Patent Application
20160272677
The present invention relates to novel crystalline forms of S-acetyl glutathione (SAG) which are useful in the preparation of pharmaceutical or nutraceutical compositions.
Glutathione (GSH) is a compound which, in its reduced form, constitutes an excellent antioxidant and therefore a defence against the damage caused by free radicals to higher organisms. S-acetyl-glutathione (SAG) (see
GSH or γ-L-glutamyl-L-cysteinylglycine is a tripeptide consisting of glutamic acid, cysteine and glycine, characterised by an atypical peptide bond, namely the bond that binds the nitrogen of cysteine to the carboxyl in y glutamic acid. It is the main thiol compound with the lowest molecular weight present in both animal and plant cells (about 95% of the total). Its function is to maintain in the reduced state the -SH groups of many enzymes and proteins whose oxidation (with the formation of S-S intra- and intermolecular disulphide bridges) leads, in most cases, to inactivation or loss of the biological function of the enzyme or protein.
GSH is considered to be one of the most important intracellular antioxidants produced naturally by the human body. However, chronic oxidative stress reduces the cell levels of GSH, and it is often appropriate to replenish its levels with the aid of diet supplements.
It is commonly believed that the GSH intake obtained from the diet or with the use of diet supplements is easily used by the issues, but in reality it is not absorbed “as is”, but hydrolysed into its three constituent amino acids by a gamma-glutamyl transpeptidase present in the intestine. After being absorbed and introduced into the bloodstream, said amino acids are distributed to the various tissues wherein they implement the pool of amino acids with which the body cells synthesise endogenous GSH. It is therefore necessary to use a high oral dose in order to guarantee significant absorption. When Witschi et al. evaluated the increase in the blood levels of glutathione, cysteine and glutamate after oral administration of GSH to seven healthy volunteers, no significant increases were observed at doses of up to 3 g per dose (Witschi A et al., J. Clin. Pharmacol. 43 (6), 667-1992).
Sublingual administration, which guarantees better bioavailability, can be used as an alternative to oral administration of GSH.
Finally, in the pharmaceutical field, prophylaxis based on GSH is used in some cases by parenteral, intramuscular or slow intravenous administration, for example as prophylaxis for neuropathy resulting from chemotherapy with cisplatin or analogues.
The use of SAG as a precursor is a good alternative to replenish the reduced GSH levels in the body. In fact, acetylation of the sulphur atom prevents the decomposition of GSH and facilitates its absorption through the intestinal wall, thus enabling the molecule to pass extensively into the cells.
The SAG thus assimilated by the tissues is hydrolysed by cytoplasmic thioesterase and, by hydrolysis of the acetyl group, produces reduced GSH which is available for all the biological functions wherein it is required.
The addition of SAG to cultures of fibroblasts originating from individuals suffering from a genetic glutathione synthetase deficiency has proved able to replenish the intracellular level of GSH effectively (Okun J G et al., J. Inherit. Metab. Dis. 27(6), 783-2004). SAG is also more stable in the plasma and has proved more effective than GSH in replenishing the cell levels of GSH impoverished by viral infections (Vogel J U et al., Med. Microbiol. Immunol. 194, 55-2005) (Fraternale A et al., Antiviral Res. 77, 120-2008). Finally, SAG exhibits an interesting non-GSH-dependent activity that induces apoptosis in some human tumour cell lines in vitro. (Locigno R et al., Int. J. Oncol. 20, 69-2002).
Identification and characterisation of the polymorphic forms, and of the experimental conditions for obtaining them, are very important parameters for a compound designed for nutraceutical and/or pharmaceutical use.
The synthesis of SAG has already been claimed in a Japanese patent (see Chemical Abstract 97-7222755s) and in WO92/00320. However, the authors only disclose a general method for obtaining it, without investigating the existence of polymorphic forms in any way.
As stated above, a number of difficulties are involved in the absorption of GSH, which are partly solved by the use of the SAG derivative. However, the absorption of said compound may be adversely affected by the existence of polymorphic forms thereof having different physicochemical characteristics that influence its dissolution rate, solubility and therefore bioavailability, not to mention the different behaviour of the powders during the preparation of the various formulations.
No experimental condition or preliminary indication for crystallisation and drying that suggests the existence of polymorphic forms of SAG has ever been disclosed in any patent or patent application.
