PT99058B - METHOD FOR PREPARING DEXTRIN SULPHATES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM - Google Patents

Abstract

The invention provides an agent against HIV and related viruses, in dosage unit form, comprising CD4 or a CD4-like material and a polyanionic anti-HIV agent, the content of CD4 or CD4-like material in the agent being less than the anti-virally effective dose of the CD4 or CD4-like material alone. It has been found that when the CD4 or CD4-like material is used together with a polyanionic anti-HIV agent, the combination is more effective against HIV infection than would be the case if the anti-viral effect was simply additive.

Description

According to the present invention, a correlated virus agent is provided, wherein the dextrin sulfate containing a maximum of two sulfate groups per glucose unit. Dextrin is a mixture of glucose polymers and glucose can be substituted in one or

2,3 and 6, by sulfate groups.

and the units of most of the positions

The present invention also provides compositions containing said agent. Additionally, the invention provides for the use of that agent or compositions against the HIV-1 virus and correlated viruses.

A dextrin sulfate for use in the present invention can have up to two sulfate groups per glucose unit, with the preferred dextrin sulfates having about 1 or between 0.5 and 1.5, preferably up to 1.2, groups sulfate per glucose unit. More preferably, the dextrin agent 2-sulfate or 3-sulfate, or a mixture thereof.

(We found that dextrin 2-sulfate has almost the same activity against the HIV-1 virus as dextrin 6-sulfate. However, the latter has superior anticoagulant activity for this reason,

Dextrin 2-sulfate is a particularly preferred agent,

We also found that dextrin 3-sulfates have relatively poor anti-HIV activity, compared to dextrin 2-sulfates and 6-sulfates. It follows that, for a given sulfate content, the anti-HIV activity of a dextrin sulfate is inversely related to the proportion of 3-sulfation. It turned out that for the most part

Ά

of the reaction conditions the 3-OH group of the glucose residue in a dextrin is less reactive than the 2-OH and 6-OH groups. Therefore, an increased anti-HIV activity can be obtained per sulfate group, keeping the relatively low degree of sulfation, thereby reducing the 3-sulfation content.

However, in the selection of a sulfated dextrin in particular as an anti-HIV agent, in this way, in general, there are: conflicting factors.

1. For a given sulfate content: a) toxicity increases with increasing molecular weight, and

b) anti-HIV activity increases with increasing molecular weight.

2. For a given molecular weight ιέ) toxicity increases with increasing sulfate content, and

b) the activity anti-HIV increases with increasing of content of sulfate. It seemed that sulfates of dextrin could in fact no be usable in practice as agents anti-HIV, a turn what it looked like the activity anti-HIV satisfactory was going side side by side with a toxicity unacceptable, either because the Weight molecular were too high or because the content of itself

sulfate was too high,

By restricting the degree of substitution to a maximum of 2, the present invention makes it possible to produce a dextrin sulfate with adequate anti-HIV activity, while maintaining toxicity within acceptable limits. With a relatively low degree of substitution, the proportion of 3-sulfation can be kept low, so that i

imported toxicity to dextrin sulfate is avoided by substitution 3. If a dextrin is completely replaced, that is, to give 2,3,6-sulfate, one third of the sulfate groups are 3-sulfate groups, which gives rise to additional toxicity, above all proportions, to which it intensifies anti-HIV activity. The extent to which 3-sulfation occurs when the degree of substitution is kept below 2, varies with the nature of the sulfation process, however, it is generally substantially less than that of 2-sulfation or 6-sulfation. The analytical techniques available at this time do not easily allow an accurate analysis of the extent of sulfation at the three available sites, but an examination of the NMR spectrum of a dextrin sulfate gives sufficient indication of this for practical purposes. The total sulfate content can naturally be assessed using conventional analytical methods, usually by determining the sulfur content.

molecular weight of the dextrin sulfate used in the present invention, can vary within wide limits. As an example, the dextrin sulfate used in the present invention can have an average molecular weight by weight between about 15,000 and 25,000, as determined in the dextrin used to prepare the dextrin sulfate. The technique used to determine the molecular weight of dextrin, is high pressure liquid chromatography, using chromatographic columns calibrated with dextrin standards, as reported by Alsop et al, Cromatography 246, 227-240 (1989).

