RU2842656C1 - Co-crystal of ketoprofen and production thereof, pharmaceutical compositions containing same and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a co-crystal of lysine ketoprofen (form 4), characterized by the presence of an X-ray diffraction pattern with characteristic peaks at 13.6; 16.0; 16.5; 17.3; 19.1; 19.4; 20.5; 21.8; 22.9; 23.5; 24.9; 25.9; 27.6° 2 θ ± 0.20 degrees. Invention relates to a pharmaceutical composition for the treatment of pain and inflammatory diseases, comprising a co-crystalline form of lysine ketoprofen (form 4) according to the invention and at least a physiologically acceptable excipient. Invention also relates to a method of producing a co-crystalline form of lysine ketoprofen (Form 4) according to the invention, wherein said method comprises the following steps: (a) preparing an unsaturated solution of ketoprofen in 2-methyl-1-propanol, wherein the unsaturated solution has a ketoprofen concentration of 50 mg/ml to 150 mg/ml; (b) mixing the unsaturated ketoprofen solution with solid lysine to obtain a mixture and stirring the resulting mixture at temperature of 25 °C to 60 °C, followed by cooling the mixture and separating the co-crystal of ketoprofen lysine (form 4).

EFFECT: lysine ketoprofen co-crystal, characterized by high crystallinity, smaller particle size, better taste and high rate of dissolution.

15 cl, 14 dwg, 11 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to a ketoprofen lysine co-crystal, called form 4, to a method for producing the same, to a pharmaceutical composition containing said co-crystals, and to their use in medicine, in particular to their use for the treatment of pain and inflammatory diseases.

BACKGROUND OF THE INVENTION

Ketoprofen, ((RS)-2-(3-benzoylphenyl)propionic acid, chemical formula C ₁₆ H ₁₄ O ₃ ) formula

is one of the non-steroidal anti-inflammatory drugs (NSAIDs) of the propionic acid class with analgesic and antipyretic effects.

Due to its high tolerability, ketoprofen is one of the non-steroidal anti-inflammatory drugs widely used in the clinic both for the treatment of serious inflammatory conditions and for use as an analgesic and antipyretic agent by inhibiting the production of prostaglandins by the body.

Currently used pharmaceutical compositions containing ketoprofen contain the racemate as an active ingredient, where the two enantiomers S(+) and R(-) are present in an equimolecular ratio.

In pharmaceutical compositions intended for oral use, the active ingredient is usually used in the form of the free acid, which is practically insoluble in water, while for alternative routes of administration, salts of ketoprofen with organic and inorganic bases, such as, for example, ketoprofen sodium, or with amino acids, such as the lysine salt of ketoprofen, are suitable.

Ketoprofen salts are successfully used in the treatment of such pathological symptoms of the rheumatoid and chronic type, which require the administration of the drug in high doses continuously and for a long time. It is important and desirable that for the treatment of acute and very painful manifestations there are pharmaceutical compositions suitable for immediate and controlled use, quickly releasing the active ingredient and having high bioavailability. Typical examples of these compositions are compositions for parenteral administration and/or oral administration, which provide fine dispersion of the active ingredient.

Solubility and dissolution rate of drugs are decisive factors after oral administration for the rate and extent of absorption.

These factors represent a key challenge for the development and formulation of effective drugs in the pharmaceutical industry. The problem of poor drug solubility, which also complicates synthesis and development, is known and is the cause of bioavailability problems.

Various strategies to enhance the solubility and dissolution time of poorly soluble drugs, such as conversion to salt, preparation of solid dispersion, microemulsion, exploitation of cosolubility, inclusion complex formation with cyclodextrin, etc., have been well documented.

It is also possible to achieve the desired properties of a particular active pharmaceutical ingredient (API) by forming a co-crystal of the API itself or a salt of the API. Pharmaceutical co-crystallization has attracted a great deal of academic, industrial and therapeutic interest by co-crystallizing two or more pure compounds with crystal engineering to create a new functional material.

Cocrystals can be defined as a stoichiometric multicomponent system formed by two or more compounds, solid at ambient conditions, linked by non-covalent and non-ionic interactions.

Pharmaceutical cocrystals are non-ionic supramolecular complexes and can be used to improve physicochemical properties such as solubility, stability and bioavailability in the development of pharmaceuticals without changing the chemical composition of the API.

Cocrystals containing APIs can be used for therapeutic delivery of APIs. New dosage forms incorporating API cocrystals with pharmaceutically acceptable co-components may in some cases have superior properties compared to existing dosage forms. However, the formation of cocrystals is unpredictable and, as such, not always possible. Moreover, it is impossible to predict the properties of a particular cocrystal of a compound before it is formed. Thus, finding the right conditions to obtain a particular cocrystal form of a compound with pharmaceutically acceptable properties may require significant time, effort, and resources.

Documents GB1497044A and BE882889 describe the preparation of ketoprofen salts with lysine of the formula

a method in which unsaturated solutions of the components are used.

However, the known ketoprofen lysine salt exhibits low crystallinity and a fairly large particle size, as shown herein in Figure 12 and Table 11. These properties of ketoprofen lysine salt may not be ideal in terms of hygroscopicity and powder flowability or dissolution profile and possibly in vivo bioavailability.

European Patent Application No. EP18215336.1, filed on 21 December 2018, describes the preparation and characterisation of a ketoprofen lysine co-crystal referred to as Form 1.

ESSENCE OF THE INVENTION

The Applicant has surprisingly found that ketoprofen and lysine, under certain process conditions, can form co-crystals (referred to herein as Form 4) that are characterized by higher crystallinity, smaller particle size, and better taste than the previous ketoprofen lysine salt. In addition, the ketoprofen lysine co-crystal Form 4 exhibits a higher dissolution rate than the ketoprofen lysine co-crystal Form 1.

Thus, the first object of the present invention relates to a co-crystal of ketoprofen lysine (form 4), characterized by the presence of an X-ray diffraction pattern with characteristic peaks at 13.6; 16.0; 16.5; 17.3; 19.1; 19.4; 20.5; 21.8; 22.9; 23.5; 24.9; 25.9; 27.6º 2theta, with an error in the value indicated for each peak of ±0.20 degrees (2 theta).

Another object of the present invention is a pharmaceutical composition comprising a co-crystal form of ketoprofen lysine (form 4) according to the present invention and at least a physiologically acceptable excipient.

Another object of the present invention is a pharmaceutical composition comprising ketoprofen lysine co-crystals (form 4) according to the present invention in combination with at least another pharmaceutically active ingredient.

Another object of the present invention relates to a ketoprofen lysine co-crystal (form 4) and to a pharmaceutical composition containing said co-crystal for use as a medicine.

Another object of the present invention relates to a ketoprofen lysine co-crystal (form 4) and to a pharmaceutical composition containing said co-crystal for use in the treatment of pain and inflammatory diseases.

Another object of the present invention is a method for producing a co-crystal according to the present invention, wherein said method comprises the following steps:

a) preparation of an unsaturated solution of ketoprofen in a solvent selected from 2-methyl-1-propanol and ethyl acetate;

b) mixing an unsaturated solution of ketoprofen with solid lysine to obtain a mixture.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Powder X-ray diffraction spectra of co-crystal form 1 of ketoprofen lysine (1A) and ketoprofen lysine salt (1B).

Figure 2. Powder X-ray diffraction spectrum of the co-crystal form 4 of ketoprofen lysine, obtained according to Example 1.

Figure 3. Powder X-ray diffraction spectra of ketoprofen lysine cocrystal 4 compared with ketoprofen lysine cocrystal 1.

Figure 4. Solid-state ^13C CPMAS NMR spectra (100 MHz) of ketoprofen lysine salt (4A), ketoprofen lysine cocrystal 4 (4B) and, overlapping, ketoprofen lysine cocrystal 1, ketoprofen lysine cocrystal 4, ketoprofen and lysine (details of the carboxyl region) (4C).

Figure 5. Solid-state ¹⁵ N CPMAS NMR spectra (40.56 MHz) of the co-crystal form 4 of ketoprofen lysine (5A) and in a mixture with lysine (5B).

Figure 6. FT-IR spectra of 4 ketoprofen lysine cocrystal.

Figure 7: FT-IR spectra of ketoprofen lysine salt.

Figure 8. FT-Raman spectrum of ketoprofen lysine cocrystal 4.

Figure 9. TGA thermograms of ketoprofen lysine cocrystal 1 (9A) and ketoprofen lysine cocrystal 4 (9B).

Figure 10. DSC thermograms of ketoprofen lysine cocrystal 4 (10A), ketoprofen lysine cocrystal 1 (10B).

