DD297166A5 - ANTI-HIV-ACTIVE - Google Patents

Abstract

The invention provides an anti-HIV active ingredient containing as an active ingredient a sea cucumber-derived sulfated polysaccharide and / or a pharmaceutically-acceptable salt thereof.

Description

The invention relates to a novel human immunodeficiency virus (hereinafter referred to as "HIV")

effective anti-HIV drug.

The "Acquired Immunodeficiency Syndrome" or AIDS is a serious one caused by the infection with HIV Immunodeficiency syndrome (Nature 321 (1986), 10). Given the very high mortality from this disease is the therapy

HIV infection and AIDS have become a serious social problem.

Among the known anti-HIV agents currently recognized as effective in clinical use Azidothymidine (AZT), which has reverse transcriptase inhibitory activity. Azidothymidine (AZT) used as anti-HIV drug shows insufficient clinical efficacy. Furthermore

causes azidothymidine severe side effects, such as. As disorders in the bone marrow (hematopoietic tissue) andneurologische complaints such. Headache, convulsions, etc. As the genome of HIV infiltrates into the chromosomes of the infected cell as a hereditary disease in the form of a provirus, it is inevitably necessary to give a pharmaceutical preparation for a long period of time. Accordingly, the side effects of AZT are a major obstacle to its use as an anti-HIV drug.

Thus, the object of the invention is to develop a new cure effective against HIV infections and AIDS,

which is substantially free of the above-mentioned side effects and is applicable for a long period of time.

The solution to this problem is achieved by the finding that obtained from sea cucumbers sulfated polysaccharides

and / or a pharmaceutically acceptable salt thereof possessing significant anti-HIV activity and without risk to the

Patients can be used over a long period of time. Thus, according to the invention an anti-HIV drug

which contains as active ingredient a sea cucumber-derived sulfated polysaccharide and / or a pharmaceutically acceptable salt thereof.

The present invention, derived from a sea cucumber sulfated polysaccharide (this polysaccharide is

hereinafter referred to as "FGAG") is extracted from the body wall of hollow animals and a related substance prepared by chemical conversion of FGAG.

FGAQ is a chondroitin sulfate grade and has the physicochemical properties given below

Appearance:

white, amorphous, highly hygroscopic powder;

Molecular weight:

from about 15,000 to about 80,000 (as determined by high performance GPC or polyacrylamide gel electrophoresis);

Analysis of the composition:

The composition (in terms of parts by weight) is the following:

Galactosamine 13 to 20% by weight

Glucuronic acid 11 to 19% by weight

Fucose 10 to 27% by weight

Sulfate 27bls38.5% by weight.

The analyzes were carried out by the following methods to determine galactosamine (hereinafter referred to as "GalN"), "glut ironsae" (hereinafter referred to as "GA"), fucose (hereinafter referred to as "Fuo") and sulfate.

GaIN: White method (Carbohydrate Research 114 (1983), 586,201.

GA: Bitter-Mulr-Veriahren (Anal Bicchem. 4 [1962], 330)

Fuc: Dische method (J. Biol. Chem. 175119481.595)

Sulfate: Dodgson & Price method (Biochem J. 84 [1962], 106).

FGAG is a known substance, the z. In Yao Hsueh Hsueh Pao (15 [5] (1980), 263-270), Zhongyao Tongbao (714 (1982), 27-29), Yaoxue Xuebao, 18 (3) [1983], 203-208). and Japanese Unexamined Patent Publication Nos. 10601/1988 and 128001/1988 FGAG is easy to prepare by the methods disclosed in these publications, more specifically, FGAG is prepared as follows: The cell wall of a hollow animal becomes tiu'oh alkali and further by pancreatin or a similar enzyme for extraction, FGAG is then isolated and purified from the extract Examples of sea cucumbers used in the preparation of FGAG:

Stichopus japonicus Selenka, Stichopus chloronoyus Brandt, Stichopus variegatus Semper, Holothuria pervicax Selenka, Holothuria atra, Holothuria argus, Holothuria edulis, Holothuria scabra, Parastichopus nigripunctatus, Thelenota pineapple, Holothuria monacaria Lesson, Holothuria leucospilota Brandt, Cucumaria chronhjelmi, Cucumaria echinata, Cucumaria frondosa Japonica, Pentacta australis, Paracaudina chilensis ransonneti, Molpadia musculus, Leptosynapta inhaerens, Polycheira rufescens, Synapta maculata, Halodeima cinerascens (Brandt), Actinopyga lacanora (Jaeger), Actinopyga echinites (Jaeger), or Microthele nobilis (Selenka).

