ES2364274T3 - INCLUSION COMPLEXES INCLUDING PYROXICAM, A CYCLODEXTRIN AND ARGININE. - Google Patents

Abstract

Process for preparing a soluble inclusion compound, which comprises one or more active substances that are poorly soluble in an aqueous medium included in one or more cyclodextrins, characterized in that it comprises the following successive steps: a. bringing one or more active substances into contact with one or more cyclodextrins, b. carry out a molecular diffusion stage by contacting, in static mode, a dense fluid under pressure with the mixture obtained in stage (a) in the presence of water, c. recovering the active substance / cyclodextrin molecular complex thus formed, d. carry out in a semi-solid medium a step consisting of adding and mixing an agent for interaction with the complex to the molecular complex active substance / cyclodextrins, the agent for interacting with the complex being an acid or a base, e. recovering the inclusion compound thus formed.

Description

The present invention relates to a process for the preparation of soluble inclusion compounds by means of the technology of dense fluids under pressure, in particular that of CO2.

Many active substances, particularly of interest in the pharmaceutical field, have very low solubility or are insoluble in water and consequently in biological liquids. This implies a low bioavailability of these active substances and a sharp increase in the doses administered to patients to achieve the therapeutic objective set and therefore an increase in possible side effects related to medical treatments.

The pharmacokinetic properties of a given substance depend, among others, on the affinity of its contact surface for the solvent in question, namely water in the case of the pharmaceutical field. Increasing the specific surface of the powders makes it possible to improve their dissolution speed. However, the bioavailability of active principles can be considerably increased if their dissolution rate improves. Likewise, the formation of molecular complexes composed of one or more active substances and of one or more host molecules judiciously selected for their strong solubility in biological liquids can therefore make it possible to increase the dissolution of the active substance (s) in biological fluids.

In the pharmaceutical field, there are a number of patents and publications that refer to the formation of complexes based on cyclodextrin, in the presence of an agent that interacts with the complex. However, most of the documents do not propose any industrial procedure, but rather the study of the improvement of the solubility of an active substance / cyclodextrin complex by means of the interaction agent with the complex. It is in fact a list of agents for interaction with the complex tested for the same active principle, and the analytical results observed. On the other hand, there are few documents that propose a complexation in a supercritical medium.

The documents that describe complexation processes with a cyclodextrin by supercritical fluid are the following:

In order to fix volatile molecules by inclusion, Kamihira M. et al. (J. of Fermentation and Bioengineering, Vol. 69, No. 6, 350-353, 1990) describe a procedure for the extraction of volatile aromatic compounds and interception by inclusion in cyclodextrins. The geraniol and mustard oil are thus extracted by a fluid under pressure, and then dynamically vaporized in a second reactor containing cyclodextrins. The influence of the different parameters is studied by measuring the percentage of inclusion of aromatic compounds in cyclodextrins. However, the inclusion stage is performed in dynamic and not static mode. On the other hand, the application claimed by the authors is completely different since it deals with the fixation of volatile molecules by inclusion. Finally, this procedure is not used with supercritical fluids, but with gases under pressure.

Van Hees et al. (Pharmaceutical Research, Vol. 16, No. 12, 1999) describe in their publication a method of inclusion of commercial Piroxicam in β-cyclodextrins by supercritical CO2. Since Piroxicam is a non-steroidal anti-inflammatory drug that is poorly soluble in water, its inclusion in the β-cyclodextrins should make it possible to increase its solubility in water. The procedure consists of placing a mixture of Piroxicam and β-cyclodextrins (1 / 2.5 molar ratio) in a pressurized autoclave, left in static mode. After depressurization, the mixture obtained is ground and homogenized. The complex is then dried prior to characterization by:

*

DSC (Differential Scanning Calorimetry)

*

Differential solubility technique

*

Spectroscopic methods.

These analyzes allow us to conclude regarding the percentage of complexation of Piroxicam with β-cyclodextrin. The importance of an agent of interaction with the complex on the dissolution of the complex thus obtained is not mentioned. On the other hand, no diffusion agent is used in the supercritical CO2 complex formation step in static mode.

Patent application WO 03/043604 describes a process for preparing a molecular complex of active substance in host molecules. The process uses a molecular diffusion step by means of the static contacting of a dense fluid under pressure in the possibly presence of a diffusion agent: water.

However, this step is followed by a mandatory supercritical CO2 scrubbing step. Furthermore, no interaction agent is used with the complex.

Different documents disclose the improvement of the solubility of an active substance by adding an interaction agent with the complex (Redenti, E. et al., J. of Pharmaceutical Sciences, Vol. 89, 1-8, 2000). The solubility of the active substance alone, of the active substance in the presence of the agent of interaction with the complex, of the binary complex of active substance / cyclodextrin and finally of the ternary complex of active substance / cyclodextrin / agent of interaction with the complex is studied. However, none of the described processes uses supercritical CO2, nor in particular a molecular diffusion step in static mode using a diffusion agent.

Thus, Buvàri-Barcza et al. (J. of Inclusion Phenomena and Macrocyclic Chemistry, Vol. 42, 209-212, 2002) study the solubility of benzoic acid / β-cyclodextrin and benzene / β-cyclodextrin complexes in the presence of acetic acid. The solubility of the benzene / β-cyclodextrin complex is independent of the acetic acid concentration whereas that of the benzoic acid / β-cyclodextrin complex increases with the increase in acetic acid. The authors' interpretation is as follows: in the ternary molecule β-cyclodextrin / acetic acid complex, the potential hydrogen bonds between the molecule and the internal cavity of the cyclodextrin are promoters of other interactions on the exterior of the cyclodextrin.

