydrate, the Log P value of 1.1 1 indicates a moderate balance between lipophilicity and hydrophilicity. The balance is crucial for the pharmacokinetic properties of the compound, influencing factors like absorption, distribution, metabolism, and excretion (ADME). While lipophilicity (as indicated by Log P) assists in crossing cellular membranes, excessive lipophilicity can lead to poor water solubility, affecting the drug's bioavailability and distribution. A moderate Log P value suggests a balance that might avoid these extremes. Furthermore, a positive Log P value, such as 1 .1 1 , indicates that the compound is more soluble in the organic phase (octanol) than in the aqueous phase. This is also evident in the concentration of the hydrate in octanol, shown in Table 7, being greater than that in water 18.31 pg / ml and 1.43 pg / ml, respectively. This hydrophobic character is essential for understanding how the compound behaves in biological systems, particularly in terms of membrane permeability. Compounds with a Log P in the range of 1 -3 are generally well absorbed, as they possess enough lipophilicity to pass through lipid bilayers in biological membranes but are not so lipophilic that they get trapped in fatty tissues. The Log P value of the hydrate suggests it should have good oral bioavailability as a result of good drug absorption, a key consideration in drug design. An intermediate Log P value, such as 1.11 , indicates a favorable balance, potentially leading to better absorption and bioavailability.

[0187] Raloxifene hydrochloride (RLX-HCI), molecular formula C28H27NO4S-HCI, molar mass 510.04 RLX-HCI is a highly lipophilic drug with a Log P value of 5.69. A higher Log P (like 5.69 for RLX-HCI) suggests that the substance is more lipophilic and less hydrophilic. This can imply greater solubility in lipids and less in water. In contrast, the lower Log P as is the case with 1.1 1 for RLX-Arg (1 :1 ) hydrate suggests it is less lipophilic and more hydrophilic, implying increased water solubility and decreased lipid solubility as compared to RLX-HCL Furthermore, the RLX-HCI with a higher Log P will generally be better absorbed through the lipid-rich cell membranes and may have a broader distribution in the body's lipid compartments. On the other hand, the hydrate with a lower Log P might be less efficiently absorbed but could have a more restricted distribution, potentially reducing its presence in lipid-rich tissues. Therefore, the hydrate, with its significantly lower Log P, indicates better water solubility, potentially leading to different pharmacokinetics and pharmacodynamics, and could be advantageous in certain therapeutic contexts.

[0188] Table 7: Area Under the curve and corresponding concentration

[0189] Solvent Retention time AUC Height

[0190] Octanol

[0191] 1 3.024 3903157 520353

[0192] 2 3.026 3900497 521224

[0193] 3 3.023 3897719 520500

[0194] Water

[0195] 1 3.032 281382 381 18

[0196] 2 3.031 285308 38284

[0197] 3 3.031 281288 38092

[0198] Solvent Mean AUC Concentration(pg / ml)

[0199] Octanol 3900438 18.31

[0200] Water 281335 1.43

[0201] EXAMPLE 9

[0202] Lipinski’s Rule of Thumb or Rule of Five (RO5)

[0203] Lipinski's Rule of Five is a set of guidelines used in drug discovery to predict the drug likeness of compounds, particularly their oral bioavailability. These rules are based on the observation that most orally active drugs have certain common physicochemical properties. It is important to note that Lipinski's Rule of 5 serves as a guideline rather than a strict rule. However, compounds that meet these criteria are more likely to have good oral bioavailability and be suitable for further development. These rules act as a quick and simple filter to prioritize compounds for further testing and optimization in drug discovery. If a compound violates one or more of these rules, it may not be disqualified as a potential drug candidate, but additional studies and modifications are usually required to address the issues related to bioavailability and permeability.

[0204] The Lipinski’s rule of five suggests that poor absorption or permeation is more likely if the molecular weight (MW) is over 500 Dalton, the number of hydrogen bond donors (the sum of O-H and N-H) is more than 5, the number of hydrogen bond acceptors (the sum of N’s and O’s) is more than 10 and the logarithm of the partition coefficient between n-octanol and water (log 10P) is greater than 5. The molecular weight of the RLX-Arg (1 :1 ) hydrate is 491 .58 Da as per the ORTEP diagram from the SCXRD. This is slightly below the 500 Da threshold, indicating that the compound may have acceptable molecular size for oral bioavailability. The compound has 3 hydrogen bond donors. This meets the rule's threshold, suggesting that the number of hydrogen bond donors is within the acceptable range for oral drugs. Furthermore, with 6 hydrogen bond acceptors, RLX-Arg (1 :1 ) hydrate is well within the acceptable limit of 10.