Sea cucumbers used as starting material may be raw or dried. Of the sea cucumbers exemplified above, Stichopus japonicus Selenka is the most preferred starting material. A particularly preferred related substance produced by chemical conversion of FGAG is D-HG obtained by depolymerization of FGAG.

D-HG is prepared by dissolving FGAG or a salt thereof in water and subjecting the solution to a depolymerization reaction. By the depolymerization reaction, a sulfated polysaccharide having a high molecular weight, such as. For example, heparin or a similar molecule is converted to a sulfated low molecular weight polysaccharide. The reaction is usually carried out in the presence of a cepolymerizing agent. Examples of suitable depolymerizing agents are hydrogen peroxide, hypochlorous acid, hypobromous acid, sodium hypochlorate and like hypohalogenated acids and salts thereof; Periodic acid, sodium periodate or similar periodic acids and salts thereof. Furthermore, ascorbic acid, iron or similar ions are useful as reaction promoters. In another embodiment, the depolymerization reaction using only radiations, such as. As ultrasonic waves, ultraviolet rays, X-rays o. Ä. Instead of the depolymerization agent or in combination with the above Depolynierisierungs V / irkstoff be performed. The

Particularly preferred depolymerization processes are carried out using hydrogen peroxide as a depolymerizing agent. Hydrogen peroxide is reacted in an amount of 1 to 31% by weight, preferably 1 to 16% by weight, with respect to the hydrogen peroxide concentration. The reaction time is usually 1 to 60 hours, preferably 3 to 40 hours, and the reaction temperature is in the range of from room temperature to about 8O ⁰ C, preferably from about 4O ⁰ C to about 60 ° C. The pH range in the hydrogen peroxide reaction is acidic or neutral in the range of 1 to 8, preferably 3 to 7. In order to maintain a constant pH, hydrogen peroxide can be dissolved in a buffer such. As acetate buffer, phosphate buffer, Tris buffer o. Ä. Be implemented. For pH control, dilute sodium hydroxide may be used in the reaction. After completion of the reaction, the pH is returned to the neutral range and isolation and purification are performed. The isolation and purification may, for example, by fractional precipitation using an organic solvent, such. For example, ethanol, acetone; Acetate, such as As potassium acetate, barium acetate, calcium acetate, ammonium acetate, or quaternary ammonium salts such as Cetyltrimethylamm ^ ni> imsalz be performed. Isolation and purification may also be accomplished by ion exchange chromatography using resins such as DEAE-cellulose (Sigma Chemical Co.), DEAE-Toyopearl (Tosoh Corporation), DEAf-Cellulofine (Chisso Corporation), Dowex-1 (Dow Chemical Co .), By gel filtration chromatography using gels such. Sephadex GJO, Sephadex G-200 (Pharmacia LKB Biotechnology), by dialysis using Spectra / Por (Spectrum Medica Industries, Inc.) or by ultrafiltration. These agents are used individually or in a suitable combination

 The D-HG thus prepared has the following physicochemical properties

Appearance:

white, amorphous, highly hygroscopic powder;

Molecular weight:

30U0 to 42000 (determined by high performance GPC);

Analysis of the composition: D-HG contains sulfates and saccharides containing GaIN, GA and Fuc in a molar ratio of GalN: GA: Fuc: sulfate =

1: 0.80 ± 0.20: 0.85 ± 0.15: 3.4 ± 0.90;

Solubility: Soluble in water but insoluble in ethanol, acetone and similar organic solvents; Specific rotation:

Ia) S ⁰ = -55 to -73 ° (C = 1%)

Color reaction; Elson-Morgan reaction + Carbazole-sulfuric acid reaction + Cysteine-sulfuric acid reaction + Orcinol hydrochloric acid reaction + AzurAMetachromasia reaction + A preferred molecular weight of D-HG is about 4,000 to about 15,000 (as determined by high-performance GPC).

