OA19672A - Therapeutic composition derived from anthostema senegalense. - Google Patents
Therapeutic composition derived from anthostema senegalense. Download PDFInfo
- Publication number
- OA19672A OA19672A OA1201900277 OA19672A OA 19672 A OA19672 A OA 19672A OA 1201900277 OA1201900277 OA 1201900277 OA 19672 A OA19672 A OA 19672A
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- OA
- OAPI
- Prior art keywords
- molécule
- molécules
- chemical composition
- hiv
- plant extract
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- 239000000203 mixture Substances 0.000 title claims abstract description 81
- 241001553164 Anthostema senegalense Species 0.000 title claims description 28
- 230000001225 therapeutic Effects 0.000 title description 2
- 230000000694 effects Effects 0.000 claims abstract description 71
- 239000000126 substance Substances 0.000 claims abstract description 57
- 150000003839 salts Chemical class 0.000 claims abstract description 25
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- 238000000338 in vitro Methods 0.000 claims description 10
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- 150000001408 amides Chemical class 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 230000001476 alcoholic Effects 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 238000001802 infusion Methods 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- 238000002955 isolation Methods 0.000 claims description 7
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- KSEBMYQBYZTDHS-HWKANZROSA-N Ferulic acid Chemical group COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 3
- 229940098465 Tincture Drugs 0.000 claims description 3
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- BXZVVICBKDXVGW-NKWVEPMBSA-N ddIno Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 3
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- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
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- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
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- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
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- QDUDLLAGYKHBNK-QPYQYMOUSA-N gammacerane Chemical compound C([C@]1(C)[C@H]2CC[C@H]34)CCC(C)(C)[C@@H]1CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CCCC1(C)C QDUDLLAGYKHBNK-QPYQYMOUSA-N 0.000 description 1
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- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
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- KZJWDPNRJALLNS-VJSFXXLFSA-N β-Sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
Abstract
The present invention relates to a chemical composition characterized in that it comprises: - the molecule A or a physiologically acceptable salt thereof, and/or: - the molecule B or a physiologically acceptable salt thereof, said molecule A having the following formula: and the molecule B having the following formula: the molecule A or a physiologically acceptable salt thereof being biologically active and stimulating immune activity, the molecule B or a physiologically acceptable salt thereof being biologically active and stimulating immune activity.
Description
LABORATOIRE MICHEL IDERNE
Therapeutic composition derived from anthostema senegalense
The présent invention fallsAvithin the field of Chemical compositions that are suitable for use in the préparation of médicinal products or food / dietary suppléments. -
In particular, the invention makes possible the préparation, of médicinal products· or food suppléments that are capable of enhancing the body's immune system and defences, in particular .against viruses, bacteria, fungi, and protozoa that are generally responsible for a number of sometimes life- threatening diseases.
The invention relates specifically to a Chemical.composition that comprises of biologically active molécules that contribute to increasing and stimulating the immune activity in the body and thus enhance the immune defences, in particular against viruses.
More particularly, the Chemical composition of the invention exhibits a biological activity against the Human Immunodéficiency Virus (HIV) and its résultant; Acquired Immune Deficiency Syndrome (AIDS), generally accompanied by 15 opportunistic infections thereof.
It should be noted that HIV is a retrovirus that infects humans and of which two 'types are known; HIV-1 and HIV-2. The HIV infection is a pandémie that is widespread across the entire planet, particularly sub-Saharan Africa which alone “ accounts for 69% of ail cases. The HIV infection evolves in a patient in multiple phases;
- The first phase, referred to as primary infection ; .
- An asymptomatic phase, referred to as latency, ' · '
- An increased immunosuppression phase, referred to as AIDS.
The primary infection phase consists of active réplication, in the body of HIV which causes destruction of the cells of the immune system in particular T4 lymphocyte 2 5 cells.
