WO2007088207A2 - A process for preparing an organic compound in the solid state and pharmaceutical formulations comprising the said organic compound - Google Patents

Abstract

A process for preparing, starting from the initial organic compound crystallised in the solid state and possessing one or several biological properties, an organic compound crystallised in the solid state and possessing one or several modified biological properties compared with the starting organic compound, said process comprising the following steps: a) supplying the original organic compound in the solid state; b) sublimating to the vapour state said starting organic compound and condensing the compound sublimated to the vapour state onto the surface of a substrate maintained at ambient temperature; c) retrieving the organic compound in the solid state obtained at the end of step b), from the said substrate.

Description

A process for preparing an organic compound in the solid state and pharmaceutical formulations comprising the said organic compound

FIELD OF THE INVENTION

The present invention relates to the field of the preparation of organic compounds possessing excellent preventive or curative pharmaceutical properties.

PRIOR ART There exists a constant need to improve preventive and curative therapeutic treatments for humans and animals, compared with treatments currently available to the public, given the great variety of disorders and diseases.

In particular, there is a need to increase the preventive or curative therapeutic efficacy of known active principles.

The improvement of the preventive or curative therapeutic efficacy of known active principles includes: (i) obtaining an optimal preventive or curative therapeutic activity with reduced quantities of active principles, compared to the quantities of the same active principle currently used, (ii) obtaining an optimal preventive or curative therapeutic effect with reduced or non-existent adverse effects, (iii) improving the stability of active principles, either by increasing their stability during their shelf life, or by increasing their half-life once they have been administered in the organism, (iv) increasing the accessibility of active principles in relation to their biological targets in the organism, for example by allowing certain active principles to reach biological sites located in the neurones, which involves them crossing the haematoencephalic barrier.

The aforementioned needs encompass research for increased preventive or curative therapeutic efficacy in active principles such as amino acids.

Numerous amino acids are known for their biological activity and are used, as an active substance, in diverse therapies including preventive or curative therapies for pathologies related to a deregulation of certain signal pathways of the central nervous system, such as epilepsy or Parkinson's disease.

Amino acids with therapeutic applications include gamma aminobutyric acid, also known as GABA, which is an important neurotransmitter that is widely distributed throughout the central nervous system (CNS) at a level at which this neurotransmitter generally possesses an inhibiting activity. Biosynthesis of GABA and receptors for this neurotransmitter have been known for a long time. There are two main classes of GABA receptors, respectively (i) receptors known as GABA_A and GABA_C, which are ionotropic receptors, and (ii) receptors know as GABA_B, which are metabotropic receptors. The stimulation of GABA receptors and the subsequent transduction of the stimulation signal from these receptors involves metabolic paths that are called GABAergic. The stimulation of GABAergic paths is sought in many medical indications, including the treatment of epilepsy, mood disorders and more recently medical indications of neuroprotection.

Thus GABA, which is the major neurotransmitter inhibitor of the central nervous system and which inhibits the pre-synaptic liberation of neurotransmitters by modifying the polarisation of the chlorine channels inside the nerve cells, could potentially be used as an active substance of a sedative or anxiolytic medication. Unfortunately, GABA in solution obtained from commercial GABA is a solid which crystallises in the monoclinic system. This amino acid cannot cross the biological membranes, in particular the haematoencephalic barrier. Consequently, today, a potentially therapeutically active amino acid such as GABA is unusable as an active substance of a medication. This aspect of the physical chemical behaviour of GABA has particularly been reported by BAC et al. (1998, The Journal of Neuroscience, vol.l8(l l):4363-4373). These authors showed that GABA, which has no activity in an experimental model of audiogenic seizures induced in magnesium deficient mice ("MDDAS" test, for Magnesium Deficiency- Dependent Audiogenic Seizures), exercises an anticonvulsive activity, in this same murine experimental model, when the mice develop among other things lesions in the haematoencephalic barrier. More generally, the therapeutic potential of a substance depends on both (i) the biological targets with which the substance can interact and (ii) the in vivo access of the substance to the target receptor cells to which it binds. A failure in the in vivo accessibility of a substance for the target can represent a limiting element in the biological activity of this substance and can constitute the cause of the therapeutic inefficacy of a substance which, however, possesses important properties in vitro.

In order to overcome the disadvantages related to the impossibility for amino acids with therapeutic potential to cross biological membranes, particularly those such as the haematoencephalic barrier, the current state of the art is to administer precursors of active amino acids to patients, precursors which are capable of crossing biological membranes, said precursors being subsequently metabolised into the therapeutically active amino acid. For example, such precursors of a zwitterionic therapeutic active substance have been synthesised for GABA. In particular the substance known as Progabide (4-(4'-chloro-5-fluoro-2-hydroxybenzhydrylidene amino) butyramide, can be mentioned here, which is a non zwitterionic substance in the solid state, and which is capable of crossing the haematoencephalic barrier, then decomposing into the GABA active molecule, close to the target receptors on the nerve cells.

In the same way, it has been shown that a hydrophilic zwitterionic substance, such as the substance L-767,679 (MERCK Research Laboratories), after oral administration, crossed the intestinal barrier with difficulty to enter the blood stream, contrary to a precursor substance, the prodrug L-775,318 (Merck Research Laboratories), non zwitterionic, (Prueksaritanont et al , 1998, Drug Metab. Dispos., vol.26, 6) :520-527).

However, the synthesis of precursors of therapeutically active organic compounds, including therapeutically active amino acids, requires long and costly research, particularly to ensure that such precursors of active substances will be effective and will indeed be metabolised into the appropriate active substance.

Moreover, the quantities of active substances that are finally bioavailable for the target cell receptors are not accurate, particularly because of the heterogeneity of physiological conditions of the different tissues targeted, and due to the great variability in the metabolisation of the active substance precursor into the therapeutically active substance.

Furthermore, by definition, the precursor compounds for active substances are fairly unstable for long-term storage of the medication, which is a further technical disadvantage.

DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the X-ray diffraction diagram of reference

GABA in monoclinic form (Figure 1 A) and GABA in tetragonal form obtained according to the process of the invention (Figure IB). On the Y- axis: relative intensity, expressed as a % of the highest intensity peak value. On the X-axis: diffraction angle (θ).

Figure 2 illustrates the results of the scanning differential calorimetry obtained respectively with a reference commercial GABA (upper curve) and a GABA obtained by the process according to the invention (lower curve).

On the X-axis, the temperature readings, expressed in degrees Celsius.

On the Y-axis, the heat flow signal, expressed in milliwatts (mW). Figure 3 illustrates the experimental thermodynamic cycle and the physical state analysis P = f(T) of GABA modified according to the process of the invention.

On the X-axis, the temperature, expressed in degrees Celsius. On the Y-axis, pressure, expressed in Pascal (Pa). Figure 4 illustrates the comparative catalytic activity of the

GABase using as a substrate (i) a GABA obtained by the process of the invention (Figure 4A) and (ii) a reference commercial GABA (figure 4B). On the X-axis, the incubation time of the GABA substrate with the GABase enzyme expressed in minutes. On the Y-axis, the difference in absorbance at 340 nanometers (ΔA₃₄₀) expressed as difference in optical density (O.D.).

In figure 4 A and figure 4B, the two lower curves represent the results obtained using 160 nanomole and 400 nanomole quantities of

GABA, incubation having been carried out in the absence of α- ketoglutarate. The middle curve represents the results obtained using a quantity of 160 nanomoles of GABA in presence of 1000 nanomoles of α- ketoglutarate.

The upper curve represents the results obtained using a quantity of 400 nanomoles of GABA in presence of 1000 nanomoles of α- ketoglutarate.

Figure 5 illustrates the results of a kinetic study on GABase activity obtained for a solution prepared from commercial GABA

(monoclinic) and a solution obtained from tetragonal GABA on solvated forms of a commercial reference GABA and a GABA obtained by the process according to the invention.

Figure 5 A illustrates the speeds of enzyme reactions depending on the GABA concentrations. On the X-axis, there is the concentration of the GABA substrate, expressed in millimoles (mM).

On the Y-axis, the speed of the enzyme reaction, expressed in nanomoles per minute.

Figure 5B illustrates the results of the Lineweaver-Burk plot obtained from results shown in Figure 5A.

On the X-axis, the inverse of the GABA substrate concentration, expressed in mM^"1. On the Y-axis, the inverse speed of the enzyme reaction, expressed in nmol^.min.

Figure 6 shows a comparison of the anticonvulsive properties of a reference commercial GABA and a GABA obtained by the process according to the invention, dissolved in a 0.9% NaCl saline solution (P/P). On the Y-axis, the percentage of animals having had convulsions.

The empty block graph at the left of figure 6 shows the results obtained with a control saline formulation not containing GABA.

On the right-hand side of figure 6, there are three groups of block graphs showing the results obtained with respectively 25 mg/kg, 50 mg/kg and 100 mg/kg of GABA. In each group, the right-hand block graph shows the results obtained with a GABA prepared according to the process of the invention; the left-hand block graph shows the results obtained by administering a reference commercial GABA.

DETAILED DESCRIPTION OF THE INVENTION The various technical problems related, particularly, to the bioavalability of an active substance, particularly an active substance that is naturally zwitterionic in the solid state, for the corresponding target cell sites or receptors, have been resolved by the invention. It has been shown according to the invention that the activity of organic compounds of therapeutic importance could be modified by treating these organic compounds by a process comprising a step of sublimating the starting substance in the solid state, then condensing the sublimated organic compound in order to retrieve, at the end of the process, the said organic compound in the solid state, the end product of the process possessing, once it is dissolved, a modified biological activity compared with the starting organic compound.

More specifically, it has been shown according to the invention that by treating organic compounds of therapeutic importance by such a process it was possible to modify the biological activity spectrum of the starting organic compound, in general by increasing its preventive or curative properties for diverse disorders or for diverse pathologies.