Hereinafter, the composition of the saccharides contained in the substance in a form in which salt is not formed, i. in free form,

GaIN 18 to 24% by weight

GA 14 to 21% by weight

Fuc 13 to 20% by weight

Sulfate 31 to 44% by weight.

As the above analysis shows, FGAG and its related substance have sulfates and carboxyl groups in the molecule which react with bases to form a salt. These sulfated polysaccharides are stable as a salt and are usually isolated as sodium and / or potassium salt. These salts of these sulfonated polysaccharides can be prepared by treatment with cation exchange resins such. B. Dowex 5OW, are converted into free-solubilized polysaccharides. The sulfated polysaccharides may also be converted to the desired salts by conventional redispersion, if appropriate. Suitable salts of sulfated polysaccharides are pharmaceutically acceptable salts comprising potassium and sodium salts or salts of similar alkali metals and salts of calcium, magnesium, barium or similar alkaline earth metals or pyridine or similar organic bases. The process for producing FGAG and its related substance will be described in detail in the reference examples given later.

From the sea cucumber-derived suifatisierte polysaccharides and / or pharmaceutically acceptable salts thereof are useful for pharmaceutical compositions used for HIV treatment. More specifically, the anti-KiV active ingredient of the present invention containing the active ingredients of the present invention and a pharmaceutically acceptable carrier is prepared in a form that varies depending on the type of HIV treatment. The dosage forms used in the present invention include tablets, capsules, powders, granules, granules, solutions, emulsions, suspensions and like forms for oral administration and injections, suppositories, ointments, patches and similar parenteral forms. The preparation of such forms may be carried out by methods of preparation commonly employed by one of ordinary skill in the art. In the preparation of a solid preparation for oral administration of the inventive anti-HIV active ingredient by mixing the invention, ι effective ingredient with a

Drug carrier with or without the addition of binders, solvents, lubricants, dyes, flavors, fragrances, etc. and processing dec mixture in tablets, capsules, powders, granules, grains o. Ä. Made in the usual way. Injectable preparations may be prepared by adding a pH-adjusting agent, buffer, stabilizer, isotonizing agent, local anesthetic, or the like. be formulated into the active ingredients and processing of the mixture in a conventional manner for intravenous, intramuscular, subcutaneous, intracutaneous or intraperitoneal injections. Suppositories may be prepared by formulating a mixture of the active ingredients, excipients and, optionally, a surfactant in a conventional manner.

Examples of excipients that can be used for solid oral preparations are lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerol, sodium alginate or gum arabic. Examples of excipients for oral preparations are polyvinyl alcohol, polyvinyl ether, ethyl cellulose, gum arabic, shellac or sucrose. Examples of the lubricant are magnesium stearate or talc. Colorants, disintegrators and other adjuvants to be added include those commonly used. Tablets can be coated by conventional methods.

Examples of suppositories. Usable raw materials include oily base materials such as macrogol, lanolin, cocoa oil, fatty acid triglycerides, Witepsol (registered trademark for the product of Dynamite Nobel).

The amount of active ingredients per dosage unit in the preparation of the invention varies, for. B with the symptoms of the patient to whom the drug should be given or the form of the drug. Usually, a preferred amount is 10 to 200 mg in an oral preparation, 1 to 100 mg for an injection or 10 to 100 mg in the suppository, each for one unit dose. The daily clinical dosage of the active ingredient of the present invention also varies with the age of the patient, sex, condition and other factors, but is usually in the range of about 10 to about 1000 mg, preferably about 50 to about 200 mg in 1 to 4 divided doses are given.

