WO1995000177A1

WO1995000177A1 - Pharmaceutical preparation and a method for inhibiting the replication of various viruses

Info

Publication number: WO1995000177A1
Application number: PCT/CA1994/000343
Authority: WO
Inventors: Thomas C. Usher; Natu Patel; Chhagan Tele; I. Louis Wolk
Original assignee: Dextran Products Limited
Priority date: 1993-06-17
Filing date: 1994-06-17
Publication date: 1995-01-05

Abstract

The invention provides for both a pharmaceutical preparation and a method for inhibiting in vivo the reverse transcriptase enzyme and the replication of human immunodeficiency virus (HIV). In one embodiment the pharmaceutical preparation is a conjugate of Dextran, Modified Dextran, Dextran Sulphate or Polysaccharides and 3'-azido-2',3'-didesoxythymidine (AZT) which may be administered via different routes in appropriate dosage forms, dosage quantities and dosage regimens to patients suffering from a viral disease such as AIDS and its related disorders. This conjugate represents a novel structure which functions as a structural unit which combines the known additive and synergistic properties of dextran or dextran sulphate with AZT and at the same time appears to ameliorate the toxic effects of AZT.

Description

PHARMACEUTICAL PREPARATION AND A METHOD FOR INHIBITING THE REPLICATION OF VARIOUS VIRUSES

This invention relates to a pharmaceutical preparation and method for inhibiting reverse transcriptase enzyme and the replication of a family of viruses known as human immunodeficiency virus (HIV) by the use of a novel conjugate of dextran, modified dextran, dextran sulphate or other polysaccharides with AZT. It also relates to a method for modifying Dextran so that it is prepared for conjugation with such a drug having a primary or secondary hydroxy group.

BACKGROUND TO THE INVENTION

Certain retrovirus infections have been known to depress immune functions in animals. In recent years, it has been discovered that a family of T-lymphotropic retroviruses causes T-cell proliferation leukaemia, helper T-cell depletion and immunosuppression in humans infected by these viruses. These viruses have become known as the HTLV family of retroviruses. A group of these viruses designated as HTLV- III has been isolated from patients with Acquired Immune Deficiency Syndrome (AIDS) and has become considered to be responsible for the development of this condition in humans. These are also known as HIV, particularly HIV-1 and HIV-2.

Since the epidemic was recognized in 1981, a rapidly increasing number of cases of AIDS have been diagnosed in the United States. Significant progress has resulted from research in selected areas including identification of the populations at risk for AIDS, the method of transmission of the causative agent, isolation and characterization of the virus that causes AIDS and development of a serologic test that identifies most infected individuals. On the other hand little progress has been made toward effective treatment of AIDS. Azidothymidine (AZT), a drug that inhibits reverse transcriptase was expected to prolong the lives of patients with AIDS but this is now in doubt. Many patients who receive AZT have temporary increases in the number of circulating helper (CD4+) T-lymphocytes. However, the drug has significant adverse affects and HIV has been isolated from the blood of patients even while they are under treatment with AZT.

Although zidovudine (3'-azido-2' , 3'-dideo- zythymidine, AZT, azidothymidine, Retrovir) is the FDA- approved drug, it has undesirable toxicity in the host (e.g. myelosuppression, neuropathy).

Progressive immunological and central nervous system disease is coupled to virus replication. The general observation is that the more the virus replicates, the more serious the disease symptoms. Prolonged virus replication results in near total destruction of immune function. This observation leads to the conclusion that the central goal of AIDS therapy is to control and hopefully eliminate virus replication.

SUMMARY OF THE INVENTION

The invention provides for a pharmaceutical preparation for inhibiting in vivo the reverse transcriptase enzyme and the replication of viruses, which comprises a conjugate of Dextran, Modified Dextran, Dextran Sulphate or Polysaccharides and AZT in appropriate pharmaceutical dosage form. The invention also provides a method for inhibiting in vivo the replication of HIV viruses comprising the different routes of administration of the aforesaid conjugates in a pharmaceutically appropriate dosage form, regimen and quantity. According to another aspect of the present invention, there is provided a method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

1 ) dissolving about 1 gram of Dextran powder having a M.W. of about 40,000 in about 10 ml of DMF:H20 (1:1) mixture;

2) adding 16% sodium carbonate solution to adjust the PH to about 8.0;

3) dissolving 0.2 to 1.0 grams of Cyanogen Bromide in 5 to 10 ml of DMF;

4) mixing 0.2 to 1.0 ml of Triethylamine in about 5 ml of DMF;

5) adding about 0.5 ml of each of the solution of step #3 and of step #4 to the Dextran solution of step #2;

6) adding about 1.0 gram of 6 - amino caproic acid after about two minutes and stirring for about eight hours at room temperature; and

7) purifying the product by precipitation with methanol.