We have now surprisingly found that SAG exists not only in the amorphous form, but also in at least two polymorphic forms characterised by different physicochemical properties, which are useful as ingredients of pharmaceutical or nutraceutical compositions.
The experimental evidence for the existence of said two polymorphic forms, described in the present invention as form A and form B, is supplied by the analyses described below, conducted both in solution and in the solid state.
The samples of the two crystalline forms subjected to 1H-NMR analysis (
Conversely, the analyses performed directly on the substance in the solid state clearly demonstrate the presence of polymorphic forms.
XRD: the analyses conducted with the X-ray diffractometer indicated significant differences in the crystallographic properties of the two samples (
Tables 1 and 2 show the best-resolved diffraction peaks, relating to polymorphic forms A and B respectively. The values shown in bold type correspond to the peaks characterising the two forms.
5.2
10.3
15.4
18.6
19.7
35.3
36.3
4.2
12.7
13.0
17.3
17.7
30.2
IR: here again, the spectra recorded by FTIR on the substances in the solid state (
Although the 1H-NMR spectra excluded the presence of solvents, the samples were also subjected to thermal analyses, namely TGA and DSC.
TGA: thermogravimetric analyses, conducted on two samples, categorically exclude the presence of “solvates” and indicate a marked weight loss, due to decomposition, at a temperature much higher than 150° C. (
DSC: The thermograms confirm decomposition at around 200° C. in both polymorphs, and although the endothermic peak, at 208.2° C. for form A and 191.4° C. for form B, presents a fairly clear start which could misleadingly indicate a fusion, it relates to decomposition with weight loss (
However, the two thermograms differ in terms of the presence, in polymorph B, of a weak endothermic event at about 135° C. Said event, which is perfectly reversible, can be seen in the cooling thermogram of the compound, as an analogous exothermic event at a slightly lower temperature (
On the basis of these data, it can therefore be concluded that SAG exists in at least two different polymorphic forms, A and B, characterised by different physicochemical properties.
One object of the present invention is therefore a crystalline form of SAG called form A, characterised by an X-ray powder diffraction spectrum, obtained with α1 (λ=1.54060 Å) and α2 (λ=1.54439 Å) copper radiation, as shown in
In the XRD diffractogram, an additional group of characterising diffraction peaks, expressed in degrees 2-theta [°], is represented by those at 20.4, 21.1, 25.1, 25.7, 27.0, 27.6, 27.9, 32.7±0.2.
Crystalline form A is characterised by an IR spectrum, obtained with a potassium bromide matrix, as shown in
Crystalline form A is also characterised by a DSC pattern, obtained with a heating rate of 10.00° C./min, having an endothermic peak between 190° C. and 210° C., connected to the decomposition of the compound, followed by other disorderly endothermic events, as shown in
Another object of the present invention is a crystalline form of SAG called form B, characterised by an X-ray powder diffraction spectrum, obtained with α1 (λ=1.54060 Å) and α2 (λ=1.54439 Å) copper radiation, as shown in
Crystalline form B is characterised by an IR spectrum, obtained with a potassium bromide matrix, as shown in
Crystalline form B is also characterised by a DSC pattern, obtained with a heating rate of 10.00° C./min, having an endothermic decomposition peak between 180° C. and 200° C., connected to the decomposition of the compound, followed by other disorderly endothermic events, and a characteristic endothermic peak at about 135° C., as shown in
A further object of the present invention is a method for the production of crystalline forms A and B of SAG with high yields and chemical purity.
Crystalline forms A and B are obtainable by crystallising SAG with mixtures of solvents such as water-acetone, water-ethanol and water-methanol, preferably water-acetone.
The most surprising finding, which in particular is not easily deducible even by the skilled person, is that all the mixtures of said solvents are able to provide both polymorph A and polymorph B, and that the discriminating factor is the conditions wherein crystallisation is triggered.
In fact, the addition of the precipitation solvent (non-solvent) before crystallisation is triggered by water gives rise to polymorphic form B, whereas if crystallisation is triggered by water alone and the non-solvent is only added to increase the yields (complete the precipitation), polymorphic form A is obtained. This behaviour is confirmed by the precipitation of both polymorphs A and B if the triggering of the crystallisation from water is allowed and the solvent (non-solvent) is added before precipitation of the product is complete.
