Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Patent
1992-07-08
1994-05-24
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
435206, C12N 936, H61K 3748, H61K 3754
Patent
active
053148169
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to a series of techniques used to prepare and purify a lysozyme dimer which will be particularly useful in the treatment of viral and bacterial infections.
BACKGROUND OF THE INVENTION
The ever growing number of bacterial strains and viral diseases which are resistant to antibiotics have made it necessary to introduce new kinds of drugs in order to treat humans and animals. Among the many present treatments and medicines used, it has been known to administer enzymes in monomeric form in order to benefit patients afflicted with various diseases. Enzymes are catalytically active proteins which perform almost all major life processes in organisms. Thus, many enzymes, either individually or in certain combinations, have been isolated for their physiochemical, physiological, or biological effects:
Among the various enzymes for which certain therapeutic effects have been documented, it is presently known that lysozyme, which has been known since 1922, can be used in various physiological and biological treatments. It has been observed that lysozyme has various therapeutic properties, such as antiviral, antibacterial, anti-inflammatory and antihistaminic effects. The antibacterial effect of lysozyme appears to be based on the hydrolysis of the beta-1-4-glycoside bond between n-acetylomuraminic acid and n-acetyloglucosamine, both contained in the bacterial wall.
Unfortunately, the tremendous potential with regard to beneficial effects possible through use of lysozyme has not been achieved primarily due to the observed cytotoxic effect of the monomeric form of this enzyme. For example, in tests with cultured fibroblasts, there has been an observed cytotoxic effect from doses of lysozyme in monomeric form even when administered at very small quantities. It thus became necessary to develop a way to maximize the potential beneficial effects which could be obtained from lysozyme by finding an effective way of controlling the cytotoxic effects associated with the monomer.
It has recently been discovered that an antiviral or antibacterial composition which does not exhibit cytotoxic effects can be constructed from lysozyme if one prepares a composition based on the dimeric form of the enzyme. Use of lysozyme in the dimeric form results in a composition useful in the treatment of a number of infectious diseases yet which does not exhibit the highly cytotoxic effects normally associated with the lysozyme monomer. The use of lysozyme dimer in various therapeutic treatments is disclosed in a co-pending application, PCT Application US88/01785.
Although ways of manufacturing the dimeric form of lysozyme from its monomeric form are known, it is now imperative that one be able to produce large amounts of the dimeric form in an inexpensive and efficient manner. It is thus highly desirable to develop a system for producing and testing great amounts of purified lysozyme dimer which does not contain the monomeric or multimeric form of the enzyme, or other contaminants.
SUMMARY OF THE INVENTION
In accordance with the present invention, an efficient method of preparing a purified lysozyme dimer product is provided which comprises the steps of:
a) preparing a lysozyme solution by adding monomeric lysozyme to a buffer solution adjusted to a pH of at least about 9;
b) adding a suberimidate coupling reagent such as dimethyl suberimidate in order to dimerize the lysozyme monomers in the solution while the solution is kept at a pH at or above about 9;
c) lowering the pH to about 7 in order to stop the dimerization reaction at a given point;
d) purifying the dimerized lysozyme solution by carrying out a first elution step in which the solution is eluted through an ion exchange column, and fractions which are substantially comprised of the dimeric form of lysozyme are collected;
e) filtering the collected fractions from the first elution step by carrying out a second elution step in which the collected fractions from the first elution step are eluted through an ion exchange column; and
f) col
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Eur. J. Diochem., vol. 124, 1982, Sorrentino: "Dimerization of Deoxyribonuclease I, Lysozyme and Papain", see p. 184, line 17-line 22; p. 184, line 47-line 57.
Chemical Abstracts, vol. 84 No. 15, Apr. 12, 1976 (Columbus, Ohio US), Wang et al.: "Preparation of Cross-Linked Dimers of Pancreatic Ribonuclease", see p. 241, Abstract 10156x, & Biochemistry 1976, 15(3), 660-5.
Chemical Abstracts, vol. III, No. 13, Sep. 25, 1989, (Columbus, Ohio), Roth, Mary R. et al.: "Analysis of Dimeric Species Derived from the Reaction of Phosphatidylethanolamine with Dimethylsuberimidate", see p. 360, abstract 111947u, & Chem. Phys. Lipids 1989, 51(1), 39-46.
Chemical Abstracts, vol. 97, No. 3, Jul. 9, 1982, (Columbus, Ohio), Hashimoto Shuichi et al.: "Dysozyme Dimer Formation On Lysozyme Oxidation with BR2", see abstract 19873M, & Int. J. Radiat. Biol. Rela. Phys., Chem. 1982, 41(3), 303-14.
Klein Peter
Uerrmann Peter
Lilling Herbert J.
Nika Health Products Ltd.
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