Likewise, Mura et al. (J. of Inclusion Phenomena and Macrocyclic Chemistry, Vol. 39, 131-138, 2001) measure the solubility of econazole in the presence of cyclodextrins (α-, β-, γ-, hydroxypropyl-β-cyclodextrins) and hydroxy acids (acids tartaric, citric, gluconic, malic and lactic). Ternary complexes are prepared by physically mixing or grinding the 3 compounds, co-evaporation or lyophilization of a solution containing the 3 compounds. Ternary complex formation is controlled by DSC. Only lyophilization makes it possible to obtain a DSC profile that no longer exhibits the econazole melting peak.

The authors conclude with the synergistic effect observed in the ternary complex, since the observed solubilities are up to 20 times higher than that of a binary econazole / cyclodextrin complex.

The same authors (Int. J. of Pharmaceutics, Vol. 260, 293-302, 2003) have also studied ternary complexes of the naproxen / hydroxypropyl-β-cyclodextrin / amino acid type. The complexes mentioned are prepared either by co-trituration or by co-evaporation of a water / ethanol solution containing the 3 compounds.

Piel et al., (J. of Pharmaceutical Sciences, Vol. 86-4, 475-480, 1997) present a study of solubility of a nimesulide / -L-lysine / β- or γ-cyclodextrin complex obtained by "spray- drying "or evaporation. The solubility of the ternary complex is, depending on the pH of the solution, up to 3,600 times more important than nimesulide alone. In this case too, the authors speak of a synergistic effect of cyclodextrin and L-lysine.

Fenivesy et al. (Proceedings of the 7th international cyclodextrins symposium, 414-418, 1994) are interested in the complexation of the active substances terfenadine, domperidone and astemizole with hydroxypropyl-β-cyclodextrin in the presence of hydroxy acids.

Two patents (EP 0 991 407 and EP 1 018 340) describe the preparation of ternary complexes active substances / interaction agent with the complex / cyclodextrin. The procedures used are kneading, spray-drying, evaporation or lyophilization. The process consists either in preparing the complex of the salt of the active substance, or in simultaneously bringing the 3 compounds into contact during the process.

A patent (EP 0 153 998 A2) filed by Chiesi et al. describes the preparation of complexes of piroxicam and beta-cyclodextrin in the presence in particular of a solution of ammonia. However, the procedure used is not performed with supercritical CO2.

The only two documents dealing with the preparation of the complex by supercritical CO2 in the presence of an agent that interacts with the complex are the following:

Following the same method as that cited above (Pharmaceutical Research, Vol. 16, No. 12, 1999), Van Hees et al. (Journal of inclusion Phenomena and Macrocyclic Chemistry, No. 44, p 271-274, 2002) describe the use of an interaction agent with the complex, L-lysine or trometamol, in the preparation of a Piroxicam / p- complex cyclodextrin by supercritical CO2.

The use of L-lysine or trometamol allows them at the same time to increase the percentage of inclusion of piroxicam in beta-cyclodextrin and to improve the dissolution of the complex formed.

The characterization is carried out by:

*

DSC (Differential Scanning Calorimetry)

*

Differential solubility technique

*

Dissolution kinetics in buffered medium.

However, no diffusion agent is used during this stage. V. Barillaro et al. (Proceeding of the 6th International Symposium on Supercritical Fluids, Versailles, p 1897-1902, 2003) have focused more on improving

5 that the addition of an acidic agent for interaction with the complex could contribute to increase the percentage of inclusion of Miconazole in some cyclodextrins. Different agents of interaction with the complex have been used (melic, maleic, fumaric, citric acid) as well as different cyclodextrins (beta-cyclodextrin, HP-betacyclodextrin, γ-cyclodextrin, HP-γ-cyclodextrin).

10 However, the inclusion stage is performed in dynamic and not static mode.

In the two documents cited above, it is therefore important to note that the complexation by supercritical fluids is carried out on the ternary mixture active substance / agent of interaction with the complex / cyclodextrin and that on the other hand the agent of interaction with the complex is not arginine.

The piroxicam molecule, represented above, has an enol function and a pyridine cycle that can be salified or not depending on the pH value of the dissolution medium.

image 1

20 Wiseman et al. (Arzneim.-Forsch.lDrug Res. 26 (7) 1976, 1300-1303) have determined in a mixture of dioxane: water 2: 1 (v / v) the value of pKa of the enol function (pKa ∼ 2) and pyridine cycle) pKa ∼ 6.3).

Depending on the pH value of the dissolution medium, the piroxicam (PX) molecule will therefore exist in different forms, namely:

pH <2

-the pyridine cycle is protonated into pyridinium ion (NH +) 30 -enol function is not ionized (-OH)

2 <pH <7

-the pyridine cycle is protonated (NH +) -the enol function is ionized in enolate (O -).

It is the zwitterionic structure of piroxicam. In terms of molecular conformation, the zwitterionic structure is flat. This flatness results from intramolecular hydrogen bonds between the enolate anion and the amide group on the one hand and the carbonyl function and the pyridinium cation on the other hand.

40 pH> 7

-enol function is ionized (O -) -the pyridine cycle is not protonated (N).

The inventors have surprisingly discovered that the separation of the complexation and addition stages of the interaction agent with the complex makes it possible to substantially improve the physicochemical properties of the complex thus obtained. In addition, they have also observed that the use of arginine as an interaction agent with the complex when the active substance is piroxicam allowed to obtain the complexes that have the

50 most interesting properties. The objective is to improve the in vivo dissolution of an active substance insoluble in water, that by including the active substance in a soluble porous support and then modifying the physicochemical properties of the system thus formed.

The present invention therefore relates to a process for the preparation of a soluble inclusion compound comprising one or more active substances that are poorly soluble in an aqueous medium included in one or more cyclodextrins, characterized in that it comprises the following successive steps:

to.

putting one or more active substances in contact with one or more cyclodextrins,

b.

carry out a molecular diffusion stage by contacting, in static mode, a dense fluid under pressure with the mixture obtained in stage (a) in the presence of water,

c.

recovering the active substance-cyclodextrin molecular complex thus formed,

d.

carry out a step in a semi-solid medium that consists of adding and mixing an agent for interaction with the complex to the molecular complex active substance-cyclodextrins, the agent for interacting with the complex being an acid or a base,

and.

recovering the soluble inclusion compound thus formed.