Figure 11. Principal component analysis (PCA) plot of ketoprofen lysine co-crystal 4 (time change) (11A), ketoprofen lysine co-crystal 1 (time change) (11B) and partial least discriminant analysis (PLS-DA) plot of ketoprofen lysine co-crystal 1 and ketoprofen lysine co-crystal 4 (11C).

Figure 12. Powder X-ray diffraction pattern of ketoprofen lysine co-crystal 4 compared with ketoprofen lysine salt.

Figure 13. DSC thermogram of ketoprofen lysine salt.

Figure 14. 1H-NMR spectrum of the co-crystal form 4 of ketoprofen lysine (onset temperature 100.53ºC).

Abbreviations: KL1 - ketoprofen lysine co-crystal 1; KL4 - ketoprofen lysine co-crystal 4; KLS - ketoprofen lysine salt; KET - Ketoprofen; LYS - Lysine

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise specified, all technical terms, designations, and other scientific terminology used herein are intended to have meanings commonly understood by those skilled in the art to which this invention pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and/or ease of reference; thus, the inclusion of such definitions in this description should not be construed as representing a material difference from what is commonly understood in the art.

The term "physiologically acceptable excipient" as used herein refers to a substance that is devoid of any pharmacological effect of its own and does not cause adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, sixth edition 2009, incorporated herein by reference.

For the purposes of the present invention, the expression “room temperature” means a temperature range from 18 to 25ºC.

For the purposes of the present invention, the term "cocrystal" means a stoichiometric multicomponent system in which the components are linked by non-covalent, non-ionic interactions and are individually solid at room conditions.

For the purposes of the present invention, the term "pain" shall mean pain caused by disorders of various natures and origins, such as, for example: headaches or cephalalgia (both primary and therefore not associated with other factors or diseases, and secondary and therefore dependent on trauma, lesions and various diseases); toothache (with abscesses or caries that create pain in the pulp of the tooth, in the presence of numerous vessels and nerves); menstrual pain (pain in the abdomen and lower abdomen and headaches caused by hormonal changes characteristic of the menstrual period); neuralgia or severe nerve pain due to deformation, injury and infection; muscle pain, or myalgia (pain at the level of the muscles when using or touching them, due to sudden contractions or injuries); bone and joint pain, such as inflammation of the joints (bones, cartilage, ligaments and tendons) after injuries, in old age, with sprains and injuries.

The terms "approximately" and "about" in this document refer to the range of experimental error that may occur in a measurement.

The term "saturated solution" should be understood as a chemical solution containing the maximum concentration of a solute dissolved in a solvent at a given temperature. In this context, unless otherwise specified, reference is made to room temperature.

The term "unsaturated solution" shall be understood as a chemical solution containing a concentration of solute dissolved in a solvent at a given temperature that is lower than the maximum concentration of solute dissolved in that solvent at the same temperature. In this context, unless otherwise specified, reference is made to room temperature.

The first object of the present invention relates to a co-crystal of ketoprofen lysine (form 4), characterized by an X-ray diffraction pattern with characteristic peaks at 13.6; 16.0; 16.5; 17.3; 19.1; 19.4; 20.5; 21.8; 22.9; 23.5; 24.9; 25.9; 27.6º 2theta, with an error in the value indicated for each peak of ±0.20 degrees (2 theta).

Figure 1 shows the powder X-ray diffraction spectra of the precursor co-crystal 1 of ketoprofen lysine (1A) and the lysine salt of ketoprofen (1B).

A typical powder X-ray diffraction pattern of the 4-ketoprofen lysine co-crystal is shown in Figure 2.

The ketoprofen lysine co-crystal 4 of the present invention is a polymorphic form of the ketoprofen lysine co-crystal 1, as shown by powder X-ray diffraction (Table 2) and ss-NMR (Table 7) analyses.

A comparison of the powder X-ray diffraction patterns of the ketoprofen lysine co-crystal form 4 of the present invention and the ketoprofen lysine co-crystal form 1 is shown in Figure 3 and Table 2.

As shown in Table 2, the powder X-ray diffraction pattern shows corresponding differences between the two polymorphs Form 1 and Form 4 of ketoprofen lysine co-crystal in the region from 15 to 28º 2theta, with peaks showing a shift of ≥0.2 degrees at 15.5; 16.0; 17.3, 19.2; 19.3; 19.4; 21.7; 24.5; 24.7; 25.9; 26.0; 26.5º 2theta and peaks with a shift of 0.3 degrees at 25.9 vs. 26.2, 26.5 vs. 26.8, 26.5 vs. 26.8 2theta.

Furthermore, the co-crystal nature of the ketoprofen lysine co-crystal form 4 of the present invention is shown in the solid state CPMAS NMR spectra ^{of 13C} (100 MHz) and ^15N (40.56 MHz), as shown in Figures 4, 5 and Table 7.

The ketoprofen-lysine co-crystal 4 of the present invention was further characterized by TGA (Figure 9B), DSC (Figure 10A), FT-IR (Figure 6 and Table 4), FT-Raman (Figure 8 and Table 5), and liquid phase 1H-NMR spectra (Figure 14).

Preferably, the molecular ratio between ketoprofen and lysine in the co-crystal form 4 of the present invention is 1:1.

Preferably, the co-crystal of the present invention is a co-crystal of D-lysine salt of (R)-2-(3-benzoylphenyl)propionic acid.

Preferably, the co-crystal of the present invention is a co-crystal of (R)-2-(3-benzoylphenyl)propionic acid L-lysine salt.

Preferably, the co-crystal of the present invention is a co-crystal of D-lysine salt of (S)-2-(3-benzoylphenyl)propionic acid.

Preferably, the co-crystal of the present invention is a co-crystal of L-lysine salt of (S)-2-(3-benzoylphenyl)propionic acid.

Advantageously, the ketoprofen lysine co-crystal form 4 of the present invention exhibits a high dissolution rate, higher than the dissolution rate of the ketoprofen lysine co-crystal form 1 (Table 10).

Thus, the high dissolution rate of the 4-ketoprofen lysine co-crystal of the present invention is a predictor of preferred use in the treatment of those pathological and chronic symptoms that require the administration of the drug in high doses continuously and over a long period of time.

In addition, the ketoprofen lysine co-crystal 4 of the present invention is characterized by a better taste compared to the ketoprofen lysine co-crystal 1 of the present invention, as shown in the electronic tongue test (see Experimental Part, item 8, and Figure 11).

Another object of the present invention relates to pharmaceutical compositions comprising the ketoprofen lysine co-crystal 4 of the present invention and at least one physiologically acceptable excipient.

The pharmaceutical composition of the present invention is preferably suitable for immediate and convenient administration and rapidly releases the API.

Preferably, said excipients are selected from the group consisting of: povidone, colloidal silicon dioxide, hydroxypropyl methylcellulose, eudragit EPO, sodium dodecyl sulfate, stearic acid, magnesium stearate, aspartame, mannitol, xylitol, talc, flavorings.

Preferably, the pharmaceutical composition of the present invention is in solid form, more preferably in the form of a solid granulate.

Another object of the present invention is a co-crystal form of 4 lysine ketoprofen and a pharmaceutical composition containing said co-crystal form for use in medicine, preferably for use in the treatment of pain and inflammatory diseases.

Preferably, the co-crystal form 4 of ketoprofen lysine and the pharmaceutical composition containing said co-crystal are used for treating pain, wherein the pain is selected from the group including: acute pain, headache, toothache, menstrual pain, muscle pain, bone and joint pain.

Preferably, the co-crystal form 4 of ketoprofen lysine and the pharmaceutical composition containing said co-crystal are used for the treatment of inflammatory diseases, wherein the inflammatory diseases are selected from the group consisting of rheumatic diseases.

Another object of the present invention is a pharmaceutical composition comprising the co-crystal form of ketoprofen lysine (form 4) according to the present invention in combination with one or more pharmaceutically active ingredients.

Another object of the present invention is a method for producing a co-crystal according to the present invention, wherein said method comprises the following steps:

a) preparation of an unsaturated solution of ketoprofen in a solvent selected from 2-methyl-1-propanol and ethyl acetate;

b) mixing an unsaturated solution of ketoprofen with solid lysine to obtain a mixture.

The present method is characterized by one or more of the following features, taken individually or in combination.

Preferably, the solvent in step a) is 2-methyl-1-propanol.

Preferably, the unsaturated solution in step a) has a ketoprofen concentration of lower than 350 mg/ml, more preferably lower than 200 mg/ml, even more preferably lower than 100 mg/ml.

Preferably, the unsaturated ketoprofen solution in step a) has a ketoprofen concentration of 50 to 150 mg/ml, more preferably 70 to 120 mg/ml, even more preferably 80 to 100 mg/ml.

