OA10332A - Compositions having antiviral properties and method of obtaining - Google Patents

Abstract

The compositions of the invention are prepared from acetic acid and coconut powder extracts, a solution of mineral salts, extracts of cactaceae, liliaceae, anacardiaceae and euphorbiaceae.

Description

) 1 010332

COMPOSITIONS HAVING ANTIVIRAL PROPERTIES

The subject of the invention is compositions having, in particular, antiviral and, in particular, antiretroviral properties. The fight against diseases caused by retroviruses mobilizes many research teams around the world. In particular, considerable efforts are being made to combat the AIDS epidemic (Acquired Immunodeficiency Syndrome). It is known that the causative agent of AIDS is antrovirus called. HIV, for human immunodeficiency virus, or most commonly HIV, according to its abbreviated designation in English.

The most widely used antiviral drugs are nucleoside derivatives, including zidovudine, or AZT, which was the first antiretroviral available. Since then, the emergence of resistant mutants has led to the search for new treatments.

The work of the inventor on a mixture of acid and naturally occurring products has led to the development of high efficiency compositions to inactivate the infectivity of HIV. The invention therefore aims to provide new antiretroviral compositions capable.

It also relates to a process for obtaining these compositions.

The compositions according to the invention are characterized in that they are prepared in part: acetic acid and extracts of coconut powder, mineral salts solution, extracts of cactacea, deliliaceae, d anacardiaceae, and euphorbiaceae.

Coconut is the fruit of coconut. According to the invention, the grated white flesh is advantageously used.

Cactaceae particularly suitable for the implementation of the invention include opuntia. It is recalled that opuntia is the generic name for racquet, nopal, prickly pear and other species native to tropical America.

We. especially uses the fruits of the racquetverte.

Suitable lilies within the scope of the invention include sarsaparilla. The root of sarsaparilla is used with advantage. .

As anacardiaceae, particularly mombin, and especially the bark of mombin.

A particularly preferred euphorbiaceae is castor oil and its seeds, especially in the grinded state and in the form of a powder, are especially used according to the invention.

Preferred compositions are made from acetic acid, grated coconut, denier water, green racquet fruit, parsley root, mombin bark, roasted castor seed powder.

These different ingredients are advantageously used in the following proportions: acetic acid: from 5 to 10 liters, in particular from 7 to 3 liters; - coconut powder (preferably, shredded chorale): equivalent to about 10 coconuts; Potassium sulphates, sea: 3 to - solution of mineral salts (in particular sodium, magnesium, calcium, phosphates, chlorides, carbonates and bicarbonates), for example water of 5 liters, in particular 4 liters; - snowshoes, in particular racket fruitsfrom 10 to 20 fruits corresponding to 1 grated: in particular1,5 kg; 2 kg, about 15 fruits, corresponding to about sarsaparilla root: 500 g to 1 kg, in particular about 750 g; - bark of mombin: from 500 g to 1.5 kg, in particular about 1 kg; - Grilled castor seeds and ground: from 500 g to 1.5 kg, in particular about 1 kg.

In another aspect, the compositions of the invention are as obtained by allowing the coconut powder to macerate with acetic acid for about 24 hours by adding to the filtrate of the maceration mixture the various ingredients listed below. above, advantageously in the proportions indicated, bringing the resulting mixture to boiling for at least 1 hour, and recovering the filtrate. The invention also relates to a process for obtaining the compositions defined above, characterized by the steps previously reported. The study of the properties of the compositions of the invention has demonstrated an inhibitory effect of the reverse transcriptase activity of HIV-1 may exceed 90% compared to the activity measured in witnesses. There is also a decrease in the cytopathogenic effect of HIV-1 in cells infected with cevirus, treated with compositions of the invention. The absence of toxicity of these compositions was verified on the mouse by intraperitoneal injection at physiological pH. The invention therefore aims to take advantage of the properties of these compositions for the development of pharmaceutical compositions.

The pharmaceutical compositions of the invention are characterized in that they contain an effective amount of at least one composition as defined above, in association with a pharmaceutical vehicle.

These compositions are particularly suitable for the prevention and treatment of AIDS, if appropriate in combination with other drugs.

These compositions are preferably administered by injection in the form of isotonic injectable solutions or suspensions with respect to blood duplasma.

The amount of active ingredient varies according to the state of the patient, the dosage in humans being most often of the order of 1 to 500 mg per day of active principle (dry extract). The invention will be illustrated hereinafter by way of example of the preparation of a composition of high efficiency with respect to the Hiv-1 virus and by the results of tests in vitro and in vivo.