The phase of latency represents a phase m which the viral load decreases slightly andis stabilisedinthebody. . G H
The AIDS phase consists-of a greater multiplication of HIV which is invasive and wherein the symptoms of AIDS as well as opportunistic diseases appear, êventuallyculminating in the death of the patient.
To date, there does not exist any effective treatment to cure AIDS and completely eliminate lhe development and spread of the virus (HIV) in the body of the patients. · ’’ ’ '
In a known matter, various types of treatments such as triple therapy treatments combining three antiviral molécules are used in order to slow the growth and multiplication of HIV in the body and its progression to the AIDS stage.
However, these treatments continue to be very expensive with a high purchase price mainly due to the long lead time for the manufacture and production thereof at industrial scale. Moreover, the latter require very rigorous storage conditions and their access is restricted in respect of patients in developing countries.
Furthermore, current treatments are not always effective in ail patients. Indeed, the rapid pace of multiplication and mutation of HIV in the body being such, these treatments lose their effectiveness over time.
In addition, even when they are effective, these treatments hâve the disadvantage of presenting many undesirable side effects on the health of the patient being treated who consequently will lose quality of life. In fact, current treatments présent a certain level of toxicity that is harmful to the patient's body, in particular due to a number of synthetic Chemical excipients of non-natural origin that may présent the risk of rejection by the body.
Thus, the présent invention is aimed at overcoming the drawbacks of the State of the art by providing a Chemical composition consisting of an alternative to the above noted solutions. This alternative must be effective and serve to enable enhancing the body’s immune defences, in particular in patients afflicted with HIV, while also minimising the side effects from administering the médication comprising the alternative Chemical composition.
In addition, this alternative solution must be accessible to patients in the developing countries, both in terms of production costs and purchase costs, but also with regard to storage of the active product, and ease of administration and use by the patient.
In addition, in order to achieve this objective, the Chemical composition needs to be of the lowest toxicity possible to the body and should target only the viruses, bacteria, fungi, and protozoa responsible for pathologies or disease conditions that weakening the immune system. In order to limit the risks of toxicity, it is advantageous for the Chemical composition to présent a maximum of éléments that are natural in origin (naturally sourced), and considered to carry no/low risk of causing harm to health and the environment.
The présent invention is aimed at overcoming the drawbacks of the state of the art, by providing a Chemical composition comprising :
- the molécule A or one of the physiologically acceptable salts thereof, and/or;
- the molécule B or one of the physiologically acceptable salts thereof, 5 - the said molécule A having the following formula :
and the molécule B having the following formula :
In a spécifie manner in the Chemical composition of the invention :
- the molécule A or one of the physiologically acceptable salts thereof is biologically active and is a stimulator of immune activity ;
- the molécule B or one of the physiologically acceptable salts thereof is biologically active and is a stimulator of immune activity.
Advantageously, the said molécule A présents :
- at position 16, as a substitute for the carbonyl function =0, either an - OH, or an aldéhyde carbonyl group - RCOH, or a ketone - RI - CO - R2, or an ester R - COO - R', or an amide of the type - R - C(=O)NH2 ; and / or
- at position 3, a glycosylation or estérification in the form of - OR, where R 20 is either a feruloyl or caffeoyl group, or a sugar or an amide.
Also advantageously, the said molécule A is conjugated with amino acids, quatemary ammonium salts, glycosides, hemiphtalates, or even carbamates.
Also in an advantageous manner, the said molécule B présents, at position 28, an estérification of an amide or a sugar.
In addition, according to other characteristic features of the invention, the Chemical composition of the invention comprises at least one of the following molécules or one of the physiologically acceptable salts thereof, or one of the possible combinations of the said following molécules, the said one or more molecule(s) being 5 selected from the following list :
- the molécule C having the formula :
wherein n is comprised between 8 and 26 :
- the molécule D having the formula :
the molécule E having the formula :
- the molécule F having the formula :
- the molécule G having the formula :
- The molécule H having the formula :
- The molécule I having the formula :
with n comprised between 14 and 26 ;
- The molécule J having the formula :
According to a first embodiment of the invention, each of the said molécules A, B, and each of the physiologically acceptable salts thereof is of synthetic origin and derived from a Chemical synthesis in the laboratory or from a hemi - synthesis.
