United States Patent:  6,011,008

Assignee:  Yissum Research Developement Company Of The Hebrew University Of (Jerusalem, IL)

Filed:  June 4, 1998

A method for producing a water-soluble polysaccharide conjugate of an oxidation-sensitive substance is described. The method comprises the following steps: (a) activating the polysaccharide to a dialdehyde by periodate oxidation; (b) purifying the dialdehyde from interfering anions and by-products; and (c) coupling the substance to the purified dialdehyde by Schiff base formation to form the conjugate. Optionally, the conjugate of step (c) is reduced to an amine conjugate by a reducing substance. The product conjugate may then be further purified from various reaction byproducts. The disclosed method results in the substance substantially retaining its biological activity. Also described are imine and amine polysaccaride conjugates of various drugs and polypeptides.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for the preparation of water-soluble oxidation sensitive substances which are also suitable for parenteral administration.

It is a further object of the present invention to provide a pharmaceutical composition comprising a water-soluble conjugate of an oxidation sensitive substance which is suitable for parenteral administration as well as for topical and oral application.

The present invention provides a solution to the problem of preparing water soluble conjugates of oxidation sensitive substances which, until now, have become oxidized during the conjugation process, thus losing a significant portion of their activity. The invention provides water soluble conjugates whose biological activity is comparable to the activity of the free substance, with the added advantage of water solubility.

According to one aspect of the present invention, there is provided a method for producing a water-soluble polysaccharide conjugate of an unoxidized, oxidation sensitive substance comprising:

(a) activating the polysaccharide to a dialdehyde by periodate oxidation;

(b) purifying the dialdehyde from interfering anions and by-products; and

(c) coupling the substance to the purified dialdehyde by Schiff base formation to form the conjugate.

According to a second aspect of the present invention, the conjugate product of step (c) above is reduced to an amine conjugate by a reducing substance.

The conjugate obtained after step (c) or after reduction may be purified from undesired byproducts and unreacted reactants to yield a purified conjugate. Purification is particularly required when the conjugate is intended for therapeutic use.

According to another aspect of the present invention, there is provided a water-soluble conjugate of a polysaccharide and an unoxidized, oxidation-sensitive substance. The substance may be conjugated to the polysaccharide via either an imine or amine bond.

According to yet another aspect of the present invention, there is provided a pharmaceutical composition for use in the treatment of various physiological disorders comprising the conjugate of the invention as the active ingredient. Examples of such disorders include cancer, microbial infection and inflammation.

In a preferred embodiment of the present invention, the substance is selected from the group consisting of low molecular weight drugs and amine drug derivatives, and low molecular weight polypeptides and polypeptide analogs. Polyene antibiotics are excluded from the substances included in the conjugates of the invention.

The term "low molecular weight drug" in this specification refers to a drug having a molecular weight of less than 6000 Daltons. Examples of such drugs include, but are not limited to 5-amino salicylic acid, aminoglucoside antibiotics, flucytosine, pyrimethamine, sulfadiazine, dapsone, trimethoprim, mitomycins, methotrexate, doxorubicin, daunorubicin and polymyxin B.

The term "amine drug derivatives" refers to oligo-peptyl esters of hydroxyl containing drugs which contain a primary amine. The oligopeptide chain can comprise identical or different amino acids and will usually contain 10 amino acids or less. Examples of such derivatives include, but are not limited to, alanyl-Taxol, triglycyl-Taxol, alanyl-glycyl-dexamethasone, glycyl-dexamethasone and alanyl-dexamethasone.

The term "low molecular weight polypeptide" in this specification refers to a peptide or polypeptide having a molecular weight of less than about 6000 Daltons. Oxidation sensitive polypeptides are those comprising one or more oxidizable amino acids such as cysteine, methionine, tyrosine, histidine and tryptophan. Examples of such polypeptides include, but are not limited to, luteinizing hormone releasing hormone (LHRH), bradykinin, vasopressin, oxytocin, somatostatin, thyrotropin releasing factor (TRF), gonadotropin releasing hormone (GnRH), insulin and calcitonine.

The term "polypeptide analogs" in this specification refers to chemical modifications of bioactive peptides including cyclic derivatives, N-alkyl derivatives, derivatives in which fatty acids are attached to the amino acid terminals or along the peptide chain, and reverse amino acid derivatives.

The polysaccharide of the invention may be natural or synthetic and may be either branched or linear. An example of a linear polysaccharide is dextran. Examples of branched natural or semisynthetic polysaccharides are arabinogalactan (AG) and branched dextran, respectively. The molecular weight of the polysaccharide useful in accordance with the invention will generally be in the range of about 5000-75,000 Dalton.

The reaction of periodate with a polysaccharide (structure A, FIG. 1) leading to an oxidized polysaccharide (OP--structure B) is well-studied. The separation of the activated polymer from the interfering anions (periodate, iodate and formate) of the reaction mixture may be achieved, for example, by applying the reaction mixture to a column filled with a strongly basic anion-exchanger in the acetate form. This process of purification is fast and provides a high yield of the purified OP at the initial concentration, the anion-exchanger being easily regenerated afterwards.

The OP may then be reacted with the amino group of a biologically active substance (in the example of FIG. 1--doxorubicin) to form a Schiff base (structure C). The imine conjugate is useful for therapeutic applications where it is desired that the conjugate be hydrolyzed within a cell to release the active substance. Optionally, the Schiff base bond may be converted into a stable amine bond by a reducing substance, preferably sodium or potassium borohydride (structure D). Such a bond is unlikely to be easily hydrolyzed in the body. It is to be understood that other reducing substances, such as NaHSO₃ can also be used.

The conjugate may then be separated from the salts, low molecular weight polymer fractions and traces of unbound water-soluble drug by dialysis, followed by centrifugation and lyophilization.

The conjugates do not contribute markedly to an increase in osmotic pressure, notwithstanding their high molecular weight. Therefore for the preparation of an parenteral solution, e.g. an intravenous composition, the lyophilized conjugate is simply dissolved in saline and sterilized by filtration or autoclaving.

If it is desired to prepare compositions in the form of eye or ear drops, appropriate preservatives such as parahydroxybenzoate ester derivatives (methyl, propyl, butyl) may be added to the composition.

Claim 1 of 32 Claims

1. A water-soluble conjugate of a polysaccharide and an unoxidized, oxidation-sensitive substance, said substance being conjugated to said polysaccharide via an imine bond,

wherein said polysaccharide is activated as a dialdehyde and purified from interfering anions and by-products on a column filled with a strongly basic anion exchanger,

with the proviso that said substance does not include polyene antibiotics.

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