Example 1 Preparation of a Composition According to the Invention

For at least 24 hours, white flesh, dried, grated, coconut is macerated with 7 to 8 liters of white vinegar or acetic acid.

The mixture is then filtered.

The following ingredients are added to the recovered filtrate: - mineral-rich solution, such as seawater: 4 liters; - grated green racquet (about 15 fruits): 1.5 kg: 750 g - sarsaparilla root 5 - mombin bark: 1 kg - roasted castor seed powder: 1 kg

The mixture thus obtained is about 1 h and then filtered. brought to a boil for 10

Example 2 Study of inactivation of HIV-1 by the composition of Example 1

FROTOCQLF ^ EXFERIMERIAL - Inactivation of the virus

The suspension of viral particles A in the RPMI 1640 culture medium (HIV-IIIB, reverse transcriptase activity 3.8 × 10 6 cpm / ml) is incubated with the composition of Example 1, hereinafter referred to as "the composition", (V / V), for 20 min at 4 ° C, then 1 hour at room temperature, finally 2 hours at 37 ° C. At the end of intubation, the mixture is diluted 1/10, 1/20, 1/40, 1/100, 1/200 in the RPMI 1640 culture medium plus 10% fetal calf serum (FCS). preheated for 30 minutes at 56 ° C. 5 x 105 MT4 cells (human cells of lymphoid origin transformed with HTLV-1) are resuspended in 250 μl of the different dilutions of the virus-composition mixture.

After 1 hour of adsorption at room temperature, with resuspension of cells every 15 minutes, the unadsorbed virus is removed from the cells. These are washed twice with RPMI and then resuspended in the RPMI 10% FCS medium, at a concentration of 2 x 10 5 / ml. The infected cells are cultured in microtiter plates (Costar 96 buckets) at a rate of of 200 pl / bucket.

All cultures are made at least endouble. The cultures are diluted with an identical volume of medium after 5 days and 8 days of infection.

A series of control cultures are infected in parallel with the mixed virus (V / V) suspension A with RPMI medium. Incubations and dilutions are carried out strictly under the same conditions as for the virus treated with the composition.

Additional control is performed with levirus preincubated with 0.15 M NaCl solution.

Virus production was measured by assaying the reverse transcriptase activity associated with virus particles released from the cells in the culture medium. Reverse transcriptase is also called reverse transcriptase or RT for short.

The RT test is carried out on 65 μl of medium according to the protocol described by Moog et al., In Antiviral Research, 24 (1994), 275-288. The results are expressed in cpm / test (table, lower part).

Cell viability is measured by the MTT test described by Moog et al., In the reference given above (table, upper patient). The intensity of the staining is expressed in milli DO. 7 010332

The production of HIV after infection of MT4 cells with virus preincubated with the composition is evaluated. After 5 or 7 days of infection, the values of RT are between 699 and 1538 cpm / test, whereas, for supernatants of uninfected cultures, they are 1400-2100 cpm / test. All cultures infected with the untreated virus produce virus from J5 to J11.

The production of HIV after infection of CEM-SS cells by HIV-1 LAi is evaluated

A suspension of virus B (HIV-1 LAi, reverse transcriptase activity 3 x 10 6 cpm / ml) is incubated (V / V) with the composition. All other steps are those described for the MT4 cell experiment.

Under these conditions, the cultures infected with the control virus (not incubated with the composition) produce virus, measurable by RT, 11 days after infection.

In the case of cells infected with levirus preincubated with the composition, the RTs are similar to those measured on uninfected supernatants (1707-2564 cpm / test) after 11 days of infection. EXPERIENCE C:

The experimental protocol followed is identical to that of experiment A. The measurements made at 15 and 17 post-infection show that no virus replication is detected, or very little, when it has been preincubated with the virus. composition (RT of uninfected control cells 972-12192 cpm / assay). EXPERIENCE D:

In order to be able to measure virus production at earlier times than in Experiment B, a 10-fold higher virus concentration was used.

Virus production after 5 and 8 days is highly inhibited by preincubation of the virus with the composition. The absence of the effect observed using the 0.15 M NaCl solution indicates that the inhibition of the virus is specific to the composition.

These results thus show that preincubation of the HIV-1 virus with the composition of the invention results in inactivation of the infectious power of the viral particles.

Example 3 Study of the Effect of the Composition of Example 1 on the Reverse Transcriptase Activity G'KIV-L

A suspension of HIV-1 virus was mixed (V / V) at different dilutions of the composition, The determination of the reverse transcriptase (RT) activity associated with the viral particles was carried out as described by Moog et al, in the reference above. above.