[0205] This suggests a favorable profile for oral bioavailability in terms of its ability to participate in hydrogen bonding necessary for drug-receptor interactions. Finally, the Log P value of 1.11 is well below the threshold of 5, indicating that the compound is lipophilic. This can be favorable for lipid membrane permeability, which is important for absorption.

[0206] REFERENCES

[0207] Mohamed, S., Tocher, D.A. and Price, S.L., 201 1. Computational prediction of salt and cocrystal structures— Does a proton position matter? International journal of pharmaceutics, 418(2), pp.187-198.

[0208] Greenhalgh, D. J., Williams, A. C., Timmins, P. & York, P. 1999. Solubility parameters as predictors of miscibility in solid dispersions. Journal of Pharmaceutical Sciences, 88, 1182- 1190.

[0209] Nagy, S., S. Pal, and A. Szechenyi, Reliability of the Hansen solubility parameters as co-crystal formation prediction tool. International journal of pharmaceutics, 2019. 558: p. 319- 327.

[0210] Thayyil, A. R., Juturu, T., Nayak, S. & Kamath, S. 2020. Pharmaceutical cocrystallization: Regulatory aspects, design, characterization, and applications. Advanced Pharmaceutical Bulletin, 10, 203.

Claims

CLAIMS1 . A method for producing a hydrate of Raloxifene (RLX), the method comprising:(a) combining RLX, or a salt thereof, with an amino acid to form a co-former mixture;(b) dissolving the co-former mixture in a solvent to form a solution; and(c) evaporating the solvent from the solution to form a hydrate of RLX.

2. The method of claim 1 , wherein the salt of RLX is a hydrochloride salt of RLX (RLX- HCI).

3. The method of claim 1 or 2, wherein the amino acid is selected from L-tyrosine, phenylalanine, L-arginine, L-tryptophan, proline and / or glycine.

4. The method of any one of claims 1 to 3, wherein the amino acid is phenylalanine, L- tyrosine, L-tryptophan, or L-arginine.

5. The method of any one of claims 1 to 4, wherein the amino acid is L-arginine.

6. The method of any one of claims 1 to 5, wherein the molar weight ratio of the RLX or salt thereof to amino acid is 2:1 , 1 :1 or 1 :2 (w / w).

7. The method of claim 6, wherein the molar weight ratio of the RLX or salt thereof: amino acid is 1 :1 (w / w).

8. The method of any one of claims 1 to 7, wherein the solvent is ethanol or methanol.

9. The method of any one of claims 1 to 8, wherein the hydrate of RLX has increased solubility compared to RLX or a salt thereof.

10. A hydrate of RLX produced according to the method of any one of claims 1 to 9.

11. A pharmaceutical composition comprising a hydrate of RLX prepared according to the method of any one of claims 1 to 9.

12. The pharmaceutical composition of claim 11 , further comprising a pharmaceutically acceptable excipient.

13. A hydrate of RLX of claim 10 or a pharmaceutical composition of claim 11 or 12 for use in a method of treating osteoporosis in a subject, the method comprising administering the hydrate of RLX or the pharmaceutical composition to the subject.

14. A hydrate of RLX of claim 10 or a pharmaceutical composition of claim 11 or 12 for use in a method of treating and / or preventing breast cancer in a subject, the method comprising administering the hydrate of RLX or the pharmaceutical composition to the subject.

15. The hydrate of RLX or the pharmaceutical composition for use of claim 13 or 14, wherein the hydrate of RLX or the pharmaceutical composition has increased solubility and / or bioavailability in the subject compared to RLX, a salt thereof, or a pharmaceutical composition comprising RLX, or a salt thereof.

16. A method of treating osteoporosis in a subject, comprising administering a hydrate of RLX of claim 10 or a pharmaceutical composition of claim 11 or 12 to the subject.

17. A method of treating and / or preventing breast cancer in a subject, comprising administering a hydrate of RLX of claim 10 or a pharmaceutical composition of claim 11 or 12 to the subject.

18. The method of claim 16 or 17, wherein the hydrate of RLX or the pharmaceutical composition has increased solubility and / or bioavailability in the subject compared to RLX, a salt thereof, or a pharmaceutical composition comprising RLX, or a salt thereof.

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