The purpose of the invention is a process for preparing, starting from the initial organic compound crystallised in the solid state and possessing one or several biological properties, an organic compound crystallised in the solid state and possessing one or several modified biological properties compared with the starting organic compound, said process comprising the following steps: a) supplying the starting organic compound in the solid state; b) sublimating to the vapour state said starting organic compound and condensing the substance sublimated to the vapour state onto the surface of a substrate maintained at ambient temperature; c) retrieving the organic compound in the solid state obtained at the end of step b), from the said substrate. As will be specified below in the specification and in the examples, by treating the starting organic compound in the solid state by the aforesaid process it was possible to prepare diverse organic compounds in the solid state, of the same chemical formula as the starting substance, and which possess modified biological properties compared with the starting organic compounds in the solid state. The term "modified biological property", for the purposes of the present specification, means that the final substance obtained by the process possesses, compared with the starting substance:

(i) the same known biological activity as the starting substance, this biological activity being increased, or on the contrary reduced compared with that of the starting substance; and/or

(ii) one or several biological activities that the starting substance does not possess.

Thus, with the process of the invention, the starting organic compound and the end product of the process both have the same basic chemical formula, that is, the same qualitative and quantitative atomic composition. As an illustration, treating typical commercial γ- aminobutyric acid of formula C₄H₉NO₂ by the aforesaid process results in the preparation of a γ-aminobutyric acid with modified biological properties but with the same chemical formula C₄H₉NO₂ as can be shown by analysing the proton and carbon 13 nuclear magnetic resonance (NMR) spectra.

For the purposes of the present specification, the end product of the aforesaid process, which has the same chemical formula as the starting organic compound in the solid state, and which possesses modified biological properties, compared with the starting substance, can also be called the "redeposited" product.

Thus, it has been shown according to the invention, in an experimental model of convulsive audiogenic seizures induced by magnesium deficiency (MDDAS), that typical commercial GABA had no effect on these intra-cerebral disorders, whereas the redeposited GABA obtained as the end product of the aforesaid process, made it possible to reduce, and even to block, the onset of audiogenic seizures in this experimental model in mice. Also, it has been shown according to the invention that redeposited dopamine had a major effect on the central nervous system (CNS), in an experimental model inducing convulsions in mice treated with a non-convulsive and non-lethal dose of cocaine, whereas typical untreated dopamine does not possess this activity. Also, it has been shown according to the invention that redeposited valproic acid possessed properties of inhibiting, and even blocking, convulsive audiogenic seizures in the MDDAS experimental model, whereas typical commercial valproic acid was inactive at the same doses.

It has also been shown according to the invention that a redeposited antibiotic such as redeposited sodium fusidate, preserves the antibacterial activity spectrum of typical commercial sodium fusidate, but acquires additional anti-inflammatory properties. Thus the process of the invention makes it possible to prepare organic compounds in the solid state of the same chemical formula as the starting substance, but which possess, after being redissolved, modified biological properties, and in particular improved therapeutic activities, compared with the starting organic compounds. According to a preferred embodiment of the aforesaid process, the sublimation of the starting organic compound to the vapour state is carried out, at step b), by heating at reduced pressure.

Preferably, step b), sublimation is carried out at a temperature ranging from 18⁰C to 25⁰C and at a reduced pressure ranging from 0.5 10^{" 3} to 2 10^"3 Pa.

According to an advantageous characteristic of the aforesaid process, first a quantity selected between 200 and 500 mg of the starting organic compound in the solid state is placed in a container, preferably a molybdenum crucible, said container being linked to a controllable means of heating. The controllable means of heating preferably comprises one or several resistances linked to a means of controlling the electrical current.

The container is placed in a airtight enclosure in which a high vacuum can be created.

The same enclosure contains, placed vertically above the container, a substrate surface, for example a rotary substrate carrier, which is situated at a preferred distance ranging from 10 to 20 cm, vertically above the said container.

Then the sublimation of the starting organic compound in the solid state into the vapour state is carried out by creating a high vacuum, comprised between 0.5 10^"3 Pa and 2 10^"5 Pa, for example from 0.5 10^"3 to 3 10^"4 Pa, including from 0.5 10^"3 Pa to 2 10^"3 Pa, in the enclosure, at a sublimation temperature between 16O⁰C and 18O⁰C.

Then the organic compound in the vapour state obtained as above is condensed on the surface of a substrate, preferably the aforesaid rotary substrate carrier.

The heating temperature of the container is adapted so as to be able to control the speed of deposit of the starting organic compound in the solid state on the substrate surface.

Preferably, the heating temperature of the container is adapted so as to obtain a speed of deposit of the end product organic compound in the solid state ranging from 0.1 to 5 nanometres per second, preferably from 0.2 to 2 nanometres per second, and still more preferably from 0.3 to 1 nanometres per second, for example a speed of deposit of 0.5 nanometres per second. Preferably, the rotary substrate carrier consists of a disc of Pyrex^® glass.

In order to implement the aforesaid process, those skilled in the art could advantageously use an evaporator device of the AUTO 306 type, commercialised by the Edwards High Vacuum International Company (Manor Royal, Crawley, West Sussex, RH 10 2LW, UK).

Thus, at step b) of the aforesaid process, the organic compound in the vapour state is condensed into the final form of the said organic compound in the solid state. What is created is a deposit of the organic compound in the solid state on the surface of the substrate, preferably the rotary substrate carrier, by condensation of the gaseous phase at a temperature of the substrate carrier comprised between 15⁰C and 25⁰C.

Thus a deposit of a chosen thickness of the organic compound in the solid state is created, ranging from 0.1 μm to 10 μm. The rise in vapour pressure by increasing the temperature of the container, for example a crucible, makes it possible to control the sublimation of the organic compound in the solid state.

The analysis of the final organic compound in the solid state which is obtained at the end of stage c) of the aforesaid process shows that this substance can have undergone detectable or measurable physical or chemical modifications, without changing the chemical formula of the starting product.

As specified subsequently in the specification, including the examples, the redeposited GABA end product of the invention process can be distinguished from commercial reference GABA by diverse physical characteristics and diverse characteristics of biological activity.

The redeposited GABA obtained according to the invention, which is a tetragonal crystalline structure GABA, which can also be called quadratic crystalline structure, had previously been described by Dobson and Gerkin (1996, Acta Cryst., Vol. C52 : 3075-3078). However, according to the process used by Dobson and Gerkin, the tetragonal GABA was obtained by (i) dissolving a monoclinic commercial GABA in water, then (ii) by slow evaporation of the aqueous solution. The process described by Dobson and Gerkin could exclusively be used for preparing a few monocrystals of tetragonal GABA, which was enough for a subsequent study of these crystals by X-ray diffraction. However, the process described by Dobson and Gerkin would be totally inappropriate for preparing tetragonal GABA in sufficient quantities for testing any possible biological activity. Moreover, the process as described in Dobson and Gerkin's article has not proved to be reproducible.

On the contrary, the process of the invention has the technical advantage of being reproducible and of making it possible to prepare quantities of redeposited GABA of the order of at least 10 mg, using a laboratory sublimation device. Thus, with an appropriate sublimation device, the invention process can be embodied in such a way as to easily produce large quantities of redeposited GABA, for example several kilograms.

The aforesaid process is preferably implemented with organic compounds of low molecular weight, advantageously with starting organic compounds with a molecular weight of less than 1000 g.mol-1, and preferably with starting organic compounds with a molecular weight of less than 600 g.mol^"1.

As an illustration, GABA, which has a chemical formula of

C₄H₉NO₂ has a molecular weight of 103.12 g.mol^"1. Dopamine, which has a chemical formula Of CgHnNO₂ has a molecular weight of 153.18 g.mol^"1. Valproic acid, which has a chemical formula Of CgHi₆O₂ has a molecular weight of 144.21 g.mol^"1. Fusidic acid, which has a chemical formula of C_3IH₄₈O₆ has a molecular weight of 516.72 g.mol^"1.

Thus, in certain embodiments of the aforesaid process, the starting organic compound has a molecular weight of under 550 g.mol^"1.

In other embodiments of the aforesaid process, the starting organic compound has a molecular weight of under 300 g.mol^"1.

In other embodiments of the aforesaid process, the starting organic compound has a molecular weight of under 200 g.mol^"1. Preferably, the starting organic compound comprises at least one group selected from the groups comprising hydroxyl, carboxyl and amino.

In particular, the preferred starting organic compounds include amino acids, which comprise at least one carboxyl group and at least one amino group, as is the case of GABA for example. By way of illustration, dopamine comprises two hydroxyl groups and one amino group. Valproic acid comprises one carboxyl group. Fusidic acid comprises two hydroxyl groups and one carboxyl group.

In a particular embodiment of the aforesaid process, the starting organic compound consists of γ-aminobutyric acid or GABA in monoclinic form.

In a further embodiment of the aforesaid process, the starting organic compound consists of dopamine.

In yet another embodiment of the aforesaid process, the starting organic compound consists of valproic acid, or a valproic acid salt, including the sodium salt.

In yet another embodiment of the aforesaid process, the starting organic compound consists of fusidic acid, or a fusidic acid salt, including the sodium salt.

As illustrated in the examples, an organic compound obtained as an end product of the aforesaid process, although it has a chemical formula identical to that of the starting organic compound, can undergo changes in conformation likely to confer on the end product of the process modified biological properties compared with the starting organic compound. Thus, an analysis of the redeposited GABA by X-ray diffraction has shown that redeposited GABA consists of a tetragonal type GABA, that is a GABA that has a different spatial configuration from typical commercial GABA used as a starting product, the latter being a GABA with a monoclinic crystalline structure. Particularly it has been shown in the examples that redeposited GABA, which has the form of a GABA with a tetragonal crystalline structure, possesses the spatial group: IA_\cd. It has also been shown that the GABA with a tetragonal crystalline structure obtained as an end product of the invention process possesses the following crystal parameters, obtained by analysis of the X ray diffraction results: (i) a = 1196.3 pm, (ii) c = 1528.2 pm, (iii) Z = 16 , and (iv) p = 1.253 Mg. m^"3 as measured at a temperature in the range between 2O⁰C and 25⁰C.