Preferably, the unsaturated ketoprofen solution in step a) has a ketoprofen concentration of lower than 200 mg/ml, preferably lower than 150 mg/ml, more preferably lower than 100 mg/ml.

Preferably, in step b) of the present process, solid lysine is mixed with the unsaturated solution of step a) by directly adding the solid powder to the solution, preferably with stirring, to obtain a mixture.

Alternatively, the solution from step a) can be added to solid lysine, preferably with stirring, to obtain a mixture.

Alternatively, solid lysine may be in the form of a suspension in a suitable antisolvent, namely: in a solvent in which lysine has low solubility, such as, for example, ethyl acetate, ethanol, methanol, 2-methyl-1-propanol or mixtures thereof, preferably in 2-methyl-1-propanol. In this case, the antisolvent is preferably used in a volume-weight ratio of from 10 to 70 ml/g, more preferably from 30 to 50 ml/g lysine.

An indicative value of low solubility is, for example, the solubility of lysine in 2-methyl-1-propanol, which is about 0.026 g/ml.

Preferably, the mixture obtained in step b) is stirred for 1 to 80 hours, more preferably 3 to 70 hours, even more preferably 5 to 60 hours.

Preferably, the mixture obtained in step b) is stirred at a temperature of from 5ºC to 70ºC, more preferably from 25ºC to 60ºC, even more preferably from 50ºC to 60ºC.

Preferably, the mixture obtained in step b) has a water content of from 0.3 to 1.5% (v/v), more preferably from 0.8% to 1.1% (v/v).

Preferably, in the present method, the molar ratio of ketoprofen:lysine is from 1.5:1 to 3.5:1, more preferably from 2:1 to 3:1.

Preferably, the mixture obtained in step b), after stirring for a suitable time and at a certain temperature, is cooled to a temperature of from -5ºC to 60ºC, more preferably from 10ºC to 50ºC, even more preferably to room temperature.

In a preferred embodiment of the present process, the unsaturated solution of ketoprofen in 2-methyl-1-propanol of step a) has a ketoprofen concentration of 80 to 100 mg/ml and a ketoprofen/lysine molar ratio of 2:1 to 3:1. In step b), solid lysine is mixed with the unsaturated solution of step a) by directly adding the solid powder to the solution. The mixture obtained in step b) is stirred for 5 to 60 hours at a temperature of 50ºC to 60ºC and then cooled to room temperature.

Advantageously, the present process provides the 4-ketoprofen lysine co-crystal of the present invention in high yield as shown in Table 1.

EXPERIMENTAL PART

The following are some non-limiting examples relating to the method for preparing the 4-ketoprofen lysine co-crystal, its yields and characterization by powder X-ray diffraction analysis, DSC/TGA analysis, NMR analysis, FT-IR analysis, RAMAN analysis, particle size distribution analysis, dissolution rate test and taste evaluation using an electronic tongue.

1. OBTAINING KETOPROFEN LYSINE COMPOUNDS

Example 1

Preparation of co-crystal form of 4 ketoprofen lysine

Ketoprofen free acid (1.9 g, 7.5 mmol) was dissolved in 20 mL 2-methyl-1-propanol (MPR) and stirred at room temperature until dissolved. Solid lysine (0.55 g, 3.75 mmol) was then added and the resulting mixture was heated at 50 °C for 3 h. The mixture was then cooled to 5 °C and filtered. Washing with MPR (6 mL˟2) was performed to recover the powder from the reactor wall. The filter cake was then washed with the mother liquor under vacuum. The filter cake was dried in an oven at 60 °C and 50 mbar vacuum for 16 h. The compound was obtained as a white powder in 63% yield.

The compound was characterized by X-ray powder diffraction, TGA, DSC, FT-IR, FT-Raman, 1H-NMR in liquid state (see sections below).

Example 2

Preparation of 4-ketoprofen lysine co-crystal (scaling up)

The method according to example 1 was repeated, using 188 g of ketoprofen.

The main parameters of the method were studied according to the conditions and the results are collected in the following table 1:

Table 1

Pr.№ Mol. ratio KET/LYS Conc. KET mg/ml Water content (% v/v) Heating temperature
stage b) Mixing time (h) Cooling temperature (ºC) Product Output % w/w 2A 3/1 87 0.3 60 24 25 Form 4 80.9 2B 3/1 87 1 60 24 25 Form 4 89.7 2C 1.5/1 87 1 60 5 25 Form 4 84.4 2D 2/1 87 1 60 24 25 Form 4 88.0

Example 3

Preparation of Form 1 Ketoprofen Lysine (Comparison)

650 mg of racemic lysine were suspended in 0.5 ml of water, then the suspension was filtered and added to 87 mg (0.34 mmol) of ketoprofen. The mixture was stirred in this manner for 24 hours at room temperature; after this time no solid separated, so the solution was left to evaporate at high temperature until the formation of a sticky solid was observed. In order to obtain a solid suitable for powder X-ray diffraction analysis, the sticky solid was suspended in isopropyl ether (IPE) for 24 hours, yielding after filtration the ketoprofen lysine co-crystal form 1.

The powder X-ray diffraction spectra of ketoprofen lysine co-crystal 1 are shown in Figure 1A.

Example 4

Obtaining ketoprofen lysine salt (comparison)

Ketoprofen 0.76 g and racemic lysine 0.44 g (equiv. ratio 1:1) were stirred in 20 ml of methanol at 40 °C for 1 hour. Ketoprofen was dissolved, while the suspended lysine was filtered (0.45 μm filter) directly into the Mettler Toledo Easymax 102 reactor. The solution was left under stirring for 5 minutes in the reactor, then 100 ml of ethyl acetate were added and the solution was cooled to -5 °C without formation of a solid. To initiate nucleation, an additional amount of ethyl acetate (20 ml) was added via pipette in two aliquots (10 ml and 10 ml). The system was left under stirring until the suspension acquired a milky color. An additional 30 minutes of stirring were used. The precipitate was then filtered and characterized by X-ray powder diffraction analysis (Figure 1B).

2. POWDER X-RAY DIFFRACTION ANALYSIS

Powder X-ray diffraction analysis was performed using an instrument having the following characteristics:
Device type: Rigaku MiniFlex600 Application Software: Miniflex Manual Measurement details Measurement type: Single scan Sample mode: Reflection Scanning Scanning range: 3,000-40,000º (2θ) Step size: 0.01º (2θ) Speed: 10.0º/min (2θ) Scan mode: Continuous Wavelength used Presumed wavelength type: Kα1 Kα1: 1.540598 Å Kα2: 1.544426 Å Intensity ratio Kα2/Kα1: 0.50 Kα: 1.541874 Å Kα: 1.392250 Å Device details X-ray generator Tube output voltage: 40 kV Tube output: 15mA High Voltage Generation Method: Cockcroft-Walton High Frequency Method Stability: Within ±0.05% for both tube voltage and tube current, relative to ±10% change in input power X-ray tube Name: Toshiba Analyx type A-26L Anode material: Cu Maximum power: 0.60 kW Focus size: 1˟10mm Kβ filter Name: Ni-filter Thickness (mm): 0.015 Material: Ni Goniometer (device for measuring angles) Type: Vertical θ/2θ Goniometer radius: 150 mm Scan axis: θ/2θ related 2θ scanning range: +2º to +140º Minimum axis pitch angle θ/2θ: 0.005º (2θ) Positioning speed: 500º/min (2θ) Scanning speed: from 0.01 to 100º/min Base angle: 2θ=10º X-ray shooting angle: 6º (fixed) The gap DS: 1.25º IHS: 10.0 mm SS: no (open) RS: no (open) Soller's gap on the falling side: 2.5º Soller gap on the receiving side: 2.5º Detector Title: Ultrafast one-dimensional D/teX detector

A sample of the ketoprofen lysine co-crystal 4, prepared according to the method of Example 1, was analyzed by powder X-ray diffraction (Figure 2).

Figure 3 shows the powder X-ray diffraction spectra of the co-crystal form 4 of ketoprofen lysine and form 1 of ketoprofen lysine.

The characteristic peaks of the powder X-ray diffraction spectrum of the co-crystal form 4 of ketoprofen lysine according to the invention are given in Table 2 in comparison with the profile of form 1 of ketoprofen lysine.

Table 2 also shows the position and assigned hkl plane, as well as the peak intensities of the two polymorphs.

