AP705A

AP705A - Compositions having antiviral properties.

Info

Publication number: AP705A
Application number: APAP/P/1996/000872A
Authority: AP
Inventors: Alix Roland Commin
Original assignee: Alix Roland Commin
Priority date: 1995-03-06
Filing date: 1996-02-13
Publication date: 1998-12-04
Also published as: JPH10506124A; EA199600087A1; DE69613190D1; AU4834096A; FR2731354A1; FR2731354B1; ATE201825T1; AP9600872A0; CA2189474C; WO1996027383A1; EP0768887A1; AU711634B2; DK0768887T3; OA10332A; ES2171653T3; DE69613190T2; EA000112B1; GR3036556T3; CA2189474A1; EP0768887B1

Abstract

Compositions of the invention are elaborated from acetic acid and extracts of coconuts in powder, mineral salts solution, extracts of cactacea, liliaceous, anacardiaceae and euphorbiaceae.

Description

COMPOSITIONS HAVING ANTIVIRAL PROPERTIES:

HOW TO OBTAIN THEM

The invention deals with compositions having antiviral properties in particular and above all antiretroviral properties.

The struggle against diseases due to retrovirus mobilizes a lot of research teams all over the world. Important efforts are particularly engaged to fight against the AIDS epidemic (Acquired Immune Deficiency Syndrome).

We know that the agent responsible for AIDS is a retrovirus called HIV (Human Immune Deficit Virus) according to an English abbreviated form.

The antiviral drugs most used come from nucleosides, including zidovudine, or AZT, which was the first antiretroviral available. Since then, the emergence of tough mutant virus has led to search for new treatments.

The tests conducted by the invetor, from a mix of acid and natural products, have led him to achieve compositions of great impact to inactivate the infectious power of HIV.

This invention is consequently aiming at providing new efficient antiviral compositions.

Its aim is also to find out how to obtain such compositions.

According to the invention, the main features of the compositions are based

AP/P/ 9 6 / 0 0 8 7 2 on the fact that they are elaborated from acetic acid, extracts of coconuts powder, mineral salts solution, extracts of cactacea, liliaceous, anacardiaceae and euphorbiaceae.

The coconut is the coconut tree fruit. According to the invention, the white grated content is largely used.

Cactacea particularly adapted to implement this invention include opuntia. As a reminder, opuntia is the generic name for nopal, prickly pear and other species from tropical America.

The fruits of the green nopal are more specifically used.

In the invention context, appropriated liliaceous include salsepareille. The use of salsepareille root is favourable.

As anacardiaceae, mombin is more particularly used and above all bark of mombin.

An euphorbiacea particularly appreciated is the castor oil plant. Its seeds, 20 when roasted and powdered, are more specifically used according to the invention.

Favourite compositions are elaborated from acetic acid, grated coconuts, sea water, green nopal fruits, salsepareille' root, mombin bark, castor oil plant roasted and powdered seeds.

The use of these different ingredients according to the following proportions is favourable:

- acetic acid: from 5 to 10 liters, specially from 7 to 8 liters;

- coconuts powder (preferably the white grated content): around 10 coconuts;

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- mineral salts solution (specially sodium, potassium, magnesium, calcium, phospates, chlorides, sulphates, carbonates and bicarbonates). For instance, sea water: from 3 to 5 liters, 4 liters in particular;

- nopal and in particular grated fruits of green nopal; from 10 to 20 fruits accounting for 1 to 2 kg and about 15 fruits in particular, accounting roughly for 1.5 kg;

- salsepareille root: from 500 g to 1 kg and about 750 g in particular;

- mombin bark; from 500 g to 1.5 kg and about 1 kg in particular;

- roasted and ground seeds of castor oil plant: from 500 g to 1.5 kg, and about 1 kg in particular.

According to another point of view, the compositions of the invention can be obtained by macerating coconut powder with acetic acid for roughly 24 hours and by adding the different above mentioned ingredients to that filtrate according to the indicated proportions. Then we bring this mixture to the boil for at least 1 hour and we finally pick up the filtrate.

The invention is also aiming at finding out how to obtain the above defined compositions, which main features have been previously mentioned.

The study of the invention compositions properties has pointed out an inhibitory effect of HIV-1 reverse transcriptase activity likely to exceed 90%, compared to the activity calculated on the pilot cells. We can also notice a drop in the cythopathogenic effect of HIV-1 on cells contaminated by the virus and treated with the compositions of the invention.

RPIPI 96/00872

The absence of toxicity of these compositions has been proved on the mouse by intraperitoneal injection with physiological pH.

The invention is consequently aiming at taking advantage of the composition 5 properties in order to develop pharmaceutical compositions.

The main characteristics of the invention pharmaceutical compositions are based on the fact that they include an efficient quantity of at least one previously defined composition, added to an inert pharmaceutical vehicle.