Equally, the redeposited GABA which is the end product of the invention process can be distinguished from typical commercial GABA by the characteristics of its infrared spectrum. Thus the redeposited GABA which is the end product of the aforesaid process, possesses an infrared spectrum with infrared absorption bands, given in cm^"1, having the following values: 1647, 1569, 1554, 1532, 1474, 1447, 1437, 1428, 1396, 1382, 1360, 1339, 1305, 1283, 1267, 1240, 1 163, 1124, 1063, 1028, 1007, 995, 949, 902, 867, 779, 754, 656. The values of the infrared spectrum bands which comprise the technical characteristics of the redeposited GABA which is the end product of the aforesaid process, can vary, for a given infrared absorption band, depending on the tests, by +/- 4 cm^"1. Equally, the redeposited GABA which is the end product of the invention process can be distinguished from typical GABA in monoclinic form by its heat capacity properties (c_p) measured between 303 kelvins and 363 kelvins as illustrated in the examples, using equation (1) as follows: C_p = A_o+ AiT (1), wherein:

C_p is the value of the heat capacity (or specific heat) at constant pressure, expressed in J.K^.mol^"1 ; A₀ = 27.423 expressed in J.K^.mol^"1; Ai = 0.3936 expressed in J.K^"2 mol^"1 T is expressed in kelvins. It has been shown, according to the invention, that the thermal capacity, which is a distinctive technical characteristic of redeposited GABA, is defined by a value of A₀ = 78.834 J.K^.mol^"1 and a value of Ai de 0.2575 J.K^.mol^"1. Another distinctive technical characteristic of the redeposited

GABA obtained according to invention process consists in its melting point, which is 216 +/- I⁰C.

A further distinctive technical characteristic of the redeposited GABA consists in its melting enthalpy which is 1147 +/- 28 J.g^"1, which corresponds to 118.28 +/- 3 kJ.mol^"1.

The results of the calorimetry analysis achieved with a redeposited organic compound obtained by the invention process, including redeposited GABA, can be carried out by any technique known to those skilled in the art, and particularly by the technique described by B. Legendre et al. (Thermochemica Acta 400, (2003) 213-219; J. of Thermal Analysis and Calorimetry vol 76, (2004) 255-264 ).

When redeposited GABA is dissolved in a 0.1 N solution of NaOH, a further distinctive technical characteristic of the redeposited GABA consists in its ultraviolet absorption spectrum. The UV absorption spectrum of a solution prepared extemporaneously with redeposited GABA in its solid state is characterised by i) the greatest absorption peak situated at wavelength λ = 217 nm with an absorbance value at this wavelength A = 0.058 σ = 0.0017 O.D. units and by (ii) an extinction coefficient of ε = 0.5644 g \L, σ = 0.0556. Typical commercial GABA and redeposited GABA obtained by the invention process can also be differentiated by diverse biological properties, both in vitro and in vivo.

In vitro, both redeposited GABA and typical commercial GABA are good substrates for GABase, which is an enzyme combination prepared from Pseudomonas fluorescens containing a GABA aminotransferase and a SSAL dehydrogenase ("succinic semialdehyde dehydrogenase"), which is commercialised among others by BOEHRINGER (Germany) and also by Aldrich/Sigma (France).

It has been shown according to the invention that redeposited GABA can be differentiated from typical commercial GABA by its apparent affinity constant for GABase, particularly by the value of the apparent Km of redeposited GABA for GABase, which is 0.48 mM +/- 0.05.

As has been mentioned previously, redeposited GABA is also differentiated by its in vivo properties and in particular by its capacity to inhibit or block the onset of audiogenic seizures, in an experimental model of audiogenic seizures induced by magnesium deficiency (MDDAS) in mice.

Redeposited GABA obtained as the end product of the invention process consists of a tetragonal GABA which is metastable and which retains its specific physical and biological properties for several days, when it is in the solid state in powder form.

On the contrary, due to the metastable nature of redeposited

GABA it is not possible to store this physical form of GABA for more than 24 hours after dissolving it in a liquid, preferably an aqueous solution.

Without wanting to be tied to a particular theory, the applicant thinks that redeposited GABA could also have a lower affinity than typical GABA for enzymes other than GABase and thus be degraded less rapidly than typical GABA, when administered to a human or animal organism. This hypothesis could explain, at least in part, the effect of redeposited GABA which was observed at the central nervous system, in physiological situations in which typical GABA has no effect.

In the same way, a change has been observed in certain physical chemical characteristics of other organic compounds of therapeutic interest, after treating them by the invention process, with no change in the chemical formulae of the substances.

Thus, for example, dopamine, which is naturally present in the form of a zwitterionic white powder in the solid state, is retrieved as the end product of the invention process in the form of brown dopamine powder that is non-ionic in the solid state.

For sodium fusidate, it has been shown that redeposited sodium fusidate can be distinguished from typical sodium fusidate at least by its solubility properties in aqueous solutions. In particular, redeposited sodium fusidate is insoluble at the final concentration of 25 mg/ml in PBS buffer, whereas the sodium fusidate used as starting product is soluble in the same conditions.

Equally, redeposited sodium fusidate is moderately soluble at the final concentrations of 2.5 mg/ml in an aqueous solution of purified water containing a final concentration of 1 mg/ml of monohydrated citric acid,

19.6 mg/ml of disodium phosphate dihydrate and 0.5 mg/ml of disodium edetate (EDTA), whereas the sodium fusidate used as starting product is soluble, including at the final concentration of 50 mg/ml, in the same aqueous solution.

Equally, redeposited sodium fusidate is soluble in a final concentration of 0.2 mg/ml of monohydrate citric acid, whereas sodium fusidate used as a starting product precipitates from solution, in identical conditions. Both for GABA and for dopamine obtained in the non-ionised form in the solid state after treatment by the invention process, a change is observed in the ability of these two redeposited products to act on the central nervous system.

As is shown in the examples, whereas natural GABA in the solid state has no anti-convulsive activity, because the target cell sites or receptors, respectively GABA_A and GABA_B receptors, are present at the surface of the nerve cells protected from the general blood circulation by the haematoencephalic barrier, redeposited GABA obtained according to the invention process possesses an anti-convulsive activity of the same order as that observed previously by BAC et al. (J. Neurosci.1998, 18,

4363-4373) in an experimental model in mice in which the lesions of the haematoencephalic barrier had been caused experimentally.

As illustrated in the examples, GABA treated according to the invention process, in the experimental MDDAS model described by BAC et al. (1998) has an ED₅₀, that is an effective dose able to protect 50% of treated animals against convulsive seizures called audiogenic, equal to 25 mg/kg.

Similar results have been obtained by redeposited dopamine, as described in the examples. Thus, in an experimental model in mice of a toxicity test for cocaine, it has been shown that natural zwitterion dopamine in the solid state injected into mice to which a 4 mg/kg dose of cocaine had previously been administered, brought about no measurable change in the onset of convulsions, compared to a control batch of mice that had not received the prescribed dose of cocaine. However, it is observed that redeposited dopamine obtained according to the invention process enters into synergy with the cocaine, in the same experimental model in mice, causing the death of 100% of the treated mice. By the observation of a potentialisation of convulsions caused by cocaine, it has been shown that redeposited dopamine exercises its biological activity on the target receptor sites of the nerve cells.

As illustrated in the examples, it is also possible using the invention process to prepare redeposited sodium fusidate that has an antibacterial spectrum identical to that of sodium fusidate used as the starting product and possessing additional biological properties, such as anti-inflammatory properties. The anti- inflammatory properties of redeposited sodium fusidate are illustrated in particular by an inhibiting action of the activation of macrophages and an inhibition of the membrane expression of ICAM-I molecules and by the major Class II histocompatibilty antigens, such as MHC I-A antigens, by previously activated macrophages, for example by LPS.

From the aforesaid facts, it transpires that the possibility, using the invention process, of obtaining among other things substances in the solid state, with modified structure or physical chemical properties compared with the starting substance of the same chemical formula, allows for the first time for the use of these substances for therapy without needing the preliminary synthesis of the precursor molecules.

After treatment by the invention process, GABA can now be used directly to exercise its diverse therapeutic activities, particularly as an active anxioloytic substance, as an active hypnotic substance, as an anti- convulsive agent, as a neuroprotector agent and also as a myorelaxant agent.

In the same way, non-ionised dopamine in the solid state, which can be obtained as an end product of the invention process, can be used for example for its positive effect, for example to reduce the symptoms of Parkinson's disease. In the same way, non-ionised valproic acid in the solid state, which can be obtained by the invention process, can be used directly and effectively, particularly for treating epilepsy.

In the same way, redeposited fusidic acid, or any one of its salts, including its sodium salt, which can be obtained by the invention process, can be used effectively as an antibacterial agent possessing anti- inflammatory properties.

Thus, thanks to the invention process, many organic compounds in the solid state can be transformed into redeposited substances in the solid state in order to potentialise, or even reveal, their therapeutic biological activity.

It has been shown according to the invention that by obtaining a non-ionised substance in the solid state it was possible to increase the bioavailability of the substance for the target cell receptor or receptors. Thus, a redeposited organic compound, which can be prepared according to the invention process, is useful as an active substance of a medication, particularly as an anticancer, antibiotic, antiparasite, antiviral, anti-atheromatous or neurological medication.

The substances concerned encompass amino acids. Furthermore, the kinetic and pharmacological profile that characterises a starting organic compound in the solid state can be improved, by using a redeposited substance in the solid state as an active substance of a pharmaceutical formulation.

From then on, any process capable of creating or improving the access of a substance to its target receptor represents a decisive therapeutic innovation, as important as the discovery of a new active substance.

Thus the invention supplies original means of reducing the differences that can exist, for a given active therapeutic substance, between high biological activity in vitro and its therapeutic inefficacy in vivo.

In other words, the therapeutic impact of the use of an organic compound, with therapeutic potential treated by the invention process in the solid state is particularly: to improve the therapeutic activity of any active substance used at present in curative or preventive therapy; to improve the therapeutic impact of a potential therapeutic substance that has not been retained at any time during development in clinical trials, for example due to poor kinetics, or to weak pharmacological activity; to make it possible to use, as an active substance, a substance having biological properties in vitro, but devoid of all therapeutic effect in vivo.

Another object of the invention consists of a pharmaceutical formulation comprising, as its active substance, a redeposited organic compound that can be obtained by the process according to the invention, in combination with one or several pharmaceutically acceptable excipients.

Another object of the invention consists of a pharmaceutical formulation comprising, as its active substance, a non-ionised amino acid compound in the solid state, the end product obtained by the process according to the invention, in combination with one or several physiologically compatible excipients.

In particular, the purpose of the invention is a pharmaceutical formulation comprising, as its active principle, at least γ-aminobutyric acid or GABA that can be obtained from the aforesaid process the said γ- aminobutyric acid or GABA being characterised in that it consists of a GABA in the form of a tetragonal crystalline structure having the following spatial group: IA_\cd. More particularly, the said redeposited GABA is characterised in that it has the following crystallographic parameters: (i) a= 1196.3 pm,

(ii) c= 1528.2 pm, (iii) Z= 16 and

(iv) p=l .253 Mg.m^"3, as measured at a temperature comprised between 2O⁰C and 25⁰C, in combination with one or several pharmaceutically acceptable excipients.