In the process according to the present invention, there is no supercritical CO2 washing step between the steps

(c) and (d).

In another advantageous embodiment, step (e) is followed by a step (f) for drying the soluble interaction compound, advantageously between 60 ° C and 80 ° C and advantageously overnight.

The process according to the present invention is thus constituted by the chaining of two phases that are:

- form an inclusion complex between an active substance and a cyclodextrin in a supercritical medium (steps (a),

(b) and (c))

- non-covalently "fix" an acid or a base in the complex obtained, in particular in order to improve its physicochemical properties (steps (d) and (e)).

The invention therefore describes a five-stage process.

* The first three so-called maturation stages essentially consist of a molecular diffusion phase in a dense medium under pressure, and in particular supercritical, which allows active substances to be included in cyclodextrins. The objective sought during this diffusion phase is to form inclusion complexes between active substances and cyclodextrin.

By the term "inclusion complex or molecular complex" is understood in the sense of the present invention, any complex that associates in a non-covalent manner the active substance and the cyclodextrin. It is the complex that comes from step (b) of the process according to the invention.

By the expression "soluble inclusion compound" is understood in the sense of the present invention, any entity formed by the association of the acid or the base on the one hand and the molecular complex on the other hand. It is the final product obtained by the process according to the invention.

The agent for interacting with the complex is an acid or a base. The choice of an acidic or basic agent will depend on the acidic or basic nature of the active substance. Thus, preferably, an acidic agent will be used with a molecular complex containing a basic active substance and a basic agent with a molecular complex containing an acidic active substance.

Advantageously, the agent for interaction with the complex is selected from an amino acid, a carboxylic acid, an acetate, a carboxylate, an amine or ammonia, advantageously in the form of a 28% ammonia solution. Still more advantageously, it is selected from acetic acid, tartaric acid, citric acid, gluconic acid, malic acid, lactic acid, maleic acid, fumaric acid, L-lysine, L- valine, L-isoleucine, L-arginine or ammonia. Even more advantageously, it is ammonia.

By the expression "dense fluid under pressure" is understood in the sense of the present invention any fluid used at a temperature or at a pressure higher than its citric value. Advantageously, it is CO2 pure or mixed with an organic solvent conventionally used by a person skilled in the art.

By the expression "active substance poorly soluble in an aqueous medium" is understood in the sense of the present invention any active substance that is poorly or not at all soluble in an aqueous medium and which in particular has a solubility of less than at least 20 µg / ml. In particular, it may be a pharmaceutical active agent (such as analgesics, antipyretics, aspirin and its derivatives, antibiotics, anti-inflammatories, antiulcer drugs, antihypertensives, neuroleptics, antidepressants, oligonucleotides that have therapeutic activity, peptides that have therapeutic activity and proteins that have therapeutic, cosmetic or nutraceutical activity. Advantageously, it is an active substance selected from the group consisting of anilide derivatives, epipodophyllotoxin derivatives, minoxidil, piroxicam, valeric acid, octanoic acid, lauric acid, stearic acid, tiaprofenic acid. , omeprazole, econazole, miconazole, ketoconazole, astemizole, cyclobenzaprine, nimesulide, ibuprofen, terfenadine, domperidone, naproxen and eflucimibo, advantageously it is piroxicam.

The host molecule is selected from the group consisting of cyclodextrins and their mixture. Advantageously, it is β-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin. Advantageously, it is β-cyclodextrin.

Cyclodextrins are molecules called "cages" because they comprise, within their structure, a relatively rigid and hydrophobic cavity that allows them to trap or encapsulate other molecules. The complexation phenomenon is the result of a multitude of interactions (substrate / solvent, solvent / solvent and cyclodextrin / solvent) that lead to the most stable thermodynamic state:

(1)

the interactions of Van der Waals;

(2)

hydrophobic interactions;

(3)

hydrogen bonds;

(4)

the release of high energy water molecules during replacement by the guest molecule;

(5)

the release of stress energy within the cyclodextrin molecule during complex formation.

Advantageously, the soluble inclusion compound consists of the association of piroxicam, a cyclodextrin and arginine, advantageously L-arginine.

The diffusion agent is water.

By the term "static mode" is meant in the sense of the present invention a reaction or a process in which all the reactants are brought into presence simultaneously and in which the reaction is allowed to proceed. For example, in step (b) of the present invention, the active substance (s), water and supercritical CO2 are autoclaved and allowed to react for several hours. The product mass does not evolve during the reaction. Conversely, in dynamic mode, the reactive agents are supplied as the reaction or production proceeds. Frequently, in the framework of a dynamic mode, there is a circulation of a fluid or agitation. Product mass evolves during production.

During stage (a) the active substance and cyclodextrin are introduced in solid or liquid form into a container into which during stage (b) the dense fluid under pressure and the water are injected in judiciously selected proportions. The pressure and temperature conditions as well as the duration of the treatment are defined, by any appropriate method, depending on the nature of the active substance (s) and the cyclodextrin (s).

Advantageously, step (b) of molecular diffusion of the process according to the present invention is carried out under stirring.

The water can be added continuously or discontinuously in an amount comprised between 1 and 50% by mass, preferably between 10 and 25% by mass.

The molar ratio of the active substance cyclodextrin / acid or base may be selected in such a way as to ensure the best inclusion of the active substance within the cyclodextrin. Thus, advantageously, the molar ratio of active substance cyclodextrin is between 1/1 and 1/10, advantageously between 1/1 and 1/5, advantageously between 1/2 and 1 / 2.5, and still more advantageous 1 / 2.5. Also advantageously the molar ratio of acidic or base active substance is between 1/1 and 1/3, advantageously 1/1, more advantageously 1 / 1.2.

The time required for molecular diffusion in step (b) is determined by any appropriate method. This step (b) can be repeated as many times as desired to obtain a satisfactory dissolution rate. Advantageously, step (b) lasts between about 1 and 16 hours.