According to another characteristic feature of the invention, each of the said molécules A, B and each of the physiologically acceptable salts thereof of the said composition of the invention is of natural origin and derived from a plant extract of Anthostema senegalense.
Advantageously, the plant extract of Anthostema senegalense :
- consists of a polar extract, that is aqueous, alcoholic or hydro - alcoholic or an apolar (or non-polar) extract or a mother tincture of Anthostema senegalense with an éthanol content of 70% V / V ;
- the said plant extract is obtained from the trunk/stem bark, in particular dried bark, of Anthostema senegalense.
Also in an advantageous manner, the said Chemical composition of the invention is intended to be used as a médicament or as a food - dietary supplément.
Even more specifically, the said Chemical composition of the invention is intended to be used in the treatment of HIV infection type 1 or type 2, AIDS and the clinical manifestations that accompany it.
Preferably, the said Chemical composition of the invention is in an oral galenic form or in the galenic form of an infusion sachet.
According to another embodiment, the said composition of the invention is in an oral galenic form of microspheres produced by a method of extrusion and spheronisation, of tablets, soft capsules, granules, gel capsules, powders, ampoules, syrups or even décoctions.
The présent invention also relates to an isolation method for isolating by means of extraction, identification and sélection of the one or more biologically active molecule(s) of the Chemical composition of the invention in which :
- A part of the Anthostema senegalense plant is dried ;
- A plant extract is prepared, from the dried part of the plant, by extraction with an apolar or polar solvent or by macération, infusion, décoction, percolation, digestion or leaching ;
- The said plant extract is concentrated under vacuum ;
- The molécules of the plant extract are separated by chromatography ;
- In vitro testing is carried out on the biological activity of the said molécules against an HIV-type viral pathogen according to the method of E de Clercq ;
- The molécules having a selectivity index against the said pathogen >50 and a 50% cytotoxic concentration CC50 > 45 pg/mL are selected and isolated ;
- It is then necessary to identify the one or more molécules that hâve been separated, selected and isolated by means of nuclear magnetic résonance and mass spectrometry.
More specifically for the operational implémentation of the said method of the invention :
- the part of the Anthostema senegalense plant which is dried consists of the bark of the trunk/ stem, or the bark of the roots, or the leaves or the fruits ;
- a plant extract is prepared by percolation with CHC13 in order to obtain an apolar plant extract ;
- after concentration under vacuum, the molécules of the plant extract are separated by means of repeated column chromatography and thin layer chromatography.
In an advantageous manner, in the said method of isolation by extraction of the one or more biologically active molecule(s) that stimulate the immune System, it is necessary to separate the molécules of the said apolar plant extract by column chromatography, using as the stationary phase a polar silica gel and, as the mobile phase, an apolar eluent constituted of a gradient of CHCI3 in hexane.
Other characteristic features and advantages of the invention shall become apparent from the detailed description that follows of non - limiting embodiments of the invention.
The présent invention relates to a Chemical composition comprising the molécule A and / or molécule B, or one of the physiologically acceptable salts thereof.
In other words, the said Chemical composition comprises the molécule A or one of the physiologically acceptable salts of the molécule A ; either in the presence or not of the molécule B or one of the physiologically acceptable salts of the molécule B.
In similar fashion, the said Chemical composition comprises the molécule B or one of the physiologically acceptable salts of the molécule B ; either in the presence or not of the molécule A or one of the physiologically acceptable salts of the molécule A.
The term physiologically acceptable is used to refer to the fact that the one or more molecule(s) as well as their salts do not adversely affect the functioning or the mechanical -, physical -, or biochemical organisation of a living organism, especially of a human.