Measurements were made on at least two samples. WH »* iwww 9 010332

The results obtained are reported in Table 1.

It is found that the use of the composition, at the highest concentrations, inhibits RT activity by more than 90%. .1

Another series of experiments, the results of which are given in Table 2, confirm the previous data. s È (? 10

These experiments show that the compounds of the invention are capable of inhibiting the transcriptase activity measured in vitro.

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Table 1 - RT activity (Cpm / test) 010332 FINAL DILUTION OF COMPOSITION / VIRUS SOLUTIONS i 1! - AVERAGE 1.164 783 973 (93%) * 1/6 759 688 724 (94%) 1; 1/8 2.453 2.875 2664 (80%) 1/54 6.875 5.949 6.412 (50%) 1/162 10.035 8.220 9.128 (29%) 1/486 11.747 12.981 12.364 20 14.318 10.743 11.764 MEDIUM TEMPER VIRUS 12.794 25 12.687 12.218 15.036

* Figures in parentheses indicate% inhibition of RT s activity 11

Table 2 - RT activity (Cpm / test) 010332 FINAL DILUTION OF SOLUTIONS COMPOSITION / VIRUSES 1/2 550 783 AVERAGE 666 (100%) * 1/4 925 524 724 (100%) 1/8 572 442 507 (100%) 1/16 910 797 854 (100%) 1/32 27.045 30.535 28.790 (82%) 1/64 55.180 71.351 63.265 (60%) 1/128 108.652 103.673 106.163 (32%) 1/256 129.555 107.540 118.548 (24%) 1/512 81,234 149,698 115,466 (26%) 1/1024 167,936 144,598 156,267 ii]

ί WITNESS VIRUS 157.233 155.059

* Figures in parentheses for inhibition of RT activity indicate%

AVERAGE 156.146

Claims (1)

12 010332 CLAIMS Μ! 4}! 1 / Compositions possessing in particular antiviral properties and. in particular antiretrovirals, characterized in that they are prepared from acetic acid and extracts of 1 coconut powder, of mineral salts solution. extracts of cactacea, liliaceae, anacardiaceae, and euphorbiaceae. 2 / Composition according to Claim 1, characterized in that the grated white flesh of the coconut is used. 3 / Composition according to Claim 1 or 2, characterized in that an opuntia, commecactaceae, is used in The composition according to one of claims 1 to 3, characterized in that sarsapleille is used as the liliaceous, in particular the root of the sarsleilleille. 5 / Composition according to one of claims 1 to 4, characterized in that, commeaneoarciacèe, mombin, especially the bark of mombin 5 / Composition according to one of claims 1 to 5. characterized in that The seeds of castor oil are used as thephosphate, in particular the roasted castor seeds, and the composition according to one of claims 1 to 6, characterized in that it is prepared from acetic acid. , nuts grated coconut, sea water, green racquet fruit, sarsaparilla root, ruffin bark, ricotta seed powder. 8 / Composition according to claim 7, characterized in that the different ingredients are used in the following proportions - acetic acid: 5 to 10 liters, especially 7 to 8 liters; - coconut powder (preferably shredded white flesh): equivalent to about 10 coconuts; - solution of mineral salts (in particular sodium, potassium, calcium, magnesium, phosphates, chlorides, sulphates, carbonates and bicarbonates), for example water demer: 3 to 5 liters, in particular 4 liters; - Snowshoeing, including racket fruits verterated: from 10 to 20 fruits corresponding to 1 to 2 kg, including about 15 fruits corresponding to about kg; sarsaparilla root: 500 g to about 750 g in particular; - bark of mombin: from 500 g to 1.5 kg, particularly about 1 kg; - Grilled castor seeds and ground: 500g to 1.5 kg, including about 1kg. 1.5 kg, of 9 / Compositions having antiretroviral properties, characterized in that they are obtained by letting macerate coconut powder with acetic acid, for about 24 hours, by adding to the filtrate of the mixture maceration method 14 - the ingredients according to one of claims 1 to 8, preferably in the proportions indicated, bringing the resulting mixture to a boil for at least 1 hour, and recovering the filtrate. 10 / Process for obtaining a composition according to any one of the preceding claims, characterized in that the coconut powder is macerated with acetic acid, for about 24 hours, the filtrate is added to the filtrate. maceration mixture the ingredients according to one of claims 1 to 8, the mixture is heated to boiling for at least 1 hour and recovers the filtrate. 11 / Medicaments having particular antiretroviral properties, characterized in that they contain an effective amount of a composition according to any one of claims 1 to 9, in association with an inert carrier. 20 r-l * 1 * '* J [^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^