Production of toxin binding biopolymers, use thereof

Details Production of toxin binding biopolymers, use thereof Production of toxin binding biopolymers, use thereof

1995-11-01

1998-09-15

Prats, Francisco C.

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

514 54, 514 59, 514 60, 435 97, 435101, 435102, 435103, 536102, 536103, 536112, 536126, 424 944, 424 9461, A61K 31715, A61K 3843, C12P 1918, C08B 3100

Patent

active

058078444

DESCRIPTION:

BRIEF SUMMARY
This invention relates to a method for production of toxin-binding, non-toxic, modified carbohydrate biopolymers.
The invention also relates to the use thereof for the treatment of septic shock, infections and toxic states, and to the use of them as carriers for transportation of biologically or therapeutically important materials (enzymes or drugs) into phagocytes or cells bearing Fc and complement receptors.
Adsorption of various toxic agents by materials of large surface as kaolin, aluminium or/and magnesium silicates (GUT 34, 51-55, 1993) can result in a detoxification effect. The disadvantage of these adsorbents is their intolerability for human organism.
Non-toxic adsorbents metabolised in the cells or in the organism--termed in the present description as toxin binding non-toxic biopolymers (TBNTBP)--can solve this contradiction.
We have found that non-toxic, structurally modified biopolymers can be prepared by synthetic way and these biopolymers are similar to the native polymers which are metabolized in the organism by physiological pathways.
According to this invention the non-toxic biopolymers may be produced from polysaccharides containing alpha-1,4 or 1,6 linkages. According to the inventive process the polysaccharides containing alpha-1,4 or 1,6 linkages are reacted with D-glucose-1-phosphate in the presence of glycogen phosphorilase enzyme, in buffer solution at pH 6,8-7,4 at the temperature of 20.degree.-30.degree. C. and the obtained biopolymers are separated by well-known methods. Polysaccharides (containing 1,4 or 1,6 linkages ) advantageously glycogen, amylopectin, dextrin or the mixtures thereof can serve as acceptor molecules, "primers" for the chain-elongation process.
Besides glycogen, amylopectin, and dextrin other polymers of homo- and heteroglycans can also be used as "primer" polysaccharides. In general, all types of glycan molecules containing glucose units with alpha-1,4 or 1,6 bond types can serve as templates for the reactions.
The inventive process for the production of biopolymers is an enzymatic elongation of the respective chains in the soluble polysaccharides (glycogen and other glycans with alpha-1,4 or 1,6 linkages or the mixture of them ) by the enzyme glycogen phosphorylase `a` or `b` (EC 2.4.1.1) and the substrate D-glucose-1-phosphate resulting in a insoluble, "coarse disperse", colloidal particles with average size (diameter) not larger than 1 micrometer.
Both phosphorilase `a` and `b` can be used as catalysts for the side-chain also required when phosphorilase `b` is used).
The biopolymers can be produced in the presence of phosphorilase with specific activity of 50-55 IU/mg in pH 6.8-7.4 buffer solution (for example Tris-HCl or sodium beta-glycerophosphate completed with EDTA, beta-mercaptoethanol or NaF etc.) at 25.degree.-30.degree. C. during 20-30 min incubation time.
After precipitation of polymer with trichloro acetic acid (TCA) and resuspension of pellet in buffer, the precipitation of final biopolymer particles is taking place on the influence of cold ethanol treatment.
Advantageously we react 1 part of weight of polysaccharide containing 1,4 or 1,6 linkages with 0,5-1,5 part of weights of D-glucose-1-phosphate in the presence of 50-200 IU of phosphorylase enzyme.
If we use a mixture of glycogen with other polysaccharides as starting material, the rate of weight of the components is between 0,2:1-2:1.
According to this invention we have found that structurally modified biopolymers with alpha-1,4 or 1,6 linkages can be used ill septic shock, infections, acute phase reactions, and toxic states it is common that a great number of pathologic substances and mediator agents as cytokines, enzymes, lipid mediators, biogen amines, acute phase proteins, toxins are simultaneously present in the collapsed circulation, representing the state of multiple organ dysfunction syndrome. In the therapy of such cases the use of one or two antidotes can not be enough. The toxin binding biopolymers, like the structurally modified derivatives of glycogen, however, can bind simul

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