The Chemical composition of the invention is physiologically acceptable in its entirety, that is to say that ail of the éléments that compose it are physiologically acceptable. The said Chemical composition of the invention is physiologically acceptable through its various different components, that is to say, it is biocompatible, entails little or no toxicity for the proper functioning of the body and with no physical or Chemical properties that render its administration and metabolism difficult and complicated.
Thus, the composition of the invention is also pharmaceutically acceptable, that is to say, it may be used as a médicament to treat a patient and that there are more positive than négative effects on the health of the patient.
The said one or more molecule(s) A and/or B are biologically active, in particular against viral, bacterial, and protozoan entities or fungi that are responsible for causing various pathologies. In other words, the molécule A or the molécule B has a single effect or act in a synergistic manner against the prolifération of the said entities that are responsible for causing the said pathologies.
Thus, thanks to the capacity to be biologically active, the said one or more molecule(s) A and / or B hâve the property and effect of stimulating the immune activity in the body and of enhancing it.
According to the invention, the molécule A, a pentacyclic triterpenoid gammacerane, has the following formula :
According to one particular embodiment of the invention, the formula of the molécule A may be modified in a manner such that :
- At position 16, the carbonyl =O cambe reduced and give an - OH, or even the aldéhyde carbonyl functional group -RCOH, a ketone -R1-CO-R2, an ester - R - COO - R’, an amide for example of the type - R - C(=O)NH2 ;
- At position 3, a glycosylation or estérification may be carried out in the form of a functional group of the type 3 - OR, wherein R may be a feruloyl or caffeoyl group, a sugar or an amide for example of the type - R - C(=O)NH2.
According to another particular embodiment, the molécule A may be conjugated with amino acids, quatemary ammonium salts, glycosides, hemiphtalates or even carbamates.
According to the invention, the molécule B is presented with the following formula :
According to one particular embodiment, the molécule B may undergo estérifications at its carbon 28 position in particular with such types of sugars as rhamnose, arabinose, glucose, amino sugars, glucosamine, galactosamine or even with amides of such type as benzylamide, heteroaromatic amide.
For example, for the molécule B, a coupling can be brought about with a side chain of amino acids or peptides among others at its carbon 28.
According to one particular embodiment, the molécule B may undergo modifications at its carbon 3 position, such as :
- estérifications with various acids such as ferulic acids or caffeic acids, hydroxycinnamic acids, diméthylsucciniques acids ;
- conjugations with nucleosides that are inhibitors of reverse transcriptase such as zidovudine, azidothymidine ;
- bonds with sugar types such as mono or oligosaccharides.
Preferably, the said Chemical composition of the invention may comprise, in addition to the molécules A and/or B, one of the following molécules, one of the physiologically acceptable salts thereof, or even one of the possible combinations of the said following molécules :
- The molécule C, a ferulic acid ester, having the formula :
with n comprised between 8 and 26 ;
The molécule D : macrocyclic polyprenoide, having the formula :
- The molécule E: 3 - 0 - acetyl - o - friedo - olean - 14-en-28-oie-acid (Oacetyl aleuritolic acid), having the formula :
- The molécule F: a (3p)-Stigmasta-5, 22, 25-trien-3-ol having the formula :
- The molécule G, a β - Stigmasterol, having the formula :
- The molécule H, a β - Sitosterol, having the formula :
The molécule I, an iso - alcohol benzoate, having the formula :
With n comprised between 14 and 26 ;
- The molécule J having the formula :
In an advantageous manner and according to one particular embodiment, each of the molécules A to J mentioned above is of natural origin, derived and selected from a plant extract of Anthostema senegalense in particular for its ability and capacity to be biologically active.
Advantageously, the said plant extract is obtained from the bark of the trunk, 15 especially dried, of Anthostema senegalense.
However, other parts of the Anthostema senegalense plant such as the leaves, root bark or fruit can also be used in order to obtain the said plant extract.