According to another aspect of the present invention, there is provided a method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

1) dissolving about 1.0 gram of Dextran powder having a M.W. of about 40,000 in about 5 ml of water;

2) adding about 7.5 ml of NAOH (40%);

3) adding 1 to 10 grams of Choloracetic acid and stirring the mixture for about 16 hours at room temperature.

4) purifying the sample with precipitation and drying the sample.

According to a further aspect of the present invention, there is provided a method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

1) dissolving about 1.0 gram of Dextran powder having a M.W. of about 40,000 in about 10 ml DMF:H20 (1:1) mixture by warming;

2) adjusting the PH to about 8.0 with 16% sodium carbonate solution and cooling to below freezing temperature;

3) separately dissolving 0.2 to 10 gram of Cyanogen Bromide in 5 to 10 ml DMF;

4) dissolving 0.2 to 1.0 ml Triethylamine in about 5 ml DMF;

5) cooling the Dextran solution of step #2 and thereafter adding solution #3 and #4;

6) after about 5 minutes, adding about 0.8 gram of N- Acetylcystein;

7) adjusting the PH to about 9.0 with 16% sodium carbonate solution and stirring the reaction mixture for about 24 hours. ;

8) purifying the sample with precipitation and drying the sample.

According to yet another aspect of the present invention, there is provided a method of preparing a conjugate of Dextran Sulphate and Cytochalasin-D, comprising the steps of:

1) dissolving 0.1 to 0.5 grams of Dextran Sulphate having a M.W. of about 8,000 in 5 to 15 ml of a DMSO and Pyridine (1:1) mixture by warming;

2) adding 5-50 mg of Cytochalasin-D and about 75.0 mg of Dicyclohexyl Carbodiimide;

3) stirring the reaction mixture for about 6 hours at about 50° and then at room temperature for about 60 hours;

4) adding about 10 ml of water and dialyzing the solution exhaustively against water, filtering through 0.45 micron filter paper and freeze drying. Detailed Description of the Preferred Embodiments

There are a number of drugs which have properties which are helpful in the treatment of severe viral diseases, and especially AIDS, but which have strong side effects. Further, some potential drugs have not yet been applied in clinical treatment, probably due to their high toxicity and metabolic instability.

It has been discovered that it is possible to conjugate drugs which have a primary or secondary hydroxy group with Dextran once the Dextran has been modified to facilitate the conjugation. It has also been discovered that Dextran Sulphate may be conjugated with such drugs. Cytochalasin and AZT are two such drugs.

Those skilled in the art will be aware of pharmaceutically appropriate dosage forms for the conjugate of Dextran, Modified Dextran, Dextran Sulphate or Polysaccharides and AZT as well as the manner in which a suitable dosage quantity, regimen and routes of administration may be derived in respect of a particular patient.

Modified Dextran may be substituted, oxidised, cationic, anionic, spacer or activated Dextran.

The demonstrated absorption qualities of Dextran, Modified Dextran, Dextran Sulphate and Polysaccharides with AZT will bring these conjugates into the endothelium system with a consequently improved effect in the treatment of viral diseases.

The Dextran conjugated with AZT may have a molecular weight within the range of 4,000 to 1,000,000. The Dextran Sulphate conjugated with AZT may have a molecular weight within the range of 8,000 to 1,000,000. Examples of polysaccharides which may be conjugated with AZT are cyclodextrin and cellulose sulphate. A useful conjugate of AZT and a polysaccharide will be one exhibiting a slow release mechanism.

The aforesaid conjugates may include a pharmaceutically acceptable carrier or diluent.

The pharmaceutical preparations the subject of this invention are not only useful in the treatment of AIDS and other viral diseases, but are also useful in the treatment of AIDS-related complex (A.R.C.).