In general, such a pharmaceutical formulation is useful for the prevention or treatment of pathologies for which the monoclinic form of GABA is active. Such a pharmaceutical formulation is also useful for the prevention or treatment of pathologies related to a gamma-decarboxylase deficit.

According to different aspects, such a pharmaceutical formulation is directed to the prevention or treatment of the following pathologies: a) for the GABA_A receptor: epilepsy; Huntingdon's chorea; dyskinesia, such as the said Parkinson's disease or hemiballismus (alterations in the balance between the dopaminergic, cholinergic and GABAergic striatal systems); anxiety and sleep disorders; b) for the GABA_B receptor: spastic states, such as those encountered particularly in cerebral palsies; c) indications of non-ionised GABA in the solid state as neuroprotection factors: in general, the treatment of any acute or chronic pathology related to neurological damage threatening the integrity of the central and/or peripheral nervous system, such as the following pathologies: epilepsy (e.g. post-seizure phase of epilepsy); deficiency of the cardiovascular and/or respiratory function that may give rise to ischemia/anoxia of the central nervous system (e.g. cardiorespiratory arrest, vascular insufficiency following infarct); degenerative chronic neurological diseases, particularly

Alzheimer's disease, Parkinson's disease, Huntingdon's chorea, multiple sclerosis, amyotrophic lateral sclerosis; cerebrovascular accidents (strokes) and transient ischemic attacks (TIA); central neurotoxicity induced during AIDS; any other cerebral neurotoxicity factor.

The object of the invention is also a pharmaceutical formulation comprising, as the active substance, a non-ionised dopamine in the solid state, in combination with one or several pharmaceutically acceptable excipients. It also relates to a pharmaceutical formulation comprising, as the active substance, a fusidic acid, or a salt of fusidic acid including the non- ionised sodium salt in the solid state, in combination with one or several pharmaceutically acceptable excipients. It also relates to a pharmaceutical formulation comprising, as the active substance, a fusidic acid, or a salt of fusidic acid, including the non- ionised sodium salt in the solid state, in combination with one or several pharmaceutically acceptable excipients.

The invention also relates to the use of a redeposited organic compound in the solid state for manufacturing a medication directed towards preventing or treating one or several disorders, pathologies or given diseases.

The invention also concerns the use of γ-aminobutyric acid or GABA characterised in that it possesses the spatial group /4icd, or the following crystallographic parameters:

(i) a= 1196.3 pm,

(ii) c= 1528.2 pm,

(iii) Z= 16 and

(iv) p=l .253 Mg.m ³, as measured at a temperature comprised between 2O⁰C and 25⁰C, for the manufacture of a pharmaceutical formulation for the prevention or treatment of a disease selected from among epilepsy, Huntingdon's chorea, dyskinesia, anxiety or sleep disorders.

The invention also concerns the use of γ-aminobutyric acid or GABA characterised in that it possesses the spatial group /4icd, or the following crystallographic parameters: (i) a= 1196.3 pm, (ii) c= 1528.2 pm, (iii) Z= 16 and (iv) p=l .253 +/- 0.010 Mg.m^"3, as measured at a temperature comprised between 2O⁰C and 25⁰C, for the manufacture of a pharmaceutical formulation for the prevention or treatment of spastic states.

In order to draw up a pharmaceutical formulation according to the invention, those skilled in the art could advantageously refer to the latest edition of the European Pharmacopoeia or the Pharmacopoeia of the United States of America (USP).

Those skilled in the art could particularly advantageously refer to the 4th edition "2002" of the European Pharmacopoeia or the 25-NF20 edition of the United States Pharmacopoeia (USP).

Advantageously, a pharmaceutical formulation as defined is appropriate for daily oral or parenteral administration of a quantity of a redeposited compound in the solid state comprised between 1 μg and

10 mg and preferably between 1 μg and 1 mg per kilo of the patient's weight.

Regarding more particularly the pharmaceutical compositions comprising a redeposited GABA, as defined herein, solid pharmaceutical compositions are preferred, wherein GABA is combined with a sodium source and a chlorine source, in a preferred GABA/Na/Cl molar ratio of 1/1/2, for which an optimal stability of the redeposited GABA, has been observed.

When the formulation according to the invention comprises at least one pharmaceutically acceptable excipient, this concerns particularly an appropriate excipient for topical administration of the formulation, an appropriate excipient for oral administration and/or an appropriate excipient for parenteral administration of the formulation.

In a preferred embodiment of a pharmaceutical formulation according to the invention, the redeposited organic compound is in powder form, if necessary in combination with one or several pharmaceutically acceptable excipients, placed in a sealed container in a partial vacuum, for example at a pressure comprised between 10^"2 et 10^"4 Pa, that is at a pressure at which the metastable form of the said redeposited organic compound can be stored for a long period of time.

Lastly, with particular reference to the biological activity study hereinafter, another object of the present invention is a non-ionised compound in the solid state as described above, for its use as a therapeutically active principle in a medication.

Another object of the invention is a method for preventing or treating a pathology in a patient, said method comprising a step during which the patient is administered a therapeutically effective quantity of a redeposited organic compound in the solid state or a pharmaceutical compound containing a redeposited compound in the solid state as described above, which can be prepared by the process according to the invention. The non-ionised compound in the solid state or the pharmaceutical formulation containing the non-ionised compound in the solid state can be administered by the oral route or parenteral route or be applied topically, locally on a patient's skin.

The redeposited GABA is preferably used in systemic administration, preferably intravenously.

The following examples are intended to illustrate the present invention and must in no circumstances be interpreted as limiting its scope.

EXAMPLES: EXAMPLES 1 to 9

A. MATERIAL AND METHODS OF EXAMPLES 1 to 9

A. I Preparation of redeposited GABA. The redeposited GABA was prepared by sublimation of a monoclinic commercial GABA powder marketed by SIGMA (Ref. : SIGMA ULTRA) in a partial vacuum at a pressure of 10^~3 Pa, using an Edwards evaporation device (auto 306).

Commercial GABA powder was placed in a molybdenum crucible which had been heated in a vacuum; then the vapour produced by sublimating the GABA was received on a rotating Pyrex® glass support, the support being maintained at ambient temperature 20 ± 5⁰C. A.2. X ray diffraction analysis

The redeposited GABA was studied by X ray diffraction analysis, using a diffractometer marketed by PHILIPS (reference PHILIPS 1050) and an X ray generator marketed by PHILIPS (reference PHILIPS 1729).

For measuring and analysing the X ray diffraction results, a computer was used loaded with the programs "Gonio" and "Rayon" (B. Fraisse : Thesis 1995 Universite des Sciences et Techniques du Languedoc, Montpelier). In order to carry out the X ray diffraction, a CuKαl (λ = 1,54051 A) anode was used marketed by Philips (France). The measurements were carried out at laboratory temperature.

A.3. Measuring mass density

The density measurements were carried out with a pycnometer marketed by Micrometrics (reference: AccuPyc 1330) in a helium atmosphere at a temperature of 24 J +/- 0.2⁰C.

The density data is expressed as the mean value of three series of ten measurements for the commercial GABA, and as the mean value of nine series often measurements for the redeposited GABA.

A.4 Measuring the infrared transmission spectrum (FTIR)

The infrared spectrum measurements were carried out with a spectrometer marketed by PERKTN ELMER (reference: Spectrum 2000 A.T.R. Diamond). The results concerning the infrared absorption bands are expressed in cm^"1, with a resolution of 4 cm^"1.

A.5 Measuring the differential calorie The thermal analysis was carried out on a device marketed by

Perkin Elmer (reference DSC 7) which had been calibrated for temperature and energy based on established melting points (i) for 5N indium at 156.634⁰C and at 231.9681⁰C (data standardised by the NIST ["National Institute of Standard and Technology"] and for 5N tin at 231.9681⁰C (Koch-light). For the melting enthalpy, the device was calibrated based on data relating to indium (28.44Jg^"1) and tin (59.22 Jg^"1). This is the data recommended by the American Society for Materials [ASM] for the melting point temperature of indium and tin, and the data recommended by the "Bulletin of Alloy Phase Diagram vol 7, 6 (1986) p 601 " for the melting points and enthalpy of indium and tin. A calibration was carried out for each heating regime.

Crucibles in aluminium alloy were used and fitted with lids containing openings for maintaining a constant pressure.

All the experiments were carried out in a dry gaseous nitrogen atmosphere, at a gas flow rate of 2 10^"2 L.min^"1. A.ό.Calorimetry tests

Heat capacity measurements (Cp) were carried out with the C80 calorimeter marketed by SETARAM (Caluire, France between 303 K and 363 K, according to the method described by B. Legendre et al.

Thermochemica Acta 400, (2003) 213-219 and J. of Thermal Analysis and

Calorimetry vol 76, (2004) 255-264).

A.7 Measuring ultraviolet spectroscopy The commercial GABA (SIGMA) and the redeposited GABA were dissolved in an aqueous solution of NaOH at a final concentration of 0.1N. Then the UV spectroscopy was carried out at a temperature of 2O⁰C, using a Jasco-V550 spectroscope, marketed by Bioserv (Thais, France).

A.8 Biochemical determinations

In order to determine the respective affinities of commercial GABA (SIGMA) and redeposited GABA, an enzymatic preparation of GABase marketed by ALDRICH/SIGMA (Fallavier, France) was used.

The two compounds were added to the GABase preparation in the presence or absence of 1 mM α-ketoglutarate in a ImL container also containing 8O mM of commercial TRIS buffer at pH 8.1, 0.4 mg of commercial GABase and 1 mM of NADP+.

The incubation temperature was 37⁰C and the absorbance values were followed at a wavelength of 340 nanometres. For each of the reference GABA and redeposited GABA compounds, the differences between the two plateaus of the curves, that is the differences obtained with the respective incubations in the presence or absence of α-ketoglutarate, made it possible to titrate the compound.