The pressure and temperature conditions of step (b) are selected in such a way as to favor molecular diffusion. Advantageously, the pressure of the supercritical fluid is between 0.5 MPa and 50 MPa, and the temperature between 0 and 200 ° C.

Advantageously, step (b) of the process according to the present invention is carried out in a closed reactor, in particular an autoclave.

The procedure can be carried out in "batch" or continuous. Advantageously, the process according to the present invention is carried out in "batch".

Advantageously, step (b) of the process according to the present invention is carried out in a closed reactor, optionally stirred, fed by the dense fluid and the active substance solution, if necessary, continuously.

* The last two steps ((d) and (e)) consist of adding and mixing an acid or a base to the active substance-cyclodextrin complex.

This acid or this base interacts according to two plausible hypotheses: strong interactions with the active substance included in the cyclodextrin during the previous steps, and / or strong interactions with the previously formed complex.

This makes it possible, in particular, to improve the dissolution properties of the complex in biological liquids, and in particular water, and to eventually increase the percentage of inclusion of the active substance in the cyclodextrin.

The improvement of the physicochemical properties, in particular in terms of dissolution of the system formed, may have as its origin

-A non-covalent interaction of the acid or the base of the active substance, the cyclodextrin or both (complexation, salification, etc.).

- A local variation of the pH of the dissolution medium.

- Obtaining a system that has a eutectic.

-A modification of the interface between the system and its dissolution medium (surfactant effect, change in granulometry).

Step (d) of the process is carried out in a semi-solid medium, the complex not being dissolved in an aqueous medium before adding the acid or base. This agent will therefore simply moisten the complex or form a paste with the complex. Advantageously, step (d) is carried out under stirring.

The present invention also describes a soluble inclusion compound comprising one or more active substances that are poorly soluble in an aqueous medium included in one or more cyclodextrins and an acid or a base, capable of being obtained by the process according to the present invention. Advantageously, the inclusion percentage of the active substance in the soluble inclusion compound according to the present invention is greater than 95% calculated by DSC analysis as will be described below, advantageously greater than 98%, advantageously close to 99%.

Advantageously, the solubility of the active principle when it is in the form of the complex according to the present invention is greater than 2.5 g / l, advantageously at least equal to 3 g / l. Advantageously, the percentage of solubilization of the active substance contained in a 4 g / l solution in water measured at 38 [deg.] C. after 5 and 120 minutes of stirring is greater than 63%, advantageously at least equal to 75%. Advantageously, the active substance is piroxicam and the solubility and the solubilization percentage are measured at pH = 6.3.

Advantageously, the host molecule is cyclodextrin, and the active substance is piroxicam.

Advantageously, piroxicam is present in the complex according to the present invention in a zwitterionic structure.

In the context of the present invention, "percentage of solubilization" is understood to mean the percentage of piroxicam solubilized after 5 to 120 minutes of stirring at 37 [deg.] C. of a mixture of water and piroxicam. Usually a mixture at 4 g / l of piroxicam in water will be used to measure this percentage. This solubilization can be measured by a solubilization test as follows (piroxicam solubilization test).

The present invention further describes a complex comprising piroxicam, a cyclodextrin and arginine, characterized in that the percentage of solubilization of the piroxicam contained in a solution at 4 g / l in water, measured at 37 ° C after between 5 and 120 minutes of stirring, is greater than 90%, advantageously greater than 95%, advantageously equal to 99%.

By the term "cyclodextrins" is meant, in the sense of the present invention, cyclodextrins, modified cyclodextrins, and mixtures thereof. Advantageously, they are β-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin. Advantageously, it is β-cyclodextrin.

Advantageously, the piroxicam-cyclodextrin-arginine complex has good crystallinity, advantageously, this complex has less than 40% by weight of amorphous phase, even more advantageously less than 30% by weight of amorphous phase, advantageously an amount of amorphous phase less than or equal to 20% by weight.

Advantageously, the percentage of inclusion of piroxicam in the piroxicam-cyclodextrin-arginine complex measured by differential calorimetric analysis as will be described below is greater than 98%, advantageously greater than 99%, advantageously close to 100%.

Advantageously, the piroxicam is present in the complex in the form of a zwitterionic structure.

The piroxicam, cyclodextrin and arginine complexes are capable of being obtained by the process according to the present invention as described above.

The present invention also describes a pharmaceutical composition comprising a pyroxycyclodextrin-arginine complex, and optionally a pharmaceutically acceptable excipient.

The present invention also describes a piroxicam-cyclodextrin-arginine complex or a pharmaceutical composition as a medicine, advantageously having an anti-inflammatory action and advantageously intended to treat inflammatory rheumatism, polyarthritis, osteoarthritis, tendinitis or post-traumatic conditions of the apparatus. locomotor.

The following examples are provided for guidance only.

The different examples proposed have been carried out with piroxicam as the active substance, beta-cyclodextrin as the host molecule and water as the diffusion agent. Ammonia or arginine have been used as interaction agents with the complex.

Description of the figures

Figure 1 represents the NMR spectrum of the complex obtained according to example 7.

Figure 2 represents the intramolecular ROESY-NOE spectrum of the complex obtained according to example 7.

Figure 3 represents the TG-DGT (thermogravimetric analysis) profile of β-cyclodextrin using the following procedure: increase in temperature from 5ºC per minute to 450ºC and then decrease in temperature by 10ºC per minute to 80ºC.

Figure 4 represents the DSC profile of β-cyclodextrin using the following method: equilibration at 0 ° C followed by a temperature increase of 5 ° C per minute to 240 ° C.

Figure 5 represents the profile of the thermogravimetric analysis of L-arginine using the same procedure as that of figure 3.

Figure 6 represents the DSC profile of L-arginine using the same procedure as that of Figure 4.

Figure 7 represents the DSC profile of piroxicam using the same procedure as that of Figure 6.

