Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-03-30
2002-06-18
Prats, Francisco (Department: 1651)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S025000, C514S042000, C514S043000, C514S054000, C514S057000, C514S060000, C435S072000, C435S073000, C435S252100, C435S253400, C536S006000, C424S244100
Reexamination Certificate
active
06407069
ABSTRACT:
TECHNICAL FIELD
This invention relates to improved methods of purification for a polysaccharide.
BACKGROUND
CM101, a GBS toxin, is a pathogenic molecule isolated from group B &bgr;-hemolytic Streptococcus (GBS) bacteria. Newborn infants may become infected with GBS, a condition known as GBS pneumonia or “early-onset disease,” and suffer from sepsis, granulocytopenia, and respiratory distress, i.e. pulmonary hypertension and proteinaceous pulmonary edema (Hellerqvist, C. G. et al.,
Studies on group
B &bgr;-
hemolytic streptococcus I. Isolation and partial characterization of an extra
-
cellular toxin., Pediatr. Res.,
15:892-898 (1981)).
Despite the harmful effects to neonates exposed to GBS, CM101 is not known to cause toxicity in older humans. In fact, research into this toxin has revealed a significant therapeutic application. See U.S. Pat. No. 5,010,062 and Hellerqvist, C. G. et al.,
Early Results of a Phase I Trial of CM
101
in Cancer Patients., Proceedings of the American Association of Cancer Research Annual Meeting
(1995), wherein CM101 is utilized to inhibit vascularization of tumors. Obtaining purified CM101 is critical, therefore, for both research and therapeutic purposes.
CM101 is a complex polysaccharide toxin having a molecular weight of approximately 300,000 Daltons and comprising N-acetyl-galactosamine, N-acetyl-glucosamine, glucose, galactose, and mannose residues. Nmr (nuclear magnetic resonance) results suggest that alditol residues may also be present. Carboxylic acid functional groups, probably galacturonic acid, are also believed to be an integral part of the molecule. Repeating active epitopes most likely play an important role in the pathophysiological response to CM101 by crosslinking receptors on target endothelium (Hellerqvist, C. G. et al.
Early Results of a Phase I Trial of CM
101
in Cancer Patients., Proceedings of the American Association of Cancer Research Annual Meeting
(1995); DeVore, R. F., et al.,
A Phase I Study of the Antineovascularization Drug CM
100,
J. Clin. Can. Res.,
3:365-372 (1997)).
U.S. Pat. No. 5,010,062 provides a method of purification of a GBS toxin. The method taught is labor-intensive. however, requiring numerous steps with continual levels of loss of biological activity.
Purification of CM100 as presently known in the art provides an end material which is only 40% pure as measured by chemical analyses and biological assays. The other 60% comprises plant and yeast polysaccharides and endogenous bacterial polysaccharides. The plant and yeast contaminants originate for the most part in the additives to the commercial culture media used for optimal growth of the GBS bacteria. The endogenous contaminants include GBS polysaccharides including group and type specific antigens (Paoletti, L. C. et al.,
Neonatal mouse protection against infection with multiple group B streptococcal
(GBS)
serotypes by maternal immunization with a tetravalent GBS polysaccharide-tetanus toxoid conjugate vaccine, Infect. Immun.
62(8):3236-43 (1994); Michon, F.,
Multiantennary group
-
specific polysaccharide of Group B Streptococcus, Biochem.,
27:5341-51 (1988)). CM100 of this 40% purity level represents the current clinical grade. There is a need, therefore, for a purification method of CM100 which results in an end product with increased overall purity, preferably with the removal of extraneous plant and yeast polysaccharides and GBS antigenic polysaccharides.
Additionally, the purification scheme known in the art includes environmentally unsound steps, such as the use of a large volume of phenol in a phenol:water extraction. Phenol is a well-known caustic material.
Therefore, objects of the present invention are to provide a purification method resulting in (i) a material of high purity, (ii) using a minimal number of steps, (iii) minimizing the use of caustic or toxic materials such as phenol, and (iv) increasing the yield of material.
SUMMARY OF THE INVENTION
The above objects have been achieved with the invention described herein. Particularly, a purification scheme including a hydrophobic interaction chromatography (HIC) resin for purification of CM101 from GBS bacterial culture media results in a product of greater than 95% purity.
One aspect of this invention is a process for purifying a polysaccharide toxin from GBS bacteria, the process including the use of an HIC resin. The present invention also includes a substantially pure polysaccharide toxin from GBS bacteria produced by the method disclosed herein, and a pharmaceutical composition comprising a substantially pure toxin and a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to treat a patient having a medical condition. For example, a tumor patient may be treated with the composition of the present invention.
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El Rassi Z, “Recent Progress in Reversed-Phase and Hydrophobic Interaction Chromatography of Carbohydrate Species,” J. of Chromatography A, vol. 720, No. 1, pp., 93-118, 1996.
Hellerqvist, C.G. et al., Anti-tumor effects on GBS toxin: a polysaccharide exotoxin from group B &bgr;-hemolytic streptococcus, “J. Canc. Res. Clin. Oncol.,” vol. 120, pp. 63-70, 1993.
Hellerqvist, C.G. et al., “Early Results of a Phase I Trial of CM101 in Cancer Patients,” Proceedings of the American Association of Cancer Research Annual Meeting, 1995.
Hellerqvist, C.G. et al., “Molecular Basis for Group B &bgr;-hemolytic Streptococcal Disease,” Proc. Natl. Acad. Sci. USA, vol. 84, pp. 51-55, 1987.
Hellerqvist, C.G. et al., “Preliminary results of a phase I trial of CM101 in cancer patients,” J. of Cellular Biochem., vol. 19B, pp. 26, 1995.
Hellerqvist, C.G. et al., “Studies on group B &bgr;-hemolytic streptococcus I. Isolation and Partial Characterization of an Extra-cellular Toxin,” Pediatr. Res., vol. 15, pp. 892-898, 1991.
Jennings, H.J. et al., “Structural Determination and Serology of the Native Polysaccharide Antigen of Type-III Group &bgr;-Streptococcus,” Canadian J. of Biochem., vol. 58, No. 2, pp. 112-120, 1980.
Michon, F., “Multiantennary Group-specific Polysaccharide of Group B streptococcus,” Biochem., vol. 27: 5341-5351, 1988.
Paoletti, L.C. et al., “Neonatal Mouse Protection Against Infection With Multiple Group B streptococcus (GBS) serotypes by Matrnal Immunization with a Tetravalent GBS Polysaccharide-tetanus Toxoid Conjugate Vaccine,” Infect. Immun., vol. 62, No. 8, pp. 3236-3243, 1994.
Schoel, B. et al., “Hydrophobic Interaction Chromatography for the Purification of Cytolytic Bacterial Toxins,” J. of Chromatography A, vol. 667, pp. 131-139, 1994.
Cooley & Godward LLP
Prats Francisco
Vanderbilt University
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