B. RESULTS OF EXAMPLES 1 to 9.

EXAMPLE 1 : Identity of chemical formulae of reference commercial GABA and redeposited GABA

It has been shown that the molecular formula of redeposited GABA was identical to the molecular formula of reference commercial GABA marketed by SIGMA/ALDRICH. The results of the NMR analysis in tables A and B indicate that the two compounds have the same chemical formula that effectively corresponds to that of GABA.

Proton and carbon NMR spectra

The NMR of proton and carbon 13 was carried out in deuterated water with a BRUCKER device at 200 MHz (AC-200).

The results obtained from the proton NMR and the carbon 13 NMR show no difference between the control GABA and the redeposited GABA.

The main characteristics of the proton NMR spectra and the carbon 13 NMR spectra, respectively for the control monoclinic GABA and the redeposited GABA are presented in Tables 1 and 2 below.

Table 1 : Proton NMR

Table 2: Carbon 13 NMR

Furthermore, as will be illustrated in example 9, the reference commercial GABA and redeposited GABA both constitute substrates of GABase.

EXAMPLE 2: Redeposited GABA consists of tetragonal GABA The X ray diffraction results presented in Figure 1 and table 3 confirm that reference commercial GABA consists of monoclinic GABA and redeposited GABA consists of tetragonal GABA.

In Figure 1, the X ray diffraction data is compared, and particularly the respective diffraction angles θ of the two compounds. In table 3, the interplane spacing values (d) for the two compounds are compared.

In the results presented in table 3, the intrareticular distances measured between the crystal network planes are expressed in 10^"10 metres

(A) and their intensity is expressed as a percentage of the highest intensity. The two crystal structures show that both forms of GABA have a zwitterionic conformation.

It is important to emphasise that the X ray diffraction data shows differences between the two solid forms of GABA and indicates that the redeposited GABA which is derived from commercial GABA in monoclinic form, consists of a tetragonal GABA.

Each of these forms of GABA has already been characterised in the literature (Dobson et Jerkin, 1996, Acta Cryst., vol. C52 :3075-3078 ; Tomita et al, 1973, Bull. Chem. Soc. Japan, vol. 46: 2199-2204).

To the applicant's knowledge, the existence of solid tetragonal GABA has previously been described only once, the tetragonal form having been obtained by a process that does not make it possible to produce quantities larger than that needed to carry out the crystal analysis (Dobson et Jerkin, 1996).

For monoclinic GABA, the known crystallography parameters are, respectively: a = 719.3 pm ; b = 1012.0 pm ; c = 826.0 pm ;

P = 111 1.05 ; Z = 4 ; σ = 1.226 Mg.m^"3

(Tomita et al. 1973).

For the tetragonal GABA, the crystallographic parameters were: a = 1196.3 pm ; c = 1528.2 pm ; Z = 16. σ = 1.253 Mg.m^"3 (calculated data). Commercial GABA in powder form is known to be GABA in monoclinic form. The X ray diffraction data of commercial GABA powder has been described previously (J. Visser, 1987, Technisch Physische Dienst, DeIf, Netherland ICDD Grant in Aid, (1987), Fiche ASTM 38-1739;Tomika, K., Higashi, H., Fujiwara, T. (1973) Bull. Chem. Soc. Jpn, 46, 2199-2204).

EXAMPLE 3: Density and infrared and NMR spectra of reference commercial GABA and redeposited GABA

Density

The densities of reference monoclinic GABA and tetragonal GABA were measured. For monoclinic GABA, the measured density was

1.2243 +/- 0.0005 Mg.m^"3, for a theoretical value of 1.226 Mg.m^"3. For tetragonal redeposited GABA, the measured density was 1.2437 +/- 0.006 Mg.m^"3 for a calculated theoretical value of 1.253 mg/m³.

/.R spectrum 1) The LR. spectrum of commercial monoclinic GABA comprises the following bands: 1660.21, 1638.70, 1574.89, 1505.56, 1448.52, 1425.77, 1398.14, 1384.85, 1338.37, 1306.88, 1281.64, 1170.33, 1124.02, 1060.43, 1005.82, 994.42, 887.00, 868.45, 787.84, 778.02, 645.79. The LR spectrum of redeposited tetragonal GABA comprises the following bands: 1646.53, 1568.70, 1554.20, 1531.74, 1473.55, 1447.26, 1436.87, 1427.65, 1395.84, 1381.97, 1360.00, 1338.72, 1304.94, 1282.55, 1266.59, 1240.39, 1162.95, 1123.59, 1063.05, 1027.88, 1006.79, 994.89, 948.52, 902.29, 866.66, 779.04, 753.84, 655.79. 2) The infrared spectra of the control GABA and the redeposited

GABA were traced with a BRUCKER Vector 22 infrared spectrometer. The spectra were produced by reflectance on the pure products.

The results obtained (Table 4) show that the spectra obtained for control GABA and the redeposited GABA are almost identical and that in both cases the GABA is in zwitterion form (no characteristic COOH or NH₂ bands, but the characteristic NH³⁺ and COO^" bands are present). The only differences obtained between the control GABA and the redeposited GABA are for the following bands:

- the band corresponding to the asymmetric valence vibration of the carboxylate group (v_as COO^") is at 1574 cm^"1 for the control GABA and at 1577 cml for the redeposited GABA.

- the bands at 1385 et 1398 cm^"1 corresponding to the symmetric valence vibration of the carboxylate group (v_s COO^"), show different intensity relations for the control GABA and the redeposited GABA. - two weak bands present for control GABA at 695 and 940 cm^"1 are non-existent for redeposited GABA. These bands perhaps correspond to deformation vibrations of the molecular skeleton.

The main characteristics of the respective infrared spectra of monoclinic GABA and redeposited GABA are summarised in Table 4 below. Table 4: main infrared bands for control GABA and rede o sited GABA

In the light of the above results, tetragonal redeposited GABA obtained by the invention process can thus also be distinguished from reference commercial GABA by its infrared spectrum.

EXAMPLE 4: Heat capacity of reference commercial GABA and redeposited tetragonal GABA.

The heat capacity (Cp) of monoclinic reference commercial GABA and redeposited tetragonal GABA obtained by the invention process was measured, between 303 K and 363 K, using the following equation (1):

C_p = A₀ + Ai. T (in which c_p is measured as a function of the temperature and is expressed in J.K^.mol^"1).

The respective values of A₀ and Ai of reference commercial monoclinic GABA and redeposited tetragonal GABA were measured.

The values obtained for reference commercial monoclinic GABA were: A₀ = 27.423 and Ai= 0.3936, which agrees with the data in the literature (Skoulika and Sabbah, 1983, Thermochemica Acta, vol.61 :203- 214.

The values obtained for redeposited tetragonal GABA were: A₀ = 72.834 and Ai= 0.2575. The above results indicate that redeposited tetragonal GABA obtained by the invention process can also be distinguished from reference commercial monoclinic GABA by its heat capacity characteristics.

EXAMPLE 5: U.V. spectrum of reference commercial GABA and redeposited GABA.

The results are given in Table 5. The results presented in Table 5 are expressed as the mean values (+/- the standard deviations) of ten successive measurements. The wavelength and absorbance values were determined by direct experimental measurement.

The extinction coefficient values were calculated from the experimental absorbance values determined by spectrophotometry and from the weight of GABA powder added to 0.1 N sodium hydroxide solutions, then transferred to the spectrophotometer tanks. It should be noted that for the contiguous measurements carried out on the solution prepared from tetragonal GABA, a variation of 18.5 nanometres was observed in the maximum absorbance peak wavelength at 217 nanometres. A concomitant variation was also noted of the absorbance value from 0.0455 to 0.062, respectively. The solid powder forms of commercial monoclinic GABA and tetragonal GABA obtained by the invention process were dissolved in an aqueous solution of 0.1 N NaOH.

The solutions containing the GABA were subjected to UV spectroscopy analysis either immediately, or 40 hours after dissolving the GABA. The results are given in Table 3.

The tests showed significant differences in the UV absorption spectra of commercial GABA and redeposited GABA immediately after dissolving in the 0.1 N aqueous NaOH solution, left-hand conformational isomer for the commercial GABA and trans for the redeposited GABA obtained by the invention process.

It can also be noted that the solution obtained from tetragonal GABA changed with time until thermodynamic equilibrium was obtained, that is until a solution was obtained that was identical to that obtained with monoclinic GABA. After 40 hours in solution, redeposited GABA can no longer be distinguished from commercial monoclinic GABA using its UV spectrum.

Without wanting to be tied to a particular theory, the applicant thinks that the trans isomer of redeposited GABA relaxes to the left-hand isomer.

EXAMPLE 6: Comparative calorimetric results of reference commercial monoclinic GABA and redeposited tetragonal GABA, in powder form in the solid state, or in solvated form in the liquid state

6.1 melting point and scanning differential calorimetrv measurement results.

The results are given in Figure 2.

Only an endothermic reaction was observed between 205⁰C and 245⁰C.

For each of the two solid forms of GABA used as a starting product, two closely placed peaks were detected, the first peak corresponding to the melting point of the GABA under consideration, and the second corresponding to its vaporisation point. It is important to note that for reference monoclinic GABA, the first peak is less intense than the second. On the contrary, for redeposited tetragonal GABA, the first peak is more intense than the second peak. The results in Figure 2 correspond to the mean value of five measurements.

For the reference monoclinic GABA, the start of the first peak was detected at 212.6 +/- 0.5⁰C. For the redeposited tetragonal GABA, the start of the first peak was observed at 216 +/- I⁰C.

For the reference monoclinic GABA, the enthalpy variation was 1 188 +/- 8 Jg^"1 (which corresponds to 122.5+/- 0.8 kJ mol^"1). For the redeposited tetragonal GABA, the enthalpy variation was 1 147 +/- 28 Jg^"1 (which corresponds to 1 18.28 kJ +/- 3 kJmol^"1).

The difference between these two values was mainly due to the difference between the melting enthalpies of the two solid forms of GABA tested. When the difference between the heat capacity value (c_p) of the liquid phase and the heat capacity (c_p) of the monoclinic phase between 212⁰C and 216⁰C is neglected, it can be estimated that the difference between these two values of melting enthalpy is of the order of magnitude of4.2 kJ mol^~1.

The melting temperature of the metastable phase is higher than that of the stable phase, which, although it is exceptional, is not impossible from a thermodynamic point of view.

Other scanning differential calorimetry experiments were carried out at different pressures, from 0.1 MPa to 350 Mpa, during which no phase transition was observed. Under these more drastic conditions, decomposition of the molecule could be noted.