Figure 8 represents the DSC profile of a piroxicam sample melted at 260 ° C and then cooled to 0 ° C using the equilibration procedure at 205 ° C, decreasing the temperature by 20 ° C per minute to 0 ° C and increasing the temperature by 5 ° C per minute to 220 ° C.

Figure 9 represents the DSC profile of the complex obtained according to example 7 using the equilibration procedure at 0 ° C, increasing the temperature by 5 ° C per minute to 240 ° C and then decreasing the temperature by 5 ° C per minute to 80 ° C.

Figure 10 represents the TG-DGT profile of the complex obtained according to example 7 using the procedure of increasing the temperature by 5 ° C per minute to 450 ° C and then decreasing the temperature by 10 ° C per minute to 80 ° C.

Figures 11 to 16 represent the molecular modeling of the minimized structure of the inclusion complex piroxicam-β-cyclodextrin 1: 2. In particular, figure 11 represents the profile view with the hydrogen atoms, figure 12 represents the profile view without the hydrogen atoms, figure 13 represents the view of the cavity on the side of the benzothiazine cycle with the atoms of hydrogen, figure 14 represents the view of the cavity from the side of the benzothiazine cycle without the hydrogen atoms, figure 15 represents the view of the cavity from the side of the pyridine cycle with the hydrogen atoms and figure 16 represents the view of the cavity on the side of the pyridine cycle without the hydrogen atoms.

Figure 17 represents the X-ray diffractogram of the complex obtained according to example 7, and of the complex obtained with the process of patent EP 0 153 998.

Analysis:

The percentage of inclusion of the active substance in the host molecule is evaluated by differential thermal analysis (DSC).

The DSC analyzes of the complexes obtained according to examples 1 to 6 were carried out as follows:

A temperature range under nitrogen flow is applied to the product to be tested, with the aid of a Perkin Elmer DSC7 apparatus.

The complexation performance is evaluated by measuring the reduction (or disappearance) of the thermal peak relative to the melting of the active principle "which has become free" in crystalline form.

The DSC analysis of the complex obtained according to example 7 has been carried out as follows:

The DSC Q100 apparatus from TA Instruments has been calibrated with the help of the indium melting signal under a nitrogen flow of 50 ml.min-1. The sample is analyzed in a sealed cabinet at 5ºC.min-1.

Thermogravimetric analyzes are performed with the aid of the TA Instruments TGA2950 HR apparatus in order to determine the field of thermal stability of the samples. The apparatus is calibrated at room temperature and with the aid of the Nickel Curie point at 360.46 ° C. The balance accuracy is verified by analyzing a sample of calcium oxalate.

The analysis is carried out under a nitrogen flow of 60 ml.min-1 from 25ºC to 450ºC.

The amount of water in the samples is determined by coulometry with the help of the Mettler KF DL coulometer.

37. The apparatus is calibrated with STANDARD sodium tartrate dihydrate (% H2O = 15.66 ± 0.05%).

X-ray powder diffraction is carried out with the apparatus and under the following conditions:

-Philips Xpert MDP diffractometer, Philips generator voltage 40kV intensity 20 mA -copper anti-cathode (Kalpha = 1.5418367 angstrom), Ni filter -Input slot 1/8 -Xcelerator detector -continuous scanning mode -sample crushed into powder on platelet - angular field (º2θ) 4 to 100 - scan duration: 80 seconds

The analysis of the phases is carried out with the help of the Visual CRYSTAL program.

Raman diffusion spectra are obtained with the apparatus and under the following conditions:

-Jobin Yvon Labram HR 800 Raman Spectrometer -Temperature: 22ºC -Samples: powder on microscope slide -Excitation wavelength: 752 nm, laser power 10 mW on the sample -Spectral resolution 2 cm-1, diffusing volume approximately 1 µm3 (600-trace network)

The proton NMR spectrum is recorded on a DPX AVANCE Bruker spectrometer at the nominal frequency of 400 MHz using a broadband reverse probe equipped with a field gradient accessory along the Z axis. The spectrometer is previously placed at the resonance frequency of the deuterium from the solubilization solvent, in this case dimethylsulfoxide-d6 (Eurisotop, ref. D 310B, batch A 2731). Chemical shifts are given in ppm relative to TMS (tetramethylsilane) used as internal standard.

The ROESY spectrum (Rotating frame Overhauser Enhancement SpectroscopY) is obtained by applying the Bruker pulse microprogram "roesytp.2". The recording is carried out in phasic mode with 1,024 increments and 72 sweeps per increment, that is, a total experiment duration of 53 hours per product. The acquisition is performed by selecting a spectral window of 6410.256 Hz, a relaxation time of 2 s and a spin closing time of 350 ms. Before recording the spectrum, the sample is properly degassed in order to observe the maximum intra and intermolecular NOE effect.

Due to the low solubility of the complexes in water and the sensitivity of the NMR spectrometer in the possession of the present inventors, it has been preferred to work on deuterated dimethyl sulfoxide in which it is possible to reach a concentration of 2% (w / v) in complex .

Molecular modeling: the optimization of the inclusion complex piroxicam-β-cyclodextrin (1: 2) is carried out with the Hyperchem® program, version 6.02 (Hypercube, Gainsbville, USA), implemented in a personal computer pentium III model HP Vectra.

The molecular structure of piroxicam used for the minimization calculations is extracted from the publication of Jon Bordner et al. (Acta Cryst., 1984, C40, 989-990).