Preferably, the said plant extract of Anthostema senegalense consists of a polar extract, that is aqueous, alcoholic or hydro - alcoholic, or an apolar extract, or even a mother tincture of Anthostema senegalense with an éthanol content of about 70% V/V.
Starting from a plant extract of natural origin in order to obtain the biologically active molécules that are stimulators of immune activity has the advantage of rendering them accessible in ternis of production costs for the developing countries.
Indeed, Anthostema senegalense is a forest tree widespread across West Africa, in particular in Guinea, Guinea - Bissau, Sénégal, Mali, Ivory Coast, Sierra Leone, Bénin, Nigeria, etc, which are countries having little means and consequently where the possibility of access to health care is often limited.
In addition, starting from a plant extract of natural origin makes it possible to limit the risks of toxicity and enters an environmentally responsible approach where the molécules of interest are drawn directly from the natural environment.
According to one other embodiment, each of the molécules A to J mentioned above is of synthetic origin, that is to say, derived from a complété Chemical synthesis in the laboratory environment or even from a hemi - synthesis.
According to one particular feature of the invention, the said Chemical composition of the invention is intended to be used as a médicament or as a fooddietary supplément.
In particular, the said Chemical composition of the invention is intended to be used by fatigued individuals whose immune System has been weakened by the presence of a pathogen whose origin is viral, bacterial, fringal, etc.
In an advantageous manner, the applicant was able to demonstrate that the Chemical composition of the invention exhibits a biological activity against HIV type 1 and type 2, AIDS and the clinical manifestations that accompany it, as well as certain protozoa in particular Trypanosoma cruzi, Trypanosoma brucei, Plasmodium falcîparum.
The biological activity of the Chemical composition of the invention is, in particular, due to the presence of the molécules of which it is composed. More specifically, the molécule A, the molécule B, and the molécules C to J are responsible and are at the origin of the biological activity of the Chemical composition of the invention. The presence of these molécules A to J in the human body generates the biological activity.
More specifically, the biological activity of the molécules A to J has been demonstrated by means of in vitro tests mentioned later.
Thus, according to the invention, the said Chemical composition can be used for the préparation of a pharmaceutical composition that helps to fight against HIV, AIDS, the associated opportunistic pathologies thereof, as well as against the résultant diseases caused by certain protozoa such as trypanosomiasis or malaria.
In addition, the présent invention also protects a pharmaceutical composition comprising the Chemical composition of the invention, the said pharmaceutical composition being in an oral galenic form.
The term oral galenic form is used to refer to the form in which the excipients and active ingrédients are to be prepared, the latter being namely the molécules A to J alone or in combination, constituting a médicament for use by oral administration.
According to one particular embodiment of the pharmaceutical composition of the invention, the Chemical composition of the invention may be in the form of infusion sachets, this being only when it is completely naturel in origin and derived from a plant extract of Anthostema senegalense.
The term infusion sachet is used to refer to the form in which the active ingrédient is presented, that is to say the molécules A to J derived preferably from a plant extract of Anthostema senegalense. More specifically, an infusion sachet is a small sachet containing a dose of dried plant extract to be infused.
Thus, in the context of the invention, the pharmaceutical composition comprising the Chemical composition of the invention may be in the form of a sachet containing a dried plant extract of Anthostema senegalense that one would be able to infuse. In this case, the plant extract of the sachet itself containing at least one of the molécules A or B, is advantageously a combination of molécules A to J.
Preferably, the pharmaceutical composition of the invention is in an appropriate oral galenic form, in particular such as microspheres. These latter are produced by a method of extrusion and spheronisation. The pharmaceutical composition of the invention may also be in a different oral galenic form other than microspheres, in particular in the form of tablets, soft capsules, granules, gel capsules, powders, ampoules, syrups or even décoctions.
The pharmaceutical composition of the invention has the advantage of presenting an effective and less expensive alternative for treating patients afflicted with HIV type 1 or type 2, AIDS or clinical diseases that resuit therefrom.