The present invention also contemplates a pharmaceutical preparation useful for the treatment of AIDS comprising a conjugate of a known anti-AIDS agent with (a) Dextran (b) Modified Dextran (c) Dextran Sulphate or, (d) a polysaccharide exhibiting a slow release mechanism.

The advantages of a drug-Dextran conjugate include the following:

(1) The drug-Dextran conjugate as a macromolecular compound has excellent metabolic stability, resulting in a more effective treatment.

(2) Because the conjugate is a large molecule and consists of polysaccharide, it is easy to be received and combined by receptors of the cell which consist of polysacharide-protein.

(3) Dextran and certain derivatives such as the sulphate are useful not only as a "transfer weapon", but also as an immunologically active material which has now been used in the treatment study of AIDS.

(4) The conjugate provides an effective weapon which will be able to attack and kill the harmful cells of several severe diseases including AIDS and Carcinoma.

(5) A Dextran-drug conjugate is a carrier or stabilizer frequently resulting in decreased drug toxicity, after biodistribution (due to the slow release of the drug from the conjugate), and mostly increases therapeutic efficacy.

(6) A Dextran Sulphate-drug conjugate increases the bioadhesiveness with increase in polyanionic character as it cannot be captured by the first pass effect and also during circulation.

(7) Macromolecules like conjugates of Dextran and a drug remain for prolonged periods of time in contact with cell receptors and appear to inhibit contact of certain viruses with cell receptors.

(8) Immunogenecity of drugs could be decreased by these conjugates.

(9) Conjugates of Dextran with a drug will be useful as macromolecular pro-drugs in drug delivery systems and suitable as nontoxiσ carriers for more effective drugs for the treatment of viral diseases.

Those skilled in the art will be aware of pharmaceutically appropriate dosage forms for a conjugate of Dextran or Dextran Sulphate and Cytochalasin-D or AZT as well as the manner in which a suitable dosage quantity and regimen may be derived in respect of a particular patient.

Methods for the preparation of Modified Dextran which may- be conjugated with a drug having a primary or secondary hydroxy group are as follows.

Producing Modified Dextran

Method A

1) About 1 gram of Dextran powder (M.W. about 40,000) is dissolved in about 10 ml of DMF:H20 (1:1) mixture.

2) PH is adjusted to about 8.0 with 16% sodium carbonate solution.

3) 0.2 to 1.0 grams of Cyanogen Bromide is dissolved in 5 to 10 ml of DMF.

4) 0.2 to 1.0 ml of Triethyla ine is mixed in about 5 ml of DMF .

5) About 0.5 ml of each of the solution of step #3 and of step #4 are added to the Dextran solution of step §2 .

6) After about 2 minutes about 1.0 gram of 6 - amino caproic acid is added and stirred overnight (i.e., for about eight hours) at room temperature.

7) The product is purified by precipitation with methanol.

Method B

1) About 1.0 gram of Dextran powder (M.W. about 40,000) is dissolved in water (about 5 ml).

2) About 7.5 ml of NAOH (40%) is added.

3) 1 to 10 grams of Choloracetic acid is added and the mixture is stirred for about 16 hours at room temperature.

4) The sample is purified with precipitation and dried.

Method C

1) About 1.0 gram of Dextran powder (M.W. about 40,000) is dissolved in about 10 ml DMF:H20 (1:1) mixture by warming.

2) The PH is adjusted to about 8.0 with 16% sodium carbonate solution and cooled to below freezing temperature.

3) 0.2 to 10 gram of Cyanogen Bromide is separately dissolved in 5 to 10 ml DMF.

4) 0.2 to 1.0 ml Triethylamine is dissolved in about 5 ml DMF.

5) After cooling the Dextran solution of step #2 , solution #3 and #4 are added.

6) After about 5 minutes, about 0.8 gram of N- Acetylcystein is added.

7) The PH is adjusted to about 9.0 with 16% sodium carbonate solution and the reaction mixture is stirred for about 24 hours. 8) The sample is purified with precipitation and dried.

Any of the foregoing Modified Dextrans may be conjugated with Cytochalasin-D. Examples in this regard follow.