Table 2

List of peaks of co-crystal form 4 and form 1 of ketoprofen lysine

Form 4 Form 1 h k l d(Å) 2θ(deg.) Intensity h k l d(Å) 2θ(deg.) Intensity 1 0 0 23.29 3.8 1592.1 1 0 0 23.2 3.8 1089,118 2 0 0 11.64 7.6 454.5 2 0 0 11.6 7.6 374,466 3 0 0 7.76 11.4 9.59 3 0 0 7.7 11.4 8.7 1 1 0 7.60 11.6 1212.2 1 1 0 7.6 11.6 1043,356 2 1 0 6.61 13.4 351.2 2 1 0 6.6 13.4 339,4742 0 1 1 6.51 13.6 1554.2 0 1 1 6.5 13.6 552,2654 1 -1 -1 6.50 13.6 921.8 1 -1 -1 6.5 13.6 1718,124 1 1 1 6.06 14.6 577.1 1 1 1 6.0 14.6 125,3324 2 -1 -1 6.05 14.6 181.4 2 -1 -1 6.0 14.7 614,0069 4 0 0 5.82 15.2 49.1 4 0 0 5.8 15.2 57,78102 1 0 -2 5.71 15.5 7,63710 1 0 -2 5.6 15.7 15,81796 3 1 0 5.58 15.9 406.6 3 1 0 5.6 15.9 280,511 0 0 2 5.55 16.0 133.6 0 0 2 5.5 16.1 435,8349 2 0 -2 5.54 16.0 389.5 2 0 -2 5.5 16.2 114,3189 2 1 1 5.38 16.5 1332,0 2 1 1 5.4 16.5 1002,419 3 -1 -1 5.36 16.5 1088.2 3 -1 -1 5.3 16.6 1037,016 1 0 2 5.13 17.3 1250.7 1 0 2 5.1 17.4 1159,756 3 0 -2 5.12 17.3 1174.2 3 0 -2 5.1 17.5 1026,988 4 1 0 4.71 18.8 175.8 4 1 0 4.7 18.8 211,0233 3 1 1 4.68 18.9 4,89460 3 1 1 4.7 19.0 86.7 4 -1 -1 4.67 19.0 235.2 4 -1 -1 4.7 19.1 0,00000 5 0 0 4.66 19.0 0,00000 5 0 0 4.6 19.1 139,0242 1 -1 -2 4.65 19.1 844.8 1 -1 -2 4.6 19.2 717,5275 2 0 2 4.61 19.2 42.7 2 0 2 4.6 19.4 344,9359 4 0 -2 4.59 19.3 400,5 4 0 -2 4.5 19.5 1910,591 0 1 2 4.57 19.4 1361.3 0 1 2 4.5 19.6 193,0019 2 -1 -2 4.56 19.4 2611.8 2 -1 -2 4.5 19.6 1821,501 1 1 2 4.33 20.5 1435,0 1 1 2 4.3 20.6 586,1714 3 -1 -2 4.32 20.6 589.5 3 -1 -2 4.3 20.7 1477,621 3 0 2 4.08 21.7 61.4 4 1 1 4.1 21.9 0,00000 4 1 1 4.07 21.8 1908.8 3 0 2 4.1 21.9 442,9748 5 0 -2 4.07 21.8 918.3 5 -1 -1 4.0 21.9 2226,067 5 -1 -1 4.06 21.9 31.0 5 0 -2 4.0 22.0 0,00042 5 1 0 4.03 22.0 1274,5 0 2 0 4.0 22.1 111,8395 0 2 0 4.02 22.1 102.2 5 1 0 4.0 22.1 1149,957 2 1 2 4.00 22.2 159.7 2 1 2 4.0 22.3 14,31045 4 -1 -2 3.98 22.3 90.5 1 2 0 4.0 22.4 396,7589 1 2 0 3.96 22.4 372.0 4 -1 -2 4.0 22.5 61,90912 6 0 0 3.88 22.9 1165.3 6 0 0 3.9 23.0 976,6281 2 2 0 3.80 23.4 0,00000 2 2 0 3.8 23.4 0,00000 0 2 1 3.78 23.5 1279.1 0 2 1 3.8 23.5 1225,846 1 -2 -1 3.78 23.5 12.6 1 -2 -1 3.8 23.5 0.00012 1 2 1 3.68 24.1 29.5 1 2 1 3.7 24.1 28,52559 2 -2 -1 3.68 24.2 48.6 2 -2 -1 3.7 24.2 5,65006 3 1 2 3.64 24.4 78.8 3 1 2 3.6 24.5 469,2507 5 -1 -2 3.63 24.5 478.4 5 -1 -2 3.6 24.7 1,84273 4 0 2 3.62 24.6 220.2 4 0 2 3.6 24.7 30,77004 6 0 -2 3.60 24.7 95.1 3 2 0 3.6 24.9 1293,778 3 2 0 3.57 24.9 1455.9 6 0 -2 3.6 24.9 86,76020 5 1 1 3.56 25.0 0,00000 5 1 1 3.6 25.0 24,81936 6 -1 -1 3.56 25.0 0,00000 6 -1 -1 3.5 25.1 18,50564 2 2 1 3.51 25.3 186.7 2 2 1 3.5 25.3 893,8421 3 -2 -1 3.51 25.4 625.9 3 -2 -1 3.5 25.4 1,49105 6 1 0 3.49 25.5 47.1 6 1 0 3.5 25.5 105,3335 1 -1 -3 3.43 25.9 620,0 1 -1 -3 3.4 26.2 641,4867 2 -1 -3 3.43 26.0 273.2 2 -1 -3 3.4 26.2 208,4548 0 1 3 3.36 26.5 64.1 0 1 3 3.3 26.8 60,35221 3 -1 -3 3.35 26.5 44.4 3 -1 -3 3.3 26.8 0,00000 7 0 0 3.33 26.8 102.2 7 0 0 3.3 26.9 146,0823 4 2 0 3.31 26.9 0,00000 4 2 0 3.3 26.9 0,00000 4 1 2 3.30 27.0 0,00000 3 2 1 3.3 27.0 132,7653 3 2 1 3.30 27.0 364.5 4 -2 -1 3.3 27.1 450,9335 4 -2 -1 3.29 27.1 226.0 4 1 2 3.3 27.1 0,00000 1 -2 -2 3.29 27.1 0,00000 1 -2 -2 3.3 27.2 0,00000 6 -1 -2 3.29 27.1 0,00000 6 -1 -2 3.3 27.3 12,30758 0 2 2 3.25 27.4 1086.9 0 2 2 3.2 27.5 1083,624 2 -2 -2 3.25 27.4 727.2 2 -2 -2 3.2 27.5 688,8038 1 1 3 3.23 27.6 309.7 1 1 3 3.2 27.8 192,3856 4 -1 -3 3.22 27.7 170.9 5 0 2 3.2 27.8 73,71219 5 0 2 3.22 27.7 23.5 4 -1 -3 3.2 27.9 289,2207 7 0 -2 3.20 27.8 35.9 7 0 -2 3.2 28.0 96,01039 1 2 2 3.16 28.2 25.2 1 2 2 3.2 28.2 38,54196 3 -2 -2 3.16 28.2 0,00000 3 -2 -2 3.2 28.3 0.00001 6 1 1 3.15 28.3 398.5 6 1 1 3.1 28.4 332,1199 7 -1 -1 3.14 28.4 231.4 7 -1 -1 3.1 28.5 347,4366 7 1 0 3.07 29.0 59.2 7 1 0 3.1 29.1 69,70481 2 1 3 3.06 29.2 149.5 4 2 1 3.1 29.2 21,12850 4 2 1 3.06 29.2 478.2 5 -2 -1 3.1 29.2 784.9 5 -2 -1 3.06 29.2 225.8 5 2 0 3.0 29.3 0,00000 5 -1 -3 3.05 29.3 0,00000 2 1 3 3.0 29.4 147,6632 5 2 0 3.04 29.3 0,00000 5 -1 -3 3.0 29.5 0,00000 2 2 2 3.03 29.5 140.4 2 2 2 3.0 29.5 467,9161 4 -2 -2 3.02 29.5 291.7 4 -2 -2 3.0 29.6 0,00006 5 1 2 2.99 29.9 53.6 5 1 2 3.0 30.0 64,18669 7 -1 -2 2.98 30.0 39.6 7 -1 -2 3.0 30.2 60,65774 8 0 0 2.91 30.7 26.5 8 0 0 2.9 30.8 42,02372

The X-ray powder diffraction patterns showed some significant differences in the region from 15 to 27º 2theta with peaks showing the same relative intensity.

Ketoprofen lysine cocrystal form 1 showed characteristic peaks at 15.7; 16.2; 17.5; 19.4; 19.5; 19.6; 21.9; 24.7; 24.9; 26.2; 26.8º 2theta, while ketoprofen lysine cocrystal form 4 showed distinctive peaks at 15.5; 16.0; 17.3, 19.2; 19.3; 19.4; 21.7; 24.5; 24.7; 25.9; 26.0; 26.5º 2theta: these peaks are shifted by ≥0.2 deg.