These compositions are particularly adapted to prevent and treat AIDS, combined with other drugs if necessary.

It is better to use these compositions by injection in solutions or isotonic 15 injectable suspensions regarding the blood plasma.

The quantity of active principles changes according to the patient health. The posology of active principle (dry extract) for a man more often changes from 1 to 500 mg a day.

The invention will be illustrated hereafter with an example of composition of great efficiency regarding VIH-1 virus and with results of testings in vitro and in vivo.

First example: Preparation of the composition according to the invention:

We macerate the white dry grated content of 10 coconuts with 7 to 8 liters of white vinegar or acetic acid for at least 24 hours.

The mixture is then filtered.

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We add to the obtained filtrate the following ingredients:

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- solution rich in minerals, like sea water : 4 liters,

- green grated nopal (about 15 fruits) : 1.5 kg

- bark of mombin : 1 kg

- roasted and powdered seeds of castor oil plant : 1 kg

The mixture we get this way is boiled for about 1 hour and then filtered.

Second example: Study of inactivation of V1H-1 with the composition of the first example.

EXPERIMENTAL PROTOCOL . Inactivation of the virus

The suspension of viral particles A in a RPMI 1640 culture (VIH-1 ill B, inverse transcriptase activity 3.8 X 10θ cpm/ml) is incubated with the composition of the first example - hereafter called the composition, (V/V) at a 4°C for 20 minutes, then at the ambient temperature for 1 hour, and finally at a 37°C for 2 hours. At the end of the incubation, the mixture is diluted at a 1/10th, 1/20th, 1/40th, 1/100th, 1/200th in the RPMI 1640 culture, with an added 10% of fetal veal serum first warmed up at a 56°C for 30 minutes.

• infection

Dottles of 5 X 10^ MT4 cells (human ceils of lymphoid origin changed by HTLV-1) are put in suspension again in 250 μΙ of the different dilutions made of a virus-composition mixture.

After one hour of absorption at an ambient temperature and after that the cells have been put in suspension again evey 15 minutes, the virus that has

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AP.00705 not been absorbed is eliminated by centrifugation of the cells. These ones are cleaned twice with RPMI and then put in suspension again in the RPMI medium with a 10% fetal veal serum concentrated at a 2 X 10^/ml. The contaminated cells are put in culture in microtitration plates (Costar 96 pots) split into 200 μΙ/pot.

Every culture is made at least in duplicate. Cultures are diluted in a same medium volume after 5 days and then after δ days of infection.

A series of pilot cultures are contaminated in the same time as the suspension of virus A is melted (V/V) with the RPMI medium. Incubations and dilutions are strictly made under the same conditions as for the virus C4 treated with the composition.

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An additional control is made with the virus preincubated with a 0.15 Μ o NaC1 solution.

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The production of virus has been estimated from a correct proportioning of © inverse transcriptase activity, associated to viral particles that the cells have released in the medium. The inverse transcriptase is also called £* reverse transcriptase or RT in its abbreviated form.

The RT test is made from 65 μΙ of medium, according to the protocol described by Moog and col., in Antiviral Research, 24 (1994), 275-288. The results are expressed in cpm/test (chart, bottom part).

The cellular viability is estimated by the MTT test described by Moog and col., as above mentioned (chart, top part). The colouring intensity is expressed in milli DO.

EXPERIENCE A;

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The VIH production is estimated after contamination of the MT4 ceils with virus preincubated with the composition. After 5 or 7 days of contamination, the RT values rank from 699 to 1,538 cpm/test, whereas for floating particles of non contaminated cultures, they rank from 1,400 to 2,100 cpm/test. Every culture contaminated with the non treated virus produces virus after 5 to 11 days.

EXPERIENCE B:

The production of VIH is estimated after contamination of CEM-SS cells with

VIH-1 LAL

A suspension of virus B (VIH-1 LAi, reverse transcriptase” activity 3 X 10θ cpm/ml) is incubated (V/V) with the composition. All the other stages are described in the experience made on MT4 cells.

Under these conditions, cultures contaminated with the pilot virus (non incubated with the composition) produce virus measurable by RT, 11 days after the contamination.

In the case of cells contaminated with virus preincubated with the composition, the RT are the same as the ones estimated on the floating particles of the non contaminated cultures (1,707 to 2,564 cpm/test) after 11 days of contamination.

EXPERIENCE C:

The experimental protocol which has been followed is the same as the one of experience A. The measurements made 5 days and 7 days after the contamination show that we can't or almost can't detect virus replication

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AP . 0 0 7 0 5 when it has been preincubated with the composition (RT of non contaminated pilot ceils 972-12,192 cpm/test).

EXPERIENCE D:

In order to estimate the production of virus more quickly than in experimentation B, a virus concentration 10 times more important has been used.