6.2 Calorimetry study

The solubilisation of the solid forms of reference monoclinic GABA and redeposited tetragonal GABA was carried out in an aqueous saline solution of 0.9% NaCl or 9 g of NaCl pour 1000 g of solution. The solubilisation phenomenon of each of the GABAs in solid form is the result of two distinct processes that can overlap, respectively (i) the rupture of the intermolecular bonds, followed by the solvation of the crystal followed by (ii) the fusion of the individual molecules in the solvation medium. In other words, the intermolecular and intramolecular interactions of the GABA molecules within the crystal are eliminated and replaced by the interactions of the GABA with the solvent, that is, in the present case by the interactions of GABA with water and GABA with the Na⁺ or CI^" ions.

As far as the dissolution enthalpy of the saline solvent is concerned (0.9% by mass of NaCl or 9 g of NaCl for 1000 g of H₂O), the solvation of the solid form of each of the GABAs leads, in similar experimental conditions, to temperature variations of the distinct solvation medium.

The dissolution enthalpies in the aqueous saline solution were measured in a type C80 calorimeter (SETARAM, Caluire, France) at 36.3

±0.3°C.

With reference commercial monoclinic GABA, the following calorimetric values were obtained: Δ_soiH= -34.1 +/- 0.1 Jg^"1 (which corresponds to -3516.4 Jmol^"1). Using redeposited tetragonal GABA as the starting product, the following results were obtained: Δ_soiH = - 49.4 +/- 1 Jg^"1 (which corresponds to -5094.1 Jmol^"1).

The above results indicate that the enthalpy necessary for transforming the reference monoclinic GABA into redeposited tetragonal GABA at 37⁰C is approximately 1574 Jmol^"1.

6.3 Sublimation enthalpy

The sublimation enthalpy at temperature T is given by the following equation (2):

Δ_subH (T) = Δ_subH(298 K) + W (c_p ^g - c_p ^s) dT (2) wherein:

Δ_subH298 = MO kJmol^"1.

The values of c_p given in the literature (Skoulika and Sabbah, 1983, Thermochemica Acta, vol. 61 : 203 - 214) are (i) 153.8 JK^"1 mol^"1 between 298 K and 350 K and (ii) 158.1 JK^"1 mol^"1 between 350 K and 485 K, for the gaseous phase.

For the solid monoclinic phase, the values of c_p are (i) 133.6 JK^"1 mol^"1 and (ii) 182 JK^{" 1}HIoI^"1, for the same ranges of temperature. From these values, the sublimation enthalpy around the melting temperature was calculated, which was 137 UmOl^"1. The sublimation enthalpy around the triple point is equal to the sum of the melting and vaporisation enthalpies.

However, when the measured value for the heat capacity (c_p) of the solid phase which was extrapolated to the melting point value is used, the sublimation enthalpy value is equal to 128.5 kJ.mol^"1, which is a similar value to the value measured experimentally of 122.5 kJmol^"1.

EXAMPLE 7: Thermodynamic cycle characterising the formation process of redeposited tetragonal GABA.

The thermodynamic cycle for understanding how redeposited tetragonal GABA is formed is given schematically in Figure 3.

Figure 3 shows a schematic version of the hypothetical state diagram of GABA, each form of GABA corresponding to the temperature and pressure conditions illustrated in Figure 3. Figure 3 also illustrates, in the form of a cycle with arrows, the thermodynamic cycle for obtaining experimentally the solid form of redeposited tetragonal GABA from commercial monoclinic GABA in the solid state. The monoclinic GABA in the solid state first goes from atmospheric pressure of 1.013 Pa (a) to conditions of partial vacuum of 10^{" 3} Pa (b). The first step is carried out at constant laboratory temperature of approximately 2O⁰C.

In a second step that is carried out at a constant pressure of 10^{" 3} Pa, the monoclinic GABA in the solid state is heated to between 160 and 18O⁰C and goes from the solid form (b) to the gaseous state (c) by sublimation.

In the third step, which is also carried out at a constant pressure of 10^"3 Pa, and which in practice overlaps with the second step, the gaseous GABA (c) is instantaneously transformed into a powder in the solid state (d) at laboratory temperature. During this step, the GABA vapour produced during the second step is deposited on a Pyrex® glass support, which is in rotation and is maintained at laboratory temperature.

During the fourth step, the powder is retrieved (e). During this fourth step, which is carried out at laboratory temperature, the pressure goes from 10^"3 Pa to atmospheric pressure (e).

It can be noted that if the pressure alone varies, going from atmospheric pressure to a partial vacuum at 10^"3 Pa, without simultaneously varying the temperature, tetragonal GABA in solid form is not produced.

At laboratory temperature and atmospheric pressure, the stable phase of GABA consists in the monoclinic phase, whereas at low pressure, the stable form consists in tetragonal GABA.

The process leading to the metastable tetragonal phase of GABA can be divided into several steps.

The first step is carried out in the sublimation device when vacuum conditions are reached; the monoclinic phase of GABA in the solid state is brought to low pressure but the GABA is not transformed into the tetragonal solid phase for kinetic reasons. In a second step, this phase is vaporised by heating. In a third step, the GABA is deposited on a cold glass support, at laboratory temperature, the vapour solidifying into the tetragonal phase of GABA.

When the vacuum is broken, the solid phase tetragonal GABA can be retrieved.

The transformation kinetics of GABA from the tetragonal phase to the monoclinic phase is too slow to be carried out during the step of breaking the vacuum.

This thermodynamic cycle, which makes it possible to go from commercial monoclinic GABA in the solid state to tetragonal GABA in the solid state is illustrated in Figure 3.

EXAMPLE 8: GABA state diagram

As results from example 7 above, the GABA state diagram was established on the basis of the experimental protocol used to produce redeposited GABA and on the basis of the identification of redeposited GABA as consisting of tetragonal GABA in the solid state. Moreover, the melting points were determined experimentally. They are, at a pressure of 1.013.105 Pa, 216 +/- I⁰C for redeposited tetragonal GABA. This data also contributes to establishing the state diagram of

GABA which is illustrated in Figure 3.

There are two solid forms of GABA, respectively the monoclinic form and the tetragonal form. Each of these forms is favoured from a thermodynamic point of view for a combination of pressure and temperature conditions.

GABA in monoclinic form in the solid state is the thermodynamic form favoured at laboratory temperature and at atmospheric pressure. The tetragonal form of GABA is the form favoured from a thermodynamic point of view in conditions of lower pressure and higher temperature. The solid forms of monoclinic GABA and tetragonal GABA become liquid at a temperature higher than their respective melting points, that is at temperatures higher than 212.6⁰C and 216⁰C respectively.

After sublimating GABA in a vacuum of 10^"3 Pa (passing from a solid phase to a gaseous phase), the gas phase deposits on the cold surface (laboratory temperature) in the solid tetragonal form. The temperature drop is large and rapid, since the temperature of GABA at this moment goes instantaneously from 25O⁰C to 2O⁰C when the GABA deposits on the glass support maintained at laboratory temperature. Since, in the experimental protocol, the temperature drop is large and rapid, the tetragonal form in the solid state adopted by the GABA is maintained, due to the absence of a sufficiently long time for tetragonal GABA to convert back to the monoclinic form.

The subsequent retrieval of the powder at atmospheric pressure thus consists in retrieving tetragonal GABA in the solid state.

EXAMPLE 9: Commercial monoclinic GABA and redeposited tetragonal GABA, as a substrate of GABase.

The experimental results given in Figure 4 show that reference commercial monoclinic GABA and redeposited tetragonal GABA are both substrates of GABase, which is a combination of enzymes comprising

GABA transaminase (GABA-T, classified EC 2.6.1.19 in international nomenclature) and NADP+-dependent succinic semialdehyde dehydrogenase (SSDH, classified EC 1.2.1.16, in the international nomenclature) of Pseudomonas fluorescens. It should be remembered that GABase can be used to test the presence of GABA in a sample.

The results given in Figure 4 show that the production of NAPDH obtained with the preparations of the two forms of GABA in the micomolar / millimolar range are strictly dependent on the presence of α- ketoglutarate, which indicates without any doubt that it is the GABA, and not for example the succinic semialdehyde, which is the substrate that generates the NAPDH formed by the GABase system.

From these GABA concentration measurement results using the test involving the GABase, which are given in Figure 4, it was noted that the NAPDH production plateau, which shows that all the GABA had reacted with the GABase, was reached with the redeposited tetragonal

GABA, later than the plateau reached with the monoclinic commercial GABA.

This investigation was followed by kinetic studies with the two forms of GABA in the GABase system. The results are given in Figure 5. In Figure 5A, there are the results of the enzyme reaction speed measurements as a function of GABA concentrations. The upper curve represents the enzyme reaction speeds obtained with commercial monoclinic GABA. The lower curve represents the results of the enzyme reaction speed measurements with redeposited tetragonal GABA.

In Figure 5B, the results of Figure 5A are represented in compliance with the Lineweaver-Burk plot.

Thus, in Figure 5, the starting speeds of the GABase enzyme reaction were measured as were the starting speeds of the NADP+ reduction, and were represented as a function of the final concentrations of GABA in the test medium.

In Figure 5, the NADP+ reduction speeds measured at a wavelength of 340 nanometres (v) were expressed as a function of the final concentrations of GABA (S). In Figure 5B, the inverse of the value of the starting speeds (1/v) was represented as a function of the inverse of the concentrations of substrate (1/S) according to a Lineweaver-Burk plot.

The values calculated for the apparent Km of the GABase system for commercial monoclinic GABA in solution were 0.12 mM. The values calculated for the apparent Km of the GABase system for redeposited tetragonal GABA in solution were 0.48 mM.

The differences in the affinity constants (Km) of the two forms of GABA can reasonably be attributed to the GABA-T step of the GABase, since the succinic semialdehyde is a product common to both forms of GABA.

The kinetic studies carried out with the two forms of GABA in the GABase system emphasise the major differences in the speeds with which each of the forms of GABA can be catabolised. It may be noted that, compared to the commercial solvated form of monoclinic GABA, the redeposited tetragonal GABA in solution is relatively resistant to catalysis by the GABase system.

