EP2456452A1

EP2456452A1 - Antiviral and retroviral composition, in particular for treating aids

Info

Publication number: EP2456452A1
Application number: EP09784289A
Authority: EP
Inventors: Alix Roland Commin
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-07-22
Filing date: 2009-07-22
Publication date: 2012-05-30
Also published as: WO2011010000A1

Abstract

The invention relates to compositions having antiviral properties, in particular antiretroviral properties for treating AIDS. The invention also relates to a method for producing same. The compositions of the invention are characterized in that they are developed from acetic acid and coconut extracts, a solution of mineral salts, and extracts from the Cactaceae, Liliaceae, Anacardiaceae and Euphorbiaceae families.

Description

ANTIVIRAL AND RETROVIRAL COMPOSITION, IN PARTICULAR FOR THE TREATMENT OF AIDS

The invention relates to compositions having in particular antiviral properties, and in particular antiretroviral.

The fight against diseases caused by retroviruses mobilizes many teams of researchers around the world. In particular, considerable efforts are being made to combat the AIDS epidemic (acquired immunodeficiency syndrome). It is known that the agent responsible for AIDS is a retrovirus called HIV, for human immunodeficiency viruses, or most commonly HIV, according to the abbreviated English name.

The most commonly used antiviral drugs are nucleoside derivatives, including zidovudine, or AZT, which was the first antiviral available. Since the emergence of resistant mutants led to seek new treatments.

The work done by the inventor on a mixture of products of natural origin led him to develop high efficiency compositions to inactivate the infectivity of HIV. The object of the invention is to provide new antiretroviral compositions capable of.

It also relates to a process for obtaining such compositions.

The compositions according to the invention are characterized in that they are prepared from coconut extract, solution of mineral salts, extracts of cactacea, liliaceae, anacardiaceae and euphorbiaceae, grass muskia, passiflora, rutaceae, artocarpea, bombaceae and lice of wood.

The coconut is the fruit of the coconut tree. According to the invention, the grated white meat is advantageously used.

Cactaceae particularly suitable for the implementation of the invention include opuntia.

It is recalled that opuntia is the genetic name for snowshoes, nopal, prickly pears and other species native to tropical America. We use more especially the fruits of the green racket.

Suitable lilies within the scope of the invention include sarsaparilla, and Paloes. Sarsaparilla root and aloe cream are used with advantage.

As anarcadiacea, mombin, particularly the bark of mombin, is particularly used.

Particularly preferred Euphorbiaceae are castor and sweet cassava. Castor seeds, especially in the toasted state and in the form of powder, are more particularly used for the invention. Cassava is used in grated form in the context of the invention.

Suitable grasses in the context of the invention include maize and vetiver. The stigmas of maize (corn hair) and vetiver roots are used with advantage.

As musacé, we particularly use rotten banana roots. A particularly preferred passiflora is the brown calabash.

Rutaceae particularly suitable for the implementation of the invention include lemon.

Appropriate artocarpés within the scope of the invention include the breadfruit. The fruit of the breadfruit is advantageously used.

As bombaceous, we particularly use mapou or baobal root. Wood lice are also used in the context of the invention. Living lice are advantageously used in their nest.

Preferred compositions are made from grated coconut, seawater, green racquet fruit, sarsaparilla root, mombin bark, ground seed powder grilled castor oil, aloe, corn, vetiver root, rotten and dried banana heart, wooden lice, brown callabasse, breadfruit lemon, mapou or baboal, grated sweet cassava.

These different ingredients are advantageously used in the following proportions: coconut powder (preferably, shredded white flesh): equivalent to about 10 coconut

-solution of mineral salts, especially sodium, potassium, magnesium, calcium, phosphates, chlorides, sulphates, carbonates and bicarbonates), for example seawater: 3 to 5 liters, in particular 4 liters

-Racket, including grated green racket fruit: 10 to 20 fruits corresponding to 1 or

2 kg

- Sarsaparilla root: 500g to IKg, in particular 75g approximately

-Minbin bark: from 500g to 1.5kg especially IKg

- roasted and ground castor seeds: from 500 g to 1.5 kg, in particular approximately 1 kg - from 100 to 300 g of rotten banana heart center, and preferably 200 g