dextrin sulfate can be prepared by first hydrolyzing starch to produce dextrin which after being sulfated to produce dextrin sulfate. For example, the use of a trimethylamine / sulfur trioxide complex in an alkaline aqueous medium gives predominantly 2-sulfate. Treatment of dextrin with cyclamic acid in dimethylformamide gives 6-sulfate. The 3-sulfate can be prepared by first acetylating dextrin, then sulfating it with a trimethylamine / sulfur trioxide complex in dimethylformamide and finally removing the acetyl groups with aqueous sodium hydroxide.

It is preferred to use dextrin sulfate in which there is a low proportion of low molecular weight material. As mentioned above, dextrin is prepared by hydrolysis of starch, typically by treating various starches with dilute acids or with hydrolytic enzyme. These methods produce glucose polymers with a long curing spectrum. The degree of polymerization (G.P.) varies from one or two to comparatively high numbers. The starch direct hydrolysis product can contain up to 60% by weight of material with a G.P. less than 12. According to a preferred aspect of the present invention, the dextrin derivative contains a relatively high proportion of glucose polymers with a G.P. greater than 12. Preferably, the dextrin derivative contains at least 50% by weight of glucose polymers with a G.P. greater than 12.

More preferably, the dextrin derivative contains less than 10% by weight of glucose polymers with a G.P. less than 12. More preferably, the dextrin derivative contains less than 5% by weight of glucose polymers with a G.P. less than 12. These dextrin derivatives are prepared from dextrin which has been fractionated to remove dextrin with a G.P. low, known fractionation techniques can be used, including solvent precipitation and membrane fractionation.

7.

A method of preparing a polymeric glucose mixture is described in GB 2132914, a method for the preparation of a polymeric mixture of glucose with a relatively low proportion of low-GP glucose polymers being described in example 2 of GB 2154469. it has an average molecular weight by weight of 23,700 and contains 91.9% polymers with a degree of polymerization greater than 12 and 7.9% of polymers with a degree of polymerization from 2 to 10.

It is also preferable that the dextrin derivative contains little or no material with a high molecular weight. Most preferably, the dextrin derivative contains little or no material with a molecular weight greater than 40,000.

dextrin sulfate is a particularly effective agent against the HIV-1 virus and correlated viruses. Although the mechanism of its action is not understood, it appears that dextrin sulfate in the sense of blocking the virus from binding to cells. It appears that due to its somewhat globular configuration, dextrin sulphate provides a carrier agent of sulphate groups, relatively close together, which can, in particular, effectively prevent the virus from binding to cells and thus virus entry.

dextrin sulfate can be effective at relatively low concentrations. In addition, the aforementioned globular configuration of dextrin sulfate allows the material to be effective against the HIV-1 virus and correlated viruses, even with a relatively low degree of sulfation. For example, it has been found to be effective at relatively low concentrations, a degree of sulfation as low as that of a sulfate group per unit of glucose, or even lower. This has the advantage that the amount of sulfation can be kept at such a low level, in order to avoid side effects and toxicity that would otherwise be revealed with highly sulfated materials.

dextrin sulfate can be taken enterally, including orally), and is preferably administered parenterally, for example, intravenously.

However, administration via the peritoneum may be more effective than intravenous administration, in the sense that it results in the entry of at least part of the dextrin sulfate directly into the lymphatic system, within which the replication system can be extended. of the virus.

The invention also provides a composition comprising an agent, as described above, together with an inert carrier or a diluent.

In addition, the invention provides for the use of the agent or the composition of the invention against the HIV-1 virus and correlated viruses, the agent or composition being preferably administered intraperitoneally. The agent of the invention is preferably adapted for intraperitoneal administration.

The invention further provides an agent as described above, to be used in the preparation of a pharmaceutical composition for the treatment of the HIV-1 virus and correlated viruses.

In addition, the invention provides a method of treating a human or animal patient who carries the virus

HIV-1 or a correlated virus, comprising the administration

said patient with a pharmaceutically effective amount of the agent of the invention.