The anti-HIV drug of the invention is effective in preventing HIV infection, hindering the onset and healing of AIDS and ARC (AIDS-related complex, R.R. Redfield & D. S. Burke, Science 18 [1988] 74-87). Furthermore, the active ingredient can be given over a long period of time since the sulfated polysaccharides, i. the active ingredient of the sea cucumber active ingredient has low cytotoxicity.

Thus, the anti-HIV drug of the present invention effectively protects normal individuals against HIV infection and is effective in hindering the occurrence and healing of potential carrier-hidden symptoms and the treatment of AIDS or ARC patients.

The invention will be described in more detail in the examples, examples and pharmacological tests. The percentages in the Reference Examples and Examples are by weight.

(1) Preparation of FGAG Reference Example 1

Production of FGAQ from Stichopus Japonlcus

1 kg of dried Stichopus japonicus was submerged overnight in 101 warm water to swell it. The meat was removed and homogenized. Potassium hydroxide was added in an amount giving a 1 -η mixture. The mixture was treated 100 minutes at 6O ⁰ C and the pH adjusted to 8.5. After addition of 50 g of pancreatin, the mixture was stirred for 3 hours at 5O ⁰ C.

After removal of impurities by centrifugation, 4.31% ethanol was added to the residue. The mixture was allowed to stand at 4 ° C and the resulting precipitate was collected. The precipitate was washed with 80% ethanol, anhydrous ethanol and acotone in this order and dried under reduced pressure to give 50 g of a crude product. 50 g of the crude product was dissolved in 3 μl of water and the solution was centrifuged to remove the insoluble particles. To the supernatant was added 5% sodium chloride and 40% ethanol to produce a precipitate. The precipitate was collected by centrifugation. After the precipitate was dissolved in 2.5 L of water, the solution was adjusted to a pH of 10.5. The solution was added dropwise with a 30% aqueous hydrogen peroxide solution. The mixture was heated in a water bath for about 3 hours at 5O ⁰ C under heat supply entfärbt- After cooling, the insoluble particles were removed by centrifugation. To the supernatant, about 490 g of potassium acetate was added and the mixture was kept at 4 ° C overnight. The following day, the resulting precipitate was dissolved in 21% water, the solution was cooled to ⁰ ° C. and the pH was adjusted to 2.8. The insoluble particles were removed from the solution by centrifugation. After neutralization of the supernatant, 196 g of potassium acetate were added. The mixture was allowed to stand at 4 ^° C. to produce a precipitate, which was then collected by centrifugation. The precipitate was redissolved in water to obtain a solution having a potassium acetate concentration of 0.5 M, and the solution was allowed to stand overnight at 4 ° C. The precipitate was collected by centrifugation, washed with 40% methanol and dissolved in 11% water. To the solution was added 5% sodium chloride and 40% ethanol to produce a precipitate. The precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and acetone in this order and dried under reduced pressure to give 17 g of a FGAG sodium / potassium salt. The physico-chemical constants of the salt are the following:

Molecular weight:

55000 (determined by high performance GPC); and

Analysis of the composition:

GaIN: 20.0%

GA: 18.6%

Fuc: 17.2%

Sulphate: 36.6%

Na: 6.2%

K: 7.4%

Reference Example 2 Production of FGAG from Holothuria leucospilota Brandt When using Holothuria leucospilota Brandt, FGAG derived from it was obtained by the same method Reference example 1 produced. Molecular weight:

45,000 to 55,000 (determined by polyacrylamide gel electrophoresis).

Composition analysis: GaIN: 16.0%

GA: 16.3%

Fuc: 12.3%

Sulphate: 31.0%

Reference Example 3 Production of FGAG from Cucumaria frondosa Japonica When Cucumaria frondosa Japonica was used, FGAG obtained therefrom was prepared by the same method as in Reference example 1 produced. Molecular weight:

60000 to 70000 (determined by polyacrylamide gel electrophoresis)

Composition analysis: GaIN: 18.0%

GA: 11.1%

Fuc: 13.7%

Sulphate: 32.0%

(2) Preparation of D-HG Reference Example 4

10 g of the FGAG sodium phosphate obtained in Reference Example 1 was dissolved in 75 ml of water, and 25 ml of a 30% aqueous solution of hydrogen peroxide was added. While maintaining a pH of about 7 V under "Using a dilute solution of sodium hydroxide as a pH control agent, the solution was heated at 6O ⁰ C for 12 hours. After cooling, 2% sodium chloride and 40% ethanol were added to produce a precipitate. The protein precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and acetone in this order and dried under reduced pressure to give 7.15 g of a D-HG sodium / potassium salt.