Indeed, the oral galenic form or the infusion sachet form of the pharmaceutical composition of the invention facilitâtes its administration by the patient, requires little or no equipment for administering it and poses no storage problem.
The activity of the one or more molecule(s) of the Chemical composition of the invention was demonstrated following the isolation by means of extraction, identification and sélection of the said one or more biologically active molécules of interest from an Anthostema senegalense plant.
Accordingly, the présent invention also relates to an isolation method for isolating by means of extraction, identification and sélection of the one or more biologically active molecule(s) of the Chemical composition of the invention.
This method makes it possible to extract from a plant of Anthostema senegalense, the one or more molecule(s) of interest which is / are then considered to be of entirely natural origin. The one or more molecule(s) of interest being the cells exhibiting biological activity against pathogens, in particular a biological activity qualified as significant.
In the présent application, a significant biological activity defines a compound having, against the pathogen, a 50% cytotoxic concentration (CC50) > 45 pg/mL and a selectivity index > 50.
The said method comprises the following steps :
- A part of the Anthostema senegalense plant is dried, for example the bark of the trunk/ stem or the leaves ;
- A plant extract is prepared from the dried part of the plant by means of cold or hot extraction, with a polar or apolar solvent, by means of aqueous extraction, either alcoholic or hydro - alcoholic, or by macération, infusion, décoction, percolation, digestion or leaching ;
- The said plant extract is concentrated under vacuum ;
- The molécules of the plant extract are separated by chromatography ;
- In vitro testing is carried out on the biological activity of the said molécules against an HIV - type viral pathogen according to the method of E de Clercq ;
- The molécules having a selectivity index against the said pathogen > 50 and a 50% cytotoxic concentration CC50 > 45 pg/mL are selected and isolated ;
- It is then necessary to identity the structure of the one or more molécules that hâve been separated and selected by means of Nuclear Magnetic Résonance and mass spectrometry.
According to one particular embodiment of the method of the invention :
- the part of the Anthostema senegalense plant which is dried consists of the bark of the trunk / stem, or the bark of the roots, or the leaves or the fruits ;
- a plant extract is prepared by percolation with CHC13 in order to obtain an apolar plant extract ;
- after concentration under vacuum, the molécules of the said apolar plant extract are separated by means of repeated column chromatography on silica gel, with use as eluent, of a gradient of CHCI3 in hexane and by means of thin layer chromatography.
The thin layer chromatography may be carried out with use, as mobile phase, of a mixture of dichloromethane/toluene or a mixture of toluene/chloroform or indeed even with chloroform.
Thus, the operational implémentation of the method of the invention has made it possible to identify and isolate the molécules having biological activity that are présent in a plant extract which is biologically active against HIV. Thereafter, the operational implémentation of the method made it possible to select, through the in vitro assay, the molécules that hâve significant biological activity and act as stimulators of the immune system.
The experiments and results detailed here below hâve provided the applicant with the means to illustrate the immune system stimulating biological activity of the one or more biologically active molecule(s) of the Chemical composition of the invention.
/ In vitro testing of the biological activity of the molécules of the Chemical composition of the invention
The anti - viral réplication efficiency of the molécules of the Chemical composition of the invention was tested in vitro against the réplication of the viruses HIV type 1 (strain 111b) and HIV type 2 (strain ROD) in the infected MT - 4 cells.
This efficiency was achieved in comparison with three positive control antirétroviral molécules : nevirapine, dideoxycitidine and dideoxÿinôsmœ
More specifically, the antirétroviral activity of the molécules was tested by effectively carrying but the method of E De Clercq in a microplate.
This method consists of producing a cell culture of MT - 4 in a microplate, containing as cell culture medium: the medium developed by the Roswell Park Memorial Institute (referred to as RPMI 1640”), comprising of foetal calf sérum, glutamine, sodium bicarbonate, and gentamicin as bacterial inhibitor.