The peaks of the co-crystal form 4 ketoprofen lysine with a shift of 0.3 degrees were found at 25.9 vs. 26.2, 26.5 vs. 26.8, 26.5 vs. 26.8.

As clearly seen in Figure 3, the X-ray powder diffraction pattern of ketoprofen lysine co-crystal Form 4 showed small but significant differences compared to the X-ray powder diffraction pattern observed for Form 1.

According to powder X-ray diffraction analysis, it appears that the co-crystal form 4 of ketoprofen lysine is a polymorph of the co-crystal form 1 of ketoprofen lysine with similar crystallinity.

The ketoprofen lysine co-crystal 4 of the present invention differs from the previous ketoprofen lysine salt as is evident from the analysis presented herein below.

In particular, the powder X-ray diffraction spectra of ketoprofen lysine salt are shown in Figure 1A.

The characteristic peaks of powder X-ray diffraction of ketoprofen lysine salt are shown in the following Table 3.

Table 3

List of powder X-ray diffraction peaks of ketoprofen lysine salt

Pos. [º2theta] Height [cts] FWHM [º2theta] interplanar distance d [Å] Rel. int. [%] 3,9325 2846,60 0.1476 22,46912 100,00 7,8614 122.69 0.1181 11,24637 4.31 8,5371 92.20 0.1181 10,35764 3.24 9,1615 141.64 0,1968 9,65313 4.98 11,0605 527.66 0.1181 7,99966 18.54 11,8024 131.06 0.2362 7,49843 4.60 13,0204 1516.34 0.0689 6,79958 53.27 14,1357 181.38 0,1968 6,26551 6.37 15,0097 72.85 0,1968 5,90258 2.56 17,4211 652.89 0.1574 5,09063 22.94 18,8604 2197,33 0.0689 4,70525 77.19 19,4898 678.61 0.1181 4,55471 23.84 20,9970 1941,40 0,0787 4,23104 68.20 21,4845 2046.59 0.1378 4,13613 71.90 22,2596 1258,41 0,1771 3,99383 44.21 22,9984 616.52 0,0787 3,86717 21.66 23,6473 222.80 0.2362 3,76250 7.83 24,6845 207.27 0.3149 3,60672 7.28 25,8298 707.10 0,0787 3,44933 24.84 26,6005 977.38 0.0886 3,35111 34,34 28,3958 134.48 0.1574 3,14320 4.72 29,2001 419,42 0,1771 3,05843 14.73 30,7454 46.23 0.3149 2,90814 1.62 31,9837 376.56 0.1378 2,79832 13.23 32,7583 225.02 0.1574 2,73389 7.90 33,5545 173.12 0.2362 2,67082 6.08 35,3568 377.39 0.1378 2,53870 13.26 36,6756 185.11 0.2755 2,45038 6.50 38,3677 278.83 0.3149 2,34612 9.80

Powder X-ray diffraction patterns showed corresponding signals in the region from 13 to 27º 2theta, in particular, ketoprofen lysine salt showed the most intense peaks at 13.0, 17.4, 18.9, 19.5, 20.9, 21.5 and 26.6º 2theta, while ketoprofen and lysine cocrystal form 4 showed distinctive peaks at 15.5; 16.0; 17.3, 19.2; 19.3; 19.4; 21.7; 24.5; 24.7; 25.9; 26.0; 26.5º 2theta.

3. TGA ANALYSIS

The analysis was performed using Mettler Toledo TGA/DSC1.

The sample was weighed in an aluminum cup, hermetically sealed with an aluminum perforated lid. The analysis was carried out by heating the sample from 25ºC to 320ºC at a rate of 10 deg/min.

Temperature data

Temperature range From 25ºC to 320ºC Accuracy of maintaining the set temperature ±1 K Temperature determination precision ±0.4 K Heating speed 10 K/min Cooling time 20 min (1100…100ºC) Sample volume ≤100 µl

Special modes

Automation Provisions 34 samples TGA-FTIR in combination with the Thermo Nicolet iS10 spectrometer Balance data XP5 Measurement range ≤5 g Permission 1.0 mcg Weighing accuracy 0.005% Weighing precision 0.0025% Inner ring mass 2 Empty curve reproducibility not worse than ±10 μg in the entire temperature range

The TGA thermogram of the co-crystal form 4 of ketoprofen lysine is shown in Figure 9 in comparison with the thermogram of form 1.

Thermogram of form 4 showed multiple mass losses:

25-130ºC (1.21% w/w) limited by EGA sensitivity, no organic solvent release observed (probably traces of water);

150-170ºC MPR follows the evolution accompanying the emission of carbon dioxide;

120-320ºC (35.53% w/w) release of decomposition gases (mainly carbon dioxide followed by the release of ammonia).

The TGA curve profile of ketoprofen lysine co-crystal 4 differs slightly from that of ketoprofen lysine co-crystal 1, with the latter showing a mass loss of 34.40% due to degradation compared to 35.53% for ketoprofen lysine co-crystal 4.

4. DSC ANALYSIS

The analysis was performed using a Mettler Toledo DSC1 DSC.

The sample was weighed into an aluminum cup, hermetically sealed with an aluminum lid. The analysis was carried out by heating the sample from 25ºC to 320ºC at a rate of 10 deg/min.

Temperature data

Temperature range from 25ºC to 320ºC Accuracy of maintaining the set temperature ±0.2 K Temperature measurement accuracy ±0.02 K Heating speed 10 K/min Cooling time 5 min (100ºC…0ºC)

Calorimetric data

Sensor type FRS5 Sensor material Ceramics Number of thermocouples 56 Signal generation time constant 1.8 s India Peak (height to width) 17 TAWN Resolution 0.12 Sensitivity 11.9 Permission 0.04 μW Digital resolution 16.8 million points

The DSC thermogram for the ketoprofen lysine co-crystal 4 is shown in Figure 10 (10A) in comparison with the ketoprofen lysine co-crystal 1 (10B). The DSC thermogram for the ketoprofen lysine salt is shown in Figure 13.

The DSC curve of the ketoprofen lysine co-crystal 4 showed multiple endothermic peaks:

1st very broad endothermic peak (7.10 J/g), start 29.54ºС, peak 70.81ºС, end 122.54ºС;

2nd endothermic peak (270.93 J/g), start 152.18ºC, peak 158.82ºC, end 166.43ºC due to the melting process, which is easily followed by decomposition stages.

In conclusion, the DSC thermogram of Form 4 CL had a different profile from that of Form 1 CL, with the latter showing an endothermic peak at a higher initial temperature (164.14ºC for Form 1 versus 152.18ºC for Form 4). Furthermore, the thermograms of both polymorphs differ from that of the predecessor ketoprofen lysine salt, which showed a 1st endothermic peak at 110.92ºC (54.67 J/g, onset 100.53ºC, final solidification 118.35ºC) and above 120ºC, multiple partially overlapping endothermic peaks due to decomposition stages.

Preferably, the present co-crystal form 4 of ketoprofen lysine is characterized by an endothermic peak at 158.8°C±2°C, determined by DSC according to the method described above.

5. FT-IR and FT-Raman ANALYSIS

FT-IR analysis was performed using a Thermo Nicolet iS50-ATR modular ATR spectrometer equipped with the following:

- Smart Performer Diamond

- DTGS KBr detector

- Source IR

- Beam splitter made of KBr

Information about data collection

Number of sample scans 32 Number of background scans 32 Collection length 47.29 sec Permission 4,000 Zero Fill Levels 2 Number of scan points 16672 Number of FFT points 65536 Laser frequency 15798.3 cm ^-1 Position of the interferogram peak 8192 Apodization N-B strong Phase correction Mertz Number of background scans 32 Background gain 1.0 Strengthening the sample 8 Diaphragm 100 Optical speed 0.6329

FT-Raman spectra were recorded on a Nicolet iS50 FT-IR spectrometer. The excitation source was a Nd-YAG laser (1064 nm) in a backscattering configuration (180º). The diameter of the focused laser beam was approximately 50 mm, and the spectral resolution was 4 cm ^-1 . The spectra were recorded at a laser power on the sample of about 100 mW.

The FT-IR spectrum and FT-Raman spectrum of the co-crystal form 4 of ketoprofen lysine according to the invention, as well as the peak lists are presented in Figures 6 and 8 and in Tables 4 and 5, respectively.

Table 4

List of peaks of the FT-IR spectrum of the co-crystal form 4 of ketoprofen lysine.