- 10 to 30 g of corn stigma or the value of 2 corn

- 300 to 700 g grated sweet cassava

Aloe can weigh from 75 to 125g, including 100g. One tablespoon of calabash is used. A lemon is used, preferably cut into at least three pieces. 15 to 35 centigrams of mapou or baobal bark are used, and preferably about 25 centigrams. Vetiver root is used at a rate of 10 to 30 g centigrams, preferably 20 g. The fruit of the barely ripe breadfruit represents a quantity in the composition of 10 to 30g. 1 to 2 liters of lustral water are used, in particular 1.51; obtained in the following manner: take the living wood lice in their nest, burn the nest with the lice; the ash obtained is placed on a cloth and passed to the rain water; the liquid thus obtained is lustral water. 400 to 600 ml of vinegar are used as acetic acid, in particular 500 ml.

The invention aims to cure AIDS and cure and relieve prostate and cyst. When it is desired to obtain a virucidal effect, 4 to 5 liters of vinegar are used.

The compositions of the invention are as obtained by macerating coconut powder with vinegar for about 24 hours, adding to the filtrate of the maceration mixture the various ingredients listed above, preferably in the proportions indicated, by bringing the mixture obtained to boiling for at least 1 h and recovering the filtrate. The solution is then subjected to a conventional active compound protocol.

The invention also relates to a process for obtaining the compositions defined above, characterized by the steps previously reported.

It was proceeded to:

l) optionally delipidation at neutral pH with pentane which was conducted, after evaporation of the solvent, to a first fraction called Fl, then with methylene chloride to give the fraction F2 after evaporation under reduced pressure.

2) The solution is then basified to pH 9 by adding ammonia and extracted with ethyl acetate and then methylene chloride which, after removal of the solvent, gives the fraction F7.

The table below gives the different masses of substrates obtained under these conditions.

Volume of solution: 200 ml

Mass of solution: 2034, 8g

Appearance: light orange

pH: 4-5

Given the identity of the IR spectra and HPLC chromatograms (attached), the fractions extracted with dichloromethane and ethyl acetate are combined. The initial fraction extracted with pentane of different characteristics.

These two fractions are purified by HPLC on a C 18 semi-separative column.

We obtain as follows:

66 mg of the fraction extracted with dichloromethane and with ethyl acetate,

- 73 mg of the fraction extracted with pentane

After complete chemical analysis by IR, UV and NMR (proton and carbon) techniques - 73 mg of fraction extracted with pentane F2

53 mg of the fraction extracted with dichloromethane and with ethyl acetate F7

The two fractions F2 and F7 were solubilized in 200 μl of dimethysulfoxide o Inactivation of the virus

The suspension of viral particles A in the RPMI 1640 culture medium (HIV-I HIB, reverse transcriptase activity 3.8 × 10 ⁶ cpm / ml) is incubated with F ^{2 and} F ⁷ for 20 min at 4 ° C. and then 1 hour at room temperature. finally 2 hours at 37 ° C. at the end of incubation, the mixture is diluted to 1 / 1O ⁰ ^"16, 1/20, 1/40, 1/100, 1/200 in the culture medium RPMI 1640 plus 10% fetal calf serum (FCS), preheated for 30 minutes at 56 ° C.

o Infection

Pellets of 5 × 10 ⁵ MT4 cells (human cells of lymphoid origin transformed with HTLV-I) are resuspended in 250 μl of the various dilutions of the virus composition mixture.

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

All crops are made at least in duplicate. 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.

The production of HIV after infection of MP4 cells with virus preincubated with the composition is evaluated.

F2 and F7 fractions inhibit HIV-I replication in MT-4 cells.

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 an inert pharmaceutical vehicle.

These compositions are particularly suitable for the prevention and treatment of AIDS, if necessary in combination with other drugs. 1538 cpm / test, whereas, for supernatants of uninfected cultures, they are 1400-2100 cpm / test. All cultures infected with the untreated virus produce J5 virus at J1. Experiment B: HIV production after infection of CEM-SS cells by HIV-I Lai is evaluated

A suspension of virus B (HIV-I Lai, reverse transcriptase activity 3x10 ⁶ cmp / ml) incubated (v / v) with the composition. All other steps are those described for the MT4 cell experiment.