The following examples illustrate the methods for preparing dextrin sulfate:

Example 1 - Preparation of dextrin 3-sulfate

16.2 g of the aforementioned dextrin from Example 2, GB 2,154,469, was stirred in dimethylformamide (150 ml) and heated until dissolved, then cooled to room temperature. Acetic anhydride (23 ml, 0.24 moles) was added slowly with stirring. Transient precipitation occurred and when it had dissolved again, triethylamine (25 ml, 0.18 moles) was added and the mixture was stirred for 2 days. The solution was then poured into a weak stream, with stirring in water (700 ml), the precipitate was filtered off, washed with water and dried to give 23 g of a white powder.

The acetylated dextrin (12.3 g) in dimethylformamide (75 ml) was stirred until it dissolved and then a trimethylamine-sulfur trioxide complex (15 g) was added and the mixture was stirred at room temperature, during the night. Additional trimethylamine-sulfur trioxide (10 g) was added and the mixture was heated at 60 ° C for 3 hours. The solution was cooled and poured into acetone (500 ml) to give a viscous residue. The supernatant was decanted and the residue was combined with fresh acetone (50 ml) and then the supernatant was decanted. The residue was dissolved in water (150 ml) and the remaining acetone was removed under vacuum. A solution of NaOH (5 g) in water (10 ml) was added, giving a trimethylamine gas. The strongly basic solution was stored for 2 hours, dialyzed with water for 4 days and freeze dried to give 10.2 g. The IR spectrum revealed peaks for acetate (1750 CM) and sulfate (1240 CM ^-1 ).

The product (10 g) was redissolved in water (150 ml) and NaOH (1 g) was added in water and the mixture was stirred for 3 hours at room temperature, the solution was poured into ethanol (300 ml), the supernatant was decanted and the viscous residue was combined with fresh ethanol (150 ml) to give a solid. The solid was filtered, washed with methanol and dried to obtain a brown powder. The powder was dissolved in water (200 ml) and decolorizing charcoal (5 g) was added. The solution was heated, then filtered twice and freeze dried to give 7.2 g of sulfate, 46.9%.

Example 2 - Preparation of dextrin 6-sulfate

10 g of the same dextrin as in Example 1 were heated in dimethylformamide (100 ml) and stirred at 78 ° C. When the dextrin had completely dissolved, cyclamic acid (22.5 g) was added and the reaction for 1.5 hours. A solution of NaOH (5 g) in water (5 ml) was added and ethanol (50 ml) was added and the mixture was poured into diethyl ether (400 ml). The solid was filtered, washed with ether and air dried. The solid was dissolved in water (100 ml), sodium acetate (50 g) was added and the solution was dialyzed with water for 4 days and then freeze-dried to give 15.4 g, sulfate 47 ,2%.

Example 3 - Preparation of dextrin 2-sulfate

40 g of the same dextrin as in example 1 were stirred in distilled water (150 ml) in a round bottom flask at 30 ° C. As soon as the dextrin had completely dissolved, trimethylamine-sulfur trioxide (51 g) was added to the solution. The reaction mixture was stirred for thirty minutes. Sodium hydroxide (62.5 ml and 40% w / v) was added dropwise to the reaction mixture over the period of one hour. The reaction mixture was then stirred for a further two hours and filtered under vacuum. The resulting solution was dialyzed for one day with tap water and one day with distilled water. The dialyzed solution was then concentrated by evaporation under reduced pressure. The concentrated solution contained 30 g of solids dissolved at 36% w / w (weight of dry solids) sulfate.

The products of Examples 1, 2 and 3 were identified as 3-, 6- and 2-sulfates, respectively, by examining their r.m.n spectra.

r.m.n spectrum. Carbon 13 of the original dextrin reveals six lines. Most of these can be attributed by reference to standard compounds, such as: 100.3, C-1; 77.6, C-4; 73.9, C-3; 72.2 and 71.8, C2 and C-5; 61.1, C-6.