Position Intensity Position Intensity 413.78 67,157 1070,62 78,202 486.69 52,203 1138,03 70,370 549.27 69,863 1175,55 71,146 619.37 59,317 1195,85 72,333 646.32 66,437 1246.58 56,277 664.47 69,634 1274,44 42,814 686.62 54,994 1284.79 44,134 695.94 62,593 1314.96 50,944 708.32 44,602 1352.19 52,273 715.08 44,879 1395,44 43,982 783.11 70,672 1447.92 62,117 796.95 71,172 1455,16 61,676 814.43 76,776 1486.37 63,923 825.28 76,814 1574.97 42,632 852.84 77,556 1630,50 57,448 881.61 61,448 1663.28 57,708 935.19 89,212 1980,26 94,860 971.30 75,020 2112,10 93,684 1002.80 76,495 2161.87 91,820 1020,87 79,134 2859.34 70,914

Table 5.

List of peaks of the FT-Raman spectrum of the co-crystal form 4 of ketoprofen lysine.

Position Intensity Position Intensity 225.36 32,070 1284.54 14,979 303.37 12,889 1314.25 14,772 310.75 10,746 1330,10 11,031 489.13 11,052 1337,82 11,315 495.84 10,146 1351,50 9,284 619.22 13,629 1405,55 12,189 708,71 20,149 1439.59 13,481 876.20 10,836 1444.25 13,587 881,70 11,685 1452.37 11,869 1002.40 76,305 1461.24 11,838 1028.24 14,328 1596.48 65,524 1075,63 11,559 1662.39 53,474 1137.46 15,818 2864,71 21,377 1160.94 10,609 2873.96 22,155 1169.59 15,780 2922,56 47,154 1182.53 16,692 2966.38 33,240 1194.32 25,557 3026,18 15,698 1251.04 10,617 3064.22 60,520

The FT-IR spectrum and FT-IR peak list of ketoprofen lysine salt are shown in Figure 7 and Table 6 below.

Table 6.

List of peaks of FT-IR spectrum of ketoprofen lysine salt

Position (cm ^-1 ) Intensity Position (cm ^-1 ) Intensity 414 51,203 1160 79,502 437 69,203 1179 81,151 463 76,990 1201 81,656 475 76,368 1248 65,816 518 63,818 1281 53,933 539 60,253 1320 58,117 621 69,700 1358 59,706 645 61,177 1393 53,834 675 72,596 1420 69,095 689 66,858 1448 68,893 706 55,062 1479 72,920 713 56,968 1532 44,203 756 82,432 1538 43,543 779 73,315 1557 42,899 802 80,054 1615 73,015 821 87,571 1652 65,459 831 88,245 2050 95,210 871 69,950 2089 95,059 911 88,295 2112 94,226 929 90,674 2324 93,704 958 84,976 2650 84,309 967 88,368 2879 75,764 1007 80,471 2942 72,790 1072 84,876 3420 95,023 1138 77,764

6. SOLID-STATE NMR

Solid-state NMR (ss-NMR) spectra were obtained using a Bruker Avance II 400 Ultra Shield instrument operating at 400.23, 100.63 and 40.56 MHz for ¹ H, ¹³ C and ¹⁵ N nuclei, respectively. Powder samples were packed into 4 mm OD 80 µL zirconia cylindrical rotors. Some sample was collected and used without further preparation to fill the rotor.

¹³ C CPMAS (cross-polarized magic angle spinning) spectra were acquired at a spin rate of 12 kHz using a linear cross-polarization pulse sequence, and ss-NMR spectra were acquired using a Bruker Avance II 400 Ultra Shield instrument operating at 400.23, 100.63 and 40.56 MHz, respectively, for ¹ H, ¹³ C and ¹⁵ N nuclei.

Powder samples were packed into cylindrical zirconia rotors with an outer diameter of 4 mm and a volume of 80 µl. Some of the sample was collected and used without further preparation to fill the rotor.

¹³ C CPMAS spectra were acquired at a spin rate of 12 kHz using a ramped cross-polarization pulse sequence with a 90º 1H pulse of 3.60 µs duration, 3 ms contact time, optimized recycle delays from 1.5 to 3.5 s, and scan counts in the range 430-640, depending on the sample.

¹⁵ N CPMAS spectra were acquired at a spin rate of 9 kHz using a linear cross-polarization pulse sequence with a 90º 1H pulse of 3.60 µs duration, contact times of 1 to 4 ms, optimized recirculation delays of 1.1 to 3.4 s, and scan counts in the range 14330-22770, depending on the sample.

For each spectrum, a dual-pulse phase modulation (TPPM) decoupling scheme with a 69.4 kHz RF field was used.

The ^13C chemical shift scale was calibrated against the methylene signal of the external standard glycine (at 43.7 ppm).

The ^15N chemical shift scale was calibrated against the signal of the external standard glycine (at 33.4 ppm relative to _NH3 ).

2D ¹ H− ¹³ C on-resonance and off-resonance (short and long, respectively) HETCOR spectra were obtained with contact times of 0.1 and 7 ms, respectively, and FSLG decoupling t1 and TPPM decoupling t2 (82 kHz RF fields).

288 and 384 scans were averaged over 88 and 128 increments, respectively, with a relaxation delay of 3.4 s. The indirect ^1H chemical shift scale in the HETCOR spectra was experimentally corrected by a scaling factor of 1/3, since ^{the 1H} chemical shift variance scales by a factor of 1/3 during FSLG decoupling.

Figure 4 shows the ^13C -NMR spectra of ketoprofen lysine salt (4A), ketoprofen lysine cocrystal 4 (4B) and enlarged overlapping spectra of ketoprofen lysine cocrystal 1, ketoprofen lysine cocrystal 4, ketoprofen and lysine (4C), obtained at room temperature with a rotation speed of 12 kHz.

The solid state ^13C CPMAS NMR spectra of the comparative ketoprofen lysine salt are also shown in Figure 4A.

As can be seen from the spectra shown in Figure 4, the resonances of ketoprofen lysine salt are different from the resonances of the present ketoprofen lysine cocrystal form 4, and both of them are different from the characteristic signals of the starting materials ketoprofen and lysine.

The characteristic ^13C ss-NMR resonances for the 4-ketoprofen lysine cocrystal are shown in Table 7 below:

Table 7

¹³ C ss-NMR resonances for lysine ketoprofen co-crystal form 4

δC (ppm) 197.2 133.0 196.1 128.6 179.4 126.8 177.6 55.1 174.5 50.2 147.4 38.8 145.8 32.2 141.0 29.6 136.4 24.7 134.8 22.3

The signals in the spectrum (4B) of ketoprofen lysine co-crystal 4 show an average full width at half maximum (FWHM) of 143 Hz. All resonances are at the same chemical shift as in ketoprofen lysine co-crystal 1, with some additional broad shoulders appearing in the carboxyl region (170-200 ppm) as well as in the aromatic (120-150 ppm) and aliphatic (20-60 ppm).

Table 8

List of ketoprofen lysine salt solid state NMR 13C CPMAS peaks.

Lysine salt of ketoprofen ¹³ C δ (ppm) ¹³ C δ (ppm) 199.8 127.2 197.8 125.9 182.2 564 181.1 55.5 180,0 20.8 176.6 49.4 175.7 48.2 174.3 38.6 144.5 38.2 143.8 32.3 142.7 31.7 138.2 26.4 134.6 25.7 131.8 22.5 129.5 22.0 128.6

Table 8 shows a list of characteristic signal peaks of ketoprofen lysine salt. The average full width at half maximum (133 Hz) corresponds to a moderately crystalline phase. The carboxyl signals suggest a 1:1 ratio between ketoprofen and lysine.

Possibly six independent molecules in the unit cell can be estimated from the solid-state ^13C CPMAS NMR spectra: carboxyl signals indicate the presence of 3 carboxylate moieties for both ketoprofen and lysine.

Figure 4C shows the carboxyl region of the overlapping ^13C ss-NMR spectra of ketoprofen lysine cocrystal 4, ketoprofen lysine cocrystal 1, ketoprofen and lysine.

As can be seen, the signals of the polymorphs of ketoprofen lysine cocrystal 1 and ketoprofen lysine cocrystal 4 are distinguishable from those of ketoprofen and lysine. The signal of the ketone C=O of ketoprofen falls at approximately 197.5 ppm and corresponds to the only carboxyl fragment (approximately 180 ppm) in the aromatic region, which is the region of 120-150 ppm filled only with signals of ketoprofen and the methyl signal of pure ketoprofen (approximately 20 ppm).