Conjugating Modified Dextran with Cytochalasin-D

Example 1

1) 1 gram of Dextran powder (M.W. about 40,000) was dissolved in 10 ml of DMF:H20 (1:1) mixture.

2) PH adjusted to 8.0 with 16% sodium carbonate solution.

3) 0.5 grams of Cyanogen Bromide was dissolved in 5 ml of DMF.

4) 0.75 ml of Triethylamine was mixed in 5 ml of DMF.

5) In the Dextran solution of step #2, 0.5 ml of each of solutions #3 and #4 were added.

6) After 2 minutes 1.0 gram of 6 - amino caproic acid was added and stirred overnight at room temperature.

7) The product was purified by precipitation with methanol.

8) 100 mg of above Modified Dextran dissolved in 15 ml of DMSO & Pyridine (1:1) mixture.

9) 10.0 mg of CYtochalasin-D was dissolved in 5 ml of DMSO:Pyridine (1:1) and added into Modified Dextran solution.

10) 0.2 grams of EDCI was added and stirred for 48 hours at room temperature.

11 ) The solution was purified by dialysis, filtered through 0.45 micron filter paper and dried.

Example 2

1) 1.0 gram of Dextran powder (M.W. about 40,000) was dissolved in water (5 ml). 2) 7.5 ml of NAOH (40%) was added.

3) 5.4 grams of Choloracetic acid was added and the mixture was stirred for 12 hours at room temperature.

4) The sample was purified with precipitation and dried.

5) 100 mg of above Modified Dextran was dissolved in 15 ml of DMSO & Pyridine (1:1) mixture by warming.

6) 10 mg of Cytochalasin-D was dissolved in 5 ml of DMSO and Pyridine mixture (1:1) and added in the solution of Modified Dextran.

7) 0.2 grams of EDCI-1 -(3-Dimethylamino propyl) - 3- ethylcarbodiimide hyddrochloride was added and the reaction mixture was stirred at room temperature for 24 hours.

8) 20 ml of water was added and purified by exhaustive dialysis against water, filtered through 0.45 micron filter paper and dried.

Example 3

1) 1.0 gram of Dextran powder (M.W. about 40,000) was dissolved in 10 ml DMF:H20 (1:1) mixture by warming.

2) The PH was adjusted to 8.0 with 16% sodium carbonate solution and cooled below freezing temperature.

3) 0.5 gram of cyanogen bromide was separately dissolved in 5 ml DMF.

4) 0.75 ml Triethylamine was dissolved in 5 ml DMF.

5) After cooling the Dextran solution, solutions §3 and #4 were added.

6) After 5 minutes, 0.8 gram of N-Acetylcystein was added.

7) The PH was adjusted to 9.0 with 16% sodium carbonate solution and the reaction mixture was stirred for 72 hours.

8) The sample was purified with precipitation and dried.

9) 100 mg of above Modified Dextran was dissolved in 15 ml DMSO & Pyridine (1:1) mixture.

10) The solution of 10.0 mg of Cytochalasin-D in DMSO & Pyridine (1:1) was added in above solution. 11) 0.2 grams of EDCI was added and stirred for 24 hours at room temperature.

12) The solution was purified by exhaustive dialysis against water.

13) The solution was filtered through 0.45 micron filter paper and dried.

Any of the foregoing Modified Dextrans may also be conjugated with AZT. Examples in this regard follow.

Conjugating Modified Dextran with AZT

Example 1

1) 1 gram of Dextran powder (MW about 40,000) was dissolved in 10 ml of DMF:H20 (1:1) mixture by warming.

2) The PH was adjusted to 8.0 with 16% sodium carbonate solution.

3) 0.5 gram of cyanogen bromide was dissolved in 5 ml of DMF.

4) 0.75 ml of triethylamine was mixed in 5 ml of DMF.

5) After cooling the Dextran solution of step #2, 0.5 ml of each solution #3 and #4 were added.

6) After 2 minutes, 1.0 gram of 6-amino caproic acid was added and stirred overnight at room temperature.

7) The sample was purified with precipitation with methanol.

8) 100 mg of above Modified Dextran dissolved in 7.5 ml of DMSO.

9) 13.5 mg of AZT was added.

10) 9.0 mg of 4-dimethyl aminopyridine was added.