After 5 to 8 days, the production of virus is strongly inhibited by the preincubation of the virus with the composition.

The absence of effect we can notice when using the 0.15 M NaC1 solution shows tha the virus inhibition is specific to the composition.

These results consequently show that the VIH-1 virus preincubated with the composition of the invention leads to an inactivation of the infectious power of the viral particles.

Example 3: Study of the composition effect of the first example on the transcriptase reverse activity of VIH-1.

A suspension of the ViH-1 virus has been melted (V/V) to different dilutions of the composition. The transcriptase reverse activity (RT) has been determinated, in association with the viral particles, as described by Moog and al. in the above reference.

The measurements have been made on at least two samples.

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The results obtained are mentioned in the first chart.

We notice that the use of the composition, when the concentrations are the more elevated, inhibite the RT activity to more than 90%.

Another series of experiences, which results are given in the second chart, confirm this information.

These experiences consequently bring to the fore that the compositions can 10 inhibite the transcriptase reverse activity measured in vitro.

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Chart 1 - RT activity (cpm/test)

FINAL DILUTION OF THE SOLUTIONS	COMPOSITION/VIRUS
1/2		AVERAGE
1,164 783	973 (93%)*
1/6	759 688	724 (94%)
1/8	2,453 2,875	2,664 (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

PILOT VIRUS	14,318 , 10,743 11,764	AVERAGE
	12,687
	12,218	12,794
	15,036

The figures in brackets indicate the % of inhibition of the RT activity.

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Chart 2 - RT activity (cpm/test)

FINAL DILUTION OF THE SOLUTIONS	I j COMPOSITION/VIRUS i i
	I	I AVERAGE
1/2	550 783	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 75,351	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%)
1/1,024	167,936 144,598	156,267

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		157,233	AVERAGE
PILOT VIRUS . ...		157,059	156,146

The figures in brackets indicate the % of inhibition of the RT activity.

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Claims

1. Compositions having antiviral properties in particular and more specifically

5 antiretroviral properties, characterized by the fact that they are elaborated from acetic acid and extracts of coconuts powder, mineral salts solution, extracts of cactacea, liliaceous, anacardiaceae and euphorbiaceae.

2. Composition according to claim 1, characterized by the fact that we use

10 the grated white content of the coconut.

3. Composition according to ciaim 1 or 2, characterized by the fact that we use an opuntia, nopal in particular and more specifically the green nopal fruits as cactacea .

4. Composition according to one of the 1 to 3 claims, characterized by the fact that we use salsepareille and in particular root of salsepareille as liliaceous.

20 5. Composition according to one of the 1 to 4 claims, characterized by the fact that we use mombin and in particular bark of mombin as anacardiaceae.

6. Composition according to one of the 1 to 5 claims, characterized by the fact that we use castor oil plant seeds and in particular castor oil plant

25 roasted and powdered seeds as euphorbiaceae.

AP . Ο Ο Ί Ο ζ

7. Composition according to one of the 1 to 6 claims, characterized by the fact that it is elaborated from acetic acid, grated coconut, sea water, green nopal fruits, salsepareille root, mombin bark, castor oil plant powdered and roasted seeds.

8. Composition according to claim 8, characterized by the fact that we use the different ingredients according to the following proportions:

- acetic acid: from 5 to 10 liters and from 7 to 8 liters in particular;

- coconut powder (in particular the white grated content): equivalent to roughly 10 coconuts;

- mineral salts solution (sodium, potassium, calcium, magnesium, 15 phosphates, chlorides, sulphates, carbonates and bicarbonates in particular), sea water, for example: from 3 to 5 liters and 4 liters in particular;

- nopal and grated fruits of green nopal in particular: from 10 to 20 fruits accounting for 1 to 2 kilos and around 15 fruits accounting for roughly 1.5 kg

20 in particular;

- salespareille root: from 500 g to 1 kg and around 750 g in particular;

- bark of mombin: from 500 g to 1.5 kg and around 1 kg in particular;

- ground and roasted seeds of castor oil piant: from 500 g to 1.5 kg and around 1 kg in particular.

9. Compositions having antiretroviral properties, characterized by the way we 30 obtain them: we macerate coconuts in powder with acetic acid for roughly 24

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00705 hours and we add the ingredients to the filtrate according to one of the 1 to 8 claims and according to the indicated proportions. We then bring the mixture to the boil for at least 1 hour and we pick up the filtrate.

5 10. How to obtain one composition according to any previous claim, taking into account that we macerate coconuts in powder with acetic acid for roughly 24 hours and we add mixture of the macerated ingredients to the filtrate, according to claims 1 to 8 and we bring the mixture to the boil for at least 1 hour and we finally pick up the filtrate.

U 10

11. Drugs having antiretroviral properties, characterized by the fact that they possess a sufficient quantity of one composition according to any revendications from 1 to 9, associated to an inert vehicle.