The spectra of r.m.n. of the 3-, and 6-sulfates of glucose have been reported (S. Honda, Y. Yuki and K. Thaiura, Carbohydrate Research (1973) Volume 28, pages 130 to 150) and compared for free sugars. Thus, it was observed that 3-0-sulfation causes a deviation from the field to

down from 8.5 or 9.5 ppm for C-3, a field deviation upwards of 1.1 ppm for C-2 and a field deviation upwards of 2.2 ppm for C-4, but it did not cause any change to the other positions. For 6-0-sulfation, a field deviation downwards of 6.2 ppm was observed for C-6 and field deviations upwards, of 1.7 ppm for C-5 and 0.3 ppm for the C-4, with a small change in the other positions.

r.m.n spectrum. of the product strong signal at 61.1 ppm deviation, unmodified New ones have appeared from Example a that show a characteristic of the C-6-0H prominent signs are found close to the values 82.2 and 82.5 ppm.

These chemical shifts from the 82.7 ppm reported for C-3 to D-glucose 3-sulfate, are therefore attributed to dextrin 3-sulfate. This assignment is supported by the virtual disappearance of the signal at 77.6 ppm in the original dextrin for C-4. The substitution at 0-3 is expected to cause the field to deviate upward from the signal to C-4, taking it from all other signals. New peaks are assigned at 70.2 and 70.8 ppm in C-2, in a 3-sulfate, by the deviation of the field upwards, from the original position at 72.2 or 71.8 ppm. The C-1 region reveals six lines with little space between them, between the values 100.1 and 98.3 ppm, slightly upwards in the original dextrin. From these data, it is revealed that the product of Example 1 is sulfated, almost entirely, in the 3-position.

r.m.n spectrum. of the product of Example 2 shows that the original C-6 peak at 61.1 ppm decreased markedly, with new peaks appearing at positions 67.5 ppm 69.3 ppm, for C-6-0 sulfate (deviation field down 6.4 ppm) and C-5 adjacent to 6-0 sulfate (field deviation up 2.5 or 2.9), respectively.

is replaced

These data indicate that the product of Example 2 is primarily in position 6.

r.m.n spectrum. of the product of Example 3, compared to that of the original dextrin, reveals a major signal for unsubstituted C-6-0H at 61, lppm,

- disturbed C-4 at 78.1 ppm, indicating the main signal C-l moves upwards, to 99.8 ppm, from the original position at 100.3 ppm. From these data, it appears that the product of Example 3 is replaced mainly in position-2.

in the free non-OH3 value, and

In the following Example, the effectiveness against the HIV-1 virus was tested on materials, namely dextran, zidovidine (AZT).

do and

sulfate compared to dextrin sulfate with other dextran and

Example 4 - Efficacy of dextrin 2-sulfate against HIV-1 dextran sulfate had a molecular weight of 500,000, having been obtained from Sigma Chemical Company and then purified by column chromatography. The dextran had a molecular weight of 90,000. The dextrin 2-sulfate (referred to as NBSD24 and prepared as described in Example 3 above), contains a sulfate group per glucose unit, as indicated by the infrared analysis.

The analysis of the elements indicated 12.7% of sulfur equivalent for 1.1 groups of sulfate per unit of glucose.

Normal human peripheral blood lymphocytes (PBL) were fractionated in Ficoll-Hypaque (Pharmacia Chemicals, Uppsala, Sweden) from venous blood collected in heparin

condom-free. They were mitogenetically (PHA) (1 µg / ml) for 3 days and kept in a medium of RPMI 1640, supplemented with 20% cow fetus serum (FCS) + interleukin-2 (IL2) (50-100 gg / ml ).

The human T-leukanic cell line C8166 was grown in RPMI 1640 with 10% FCS.

Several strains of HIV-1 (R.F. III B, CBL-4, Z84 and Z129) were produced in chronically infected H9 cells. Five times in excess of uninfected H9 cells were added to the culture five days before harvesting the supernatant, which is clarified by centrifugation and stored in liquid nitrogen.

The induction of antipathetic effects on CD4 ⁺ C8166 by HIV was tested by the formation of multi-nuclear giant cells (syncytia).