The molecular structure of β-cyclodextrin comes from the Cambridge Crystallographic Database (M.R. Caira et al.,

J. Chem. Soc., Chem. Commun, 1994, 1061-1062).

The energy minimization of the geometry and the conformation of the complex is carried out with the help of the force field MM2. The simulation of the physicochemical properties (pKa, LogD and solubility) from the piroxicam molecule is

performed with the ACD / logD suite program (ACD / labs software, Toronto, Canada). To measure the dissolution properties of the powder, the equivalent of 4 g / l of piroxicam is dissolved in a

aqueous solution, at 37 ° C. After 15 minutes, an extraction is carried out, then the amount of piroxicam dissolved, and the result is expressed in grams of piroxicam dissolved per liter of water. According to this method, the solubility of pure piroxicam in water is less than 0.2 g / l. PYROXICAM SOLUBILIZATION TEST Implementation protocol: The dosage of the Piroxicam in the dissolution solution is carried out by means of HPLC: Apparatus used: HPLC WATERS system: -separation module 2695,

-UV 2487 detector. Chromatographic conditions: Column: WATERS X-Terra MS C18 250x4.6 mm.

1.1.1.1.1.1 Mobile phase: Route A

60% buffer pH = 3.5 KH2PO4 6.81 g / l adjusted to pH with dilute H3PO4 R, 40% acetonitrile Flow rate: 1 ml / min. Detector wavelength: 230 nm Detector sensitivity: 2 AUFS Injected volume: 20 µl Oven temperature: 40ºC Analysis time: 12 minutes

Preparation of control solutions:

Control solution: SM: Introduce 50 mg of Piroxicam control in a 100 ml bottle. Dissolve with 20 ml of dimethylformamide and make up to volume with methanol.

Interval:

T1: 1 / 20th dilution of T3 in 40 acetonitrile / 60 water. T2: 1 / 10th dilution of T3 in 40 acetonitrile / 60 water. T3: 1 / 100th dilution of SM in 40 acetonitrile / 60 water. T4: 1 / 50th dilution of SM in 40 acetonitrile / 60 water. Tus: 1 / 20th dilution of SM in 40 acetonitrile / 60 water.

Conditions of realization of the dissolution kinetics at 4 g / l: Carrying out the test: Conditions of realization: In a 100 ml Erlenmeyer flask, place a test sample equivalent to 200 mg of Piroxicam. Add 50 ml of

Water. Put under magnetic stirring at 400 rpm, in a thermostated bath at 37ºC +/- 2ºC. Carry out a 2 ml extraction under magnetic stirring at 5, 30, 60, and 120 minutes. Filter the extractions on a 0.45 µm Gelman GHP Acrodisc polypropylene filter. The solution must be clear.

Dilute the extraction to 1 / 200th in the mobile phase. Methodology, expression of results: Inject 20 µl of each control solution. Carry out a linear regression of the surfaces of the Piroxicam peaks with respect to the concentrations. The

correlation coefficient must be greater than 0.995. Inject 20 µl of the solutions to be examined. Measure the area of the Piroxicam peak in each solution to be examined. Deduct the concentration X in µg / ml according to the regression line of the controls. Calculate the concentration in µg per ml of solubilized Piroxicam multiplying by the inverse of the dilution made

(ie: 200). The percent solubilization of piroxicam is calculated by dividing the concentration of solubilized piroxicam by the total piroxicam concentration of the starting solution.

Example 1: Results obtained using the procedure according to the present invention

8 grams of piroxicam, 75 grams of beta-cyclodextrin, and 25.2 g of water are mixed and placed in a two-liter reactor. Carbon dioxide is then introduced into the reactor under a pressure of 200 bar and at a temperature of 150 ° C. The assembly is kept in these production conditions for a period of two hours.

After the so-called maturation stage, a part of the collected powder (12 g) is mixed with 2.11 g of a 28% ammonia solution, and then placed in a ventilated autoclave at 60 ° C, overnight.

The DSC analysis reveals a percentage of inclusion of the piroxicam in the cyclodextrin of 99%, the solution of the piroxicam being 3.019 g / l.

Comparative Example 2: Results obtained after the maturation stage without the addition of any interaction agent with the complex

4 grams of piroxicam, 38 grams of beta-cyclodextrin, and 8.95 grams of water are mixed and placed in a two liter reactor. Carbon dioxide is then introduced into the reactor under a pressure of 150 bars and at a temperature of 150 ° C. The assembly is kept in these production conditions for a period of two hours.

After the medium has settled, a part of the collected dust is placed in a ventilated oven at 60 ° C, overnight. The DSC analysis reveals a percentage of inclusion of the piroxicam in the cyclodextrin of 80%, the solution of the piroxicam being 0.246 g / l.

Comparative Example 3: Results obtained if the interaction agent with the complex is present during the maturation stage (b)

2 g of piroxicam, 19 g of beta-cyclodextrin, 3.75 g of water and 1.5 g of 28% ammonia solution are mixed and placed in a two-liter reactor. Carbon dioxide is then introduced into the reactor under a pressure of 200 bar and at a temperature of 160 ° C. The assembly is kept in these production conditions for a period of two hours.

After the medium has settled, the collected dust is placed in a ventilated oven at 60 ° C, overnight.

The DSC analysis reveals a percentage of inclusion of piroxicam in the cyclodextrin of 50%, the solution of piroxicam being 1.075 g / l.

Comparative Example 4: Results obtained if an attempt is made to bind the interaction agent to the host molecule before the maturation stage (b)

19 g of beta-cyclodextrin, 2.11 g of 28% ammonia solution and 3.15 g of purified water are mixed, and placed in a ventilated oven at 60 ° C, overnight. 2 g of piroxicam and 6.57 g of water are then added. The mixture is then introduced into a two-liter reactor. Carbon dioxide is then introduced

10 in the reactor under a pressure of 200 bars and at a temperature of 160 ° C. The assembly is kept in these production conditions for a period of two hours.

The DSC analysis reveals a percentage of inclusion of the piroxicam in the cyclodextrin of 92%, the solution of the piroxicam being 0.23 g / l. fifteen

Comparative Example 5: Results obtained if an attempt is made to bind the interaction agent to the active substance before the maturation stage (b)

2 g of piroxicam and 2 g of 28% ammonia solution are mixed, and placed in a ventilated oven at 60ºC,

20 for one night. Then 14.3 g of beta-cyclodextrin and 3.3 g of water are added. The mixture is then introduced into a two-liter reactor. Carbon dioxide is then introduced into the reactor under a pressure of 150 bars and at a temperature of 150 ° C. The assembly is kept in these production conditions for a period of two hours.