In conclusion, the marked differences in the two forms of GABA to be catabolised by the GABase system can be noted, which is a characteristic that could lead to better stability, in particular a longer half- life of redeposited tetragonal GABA, when this GABA is exposed to GABA degradation enzymes, compared to the typical commercial monoclinic form.

In particular, this property of resistance of redeposited tetragonal

GABA in solution to GABA degradation enzymes could lead to an intracerebral half-life of redeposited tetragonal GABA longer than that of typical monoclinic GABA.

These properties of redeposited tetragonal GABA could, at least in part, explain the results of the in vivo redeposited tetragonal GABA which are presented in example 10, which make it possible to seriously envisage the use of redeposited tetragonal GABA for treating neurological disorders affecting the central nervous system, such as epilepsy, mood disorders and cerebral attacks, panic disorders, Alzheimer's disease and pathogenesis of development, including Rett's syndrome, Angelman's disease and autism.

EXAMPLE 10: Biological activity in vivo compared with natural zwitterion GABA in the solid state and non-ionised solid GABA according to the invention.

A. MATERIAL AND METHOD For this experiment, OFl female mice (IFFA CREDO) deprived of magnesium for 42 days (deprived medium at 50 ppm of Mg²⁺) were used. The untreated control animals received a UAR diet at 1500 ppm of

Mg²⁺.

A.2. Selection of audiosensitive mice:

At the end of the deprivation period, the animals were subjected to audiogenic stimuli; only the animals presenting convulsions were used for the following experiments.

A.3. Parameters of audiogenic stimulation

As described in the publication by BAC et al. (1998, J. Neuroscience, vol. 18(11) :4363-4373).

A.4. Molecule used

Sigma GABA (Ref. A 5835) was used. A part of the GABA was subjected to the redeposition process and designated as redeposited GABA.

A.5 Treatment

Intraperitoneal (IP) injection of GABA or redeposited GABA was carried out 45 minutes before the audiogenic test.

A.6. Observed parameters The number of mice was determined presenting complete audiogenic convulsions (wild running, clonic convulsions and tonic convulsions); according to the technique described by Bac et al. (1998, J. Neuroscience, vol. 18(1 1): 4363-4373).

B. RESULTS

B.I . Determination of the number of audiogenic seizures

The results obtained are represented in the table 6 below.

Table 6

(n = number of animals used for each experiment).

The figures indicate the number of animals that presented convulsive audiogenic seizures.

The results show that redeposited GABA alone is capable of totally abolishing the convulsive audiogenic seizures.

B.2. Determination of the percentage of animals having convulsive seizures

The results are given in Figure 6.

As was shown by Bac et al. (1998), audiogenic seizures dependent on magnesium deficiency (MDDAS) show a good response to anticonvulsive and neuroprotection substances. This in vivo model was used to test the comparative effects of commercial monoclinic GABA and redeposited tetragonal GABA obtained by the invention process.

As illustrated in Figure 6, commercial monoclinic GABA, when administered by intraperitoneal route up to doses of 100 mg/kg, provides animals with no protection against MDDAS.

On the contrary, when redeposited tetragonal GABA is used by the intraperitoneal route, a protective anticonvulsive activity is observed in the MDDAS model, which effectively confirms the physical differences observed between commercial monoclinic GABA and the redeposited tetragonal GABA that also lead to distinct biological properties, both in vitro as illustrated in example 9 and in vivo as illustrated in the present example.

EXAMPLE 11 : Comparative biological activity of natural dopamine in the solid state and dopamine as end product of the invention process A. Introduction

Cocaine, an inhibitor of dopamine recapture, leads, in mice, depending on the dose used, to clonic-tonic convulsions and mortality.(M. Hummel et al. J. Cell. Physiol. 2002, 17-27). The D2 receptors antagonist haloperidol of natural dopamine in the solid state and redeposited dopamine (in solution) reduces the number of convulsions induced by cocaine, the lethal effects of the cocaine are abolished by a Dl antagonist (SCH 23390). (Kazuaki Shimosato et al. Pharmacol. Biochem. Behav. 1995, 51, 781-788). All these toxic effects are the result of an increase, by inhibiting the recapture, of the synaptic levels of dopamine. The aim of this example is to:

1 - show that redeposited dopamine, according to our process, on crossing the haematoencephalic barrier, increases the toxic effects of cocaine.

2 - show the effect of natural GABA and redeposited GABA on the toxicity of cocaine.

A. MATERIAL AND METHOD For this experiment, OFl female mice (20 + 3g) (IFFA CREDO) were used.

A.1.Molecules used

Cocaine (Sigma), Dopamine (Sigma, ref. H 85 02). A part of the Sigma dopamine was subjected to the redeposition process and designated as redeposited DA, the natural dopamine being designated as DA.

GABA Sigma (Ref. A-5835). A part of the GABA was subjected to the redeposition process and designated as redeposited GABA. A.2. Treatment

The dopamine (DA or redeposited DA) was administered by the intraperitoneal route (IP) 30 minutes before the cocaine. Two doses of DA or redeposited DA were used (100 mg/kg, 50 mg/kg). The GABA (natural GABA or redeposited GABA) was administered intraperitoneally (IP) 30 minutes before the cocaine.

The cocaine was injected (IP) at doses of 40, 60 or 100 mg/kg.

Each batch of animals received only a single injection of cocaine or dopamine (natural or redeposited).

A.3. Observed parameters

Number of animals presenting clonic and tonic convulsions. Number of mice dead in 24 hours. For the 100 mg/kg dose of cocaine, the latency to convulsions (in seconds) was also determined.

B RESULTS

B.I . Effect of dopamine redeposited according to our process, on the toxic effects of cocaine

At a non-convulsive and non-lethal dose of cocaine (40 mg/kg) redeposited dopamine alone (100 or 50 mg/kg) induces convulsions in a large number of animals (Table 7).

At a convulsive but non-lethal dose of cocaine (60 mg/kg) redeposited dopamine alone induces mortality in almost all the animals (Table 7). At a convulsive and lethal dose of cocaine (100 mg/kg) redeposited dopamine reduces the latency to convulsions and to mortality (Table 8).

Table 8

Latency (in seconds) to convulsions and to mortality induced by 100 mg/kg of cocaine

Conclusion:

The toxicity of the cocaine is increased solely by the administration of redeposited dopamine.

2 - Effect of natural GABA and redeposited GABA on the toxicity of cocaine

Table 9

(n = number of animals used for each experiment).

The figures indicate the number of animals that presented convulsions or the number of animals dead in 24 hours.

CONCLUSION Only the redeposited GABA according to the invention abolished the mortality induced by a large dose of cocaine and caused a significant decrease in the number of convulsions. One potential application would be treating overdoses of cocaine in humans (Maciej Gasior et al. J. Pharmacol. Exp. Ther. 1999, 290, 1 148-1156). EXAMPLE 12:

Comparative biological activity of natural valproic acid in the solid state and valproic acid in the liquid state according to the invention

A - MATERIAL AND METHOD

For this experiment, OFl female mice (IFFA CREDO) deprived of magnesium for 42 days (deprived medium at 50 ppm of Mg⁺⁺) were used; the untreated control animals received a UAR diet at 1500 ppm en

Mg⁺⁺.

A-I . Selection of audiosensitive mice:

At the end of the deprivation period, the animals were subjected to audiogenic stimuli; only the animals presenting convulsions were used for the following experiments.

A-2 Parameters of audiogenic stimulation:

See attached publication. (P. Bac et al. J. Neurosc. 1998, 18, 4363-4373).

A-3 Molecule used.

Sigma valproic acid was used. A part of the valproic acid was subjected to the redeposition process and designated as redeposited valproic acid..

A-4 Treatment.

Intraperitoneal (IP) injection of valproic acid or redeposited valproic acid was carried out 45 minutes before the audiogenic test.

A-5 Observed parameters.

The number of mice presenting complete audiogenic seizures was determined (P. Bac et al. J. Neurosc. 1998, 18, 4363-4373) (wild running, clonic convulsions and tonic convulsions). B. RESULTS

The results are given in Table 10 below.

Table 10

(n = number of animals used for each experiment).

The figures indicate the number of animals that presented audiogenic convulsions. Conclusion:

Redeposited valproic acid seems to have a greater efficacy for protecting the animals in the MDDAS test.

EXAMPLE 13: Comparative biological activity in vitro of natural sodium fusidate and sodium fusidate according to the invention. 13.1 General characteristics of sodium fusidate Sodium fusidate is a steroid structure antibiotic of the fusidane family.

From a chemical point of view, it is the sodium salt of acetoxy 16β-dihydroxy 3α,l lα-diene 17 (20), 24 (25)- fusidic - 21 acid. It does not possess the groups that seem to be responsible for the anti- inflammatory activity of the glucocorticoids. It is a white powder. Its molecular weight is 538.7 g.mol^"1. The molecule is liposoluble and hydrosoluble.

It is a major antistaphylococcic that exercises its bacteriostatic effect by inhibiting protein synthesis after penetrating the bacteria and fixing on the elongation complex EF-G coupled with the GTP-GDP- GTPase energy system.

Sodium fusidate acts selectively on staphylococci, particularly S. aureus, whether or not they are penicillinase producers (CMI of 0.06 to 0.12 mg/1), including methicillin-resistant strains (CMI90 < 2 mg/ml). On the other hand, with the exception of Legionella and

Haemophilus, the gram-negative bacilli are resistant to sodium fusidate (MIC usually over 100 mg/1).

Sodium fusidate has no hormonal activity.

13.2 Solubility of sodium fusidate 13. 2-1 -Fusidate:

- instantaneous solubility at 10 mg/ml in saline phosphate buffer pH 7.2 (PBS) giving a final pH of 7.5 and in water for injection;

- instantaneous solubility at 50. mg/ml in purified water containing 1 mg/ml of acid citric monohydrate, 19.6 mg/ml of disodium phosphate dihydrate and 0.5 mg/ml of disodium EDTA;

- instant precipitation in distilled water at 0.2 mg/ml of citric acid monohydrate giving a final pH of 7.3.

13.2-2 Redeposited fusidate:

- insoluble at 10 mg/ml in PBS, in water for injection;

- moderately soluble at 2.5 mg/ml in the solvent containing citric acid, disodium phosphate dihydrate and disodium edetate, needing the use of a Potter grinder and giving a final pH of 7.0; - soluble in purified water at 0.2 mg/ml of citric acid.