Reference Example 5

6.95 g of a D-HG sodium / potassium salt were prepared by the same method as in Reference Example 4 except for the 24-hour treatment with hydrogen peroxide.

Referenzbelsplel β

A D-HG sodium / potassium salt was prepared by the same procedure as in Reference Example 4, except that the reaction was conducted while maintaining the pH of about 4 by using a dilute alkali solution. Yield 6.4g.

Reference booklet 7

10 g of the FGAG sodium salt obtained in Reference Example 1 was dissolved in 83.3 ml of a 0.2 M phosphate buffer (pH 7.0). To the solution was added 16.7 ml of a 30% aqueous hydrogen peroxide solution. The mixture was treated at 60 ^° C. for 12 hours. After cooling, 2% sodium chloride and 40% ethanol were added to produce a precipitate. The precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and acetone in that order and dried under reduced pressure to give 7.18 g of a D-HG sodium / potassium salt.

Reference bullet 8 A D-HG sodium / potassium salt was prepared by the same procedure as in Reference Example 7, except that

the reaction was carried out using a 0.2 M acetate buffer (pH 3.5). Yield 7.05g.

Reference Examples 9 and 10

2 g of the FGAG sodium hydroxide / potassium salt obtained in Reference Example 1 were dissolved in 15 ml of water. To the solution were added 5 ml of a 30% wäßriyen hydrogen peroxide solution and the mixture was 14 hours (Reference Example 9) or 40 hours (Reference Example 10) at 6O ⁰ C. After cooling, the mixture was adjusted to a pH of 7 to 8 and extensively dialyzed against water using Spectra / por 3. The mixture was freeze-dried and dried under reduced pressure. In this way, 1.62 and 1.76 g of D-HG sodium / potassium salts were prepared.

Reference sheet 11

2 g of the FGAG sodium / potassium salt obtained in Reference Example 1 were dissolved in 16.7 ml of water. To the solution 3,3ml of a 30% aqueous hydrogen peroxide solution were added and the mixture was treated for 24 hours at 45 ⁰ C. After cooling, the mixture was returned to a pH of 7, followed by 2% sodium chloride and 40%.

Ethanol added to provide a precipitate. The precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and acetone in this order and dried under reduced pressure to give 1.41 g of a D-HG sodium / potassium salt.

Reference examples 12 bit 15

2 g of the KGAG sodium / potassium salt obtained in Reference Example 1 were dissolved in 15 ml of water. To the solution was added 5 ml of a 30% aqueous solution of hydrogen peroxide, and the mixture was treated at 60 ° C for 4, C, 12 or 24 hours. After cooling, the mixture was adjusted to a pH of 7 to 8 and dialyzed completely against water using Spectra / por 3. The same treatment as in Reference Example 11 followed. In this manner, 1.42 g, 1.35 g, 1.35 g and 1.2 g of D-HG sodium / potassium salts were prepared.

Reference Examples 16 and 17

2 g of the obtained according to Reference Example 1 FGAG Natriurtv / potassium salt were dissolved in 14.7 ml of water. To the solution was added 5.3 ml of a 30% aqueous hydrogen peroxide solution. The mixture was treated at 45 ^° C. for 14 or 40 hours. After cooling, the mixture was readjusted to a pH of about 7 and then 2% sodium chloride and 40% ethanol were added to make a precipitate. The same treatment as in Reference Examples 9 to 11 followed. In this manner, 1.64 g and 1.62 g of D-HG sodium / potassium salts were prepared.