The DSC analysis reveals a percentage of inclusion of piroxicam in the cyclodextrin of 56%, the solution of piroxicam being 1,370 g / l.

Example 6: Results using the procedure according to the present invention on batches of semi-industrial size: 12.5 kg

1.1 kg of piroxicam, 10.6 kg of beta-cyclodextrin, 1.3 kg of water are mixed and introduced into a 50 liter reactor. Carbon dioxide is then introduced into the reactor under a pressure of 150 bar and at a temperature of 100 ° C. The assembly is kept in these production conditions for a period of two hours.

35 A part of the powder is placed in an oven at 80 ° C overnight. The DSC analysis reveals a percentage of inclusion of the piroxicam in the cyclodextrin of 89%, the solution of the piroxicam being 1,937 g / l.

The collected dust (12.6 kg) is mixed with 2.6 kg of a 28% ammonia solution, and then placed in a ventilated oven at 60 ° C.

The DSC analysis reveals a percentage of inclusion of the piroxicam in the cyclodextrin of 100%, the solution of the piroxicam being greater than 3 g / l.

Summary of the results obtained

Table 1 below gathers the results of the different examples 1 to 6 indicated above and allows to verify the added value of the process according to the present invention.

Table 1: 50

Procedure / Subjects

/ Ex. 1 Ex Comp. 2 Ex Comp. 3 Ex Comp. 4 Ex Comp. 5 Ex. 6

Materials used:

• Piroxicam

X X X X X X

• Piroxicam previously mixed with the complex interaction agent

X

• β-cyclodextrin

X X X X X

• β-cyclodextrin previously mixed with the complex interaction agent

X

1st phase: Complexation in supercritical CO2 medium

X X X X X

2nd phase: addition of the interaction agent with the complex

X X

1 single phase: Complexing with the agent of interaction with the complex in supercritical CO2 medium

X

Percentage of complexation (DSC)

/ 99% 80% fifty% 92% 56% 100%

Dissolution (g / l)

<0.2 3.02 0.25 1.07 0.23 1.37 > 3

5

fifteen

25

35

Four. Five

55

Comparative Example 7: Piroxicam Complex, image2 -cyclodextrin, arginine

43 grams of piroxicam, 384 grams of β-cyclodextrin and 25 grams of arginine, as well as 61 grams of diffusion agent (water) are introduced into a reactor. Carbon dioxide is then introduced into the reactor under a pressure of 15 MPa and at a temperature of 100 ° C. The set is kept in these realization conditions for a period of one hour.

On the complexes obtained, the dissolution kinetics and the solubilization percentage are measured as previously indicated in "PIROXICAM solubilization test". The results are compiled in Table 2 below.

Time (minutes)

Concentration in piroxicam (µg / ml) Solubilization percentage (%)

5

3863 96.6

30

3854 96.4

60

3941 98.5

120

3962 99

NMR:

The 1H NMR spectrum of a complex obtained according to example 7 (figure 1) is in agreement with the chemical composition of this compound.

The signal observed at 13.5 ppm is attributed to the pyridinium proton which provides additional evidence of the existence of a zwitterionic structure of piroxicam in the complex obtained according to example 7.

ROESY spectrum analysis (figure 2)

This study, which makes it possible to study magnetization transfers between protons close to each other in space, will explain the mode of inclusion of piroxicam in β-cyclodextrin and will thus allow the corresponding complex to be modeled.

From the ROESY map, intramolecular NOE effects (between protons of the same molecule) are distinguished from intermolecular effects (between protons belonging to different molecules). If the intramolecular NOE effects are obviously instructive in terms of conformational analysis, however, only the intermolecular NOE effects demonstrating an encapsulation of piroxicam in β-cyclodextrin will be cited. Analysis of the ROESY spectrum recorded in a complex obtained according to example 7 (figures 2) clearly shows cross relaxation peaks between H-9/10 of piroxicam and H-5 'of βCD and then between H-7, H- 6, H-3 of piroxicam and H-3 'of β-cyclodextrin. These results are in agreement with a 1: 2 inclusion complex, piroxicam: β-cyclodextrin.

Thermal analysis and water dosage: Thermogravimetric analysis of β-cyclodextrin (figure 3) shows two transitions at 70.6ºC and 313ºC. The first corresponds to a weight loss of 13.4% according to the amount of water determined by coulometry (14.1%), and the second transition is attributed to the thermal decomposition of β-cyclodextrin.

DSC analysis (figure 4) shows a broad endotherm centered at 162 ° C corresponding to the dehydration phenomenon. Thermogravimetric analysis of arginine (Figure 5) shows that arginine is stable up to about 230 ° C before decomposing. Two endotherms are observed in the DSC profile (figure 6), the first at 215.6ºC (melting or phase transition) and the second at 234ºC related to the decomposition phenomenon.

The DSC profile of piroxicam shows that melting occurs at 201 ° C with an enthalpy of 104 J.g-1 (figure 7).

Heating cooling cycles are carried out, in which a sample of piroxicam is melted at 205 ° C and then cooled to 0 ° C. The DSC profile of this sample (Figure 8) reveals the existence of a glass transition at 62 ° C, characteristic of the amorphous state that crystallizes at 122 ° C. A weak endotherm at 178 ° C followed by an exotherm and then a last endotherm at 197 ° C reflects the existence of several crystalline forms of piroxicam (polymorphism).

The two endotherms at 178ºC and 197ºC would be attributed respectively to the fusion of forms III and II while the initial sample before heat treatment would correspond to form I (fusion at 201 ° C) (F. Vrecer, S. Srcici and J Smid-Korbar, International Journal of Pharmaceutics 68 (1991) 35-41).