The molécules of the Chemical composition of the invention are added at different concentrations into the cell growth medium. Each concentration of molécules to be tested is prepared in triplicate. The MT4 cells placed in the presence of the molécules to be tested are referred to as treated cells.
In each cell growth well, a defined amount of HIV -1 or HIV - 2 is added in order to obtain the same concentration of virus in each well.
The cells placed in the presence of the HIV are referred to as infected cells.
Five days after the start of the process of infecting the MT4 cells with HIV -1 or HIV - 2 in the presence of the molécules to be tested at different concentrations, a count is taken of the number of living cells by means of an MTT (tétrazolium sait) colorimétrie assay by measuring the optical density at 540 nm.
As négative Controls, use is made of cells without HIV 1 or HIV 2, referred to as non - infected cells, as well as of cells without the molécules to be tested referred to as untreated cells.
Thus, succinctly stated, the method of E De Clercq provides the means to obtain a density measurement at 540 nm for :
- the cells infected with HIV -1 or HIV - 2, treated with the molécules of the composition of the invention ;
- the cells not infected with HIV, treated with the molécules of the composition of the invention ;
- the cells infected with HIV - 1 or HIV - 2, not treated with the molécules of the composition of the invention ;
- the cells not infected with HIV, not treated with the molécules of the composition of the invention.
In particular, the effectiveness of the molécules of the composition of the invention was tested :
- by measurement and calculation of the (médian) 50% inhibitory concentration GCso);
- by measurement and calculation of the 50% cytotoxic concentration (CC50) ;
- by measurement and calculation of the 50% effective concentration dose (EC50) ; and
- by measurement and calculation of the selectivity index (SI), of the fractions containing the said molécules in the presence of the two strains ofHIV.
The IC50 is the concentration of the molécule that makes it possible to decrease by 50% the viral prolifération of the négative control in vitro for a given time period, that is to say of the HIV without substance, only in its growth culture medium.
The CC50 is the concentration of active molécules that makes it possible to decrease by 50% the cytopathic effect ofHIV on the infected cells. In other words, it is the concentration of active molécules that makes it possible to decrease by 50% the viability of cells.
The EC50 corresponds to the concentration of molécules necessary in order to ensure that 50% of the cells growing in the medium do not submit to the action of the virus, that is to say, that they are protected from the cytopathic effect of the virus.
The EC50 is calculated using the following formula :
[(OD r)HIV+] - [(OD c) HIV*] / [(OD T) HIW] - [(OD c) HIW]x 100
Where:
• (OD t)HIV+ represents the optical density measurement at 540 nm, 5 days after infection, for the cells infected with HIV and treated with the composition of the invention, that is to say in the presence of molécules of the composition of the invention ;
• (OD c) HIV* represents the optical density measurement at 540 nm, 5 days after infection, for the cells infected with HIV in the absence of the molécules of the composition of the invention ;
• (OD t) HIW represents the optical density measurement at 540 nm, 5 days after infection, for the cells not infected with HIV and treated with the composition of the invention, that is to say in the presence of the molécules of the composition of the invention ; i(OD c) HIV represents the optical density measurement at 540 nm, 5 days after infection, for the cells not infected with HIV in the absence of the molécules of the composition of the invention.
The selectivity index represents the ratio CC50/EC50.
In the context of this application, it will be considered that the biological activity against the pathogen, preferably HIV -1 and HIV - 2, is significant and that there is a biological activity that stimulâtes immune activity, when the selectivity index is > 50 and the CCso> 45 pg/ml.