11) 13.0 mg of N, N-dicyclohexyl carbodiimide was added and the reaction mixture was stirred at room temperature for 16 hours at anhydrous conditions.

12) The solution was purified by dialysis, filtered with 0.45 micron filter paper and dried. Example 2

1) 1.0 gm of Dextran (MW about 40,000) powder was dissolved in water (5 ml).

2) NAOH (40%, 7.5 ml) was added.

3) Chloroacetic acid (5.4 g) was added and reacted at room temperature for 16 hours.

4) The sample was purified with precipitation and dried.

5) 100 mg of above Modified Dextran dissolved in 7.5 ml of DMSO.

6) 13.5 mg of AZT was added.

7) 9.0 mg of 4-dimethyl amino pyridine was added.

8) 13.0 mg of N, N-dicyclohexyl carbodiimide was added and reaction mixture was stirred at room temperature for 16 hours at anhydrous conditions.

9) The solution was purified by dialysis, filtered with 0.45 micron filter paper and dried.

Example 3

1) 1.0 gm of Dextran (M.W. about 40,000) powder dissolved in 10 ml of DMF:H20 (1:1) mixture.

2) PH adjusted to 8.0 with 16% sodium carbonate solution.

3) Solution cooled below freezing temperature.

4) 0.5 gm of cyanogen bromide was dissolved in 5 ml of DMF.

5) 0.75 ml of triethylamine was mixed with 5.0 ml of DMF.

6) After cooling Dextran solution of step #3, 0.5 ml of each solution #4 and 5 were added.

7) After 5 minutes, 0.8 gm of N-acetyl cysteine was added.

8) PH of solution was adjusted to 9.0 with 16% sodium carbonate solution.

9) Solution was stirred for 24 hours at room temperature.

10) Sample was purified with precipitation and dried.

11) 100 mg of above Modified Dextran was dissolved in 10 ml of D ΞO. 12) 13.5 mg of AZT was added.

13) 9.0 mg of 4-dimethyl amino pyridine was added.

14) 13.0 mg of N, N-dicyclohexyl carbodiimide was added and reaction mixture was stirred at room temperature for 16 hours.

15) Solution was purified by dialysis, filtered with 0.45 micron filter paper and dried.

Structural diagrams relating to the aforedescribed examples are set out hereinafter.

EXAMPLE 1

SPACER DEXTRAN n

DEXTRAN -AZT CONJUGATE n EXAMPLE 2

DEXTRAN

CHLOROACETIC ACID

SUBSTITUTE SHEET EXAMPLE 3

SUBSTITUTE SHEET Dextran Sulphate may also be conjugated with a drug having a primary or secondary hydroxy group. Methods for conjugating Dextran Sulphate with Cytochalasin-D are as follows.

Conjugating Dextran Sulphate with Cytochalasin-D

Method A

1) 0.1 to 0.5 grams of Dextran Sulphate having a M.W. of about 8,000 is dissolved in 5 to 15 ml of a DMSO and Pyridine (1:1) mixture by warming.

2) 5-50 mg of Cytochalasin-D and about 75.0 mg of Dicyclohexyl Carbodiimide is added.

3) The reaction mixture is stirred for about 6 hours at about 50° and then at room temperature for about 60 hours.

4) About 10 ml of water is added and the solution dialyzed exhaustively against water, filtered through 0.45 micron filter paper and freeze dried.

Method B

B) As an option to method A) , the Dextran Sulphate may have a M.W. of about 500,000.

Specific examples for conjugating Dextran Sulphate with Cytochalasin-D follow.

Example 1

1) Dissolve 0.2 grams of Dextran Sulphate (M.W. about 8000) in 10 ml of DMSO & Pyridine (1:1) mixture by warming.

2) Add 15.0 mg of Cytochalasin-D and 75.0 mg of Dicyclohexyl Carbodiimide.

3) The reaction mixture was stirred for 6 hours at 50° and then at room temperature for 60 hours.

4) 10 ml of water was added and dialyzed exhaustively against water, filtered through 0.45 micron filter paper and freeze dried.

Example 2

The steps undertaken in the first example conjugate were repeated for conjugation of Cytochalasin-D with Dextran Sulphate (M.W. about 500,000).

