Process for preparing a charcoal-GM1 complex

Details Process for preparing a charcoal-GM1 complex Process for preparing a charcoal-GM1 complex

2002-06-05

2003-08-05

Lilling, Herbert J. (Department: 1651)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C514S025000, C514S053000, C514S054000, C536S004100, C536S123000, C536S124000

Reexamination Certificate

active

06602991

ABSTRACT:

TECHNICAL FIELD
This invention refers to a process for the preparation of a charcoal-GM1 complex and to an analytical method designed to determine underivatized GM1 in aqueous solutions by high performance liquid chromatography (HPLC).
This complex may be utilized as adjutant in the treatment for cholera, in the sense that it contributes to the neutralization of the correspondent toxin [STOLL et al. Lancet, p.888-891, 1980]. Moreover, in vitro tests demonstrated its potential as antidote for cases of ferrous ions intoxication.
In order to assess the effectiveness of the process for the preparation of the activated charcoal-GM1 complex, a analytical method has been developed in order to determine the GM1 by HPLC, using a new solvent system for the analysis of the ganglioside in the underivatized form.
This isocratic system has made possible the execution of the analysis with some advantages to the methodology as described in the literature.
The process for the attainment of the charcoal-GM1 complex described herein was developed seeking the stability of the product obtained in the conditions of pH as those of gastrointestinal tract, therefore helping in its safety.
BACKGROUND ART
The bacterial caused enteric infections, that result in diarrhea, dysentery and enteric fevers, accounts for serious health problems [BLACK., R. E., BROWN, K. H., BECKER, S., ALIM, A.R.M.A., HUQ, I. Am. J. Epidemiol., v. 115, p.315-324, 1982].
Among the most important pathologies of this type, we have cholera, pathology that results from the action of an enterotoxin developed from the
Vibrio cholerae
01, a gram-negative bacteria. The production of this toxin takes place as the microorganism successfully couples with the mucous of the proximal portion of the small intestine. [LEVINE, R., KAPER, J. B., BLACK, R. E., CLEMENTS, M. L., Microbiol. Rev., v.47, n.4, p.510-550, 1983].
This toxin presents sub unity A separated from the ring plane formed by 5 minor sub units that are identical (Sub units B). Its molecular weight is of approximately 84,000, and each sub unity B has molecular weight equivalent of 11,000 and the sub unity A is divided into A1 (PM=24,000) and A2 (PM=5,000) [LEVINE, R., KAPER, J. B., BLACK, R. E., CLEMENTS, M. L. Microbiol. Rev., v.47, n.4, p.510-550, 1983].
The B pentamer is connected with GM1 ganglioside in the membrane of the intestinal epithelium cells [SUREWICZ, W. K., LEDDY, J. J., MANTSCH, H. H. Biochemistry, v.29, p.8106-8111, 1990]. The sub unity A is inserted into the cytosol, thereby liberating the A1 fragment that will activate the latent adenylate-cyclase. The result is a rapid increase in the production of the cyclical-AMP [LEVINE, R., KAPER, J. B., BLACK, R. E., CLMENTS, M. L. Microbiol. Rev., v.47, n.4, p.510-550, 1983]. The consequence of these and other factors is an excessive accumulation of salts and water in the intestinal lumen and the death of the cell [FIELD, M., RAO, M. C., CHANG, E. B., N. Engl. J. Med., v.321, p.800-806, 1989].
The ganglioside-GM1 is the specific receptor for the cholera toxin [WU, G., LEDDEN, Anal R.. Biochem., v.173, p.368-375, 1988]. Also known as monosialoganglioside, the GM1 belongs to the class of glicosphingolipids, and is differentiated (together with other gangliosides) from other components of this class by the presence of sialic acid [HADJICONSTANTIONOU, M., NEFF, N. H. J. Neurochem., v.70, n.4, p.1335-1342, 1998].
Its structure, as represented below, shows hydrophobic and hydrophilic regions. The first one consists of ceramide, constituted of sphingosine and stearic acid and the hydrophilic portion is represented by the sialoligosacaridic chain. These features may provide the formation of micelles, and it is believed that the sialic segment may be involved with the reactivity of the ganglioside with the toxin.