13.3- In vitro antibacterial activity 13.3-1- Material and method

Minimum inhibiting concentrations (MIC) of the antibiotic were determined for 6 clinically isolated strains of bacteria and one ATCC strain, by the official standardised method of microdilution in liquid medium in microplates, using dilutions carried out 2 by 2.

The commercial fusidic acid standard for creating the MICs was included in the determinations.

The results of the MICs were expressed in mg/1 as per the official standards. 13.3-2- Results

The MICs of sodium fusidate and redeposited fusidate (R fusidate) were determined on 3 strains of clinically isolated Staphylococcus, one strain of S. aureus ATCC, and on 3 strains of clinically isolated gram- negative bacilli: Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.

Table 11

The MIC results show that the bacteriostatic activity of the fusidate is not modified after redeposition.

13.4- Inhibiting activity of the activation of mouse macrophages in vitro

The effect of sodium fusidate on the vitality of macrophages after 24 hours of culture in presence of LPS (bacterial lipopolysaccharide extract of E. coli) as a stimulating agent was studied. The choice of this macrophage activator is dictated by the clinical use of sodium fusidate as antibacterial agent. So the choice was made to reproduce in vitro a bacterial stimulus for the macrophages, so as best to mimic the clinical situation.

13.4-1 Material and methods Peritoneal macrophages collected by three wash-outs (2 ml of complete RPMI with 10% foetal calf serum added) of the peritoneal cavity of mice euthanised 2 days after intraperitoneal injection of 2 ml of nutrient broth; the aspiration liquids were grouped, centrifuged at 300 g for 10 minutes at +4⁰C; the peritoneal macrophages contained in the liquids were counted and the vitality assessed by staining with trypan blue.

The macrophages (lxlO6/ml) were put into culture in sterile plates of 24 wells (in a final volume of 2 ml) in presence of LPS (10 μg/ml) and sodium fusidate at different concentrations (0 to 50 μg/ml).

After 24 hours at +37⁰C in a 5% CO₂ atmosphere, the culture supernatants were harvested and centrifuged, then frozen at -8O⁰C by aliquots for subsequent dosing of inflammation mediators; the packed cells are stored.

The cells were placed in the wells by pipetting up and down in complete medium, washed then isolated by centrifugation. Half the retrieved cells were used immediately for phenotypic analysis by flow cytometry after marking, the other half (about 1x10⁶ cells) was used for the quantitative determination of mRNA of inflammation mediators: the cells were stored frozen at -8O⁰C in 400 μl of RNAzol, a solution which lyses the cells and protects the mRNA from enzyme degradation. Before putting into culture and after the 24 hours in culture, the phenotypic analysis of the cells was carried out to assess the apoptopic and necrotic cells by double staining with annexin-V and propidium iodide. Fluorescein isothiocyanate-conjugated annexin-V fixes on the phosphatidylserine residues which are exposed at the surface of the apoptopic or necrotic cells. The propidium iodide penetrates inside the necrotic cells only and fixes on the DNA: the double marked cells are necrotic cells, whereas the cells stained only with annexin-V are apoptopic cells. The results are expressed in percentage of positive cells.

The membrane expression of the ICAM-I (CD54) adhesion molecule and class II antigens is increased after activation of the macrophages. It was visualised by staining respectively with a fluorescein isothiocyanate-conjugated monoclonal anti-CD54 mouse antibody and with a biotin-conjugated anti-I-A^b mouse antibody (class II molecule from the mouse strain C57BL/6), then revealing with fluorescein isothiocyanate conjugated strep tavidine. The results are expressed in percentage of positive cells and in mean fluorescent intensity.

The fluorescence data was acquired in a window of 10,000 cells selected by their size and granulometry, in a FACScan cytometer (Beckton Dickson) with an argon laser beam λ= 488 nm. The data analysis was carried out using the Lysis II software.

The production of TNFα by the activated cells in culture is measured in the supernatants after 24 hours in culture by ELISA

(Quantikine® M MURINE, mouse TNFα, R&D Systems). The detection sensibility is under 5.1 pg/ml. The linearity range is from 23 to 1500 pg/ml

13. 4-3 Results

A. Viability of macrophages after 24 hours of culture

This is evaluated in percentage of live cells by counting the macrophages in presence of trypan blue: the blue stained macrophages are dead cells. The results obtained are given in Table 12.

Table 12

Contrary to fusidate, the redeposited fusidate had a dose- dependent toxic effect on the macrophages in culture, the LD50 was observed between 6 and 12 μg/ml.

B- Apoptosis and necrosis of macrophages after 24 hours of culture

These two parameters are evaluated by double staining with annexin V and propidium iodide. The results are expressed in percentage of apoptopic and necrotic cells.

The results are given in Table 13 below.

Table 13

The LPS stimulation of macrophages in culture induces, after 24 hours, mean apoptosis of 50% and mean necrosis of 40% of the macrophages: fusidate induces no further apoptosis or necrosis neither does it protect the macrophages from the activation effects.

On the contrary, the repedo sited fusidate had a dose-dependent toxic effect on the macrophages stimulated in culture, with progressive increase of necrosed cells, the LD50 being observed at a concentration close to 6 μg/ml.

C-Membrane expression of ICAM-I and I-A^b molecules The expression of these molecules, induced by macrophage stimulation, is expressed both by the percentage of positive cells (%), which makes it possible to evaluate the induction of the expression of these molecules on cells that do not express them at rest, and by mean fluorescence intensity (MFI). This last value makes it possible to evaluate the intensity of the expression of these molecules, thus their number, at the surface of a cell population.

The results are given in Table 14.

Table 14

The expression of the ICAM-I and I-A^b molecules was increased on the macrophages after 24 hours of activation in culture by the LPS, both in terms of percentage of cells expressing them and in terms of membrane density. Fusidate had no effect on the expression of these molecules.

On the other hand, redeposited fusidate strongly inhibited the membrane expression of ICAM-I and class II molecules.

D- TNFα production by macrophages after 24 hours of LPS stimulation in culture.

The results are expressed in pg/ml in Table 15 below.

Table 15

Fusidate had no effect on the TNFα production by the activated macrophages.

On the other hand, redeposited fusidate strongly inhibited TNFα production by macrophages after 24 hours of LPS stimulation in culture.

13.5 General conclusion concerning the results obtained in example 13

These results show that sodium fusidate acquired new properties after redeposition, while keeping all of its initial antibacterial activity. Since it now combines an anti-inflammatory activity proven in vitro on the key cell of inflammation, with a major anti-staphylococcic activity, this antibiotic has acquired a considerable clinical potential.

Table 3

Table 5

Table 7: Effect of natural dopamine and redeposited dopamine on toxicity induced by cocaine

Cn

(n = number of animals used for each experiment). The figures indicate the number of animals that presented convulsions or the number of animals dead in 24 hours.

Claims

1. A process for preparing, starting from the initial organic compound crystallised in the solid state and possessing one or several biological properties, an organic compound crystallised in the solid state and possessing one or several modified biological properties compared with the starting organic compound, said process comprising the following steps: a) supplying the original organic compound in the solid state; b) sublimating to the vapour state said starting organic compound and condensing the compound sublimated to the vapour state onto the surface of a substrate maintained at ambient temperature; c) retrieving the organic compound in the solid state obtained at the end of step b), from the said substrate.

2. A process according to claim 1 , characterised in that the sublimation of the said starting organic compound in the vapour state is carried out, at step b) by heating at reduced pressure.

3. A process according to claim 2, characterised in that step b) sublimation is carried out at a temperature ranging from 18⁰C to 25⁰C and at a reduced pressure ranging from 0.5 10^~3 to 2 10^~5 Pa.

4. A process according to any one of claims 1 to 3, the starting organic compound consists of an organic compound with a molecular weight of under 1000 g.mol^"1, preferably under 600 g.mol^"1.

5. A process according to any one of claims 1 to 4, characterised in that the starting organic compound comprises at least one group selected from the groups hydroxyl, carboxyl and amino.

6. A process according to any one of claims 1 to 5, characterised in that the starting organic compound in the solid state comprises γ- aminobutyric acid or GABA in monoclinic form.

7. A process according to any one of claims 1 to 5, characterised in that the starting organic compound consists of dopamine.

8. A process according to any one of claims 1 to 5, characterised in that the starting organic compound consists of valproic acid.

9. A process according to any one of claims 1 to 5, characterised in that the starting organic compound consists of fusidic acid.

10. A pharmaceutical formulation comprising, as its active principle, at least γ-aminobutyric acid or GABA that can be obtained from the process according to any one of claims 1 to 6, the said γ-aminobutyric acid or GABA being characterised in that it consists of a GABA in the form of a tetragonal crystalline structure having the following spatial group: IA _\ cd.

1 1. A pharmaceutical formulation according to claim 10, characterised in that the GABA in the tetragonal crystalline form has the following crystallographic parameters: (i) a= 1 196.3 pm,

(ii) c= 1528.2 pm, (iii) Z=I 6 , and (iv) p =1.253 Mg.m^"3; in combination with one or several pharmaceutically acceptable excipients.

12. A pharmaceutical formulation comprising, as its active principle, at least one non-ionised organic compound in the solid state that can be obtained from the process according to any one of claims 1 to 6 and selected from dopamine, valproic acid and fusidic acid, in combination with one or several pharmaceutically acceptable excipients.

13. The use of γ-aminobutyric acid or GABA characterised in that it consists of GABA in the form of a tetragonal crystalline structure having the spatial group IA_\cd, for the manufacture of a pharmaceutical composition for preventing or treating a disease selected from the group consisting of epilepsy, Huntington's disease, dyskinesia, anxiety and sleep disorders.

14. The use according to claim 13, characterised in that the GABA in the tetragonal crystalline form has the following crystallographic parameters:

(i) a= 1196.3 pm, (ii) c= 1528.2 pm, (Ui) Z= 16 and

(iv) p=1.253 Mg.m^~3.

15. The use of γ-aminobutyric acid or GABA characterised in that it consists of GABA in the tetragonal crystalline form having the spatial group IA_\cd., for the manufacture of a pharmaceutical composition for preventing or treating spastic states.

16. The use according to claim 15, characterised in that the GABA in the tetragonal crystalline form has the following crystallographic parameters:

(i) a = 1 196.3 pm, (ii) c = 1528.2 pm, (iii) Z = 16 and

(iv) p = 1.253 Mg.m -3