Reference bullet 18

"> Η obtained in Reference Example 1 FGAG-sodium / potassium salt were dissolved in 30 ml of water and 12 hours 'on this."'"·<Β treated as in Reference Example 11. The solution was treated with a sodium salt solution on a Sephadex G-50T Under supervision of uronic acid, the peaks were divided into three, the last obtained eluate was collected, dialyzed completely against water, freeze-dried and dried under reduced pressure to give 0.2 g of a D-HG sodium salt ,

Reference Example 19

0.5 g of the D-HG sodium / potassium salt obtained in Reference Example 11 was fractionated in the same manner as in Reference Example 18 to give 0.18 g of a D-HG sodium salt.

Figure 1 shows an infrared absorption spectrum of the D-HG sodium salt (measured with a KBr tablet) and Figure 2 shows a proton NMR spectrum (in DjO, 90 MHz, 7O ⁰ C) thereof.

Referentials 20

2 g of an FGAG-sodium compound obtained in Reference Example 1 was dissolved in 16.7 ml of water, and 3.3 ml of a 30% aqueous solution of hydrogen peroxide was added thereto. The mixture was treated at 45 ^° C. for 5 hours. After cooling, the mixture was returned to a pH of about 7 and then 2% sodium chloride and 40% ethanol were added to make a precipitate. The precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and acetone in this order and dried under reduced pressure to give a D-HG sodium / potassium salt. Yield 1.60g.

Reference Example 21

2 g of the FGAG sodium / potassium salt obtained in Reference Example 1 was treated for 9 hours by the same method as in Reference Example 20, giving 1.54 g of a D-HG sodium / potassium salt.

Reforenzbelsplel 22

2 g of the FGAG sodium / potassium salt obtained in Reference Example 1 was treated for 12 hours by the same method as in Reference Example 20, giving 1.52 g of a D-HG sodium / potassium salt.

Reference Example 23

1 g of the FGAG sodium / potassium salt obtained in Reference Example 1 was dissolved in 8.7 ml of 0.2 M phosphate buffer (pH 7.0). To the solution were added 1.3 mL of a 30% aqueous hydrogen peroxide solution and the mixture was treated for 3 hours at 60 ⁰ C. After cooling, 5% sodium chloride and 66% ethanol were added to the mixture to produce a precipitate. The precipitate was collected by centrifugation, washed with 80% ethanol, anhydrous ethanol and diethyl ether in this order and dried under reduced pressure to give a D-HG sodium / potassium salt. This salt was dissolved in 10 ml of a 20 mM Tris-HCl buffer (pH 7.0) and mixed thoroughly with DEAE-Toyopearl (Tosoh Corporation) equilibrated with this buffer. Elution was performed in this buffer with a linear sodium chloride concentration gradient (0 to 1 M). Under monitoring of uronic acid, the fractions were collected with the peaks and precipitations carried out with the addition of a two-fold amount of ethanol. The precipitate was collected by centrifugation, washed with 80% ethane, free ethanol and diethyl ether in this order and dried under reduced pressure to give a D-HG sodium salt. Yield 0.39g. Table I shows physicochemical properties of D-HGs obtained in Reference Examples 4 to 23.