Crystalline form A of SAG can be prepared by a process comprising the following steps:
a) dissolution of SAG in water at a temperature ranging between 75° C. and 80° C.;
b) immediate cooling of the solution obtained in step a) to a temperature of below 55° C., preferably to a temperature ranging between 45° C. and 55° C., followed by further cooling until incipient crystallisation;
c) cooling to 20-25° C. of the mass obtained in step b) in the presence of minimal stirring (60-120 rpm), followed by continued stirring of the mass at 20-25° C. for between 2 and 12 hours;
d) slow addition to the suspension obtained in c) of a solvent selected from the group containing acetone, ethanol and methanol, preferably acetone, followed by cooling of the resulting suspension to a temperature ranging between 3° C. and 7° C.;
e) isolation of the solid that separates in step d), to give crystalline form A of SAG.
Crystalline form B of SAG can be prepared by a process comprising the following steps:
a) dissolution of SAG in water at a temperature ranging between 75° C. and 80° C.;
b) immediate cooling of the solution obtained in step a) to a temperature of 55° C., followed by addition of a solvent selected from the group containing acetone, ethanol and methanol, preferably acetone;
c) spontaneous cooling to 20-25° C. of the mass obtained in step b) in the presence of minimal stirring (60-120 rpm), followed by continued stirring of the mass at 20-25° C. for between 2 and 12 hours;
d) cooling of the suspension obtained in step c) to a temperature ranging between 3° C. and 7° C.;
e) isolation of the solid that separates in step d), to give crystalline form B of SAG.
Conversely, the amorphous form can be obtained by spray-drying of an aqueous solution of the product.
The two polymorphic forms A and B and the amorphous form present different physicochemical properties, in particular as regards the quality of the product, its stability, its dissolution rate in water, and the density and flowability of the powders. Quality, assay value and stability of the various forms—Crystalline forms A and B differ due to the presence of different quantities of oxidised GSH (GSSG), because the crystallisation of polymorph A gives rise to an increase in GSSG (about 1% more). This GSSG does not only derive from oxidation of the residual GSH present in the reaction environment, but also of that deriving from hydrolysis of SAG during crystallisation, albeit in minimal quantities. The percentage of GSSG is much higher in the amorphous form due to the drying conditions, which increase hydrolysis and the corresponding oxidation. This does not affect the quality of the product, because GSSG, like SAG, is able to replenish GSH after absorption.
When samples of polymorphs A and B were subjected to heat and mechanical stresses, the possibility of conversion of one polymorph to the other under the conditions used was not found.
The stability of the various solid forms was tested as described in the European Pharmacopoeia (EP), by conducting accelerated stability tests at 50° C. for 6 months. The results are set out in Table 3 as internal standardisation (% areas of ingredients) and as SAG assay value.
As will be seen from the data in Table 3, the amorphous form is much less stable than the crystalline forms, and of the latter, polymorphic form B is characterised by a higher purity and assay value.
Dissolution rate - Of the two crystalline forms, form B has the most rapid dissolution rate, and is therefore the most suitable for oral formulations, whose dissolution rate influences the absorption rate. Only the amorphous form dissolves more rapidly, but the quality and stability of the product are unsuitable for its use.
Powder density—As regards this aspect, study of the two crystalline forms demonstrates that polymorphic form B has a higher density (0.4 g/mL) than form A (0.2-0.25 g/mL) This parameter influences the flowability and compressibility of the powder, and therefore its use for the preparation of solid formulations, especially tablets. The powders of polymorphic form A therefore present better flowability.
Crystalline forms A and B of SAG can be formulated in pharmaceutical or nutraceutical compositions suitable for oral or parenteral administration, using conventional techniques and excipients.
A further object of the present invention is therefore pharmaceutical or nutraceutical compositions containing crystalline forms A and B of SAG.
A further object is the use of crystalline forms A and B of SAG for the preparation of medicaments or diet supplements.
A further object is the use of crystalline forms A and B of SAG for the preparation of vials containing powdered SAG for injectable parenteral administration.
The following examples further illustrate the invention.
The XRD spectra were obtained with a RIGAKU-MINIFLEX diffractometer. The radiations used were α1 and α2 (λ=1.54060 Å and λ=1.54439 Å, respectively) copper radiation.
The FTIR spectra were obtained with a Perkin-Elmer FTIR Spectrum-one instrument. The samples were analysed as KBr tablets without vacuum, with a 1:100 dilution.