Under these conditions, cells infected with the virus preincubated with the composition, the RTs are similar to those measured on supernatants of well infected cultures (1707-2564) after 11 days of infection. Experience C:

The experimental protocol followed is identical to that of experiment A. The measurements said on day 5 and day 7 post-infection show that little or no replication of the virus is detected when it has been preincubated with the composition ( RT uninfected control cells 972-1292 cpm / test)

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 strongly inhibited by preincubation of the virus with the composition.

The lack of effect observed using the 0.15 M NaCl solution that the inhibition of the virus is specific to the composition.

These results therefore show that the preincubation of the HIV-1 virus with the composition of the invention leads to an 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 of HIV-1

An HIV-virus suspension was mixed (v / v = at different dilutions of the composition.

The determination of the reverse activity was carried out as described by Moog and AL, in the reference above.

The measurements were performed on at least two samples.

The results obtained are reported in Table 1.

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

Another series of experiments, the results of which are given in Table 2, confirm the previous data.

These experiments therefore show that the compositions according to the invention are capable of inhibiting the activity of the reverse transcriptase in vitro. These compositions are preferably administered by injection in the form of isotonic injectable solutions or suspensions with respect to the blood plasma.

The study of the properties of the compositions of the invention has demonstrated an inhibitory effect of the HFV-I reverse transcriptase activity that may exceed 90% relative to the activity measured in the controls. There is also a decrease in the cytopathic effect of HIV-I in cells infected with this virus, 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 aim of the invention is to make use of the properties of these compositions for the preparation of pharmaceutical compositions.

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 ingredient (dry extract).

The invention will be illustrated hereinafter by an example of preparation of a composition of high efficiency with respect to the HIV-I virus and by the results of in vitro and in vivo tests.

Example 1 Preparation of a Composition According to the Invention

The following ingredients are added to the recovered filtrate:

-solution rich in minerals, such as seawater: 41;

grated green racquet: 1, 5 Kg

Sarsaparilla root: 750g

-Minbin Bark: IKg

-Grilled castor seed powder: 1 Kg

-20 g of corn stigma or the value of 2 corn

-400 g grated sweet cassava

-lOOg of aloe

-1 tablespoon of calabash

-a lemon, preferably cut into at least three pieces

mapou or baobal bark: 25 centigrams

vetiver root: 20g

- the fruit of the barely ripe breadfruit: 20g

- Lustral water: 1,51

The mixture thus obtained is boiled for approximately 1 h and then filtered.

Example 2: Study of HIV-inactivity by the composition of Example 1 Virus production was measured by an assay of the reverse transcriptase activity associated with the viral particles released by the cells into 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, bottom).

Cell viability is measured by the MTT assay described by Moog et al. In the reference given above (table, top). The intensity of the coloration is expressed in mili DO.

Experience A:

The production of HIV after infection of MT4 cell with virus preincubated with the composition is evaluated. After 5 or 7 days of infection, the RT values are between 699 and

Claims

1 / composition having in particular antiviral properties and, in particular, prepared from acetic acid and extracts of coconut powder, mineral salts solution, extracts of cactacea, liliaceous, anacardiaceae, and euphorbiaceae.

2 / composition according to claim 1, characterized in that the shredded white flesh of the coconut is used.

3 / composition according to claim 1 or 2, characterized in that an opuntia is used, such as cactacea, especially racket, especially fruits of the green racket.

4 / composition according to one of claims 1 to 3, characterized in that used as liliaceous, sarsaparilla, in particular of the sarsaparilla root.

5 / composition according to one of the claims of 4 characterized in that as anacardiaceae, mombin, especially mombin bark.

6 / compositions according to one of claims 1 to 5, characterized in that, as Euphorbiaceae, used castor seeds, in particular grilled castor seed powder;

7 / composition according to one of claims 1 to 6, characterized in that it comprises:

coconut powder (preferably shredded white meat): equivalent to about 10 coconut, solution of mineral salts (especially sodium, potassium, magnesium, calcium, phosphates, chlorides, sulphates, carbonates and bicarbonates) eg water of coconut sea: 3 to 5 liters, especially 4 liters.

Snowshoes, including grated green racket fruits: 10 to 20 fruits corresponding 1 or 2 kg

Sarsaparilla Root: 500g to 1.5kg, including about 75g of mombin bark: 500g to

1, 5 kg especially 1 kg

grilled castor seed and ground: from 500g to 1.5kg, in particular about 1kg from 100 to 300g of rotten banana heart center, and preferably 200g

10 to 30 stigmas of corn or the value of two 300g corn, 700g grated sweet cassava

Aloe can weigh 75 g to 125 g, including 100g. One tablespoon of calabash is used. A lemon is used, preferably cut at least three pieces.