Table I

Ref. MGX10 ³ [Α] 8 ° composition fuc GA sul » N / A K molar ratio Ex. 10.2 -71.2 GaIN 16.7 16.5 29.5 7.1 6.4 GalN: Prob: Huc: Sul · 4 9.7 -67.1 21.7 15.8 15.1 30.2 7.2 7.9 5 14.1 -71.5 19.8 17.6 15.6 33.1 6.2 6.7 6 4.7 -55.4 20.2 15.1 13.3 31.4 8.2 4.8 7 6.5 -61.2 19.8 15.6 15.8 32.8 7.8 4.4 8th 12.0 -70.8 20.6 16.5 17.1 38.6 4.7 6.1 9 7.8 -68.0 18.6 13.1 16.1 37.1 5.4 6.4 10 13.4 -70.1 17.5 14.6 14.6 32.7 8.1 5.5 11 14.1 -69.6 17.1 14.3 14.8 34.0 7.6 5.2 1: 0.70: 0.84: 2.5 12 8.6 -66.3 19.9 15.1 14.3 34.8 8.5 5.5 1: 0.70: 0.87: 2.8 13 7.5 -66.0 20.8 13.2 13.2 32.9 6.4 5.1 1: 0.71: 0.95: 3.0 14 5.6 -64.8 17.8 14.7 13.0 33.4 4.1 5.1 1: 0.62: 0.83: 2.9 15 10.8 -72.2 20.1 14.8 16.7 37.1 5.0 6.5 1: 0.71: 0.83: 2.9 16 6.6 -68.4 18, Ί 12.8 15.9 35.3 5.6 6.7 1: 0.85: 0.97: 3.8 17 10.2 -68.2 17.3 15.8 14.4 32.9 8.6 0 1: 0.85: 0.82: 3.9 18 7.7 -67.5 19.1 13.9 14.1 32.1 4.4 0 1: 0.79: 0.93: 3.5 19 41.4 -70.7 17.6 14.7 15.8 29.9 5.9 5.5 1: 0.69: 0.79: 3.1 20 24.3 -72.6 19.8 15.9 16.6 33.4 6.2 5.2 1: 0.64: 0.79: 3.1 21 20.1 -71.1 18.0 14.4 14.8 33.0 7.5 5.0 1: 0.68: 0.81: 3.4 22 12.8 -62.4 18.3 14.5 16.2 34.3 10.7 0 1: 0.60: 0.80: 3.0 23 19.3 1: 0.85: 0.89: 3.8 1: 0.85: 0.81: 3.7 1: 0.69: 0.90: 3.1 1: 0.74: 0.86: 3.3 1: 0.73: 0.81: 2.7 1: 0.85: 0.96: 3.4 1: 0.75: 0.86: 3.3 1: 0.78: 0.81: 3.3

Note: The molecular weights given in Table I are determined by high performance gel permeation chromatography. The other eigenvalues are determined as described above. SuI * means sulfate

The sulfated polysaccharides obtained in Reference Examples 1 to 23 showed single dots in electrophoresis (Dietrich, C.P., J. Chromatogr., 130 (1977), 299).

Preparation of the anti-HIV active substance example 1 Preparation of an injection solution The FGAG sodium / potassium salt prepared in Reference Example 1 was dissolved in distilled water to be injected To give E% aqueous solution. The solution containing 50mg of the reconstituted polysaccharide obtained from sea cucumber

was filled in a Giasfiäschchen for a freeze-drying. 2 ml of physiological saline was added as a solvent.

example Preparation of an injection solution An injection solution was prepared according to the formulation shown below. FGAG-Natriunv / potassium salt

(Reference Example 1) Physiological saline

40 mg q.s.

Per ampoule

2ml

Example 3

tablet

Tablets were prepared according to the formulation shown below.

D-HG sodium salt 10mg (Reference Example ^ 9 '. corn starch 65rr.g carboxymethylcellulose 20 mg polyvinylpyrrolidone 3 mg magnesium stearate 2 mg Per tablet 100 mg

Step Example! 4 Suppositories A suppository was prepared according to the formulation shown below.

D-HG sodium / potassium salt 60 mg

(Reference Example 17) Witepsol W-35 950 mg

(Dynamite-NobeiAG)

-Propfäpfchen 1000mg

Pharmacological test

(1) Anti-HIV Act

ΜΤ-4-ΙΙθββη (Miyoshl, I. et al., Gann Monogr., 28 (1982) 219-228) were adjusted to 2 x 10 ⁵ cells per ml of medium (RPM 11640 + 10% FCS) to 500μΙ of the medium each 50μΙ of a solution obtained in Reference Examples 1,19 and 23, obtained from sea cucumbers sulfated polysaccharides obtained in different concentrations.