Test results

DRUG CONCENTRATION # OF SYNCYTIA PER WELL (ug/ml)

1 . AD 042793 100 0 0

Dextran-AZT 10 40 42 conjugate 1 53 55 0.1 52 49

AZT standard 100 0 0 10 0 0 1 0 0 0.1 10 8

AZT 041493 100 13 12 Dextran-AZT 10 48 50 conjugate 1 54 50 (second batch) 0.1 52 51

CONTROLS CONCENTRATION # OF SYNCYTIA PER WELL

(ug)

CELLS (NO VIRUS/ NO DRUG) 0 0 0

(VIRUS/NO DRUG) 54 49 53

AZT POSITIVE CONTROL 10.0 0 0 1.0 0 0 0.1 10 11

Claims

CLAIMS :

1. A pharmaceutical preparation comprising a conjugate of AZT and Dextran.

2. A pharmaceutical preparation comprising a conjugate of AZT and Modified Dextran.

3. The pharmaceutical preparation of claim 2 wherein the Modified Dextran is substituted, oxidised, cationic, anionic, spacer-Dextran or substituted, oxidised, cationic, anionic activated Dextran.

4. A pharmaceutical preparation comprising a conjugate of AZT and Dextran Sulphate.

5. A pharmaceutical preparation comprising a conjugate of AZT and a Polysaccharide exhibiting a slow release mechanism.

6. The pharmaceutical preparation of claim 1 wherein the molecular weight of Dextran is about 40,000.

7. The pharmaceutical preparation of claim 4 wherein the molecular weight of Dextran Sulphate is within the range of about 8,000 to 1,000,000.

8. The pharmaceutical preparation of claim 5 wherein the Polysaccharide is Cyclodextrin or Cellulose Sulphate.

9. The pharmaceutical preparation of claim 1, 2, 3, 4, 5, 6 or 7 useful for the treatment of AIDS.

10. The pharmaceutical preparation of claim 1, 2, 3, 4, 5, 6 or 7 useful for the treatment of viral diseases.

11. The pharmaceutical preparation of claim 1, 2, 3, 4, 5, 6 or 7 including a pharmaceutically acceptable carrier or diluant.

12. A conjugate of AZT with (a) Dextran (b) Modified Dextran (c) Dextran Sulphate, or (d) a Polysaccharide exhibiting a slow release mechanism for use in the treatment of humans suffering from AIDS or AIDS - related complex (A.R.C. ).

13. A process for the preparation of a pharmaceutical preparation comprising the conjugation of AZT with (a) Dextran (b) Modified Dextran (c) Dextran sulphate, or (d) a Polysaccharide exhibiting a slow release mechanism.

14. A pharmaceutical preparation useful for the treatment of AIDS comprising a conjugate of a known anti-AIDS agent with

(a) Dextran (b) Modified Dextran (c) Dextran Sulphate or, (d) a Polysaccharide exhibiting a slow release mechanism.

15. A process for the preparation of a pharmaceutical preparation comprising the conjugating of AZT with (a) Dextran

(b) Modified Dextran (c) Dextran Sulphate, or (d) a Polysaccharide exhibiting a slow release mechanism.

16. A method for inhibiting the replication of HIV viruses which comprises the administration of a conjugate of AZT with a polysaccharide such as dextran having a molecular weight in the range of 8,000 - 1,000,000.

17. A method according to claim 16 wherein the polysaccharide is dextran sulphate.

18. The pharmaceutical preparation of claim 1 wherein the molecular weight of dextran is within the range of 4,000 to 1 ,000,000.

19. A method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

2) adding 16% sodium carbonate solution to adjust the PH to about 8.0;

3) dissolving 0.2 to 1.0 grams of Cyanogen Bromide- in 5 to 10 ml of DMF;

4) mixing 0.2 to 1.0 ml of Triethylamine in about 5 ml of DMF;

5) adding about 0.5 ml of each of the solution of step §3 and of step #4 to the Dextran solution of step #2;

7) purifying the product by precipitation with methanol.

20. A method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

2) adding about 7.5 ml of NAOH (40%);

4) purifying the sample with precipitation and drying the sample.