The following table illustrates the sensitivity of HIX-1 to the two sulfated materials, and the non-sulfated dextran and AZT were determined in ineffectiveness by syncytial induction;

compared to the C8166 test,

Drug Concentration Qig / ml)

0 0.1 1.0 10 100 NBSD24 -6 ND -4 -2 -2 Dextran Sulf act -6 -6 -6 -3 dead cell Dextran -6 -6 -6 -6 AZT -6 -4 -3.5 -3 -3

15,

what are they represent

HIV-1 (RF) was titrated in 10-fold dilutions on G8166 cells, in the presence of varying drug concentrations. The final dilutions of the virus produced are shown. The figures in Table are a logarithm for base 10 of the figure actually determined. For example, a figure of -6 in the table represents a given result of 10 ^- ^. ND means that a result has not been determined for the particular drug concentration. At a concentration of μg / ml, dextrin 2-sulphate and dextran sulphate, each reduced the infectious titer by 3 logarithmic units, which compare favorably with the reduction of 3 logarithmic units made by AZT. Dextran was ineffective against HIV replication. At 1 μg / ml, inhibition occurs with dextrin sulfate (2 log units) and AZT (2.5 log units). These results were confirmed by calorimetric testing, as shown in the following Table:

Damn it

NBST24 +

Dextran Sulphate +

Dextran +

AZT +

Concentration ^ g / ml)

0.1 +

+ +

The inhibition of HIV-1 living cells (C8166) is seen by one less.

(RF), tested by the reduction of MTT in was visually evaluated and is represented.

Damn it (^ g / ml) RF IIIB CBL-4 NBSD24 10 5 3 0 50 6 5 4 Dextran Sulfate 10 3 2 0 50 1 0 0 AZT 10 3 4 4 50 3 4 4 These results indicate what 2-sulfate dextrin

syncytial induction in C8166 cells, with a greater effect than either AZT or dextran sulfate.

Example 5 - Toxicity of dextrin 2-sulfate

The relative toxicities of the materials tested in Example 4 were assessed by reference to the viability of the cells in the presence of the drugs, as measured by the relative rise in [H] -thymidine. Human PBL (10 cells / well) was stimulated in 96 microtiter plates with PHA in the presence of a growing drug concentration for 3 days. Following a 5-hour trial with [HJ-thymidine (0.5uci / well), cells were harvested and counted. The results shown represent the average of quadruplicate experiments being presented as percentage incorporation of [Hj-thymidine, in the presence of the drug, compared to the control experiences without the drug. The results are given below:

'h tf

Ί

Thymidine rise (% control without any drug)

Concentration (μβ / ιηΐ) 5 10 25 50 71 100 200 AZT 20 15 5 2 1 1 0 NSBD24 65 120 105 90 90 85 78 Dextran 95 97 80 70 67 78 60 Dextran Sulfate 105 113 110 66 57 30 15 Dextran had a Weight molecular of 90. 000 and the sulfate dextran had a Weight molecular of 500 .000. In practice, there would be a need to use the drug particular in concentration around in 200 μ§ / πι1. You can see what , in this

type of concentration, AZT is highly toxic and dextran sulfate is also highly toxic. By comparison, dextrin 2-sulfate is slightly less toxic than non-sulfated dextran material.

Example__6 - Comparison__of_effectiveness__of__2-sulfate, __ 3-sulfate and 6-dextrin sulfate

50 μΐ samples of dextran sulfate and derivatives of dextrin 2-sulfate, 3-sulfate and 6-dextrin derivatives of Examples 3, 1 and 2, respectively, were pre-incubated at 37 ° C in duplicate cultures of JH11 M8166 4x10 cells per ml (50 μΐ) in final concentrations of 50, 5, 0.5 and 0.05 μ§ / μ1 for one hour.