2 hours after the addition, the medium was infected with HIV in a ratio of 5 × 10 ³ pfu / well. On the 4th day post-infection, a swab of the ΜΤ-4 cells was prepared and the cells positive for the HIV antigen were amplified by a fluorescent antibody method (S. Harada et al., Science 229 [1985], 563 566; Y. Takeuchi et al., Jpn. J. Cancer Res. (Gann, 78: 11-15, 1987).

Tables II, III and IV below show percentages (%) of HIV-infected cells (cells positive for the HIV antigen).

tables

FGAG concentration (pg / ml) (Reference Example 1) Table III D-HG concentration (pg / ml) (Reference Example 19) Table IV D-HG concentration (μρ / ιπΙ) (Reference Example 23) 10 1 1 0.1 0 (control) Percentage (%) Percentage (%) Percentage (%) 0 15 10 95 95 30 3 0.3 0.03 0 (control) 0 8th 25 95 95 10 0.1 0 (control) 0 95 95

The results show that the anti-HIV agents according to the invention effectively prevent HIV infections.

(2) cytotoxicity

The sea cucumber-derived sulfated polysaccharides were evaluated for cytotoxicity in the same manner as in the item (1). More specifically, ΜΤ-4 cells set to 1 × 10 ⁵ cells / ml of medium were incubated at different concentrations for 4 days in the presence of one of the sea cucumber-derived sulfated polysaccharides obtained in Reference Examples 1 to 19.

After the incubation, the number of cells was counted to estimate the cytotoxicity in terms of the indexes calculated by the concentrations listed when the control had 100 cells. Tables V and VI below show the results.

Table V

FGAG concentration (Mg / ml) (Reference Example 1) Table VI D-HG concentration (pg / ml) (Reference Example 19) 100 10 1 0 (control) index index 77 105 104 100 100 30 3 0,3 0 (control) 83 90 102 104 100

The results show that the anti-HIV drugs according to the invention have no significant cytotoxicity.

(3) Acute toxlactate

The FGAG sodium potassium salt prepared in Reference Example 1 was subjected to an acute toxicity test in mice. The test results show that the salt has an LD ₆₀ of 337.9 mg / kg (ip) and 335.2 mg / kg (iv) and is remarkably safe.

Claims (5)

An anti-HIV active ingredient containing as active ingredient a sea cucumber-derived sulfated polysaccharide and / or a pharmaceutically acceptable salt thereof. 2. An anti-HIV active ingredient according to claim 1, containing as an active ingredient at least one substance containing a sulfatized polysaccharide extracted from the body wall of sea cucumbers, a pharmaceutically acceptable salt davi n, a sulfated by Depolymerislerung the sulfated Polysacarides or its salt Polysaccharide or a pharmaceutically acceptable SaL Javon. The anti-HIV active ingredient according to claim 1 or 2, wherein the sulfated polysaccharide has the following physicochemical properties: Appearance: white, amorphous, highly hygroscopic powder; Molecular weight: about 15,000 to about 80,000 (as determined by polyacrylamide gel electrophoresis); and composition (in terms of parts by weight) Galactosamine 13 to 20% by weight Glucuronic acid 11 to 19% by weight Fucose 10 to 27% by weight Sulfate 27 to 38.5% by weight. The anti-HIV active ingredient according to claim 1 or 2, wherein the sulfated polysaccharide has the following physico-chemical properties: Appearance: white, amorphous, highly hygroscopic powder; Molecular weight: 3000 to 42000 (determined by high performance GPC); Molar ratio: galactosamine: glucuronic acid - fucose: sulfate = 1: 0.80 ± 0.20: 0.85 ± 0.15: 3.4 ± 0.90; Solubility: Soluble in water but insoluble in ethanol, acetone and similar organic solvents; Specific rotation: (a ⁰ = -55 to -73 ° (C = 1%), and Color reaction: Elson-Morgan reaction + Carbazole-sulfuric acid reaction + Cysteine-sulfuric acid reaction + Orcinol hydrochloric acid reaction + AzurA metachromasia reaction + Use of a sea cucumber-derived sulfated polysaccharide and / or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the treatment of diseases caused by HIV infection.