A ¹³ C T1-1H experiment (not shown) was performed to evaluate the presence of various heterogeneous phases within the analyzer sample by their T1-1H relaxation times. The analysis revealed very similar T1-1H values for all resonances, including broad shoulders between 1.8 and 1.9 s; thus, form 4 of ketoprofen lysine can be defined as a homogeneous phase.

Figure 5A shows the solid state ^15N CPMAS NMR spectra of 4-ketoprofen lysine co-crystal obtained at room temperature with a spin rate of 9 kHz, with characteristic ^15N resonances of 4-ketoprofen lysine co-crystal, in particular the signal at about 43 ppm.

Figure 5B shows the overlap of the ^15N CPMAS solid state NMR spectra of ketoprofen lysine and lysine cocrystal 4.

It is clear from the spectra that the co-crystal form of 4 ketoprofen lysine does not contain pure lysine.

In conclusion, according to the above discussed ¹³ C-CPMAS and ¹⁵ N CPMAS spectra, it is evident that:

A new homogeneous crystalline phase of ketoprofen lysine cocrystal was discovered: in ¹³ C CPMAS all resonances differ from the characteristic signals of pure ketoprofen and pure lysine. 1H T1 measurements take into account the homogeneity of the adduct.

From the ^13C and ^15N CPMAS spectra it was found that the stoichiometric ratio of ketoprofen:lysine is 1:1: one set of ^13C signals for both ketoprofen and lysine; only one set of ^15N signals for lysine (ketoprofen does not contain nitrogen atoms).

^{The 13C} and ^15N CPMAS spectra revealed two independent molecules per unit cell: one set of ^13C signals for both ketoprofen and lysine; only one set of ^15N signals for lysine (ketoprofen does not contain nitrogen atoms).

The adduct is a cocrystal based on ¹³ C and ¹⁵ N CPMAS:

- the ^13C carboxyl signal of ketoprofen falls at 177.6 ppm, which corresponds to the typical chemical shift of the neutral carboxyl group involved in hydrogen bond interactions (for example, COO- in the sodium salt of ketoprofen falls at higher chemical shifts of 181.4/180.5 ppm);

- as for the ¹⁵ N signals, ε-NH ₂ of lysine resonates at 32.8 ppm, which is a characteristic chemical shift for neutral amino groups involved in hydrogen bonding interactions (the proton group of NH ³⁺ will fall above 35 ppm).

Based on ^{the 13} C and ¹⁵ N CPMAS spectra, lysine is in its zwitterionic form: the ¹³ C signal of the carboxylate group of lysine resonates at 174.5 ppm ^, consistent with the ionic nature of the fragment; the ¹⁵ N signal of the α-NH ₃ ⁺ fragment of lysine falls at 43.0 ppm, which corresponds to the protonated state of the ammonium group (the α- _{NH 2} group of lysine falls below 35 ppm).

The phase is very similar to form 1 of ketoprofen lysine with additional peaks associated with disturbances/defects; the additional resonances associated with defects have an intermediate salt/co-crystal character (from the ¹³ C CPMAS spectra): in the ¹³ C CPMAS ss-NMR spectrum (Figure 4C) this is clearly visible with broad additional peaks at approximately 196 and 180 ppm.

7. DISSOLUTION RATE AND SOLUBILITY IN SIMULATED GASTRIC FLUID (SGF)

The dissolution rate and solubility of ketoprofen lysine cocrystal 4 and ketoprofen lysine cocrystal 1 were determined in simulated gastric fluid (SGF) and simulated salivary fluid (SSF).

Methods

Solubility in artificial gastric juice (GSF) without pepsin (USP41) at 25ºC

Excess solid was allowed to equilibrate under magnetic stirring. At predetermined time points, an aliquot of the supernatant was removed, filtered, and analyzed by HPLC for ketoprofen concentration.

Intrinsic Dissolution Rate: A 150 mg powder sample was pressed into a 11 mm diameter round die using a hydraulic press at a force of 3 tons for 3 minutes. The resulting compacts were maintained inside the die and analyzed in a USP41 Apparatus 2 (Distek Dissolution System 2100B) under the following conditions: 500 ml simulated gastric fluid (GSF) without pepsin or USP phosphate buffer pH 6.5 (PB6.5) at a temperature of 37ºC and a paddle speed of 30 rpm. The amount of solid dissolved at each time point was determined spectrophotometrically at 260 nm or by HPLC. The test was performed in 6 replicates in SGF and in 3 replicates in phosphate buffer pH=6.5.

Statistical analysis of the data was performed using Excel.

Solubility

The solubility of ketoprofen lysine co-crystal 4 in SGF compared to that of ketoprofen lysine co-crystal 1 is shown in the following Table 9.

Table 9

Solubility of ketoprofen from ketoprofen lysine cocrystal 1 containing ketoprofen and from ketoprofen lysine cocrystal 4 at 25ºC (mean±standard deviation, n=3)

Solubility (mg/ml) Co-crystal form 1 ketoprofen lysine 0.243±0.078 Co-crystal form of 4 ketoprofen lysine 0.253±0.089

The solubility of ketoprofen lysine cocrystal 4 was similar to that of ketoprofen lysine cocrystal 1.

Internal dissolution rate

The intrinsic dissolution rates (IDRs) of ketoprofen lysine co-crystal 1 and ketoprofen lysine co-crystal 4 in simulated gastric fluid (SGF) at pH 1.2 and in simulated salivary fluid (pH 6.5) are shown in the following Table 10.

Table 10

Intrinsic dissolution rates of ketoprofen lysine cocrystals form 1 and form 4

Sample GSF (pH 1.2) IDR (mg∙cm ^-2 ∙min ^-1 ) Confidence interval (95%) CL form 1 (n=6) 0.94 0.7843-1.1000 CL form 4 (n=6) 1.16 0.9871-1.3310 Sample Simulated Salivary Fluid Sample pH 6.5 IDR (mg∙cm ^-2 ∙min ^-1 ) Confidence interval (95%) CL form 1 (n=3) 4.23 3.5956-4.8655 CL form 4 (n=3) 5.16 4.8175-5.4955

From the above data it follows that the release of ketoprofen at pH 1.2 (SGF) from the ketoprofen lysine co-crystal 4 is faster than from the ketoprofen lysine co-crystal 1 (1.16 versus 0.94). At pH 6.5 (saliva pH) this difference was more noticeable (5.16 versus 4.23).

8. ELECTRONIC LANGUAGE

The analysis was performed using technologically advanced taste sensors equipped with an ion-selective potentiometric electrode (ISPE) detection system, which provides results with high correlation to sensory assessments. This system provides a comprehensive and objective assessment of the taste of food products and pharmaceuticals with high accuracy and reliability.

The response of sensory matrices was processed using pattern recognition methods: principal component analysis (PCA) and partial least discriminant analysis (PLS-DA) in order to 1) compare sample solutions with reference solutions of salty, sweet and bitter taste; 2) detect similarity or difference from time t0 (freshly prepared) to time t48h (2 days after preparation).

In Figures 11A and 11B, a comparison of the organoleptic analysis of samples of ketoprofen lysine co-crystal 4 and ketoprofen lysine co-crystal 1 at different time points after dissolution is presented as PCA scores.

In these figures, the inventors observed a different trend over time for the two samples. The greatest change over time was observed at 6 h, indicating a different taste of ketoprofen lysine co-crystal 1 compared to ketoprofen lysine co-crystal 4. Minor variations were noted at the remaining time points.

To highlight the different behavior in this assay observed for the ketoprofen lysine cocrystal 4, a PLS-DA(11C) model was created and plotted.

From the graph in Figure 11C, it can be seen that the data for the ketoprofen lysine co-crystal 4 are closer to the sweet taste region than the data obtained for the ketoprofen lysine co-crystal 1, thus confirming the expectation of a better taste of the present ketoprofen lysine co-crystal 4.

9. PARTICLE SIZE DISTRIBUTION

The co-crystal form 4 ketoprofen lysine and ketoprofen lysine salt were analyzed using Mastersizer laser diffraction. Particle size analysis is related to the rheological behavior of the powder and the dissolution rate of the product.

Device

Brand of the device: Malvern

Device type: Morphology G3S

Application Software: Morphology Software 8.20

Microscope

Light source: White light, brightfield diascopic and episcopic polarizer/analyzer and DIC contrast enhancement options

Detector: CCD matrix with a resolution of 2592 x 1944 pixels, 5 megapixels, color CCD matrix

Pixel Width: 2.78 µm x 2.78 µm

Optical system: Nikon CFI 60 bright/dark field system

Lens: 2.5˟: 13µm-1000µm (nominal)

5˟: 6.5 µm-420 µm (nominal)

10˟: 3.5 µm-210 µm (nominal)

20˟: 1.75 µm-100 µm (nominal)

50˟: 0.5 µm-40 µm (nominal)

A significant change in particle size was observed between the co-crystal form 4 ketoprofen lysine and the ketoprofen lysine salt, as shown in the following Table 11.