50 μΐ of TCID 10 ³ (illb, RF, CBL20) or TCID 2 (RUT) were added and cultures were incubated until control cultures (no cause of effect) revealed 90% + syncytia (2- 3 days). The inhibitory concentration was taken as the minimum concentration, in which the formation of syncytia was found to be <10% of that of the controls at the end point. The results are shown in the following table:

Illb RF RUT CBL20 Sulfate Dextran 5 50 50 50 2-sulfate dextrin 5 5 5 50 3-sulfate dextrin 50 5 50 > 50 6-sulfat dextrin 5 5 50 > 50 0 sulfate dextran had one molecular weight of 8,000. These results indicate a greater effectiveness general of 2- -sulfate and 6-sulfate dextrin, in comparison how sulfate dextran and 3-sulfate dextrin (being the last most effective that sulfate dextran against RF) • Example 7 - Comparison of anti- coagulant of sulfate dextrin

The materials to be tested were each dissolved in barbial buffer to a concentration of 1 mg / ml = 1000 μ§ / πι1, with a further dilution of 100 μ§ / ιη1, by means of a 1:10 dilution. in barbital buffer. The stored normal plasma (1 ml) was then placed in each of the nine plastic tubes. The solutions containing the materials to be tested were then added to the plasma to obtain an average final concentration of 1-300 μg / ml, in each case the volume being brought up to 1.5 ml with the barbital buffer. The resulting mixtures were incubated at 37 ° C for 30 minutes, after which two 0.2 ml aliquots were extracted from each sample into tubes.

Claims (10)

there. Process for the preparation of dextrin sulphates, namely dextrin 2-sulphate, dextrin 3-sulphate and dextrin 6-sulphate, characterized by the fact that starch is hydrolyzed in order to obtain dextrin, preferably with 50 % of molecules with a degree of polymerization greater than 12 and then; a) for the preparation of dextrin 3-sulfate, if the dextrin is dissolved in dimethylformamide, if it is added acetic anhydride and triethylamine, if it is stirred, if the reaction mixture is poured into water, if the acetylated derivative is separated by filtration, if the filtrate is dissolved in dimethylformamide, reacting with trimethylamine / sulfur trioxide complex, pouring the reaction mixture in acetone, decanting the supernatant, treating the residue with acetone and decanting, dissolving the residue in water and adding aqueous hydroxide solution sodium, dialysis and lyophilize the dialysed product; b) for the preparation of dextrin 6-sulphate, the dextrin is dissolved in dimethylformamide, successively added cyclical acid and an aqueous solution of sodium hydroxide and ethanol, the reaction mixture is poured into ethyl ether, the solid is separated by filtration, dissolve in water, add sodium acetate, dialysis and lyophilize the dialysed product; and c) for the preparation of dextrin 2-sulphate, if the dextrin is dissolved, if the trimethylamine / sulfur trioxide complex solution is added to the reaction mixture, if it is added dropwise to the reaction mixture, if it is stirred for a while, if filter, dialysis the solution and concentrate the dialysis product. 2a. Process for the preparation of pharmaceutical compositions for the treatment of infections by the HIV-1 virus and related viruses, in particular, for peritoneal administration, characterized by the fact that dextrin sulfates containing at least two sulfate groups are mixed per unit of glucose with the pharmacologically acceptable carrier ingredients or diluents. 3a. Process according to claim 2, characterized in that the dextrin sulfate contains 0.5 and 1.5 sulfate groups per glucose unit. 4th. Process according to claim 3, characterized in that the dextrin sulfate contains up to 1.2 sulfate groups per glucose unit. 5th. Process according to any of claims 2 to 4, characterized in that dextrin 2-sulfate, dextrin 6-sulfate or mixtures thereof are used. 6th. Process according to any of claims 2 to 5, characterized in that a dextrin sulfate is used which contains at least 50% glucose polymers with a degree of polymerization greater than 12. 7th. Process according to any of claims 2 to 7, characterized in that said dextrin sulfate contains less than 10% by weight of glucose polymers with a degree of polymerization less than 12. 8th. Process according to any of claims 2 to 7, characterized in that said dextrin sulfate contains less than 5% by weight of glucose polymers with a degree of polymerization less than 12. 9a. Process according to any of claims 2 to 8, characterized in that the average molecular weight of the dextrin sulfate employed is between 15000 and 25000. 10th. Process according to any of claims 2 to 9 _r wherein said dextrin sulphate is substantially free of polymers having a molecular weight of 40 , 000.