21. A method for modifying Dextran so that it is prepared for conjugation with a drug having a primary or secondary hydroxy group comprising the steps of:

3) separately dissolving 0.2 to 10 gram of Cyanogen Bromide in 5 to 10 ml DMF;

4) dissolving 0.2 to 1.0 ml Triethylamine in about 5 ml DMF;

5) cooling the Dextran solution of step §2 and thereafter adding solution §3 and #4;

6) after about 5 minutes, adding about 0.8 gram of N- Acetylcystein;

8) purifying the sample with precipitation and drying the sample.

22. A method for modifying Dextran so that it is prepared for conjugation with AZT or Cytochalasin comprising the steps of: 1 ) dissolving about 1 gram of Dextran powder having a M.W. of about 40,000 in about 10 ml of DMF:H20 (1:1) mixture;

2) adding 16% sodium carbonate solution to adjust the PH to about 8.0;

3) dissolving 0.2 to 1.0 grams of Cyanogen Bromide in 5 to 10 ml of DMF;

4) mixing 0.2 to 1.0 ml of Triethylamine in about 5 ml of DMF;

5) adding about 0.5 ml of each of the solution of step #3 and of step §4 to the Dextran solution of step §2 ;

7) purifying the product by precipitation with methanol.

23. A method for modifying Dextran so that it is prepared for conjugation with AZT or Cytochalasin comprising the steps of:

2) adding about 7.5 ml of NAOH (40%);

4) purifying the sample with precipitation and drying the sample.

24. A method for modifying Dextran so that it is prepared for conjugation with AZT or Cytochalasin comprising the steps of:

3) separately dissolving 0.2 to 10 gram of Cyanogen Bromide in 5 to 10 ml DMF;

4) dissolving 0.2 to 1.0 ml Triethylamine in about 5 ml DMF;

5) cooling the Dextran solution of step §2 and thereafter adding solution and #4;

6) after about 5 minutes, adding about 0.8 gram of N- Acetylcystein;

7)' adjusting the PH to about 9.0 with 16% sodium carbonate solution and stirring the reaction mixture for about 24 hours. ;

8) purifying the sample with precipitation and drying the sample.

25. A method of preparing a conjugate Dextran and Cytochalasin-D comprising the steps of claim 22 and including the following additional steps:

8) dissolving 100 mg of the Modified Dextran of step #7 in 15 ml of DMSO & Pyridine (1:1) mixture;

9) dissolving 10.0 mg of Cytochalasin-D in 5 ml of DMSO:Pyridine (1:1) and adding same into the Modified Dextran solution of step #8; 10) adding 0.2 grams of EDCI and stirring for 48 hours at room temperature; and

11) purifying the solution by dialysis, filtering through 0.45 micron filter paper and drying.

26. A method of preparing a conjugate of Dextran and Cytochalasin-D comprising the steps of claim 23 and including the following additional steps:

5) dissolving 100 mg of Modified Dextran from step §4 in 15 ml of DMSO & Pyridine (1:1) mixture by warming;

6) dissolving 10 mg of Cytochalasin-D in 5 ml of DMSO and Pyridine mixture (1:1) and adding same into the solution of Modified Dextran of step #5;

7) adding 0.2 grams of EDCI-1-(3-Dimethylamino propyl) -

3-ethylcarbodiimide hyddrochloride and stirring the reaction mixture at room temperature for 24 hours;

8) adding 20 ml of water and then purifying by exhaustive dialysis against water, filtering through 0.45 micron filter paper and drying.

27. A method of preparing a conjugate of Dextran and Cytochalasin-D comprising the steps of claim 24 and including the following additional steps:

9) dissolving 100 mg of Modified Dextran of step #8 in 15 ml DMSO & Pyridine (1:1) mixture;

10) adding the solution of 10.0 mg of Cytochalasin-D in DMSO & Pyridine (1:1) to the solution of step #9;

11) adding 0.2 grams of EDCI and stirring for 24 hours at room temperature;

12) purifying the solution by exhaustive dialysis against water;

13) filtering the solution through 0.45 micron filter paper and drying.

28. A method of preparing a conjugate of Dextran Sulphate and Cytochalasin-D, comprising the steps of:

4) adding about 10 ml of water and dialyzing the solution exhaustively against water, filtering through -0.45 micron filter paper and freeze drying.

29. The method of claim 28 wherein the Dextran Sulphate has a M.W. of about 500,000.