The TGA patterns were obtained with a Universal V2.6D TA instrument. The temperature range explored was 0° C.→300° C., with a scanning rate of 10° C./min.
The DSC thermograms were obtained with a Perkin Elmer DSC6 instrument. The temperature range explored was 30° C.→350° C., with a scanning rate of 10° C./min. In the case of
The 1H-NMR spectra were obtained with a Varian Gemini 200 instrument operating at 200 MHz, using D2O as solvent.
Preparation of SAG in Crystalline form A 5 g of crude SAG is placed under stirring and heated to 75° C. in 40 mL of demineralised water. The reaction mass is heated to 75° C.-80° C. After dissolution, the solution is immediately cooled to a temperature of under 55° C., preferably between 45 and 55° C. Cooling continues until crystallisation begins. Stirring is minimised and the solution is cooled to 20-25° C., at which temperature it is left under stirring for 2-12 h until precipitation is complete. Subsequently, again with minimal stirring, 40 mL of acetone is added in about 30-50 min. The addition is slow to prevent the formation of even a few crystals of polymorph B. The resulting suspension is then brought to 5° C.±2° C. and maintained under slow stirring (60-120 rpm) for about 1 h. At the end of that time the reaction mass is filtered to obtain a white solid, which is washed with anhydrous acetone (2×10 mL) 8.4 g of wet solid is thus obtained, which is left to dry at 50° C., 5 mbar of residual vacuum for 14-18 h. 4.3 g (86%) of white crystalline solid corresponding to crystalline form A is obtained after drying.
The analytical profile of the product thus obtained is:
Assay value: 98.6% (as is)
Impurities: Total: 1.0%; Single known impurities: GSH (0.1%), GSSG (2.2%);
Water 1.4%
Residual acetone: <500 ppm
Residual acetic acid: 0.4%
Apparent density: 0.15-0.25 g/mL
The product thus obtained presents the 1H-NMR spectra shown in
Preparation of SAG in Crystalline Form B
35 mL of demineralised water is heated to 75° C. When said temperature is reached, 5 g of crude SAG is added in a single addition with a hopper, and the reaction mass is returned to the temperature of 75° C., without exceeding the maximum temperature of 80° C. When the product has dissolved, which is checked by taking a sample under stirring, the solution is immediately cooled to 55° C., and 40 mL of acetone is added at the same temperature in about 10 min. The temperature is left to fall spontaneously to 20-25° C. under minimal stirring (60-120 rpm). The mixture is left under stirring at the same temperature for 2-12 h. It is then cooled to 5° C. and left under stirring at the same temperature for about 1 h.
The reaction mass is filtered, and washed with anhydrous acetone (2×10 mL) After drying for 14-18 h at 50° C. and 5 mbar of residual vacuum, 3.8 g of white crystalline solid corresponding to crystalline form B is obtained (yield 76%).
The analytical profile of the product thus obtained is:
Assay value: 99.3% (as is)
Impurities: Total: 0.8%; Single known impurities: GSH (0.2%), GSSG (1.1%)
Water 1.2%
Residual acetone: <0.1%
Apparent density: 0.25-0.40 g/mL
The product thus obtained presents the 1H-NMR spectra shown in
Preparation of SAG in Crystalline Forms A and B
35 mL of demineralised water is heated to 75° C., and 5 g of crude SAG is then added in a single addition with a hopper. The reaction mass is heated to 75° C., without exceeding the temperature limit of 80° C. When the product has dissolved, which is checked by taking a sample under stirring, the solution is cooled to a temperature ranging between 35° C. and 45° C. 40 mL of acetone is added to the solution at said temperature in about 10 min, under minimal stirring (60-120 rpm). The temperature is left to fall spontaneously to 20-25° C. At the end of the addition, the solution will be cloudy. The solution is maintained under stirring at the same temperature for 2-12 h, after which it is cooled to 5° C. and left under stirring for about 1 h. The reaction mass is filtered, and washed with anhydrous acetone (2×10 mL) After drying for 14-18 h at 50° C. and 5 mbar of residual vacuum, 4.0 g of white crystalline solid is obtained, corresponding to isomorphic crystalline forms A and B.
Apparent density: 0.35 g/mL.
| Number | Date | Country | Kind |
|---|---|---|---|
| MI2013A001856 | Nov 2013 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/073957 | 11/6/2014 | WO | 00 |