Table 11

Comparison of PSD of co-crystal 4 ketoprofen lysine and lysine salt of ketoprofen

PSD Co-crystal form of 4 ketoprofen lysine Lysine salt of ketoprofen D10 (µm) 33.68 83,82 D50 (µm) 84.26 130.78 D90 (µm) 142.94 347.74

As shown in Table 11, a significant difference in particle size distribution was observed between 4-ketoprofen lysine co-crystal and ketoprofen lysine salt, with the latter showing a PSD D90 2.43 times larger than 4-ketoprofen lysine co-crystal.

Preferably, the present ketoprofen lysine co-crystal 4 is characterized by a particle size distribution in which the D90 is less than 220 μm, preferably less than 150 μm.

The particle size of the present ketoprofen lysine co-crystal 4, which is smaller than the particle size of the previous ketoprofen lysine salt, may provide many advantages. Indeed, the particle size distribution plays an important role in the preparation process (quality control) and in the development of suitable manufacturing methods. Smaller final average particle sizes may improve content uniformity, solubility, dissolution, absorption rate and bioavailability.

Another advantage of the present ketoprofen lysine co-crystal 4 over the previous ketoprofen lysine salt is that it can be obtained directly from the crystallization step with the desired smaller particle size, which minimizes or even eliminates the subsequent micronization of the powder to obtain the desired PSD, with potential reductions in processing steps, time and, ultimately, manufacturing costs.

10. CRYSTALLIZATION

As shown by powder X-ray diffraction analysis, the co-crystal of 4 ketoprofen lysine has a crystallinity significantly higher than the lysine salt of ketoprofen, where the presence of an amorphous phase is obvious (Figure 12). This property is generally associated with the better stability of the co-crystal of 4 ketoprofen lysine, especially in terms of lower hygroscopicity compared to the lysine salt of ketoprofen.

The lower particle size distribution in combination with the higher degree of crystallinity of the ketoprofen lysine co-crystal 4 is an advantage over the previous ketoprofen lysine salt both with respect to the adjustment of the hydrodynamic parameters during the formulation process and with respect to the preparation and formulation of the coated ketoprofen lysine granulate. The assessment of the dynamic flowability and shear properties as well as the powder stability using tests such as the basic stability energy, stability index, specific energy and conventional bulk density show a higher friction phenomenon in the powder bed for the ketoprofen lysine salt compared to the ketoprofen lysine co-crystal 4. This phenomenon may be associated with a higher degree of amorphousness, irregular shape and higher hygroscopicity of the ketoprofen lysine salt compared to the ketoprofen lysine co-crystal 4.

In addition, oral formulations of ketoprofen are intended to act systemically via absorption through the gastrointestinal tract. Both drug dissolution and gastrointestinal permeability must be good enough to reach the bloodstream. The smaller particle size of ketoprofen lysine co-crystal 4 compared to ketoprofen lysine salt may indicate improved dissolution rate and bioavailability (study ongoing).

Reducing particle size increases the surface area of particles in contact with gastrointestinal juices, facilitating dissolution, improving safety and efficacy, and providing better compliance and dose tolerability.

In conclusion, the 4-ketoprofen lysine co-crystal was synthesized, isolated and characterized by X-ray powder diffraction, TGA, DSC, FT-IR, FT-Raman and NMR.

As discussed above, the X-ray powder diffraction pattern showed small but significant differences compared to that observed for the ketoprofen lysine co-crystal 1. The ketoprofen lysine co-crystal 1 has similar crystallinity (by X-ray powder diffraction analysis) and comparable but different thermal properties (similar decomposition profile - TGA/EGA and melting point at slightly lower temperature - DSC analysis).

Solid-state NMR revealed a homogeneous system characterized by a short-range structure nearly identical to that of the ketoprofen lysine cocrystal 1, characterized by site-specific disorder. Furthermore, ketoprofen and lysine were found to be present in a 1:1 stoichiometric ratio, consistent with the thermal analysis results.

This result confirmed that the different final crystallization temperature did not affect either the crystalline phase or the resulting stoichiometric ratio of ketoprofen:lysine in the solid state. Accordingly, ketoprofen lysine cocrystal 1 and ketoprofen lysine cocrystal 4 are polymorphs of the same cocrystal.

In addition, the present 4-ketoprofen lysine co-crystal is clearly different from the previous ketoprofen lysine salt, as confirmed by powder X-ray diffraction, ^13C -ss-NMR, FT-IR, FT-Raman and DSC analyses.

With regard to the intrinsic dissolution rate (IDR), the release of ketoprofen at pH 1.2 (SGF) from ketoprofen lysine cocrystal 4 was faster than that of ketoprofen lysine cocrystal 1 (1.16 vs. 0.94 mg cm-2 min ^-1 ). At pH 6.5 (saliva pH), this difference was more noticeable (5.16 vs. 4.23).

The solubility of ketoprofen lysine cocrystal 4 was similar to that of ketoprofen lysine cocrystal 1.

In terms of particle size distribution, the present 4 ketoprofen lysine co-crystal exhibits a smaller particle size than ketoprofen lysine salt, which may predict improved application, particularly in oral dosage forms.

Finally, in the electronic tongue test, ketoprofen lysine co-crystal 4 showed a superior taste (i.e., close to sweet) compared to ketoprofen lysine co-crystal 1.

Claims (17)

1. Ketoprofen lysine co-crystal (form 4) characterized by the presence of an X-ray diffraction pattern with characteristic peaks at 13.6; 16.0; 16.5; 17.3; 19.1; 19.4; 20.5; 21.8; 22.9; 23.5; 24.9; 25.9; 27.6° 2 theta ± 0.20 degrees. 2. A cocrystal according to claim 1, characterized in that it has an X-ray diffraction pattern shown in Figure 2. 3. A co-crystal according to claim 1 or 2, wherein the molecular ratio between ketoprofen and lysine is 1:1. 4. A co-crystal according to any one of claims 1 to 3, characterized by an endothermic peak at 158.8°C ± 2°C, determined by DSC in accordance with the method presented in the description. 5. A co-crystal according to any one of claims 1 to 4, characterized by a particle size distribution in which D90 is less than 220 μm, preferably less than 150 μm, when determined in accordance with the method presented in the description. 6. A pharmaceutical composition for the treatment of pain and inflammatory diseases, containing a co-crystalline form of ketoprofen lysine (form 4) according to any of the preceding claims and at least a physiologically acceptable excipient. 7. The pharmaceutical composition according to claim 6, wherein said excipients are selected from the group comprising povidone, colloidal silicon dioxide, hydroxypropyl methylcellulose, Eudragit EPO, sodium dodecyl sulfate, stearic acid, magnesium stearate, aspartame, mannitol, xylitol, talc, and flavorings. 8. A pharmaceutical composition according to claim 6 or 7, wherein said pharmaceutical composition is in the form of a solid granulate. 9. Ketoprofen lysine co-crystal (form 4) according to any one of claims 1-5, intended for the treatment of pain and inflammatory diseases. 10. The cocrystal according to claim 9 and the pharmaceutical composition according to any one of claims 6-8, wherein said pain is selected from the group comprising acute pain, headache, toothache, menstrual pain, muscle pain, bone and joint pain. 11. The cocrystal according to claim 9 and the pharmaceutical composition according to any one of claims 6-8, wherein said inflammatory diseases are selected from the group including rheumatism. 12. A method for obtaining a co-crystal form of ketoprofen lysine (Form 4) according to any one of paragraphs 1-5, wherein said method comprises the following stages: a) preparation of an unsaturated solution of ketoprofen in 2-methyl-1-propanol, wherein the unsaturated solution has a ketoprofen concentration of 50 to 150 mg/ml; b) mixing an unsaturated solution of ketoprofen with solid lysine to obtain a mixture and stirring the resulting mixture at a temperature of 25 to 60°C, followed by cooling the mixture and isolating the ketoprofen lysine co-crystal (form 4). 13. The method according to claim 12, wherein the unsaturated solution in step a) has a ketoprofen concentration of 70 to 120 mg/ml, more preferably 80 to 100 mg/ml. 14. The method according to claim 12 or 13, wherein the molar ratio of ketoprofen:lysine is from 1.5:1 to 3.5:1, more preferably from 2:1 to 3:1. 15. The method according to any one of claims 12 to 14, wherein the mixture obtained in step b) has a water content of from 0.3 to 1.5% (v/v), more preferably from 0.8 to 1.1% (v/v).