In view of its specificity by the cholera toxin, the GM1 could be used for the treatment of the toxinfection by neutralisation of the toxin in the intestinal lumen. This was originally tested with the development of the charcoal-GM1 adsorption complex by STOLL et al. [STOLL et al. Lancet, p.888-891, 1980]. Despite this, it is important to point out that the authors cite the process for obtaining this complex without, however, describing it, neither the protocol for study of stability under which the complex was submitted. There is, therefore, no description of a process for obtaining this complex in the literature, and the above work concentrates on the analysis of the therapeutic benefits made available by this complex to or for the conventional treatment of cholera.
Currently, cholera treatment covers the disinfection by antibiotictherapy, the utilization of vaccines, besides different measures for the relief of the symptoms caused by that infection. In this connection, the use of this complex accounts for an important complement for the therapy that is applied.
Aiming at the safety of the utilization of this compound, it is essential that the process used for its development results in the stability of the same under pH values that are similar to the ones found in the gastrointestinal tract. This is due, particularly to the fact that the incorporation of the GM1 in the membrane of the intestinal cells increases the biological response to the cholera toxin by an increase in the number of receptors [HOLMGREN, J., LONNROTH, J., SVENNERHOLM, L. Proc. Nat. Acad. Sci. USA 72, p.2520-2524, 1975].
For the execution of a process for the preparation of the charcoal-GM1 complex, one should obviously be certain of the quantities and degree of purity of the basic components of that complex; the charcoal and the GM1, separately.
This invention, therefore, also comprises a new method for the determination of the GM1 in aqueous solutions.
The previous technique has used HPLC, by utilizing gradient systems for elution, which at times, would generate limitations concerning the resources made available by the equipment.
Table 1 hereinbelow summarizes the methodologies described in the literature.
TABLE 1
Parameters for the chromatographic analysis of non derived
gangliosides. Comparative table of analytical methodology (HPLC) as the
described in the literature.
Column
(compr. × D.f. ×
Solvent System
Averaged time of
D. part.)
(isocratic/gradient)
&lgr; for detection
analyses
Reference
Amine column
Acetonitrile-buffer
215 nm
80 min.
GAZZOTI et al.
3
(LiChrosorb-NH2)
Phosphate mM, pH
(25 cm × 4 mm × 7 &mgr;m)
5.6
(gradient)
Silicon (aquasil SS-
n-hexano-isopropanol-
208 nm
25 min.
ANDO et al.
1
542N)
potassium chloride
(20 cm × 6 mm × 5 &mgr;m)
50 mM
(gradient)
Silicon (Aquasil SS)
Acetonitrile-
208 nm
30 min.
ANDO et al.
2
(20 cm × 6 mm × 5 &mgr;m)
isopropanol-potassium
chloride 50 mM
(gradient)
Amine column
Acetonitrile-dissodic
215 nm
1 h 20 min.
PREVITI et al.
4
(LiChrosorb-NH2)
phosphate buffered
(25 cm × 4 mm × 5 &mgr;m)
with phosphoric acid
pH 5.6
(gradient)
1
ANDO, S., WAKI, H., KON, K. High-performance liquid chromatography of underivatized gangliosides. J. Chromatogr., Amsterdam, v. 408, p. 285-290, 1987.
2
ANDO, S., WAKI, H., KON, K. New solvent system for high-performance thin-layer chromatography and high-performance liquid chromatography of gangliosides. J. Chromatogr., Amsterdam, v. 405, p. 125-134, 1987.
3
GAZZOTI, G., SONNINO, S., GHIDONI, R. Normal-phase high-performance liquid chromatography separation of non-derivatized ganglioside mixtures. J. Chromatogr., Amsterdam, v. 348, p. 371-378, 1985
4
PREVITI, M., DOTTA, M., PONTIERI, G. M., DIMARIO, U., LENTI, L., Determination of gangliosides by high-performance liquid chromatography with photodiode-array detection. J Chomatogr., Amsterdam, v. 605, p. 221-225, 1992.
It can be verified that the techniques indicated in the table above show times of analysis that vary from 20 minutes to 1 hour and 20 minutes. The proposed method has retention times somewhere around 8 minutes.
In addition, as

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