15 to 30 centigrams of mapou or baobal bark are used

And preferably about 25 centigrams;

Vetiver root is used at a rate of 10 to 30 centigrams

Preferably at 20 g

The barely ripe breadfruit represents a quantity

In the composition 10g to 30g

1 to 2 liters of lustral water are used, in particular 1. 51 liters are obtained

As follows :

Burn the nest with the lice; the ash obtained and arranged on the

Linen and spent in rainwater;

400 to 600ml of vinegar is used as acetic acid

In particular 500ml. 8 / composition according to one of claims 1 to 6, characterized in that it is prepared from acetic acid, grated coconut, sea water, green racquet fruit, root sarsaparilla , mombin bark, grilled castor seed powder.

9 / composition according to claim 8 characterized in that the different ingredients are used in the following proportions;

- 400 grams of vinegar and 3 liters or 4 liters of acetic acid

- coconut powder (preferably, shredded white flesh) equivalent to about 10 coconuts.

Solutions of mineral salts (in particular sodium, potassium, calcium, magnesium, phosphates, chlorides, sulphates, carbonates and bicarbonates)

For example seawater; 3 to 5, in particular 4 liters,

snowshoeing, including grated green racquet fruit of 10 to 20 fruits corresponding to 1 to 2 kg, in particular about 15 fruits corresponding to about 1, 5 kg.

Sarsaparilla Root: 500g to 1.5, in particular about 750g

Bark of mombin; 500g to 1kg, especially about 1kg,

Castor seed grilled and ground; 500g to 1.5kg including about 10kg

10 / Process for obtaining a composition according to any one of the preceding claims, characterized in that coconut powder is macerated with acetic acid for about 24 hours,

The ingredients according to one of claims 1 to 8 are added by filtering the maceration mixture, the mixture is boiled for at least one hour and the filter is recovered. 11 / Medicines having in particular antiviral properties; characterized in that they contain an effective amount of a composition according to a composition according to any one of claims 1 to 9 in combination with an inert carrier.

Table 2 - RT activity (Cpm / test)

Average 157.233

Witness virus 155.059 156.146

* Numbers in parentheses indicate% inhibition of RT activity. Table 1 Activity RT (Cpm / eεsai)

FINAL DILUTION COMPOSITION / VIRUS SOLUTIONS

AVERAGE

1/2 1.164

783,973 (93%)

759

1/6 688 724 (94%)

2453

1/8 2.875 2664 (80 *)

6875

1/54 5,949 6,412 (50%)

10.035

1/162 8.220 9.128 (29%)

11747

1/486 12.981 12.364

14,318 AVERAGE

10743

11764

WITNESS VIRUS 12.794

12687

12.218

15036

* Numbers in parentheses indicate% inhibition of RT activity Table 2 Activity RT (Cpm / test)

* Numbers in parentheses indicate% inhibition of RT activity Mass of initial solution = 61g Volume of initial solution = 60-65mt pH initial solution% 6

Following these results, the fractions F4 and F5 are assembled in ethyl acetate F7 = 0.009 g

Mass of initial solution = 61g Volume of initial solution = 60-65ml pH initial solution - 6

The F6 fraction is taken up in Tethanol. After filtration, the cake is precipitated.

The ethanoic phase is evaporated F8 is the solid obtained

πifH) = 0.728 g

m _F = 0.965 g

We do the same thing with F6 "

nV (r = 0,780g

nv _r = 0.839g Fractions of Project 1.1 are grouped together with those corresponding to Project 2

Summary of fractions and masses obtained

Fraction obtained after delipidation (extraction with Pentane)

Fl mass≈ 0.6mg

Fraction obtained after delipidation (extraction with methylene chloride)

F2 mass≈ lmg

Fraction obtained after acidification

F3 mass≈ 14mg

Fraction obtained after basification

F7 mass ≈ lmg

Fraction obtained after a second lyophilization

F7 'mass = 0.480g

Fractions obtained after Pethanol recovery:

After filtration, the precipitate named FO

After evaporation the paste named F8

FO mass = 1 -405g

F8 mass = 1.596g