The water content of the complex obtained according to example 7 is of the order of 10% w / w, as indicated in Table 3 below:

Table 3 - Result of the water dosages of the complex of example 7

Complex H2O content (TGA) H2O content (coulometry)

Obtained according to example 7 9.99% 10.14%

The DSC profile of the complex obtained according to example 7 (figure 9) does not show the transitions at 198-200 ° C and at 218 ° C, which suggests an inclusion close to 100%.

The TG DTG profile of the complex obtained according to example 7 (figure 10) shows a transition at 191 ° C. It is attributed

10 this transition to the breakdown of arginine. Indeed, the first stage of decomposition of arginine corresponds to a weight loss of the order of 39.2% and if the content of arginine in the complex is taken into account, approximately 6% w / w (determination by NMR), this weight loss should be of the order of 2.35% w / w, in excellent agreement with that observed in the TG and DGT profiles at 191ºC (2.40%).

15 Molecular modeling: The minimized structure of the inclusion complex piroxicam: β-cyclodextrin (1: 2) is depicted in Figures 11 to 16. This optimized structure takes into account the spatial interactions observed by ROESY spectrometry.

As indicated in the publication of G.M. Escandar (Analyst, 1999, 124, 587-591), the piroxicam molecule is

Too bulky (approximately 6 x 13.7 Å) to be fully encapsulated in a β-cyclodextrin cavity. With a PX: ((βCD) 2 complex the piroxicam molecule is fully enclosed.

Simulation of the physicochemical properties: variation of piroxicam solubility as a function of the pH of the dissolution medium. The zwitterionic structure present between pH 2 and 6, the lowest solubility. The value of logD evolves

25 in the reverse order. In the pH 2-6 range, the molecule is relatively hydrophobic, which represents another favorable interaction for encapsulation. Between pH 6 and pH 7.5, the zwitterion is still present and the solubility value increases substantially as a function of pH. Hence the interest in preparing complexes in this pH zone in order to add the two solubilizing effects that are encapsulation on the one hand and the salification of piroxicam on the other hand. The measured pH values are listed below: complex

30 according to example 7 at 13.9 mg / 10 ml H2O = pH 7.87.

X-ray diffractions of the complex obtained according to example 7 and of the complex obtained according to the procedure described in document EP 0 153 998: the diffractogram is represented in figure 17. The powder diffractogram of the complex according to example 7 shows lines of strong, good-resolution diffraction demonstrating a

Better crystallinity of this sample with respect to that of the complex according to the method of patent EP 0 153 998. According to the Visual CRYSTAL program, the complex obtained according to example 7 has between 16 and 20% by weight of amorphous phase.

Comparative example 8: piroxicam complex, image2 -cyclodextrin, arginine

400 grams of piroxicam, 3,832 grams of β-cyclodextrin and 253 grams of arginine, as well as 613 grams of diffusion agent (water) are introduced into a reactor. Carbon dioxide is then introduced into the reactor under a pressure of 15 MPa and at a temperature of 100 ° C. The set is kept in these realization conditions for a period of one hour.

On the complexes obtained, the dissolution kinetics and the solubilization percentage are measured as previously indicated in "PIROXICAM solubilization test". The results are compiled in Table 3 below.

Time (minutes)

Concentration in piroxicam (µg / ml) Solubilization percentage (%)

5

3,953 98.8

30

3,895 97.3

60

3,952 98.8

120

4,041 * 100

* Result consistent with the uncertainty related to the measurement.

Claims (9)

1. Process for preparing a soluble inclusion compound, which comprises one or more active substances that are poorly soluble in an aqueous medium included in one or more cyclodextrins, characterized in that it comprises the following successive steps:

to.

putting one or more active substances in contact with one or more cyclodextrins,

b.

carry out a molecular diffusion stage by contacting, in static mode, a dense fluid under pressure with the mixture obtained in stage (a) in the presence of water,

c.

recovering the active substance / cyclodextrin molecular complex thus formed,

d.

carry out a step in a semi-solid medium that consists of adding and mixing an agent that interacts with the complex to the molecular complex active substance / cyclodextrins, the agent that interacts with the complex being an acid or a base,

and.

recovering the inclusion compound thus formed.

2.

Process according to claim 1, characterized in that the agent for interacting with the complex is an amino acid, a carboxylic acid or ammonia, advantageously ammonia.

3.

Process according to any of claims 1 or 2, characterized in that the dense fluid under pressure is carbon dioxide.

Four.

Process according to any of claims 1 to 3, characterized in that the active substance is a pharmaceutical active agent, preferably selected from the group consisting of analgesics, antipyretics, aspirin and its derivatives, antibiotics, anti-inflammatories, antiulcers, antihypertensives, neuroleptics, antidepressants, oligonucleotides exhibiting therapeutic activity, peptides exhibiting therapeutic activity and proteins exhibiting therapeutic activity, and a cosmetic or nutraceutical active agent.

5.

Process according to claim 4, characterized in that the active substance is selected from the group consisting of anilide derivatives, epipodophyllotoxin derivatives, minoxidil, piroxicam, valeric acid, octanoic acid, lauric acid, stearic acid , thiaprofenic acid, omeprazole, econazole, miconazole, ketoconazole, astemizole, cyclobenzaprine, nimesulide, ibuprofen, terfenadine, domperidone, naproxen and eflucimibe, advantageously it is piroxicam.

6.

Process according to any of claims 1 to 5, characterized in that the pressure of the dense fluid is between 0.5 MPa and 50 MPa, and the temperature between 0 and 200 ° C.

7.

Process according to any of claims 1 to 6, characterized in that step (b) of molecular diffusion is carried out with stirring.

8.

Process according to any of claims 1 to 7, characterized in that the water is added continuously

or discontinuously in an amount comprised between 1 and 50% by mass, preferably between 10 and 25% by mass. Process according to any one of claims 1 to 8, characterized in that step (b) of the process is carried out in a closed reactor, optionally stirred, fed by the dense fluid and the active substance solution, if necessary, continuously.