The results are illustrated in Table 1 here below which shows the average values obtained over the duplicates for IC50, CC50 and SI :
| HIV-1 | HIV-2 | |||||
| IC 50 (ug/mL) | CC 50 (ug/mL) | SI | IC 50 (pg/ml) | CC 50 (pg/ml) | SI | |
| sample tested | ||||||
| AsC4.2 | 4.130 | 47.83 | 12 | 0.467 | 47.83 | 102 |
| Asc4.5 | 0.866 | 58.48 | 68 | 0.065 | 58.48 | 904 |
| AsC4.6 | 2.350 | 53.33 | 23 | 0.142 | 53.33 | 376 |
| AsC4.7 | 7.670 | 54.08 | 7 | 0.539 | 54.08 | 100 |
| Nevirapine | 0.050 | 0.011 | >4 | >4 | >4 | <1 |
| Dideoxycitidine | 0.16 | 0.12 | >20 | 0.19 | 0.11 | >20 |
| Dideoxyinosine | 2.09 | 0.68 | >50 | 3.78 | 1.22 | >50 |
It is found that the molécules présent in the fractions AsC4.2, AsC4.5, AsC4.6, and AsC4.7 possess a significant biological activity against HIV - 1 and HIV - 2.
In particular, the molécules présent in the fraction Asc4.5 exhibit a significant activity against HIV type 1 and type 2.
The molécules présent in each of the fractions AsC4.2, AsC4.5, AsC4.6 and AsC4.7 hâve significant biological activity against HIV type 2. The IC50 of the molécules contained in each of these fractions is better than that of the positive Controls of Nevirapine and Dideoxyinosine. Thus, the molécules of these fractions act synergistically to fight against viral multiplication.
In particular, the molécules of the fractions AsC4.5 Asc4.6 and hâve a high biological activity, that is better than those of the antiretrovirals Nevirapine, Dideoxycitidin and dideoxyinosine, against HIV type 2.
The isolation, identification and sélection of the molécules with significant biological activity of the Chemical composition of the invention, derived from the said biologically active fractions are described here below.
2/Preparation of the fractions, isolation and sélection of the biological activity exhibiting molécules of the invention :
From a crude extract of bark of the trunk of an Anthostema senegalense plant, a polar plant extract is prepared by means of percolation with liquid CHCI3. The said polar plant extract is concentrated under vacuum.
Then column chromatography is carried out with, use as eluent, of a polarity gradient of CHCI3 in hexane, in order to separate the component constituents according to their charge. At the conclusion of the chromatography, ten fractions are obtained which are respectively named AsCl to AsClO.
A thin layer chromatography process is carried out with each of the ten fractions ASC1, ASC2, ASC3, AsC4, AsC5, AsC6, AsC7, AsC8, AsC9, AsclO mentioned above, in order to detect the number of potentially different molécules of each of the fractions.
In order to demonstrate in respect of each of the fractions, their biological activity, in particular acting to stimulate the immune system, each of the fractions was tested in vitro vis - à - vis HIV type 1 and HIV type 2, according to the method explained later.
The fraction AsC4 showed the best anti - HIV activity.
This fraction AsC4 therefore contains an assembly of molécules with significant biological activity against HIV.
This fraction AsC4 has, as a conséquence, been separated by means of column chromatography until 8 fractions were obtained, labelled respectively from AsC4.1 to AsC4.8.
The anti - HIV activity was tested in vitro according to the method of Clerq E on each of the abovementioned fractions AsC4.1, AsC4.2, AsC4.3, AsC4.4, AsC4.5, AsC4.6, AsC4.7 and AsC4.8.
The fractions AsC4.5 ; AsC4.6 ; AsC4.2 and AsC4.7 showed the best antiviral activities as illustrated in Table 1 mentioned above.
Thus, the molécules présent in majority in these fractions are responsible for the anti - HIV biological activity.
More specifically, the fraction AsC4.5 exhibits a significant biological activity pursuant to the meaning of the invention against HIV-1 and greater significant biological activity against HIV - 2.
Also more specifically, against HIV-2, ail of the fractions AsC4.5; AsC4.6; AsC4.2 and AsC4.7 exhibit a significant biological activity pursuant to the meaning of the invention.
A sample of each of these fractions AsC4.5 and AsC4.6 was separated by silica gel (SiO2) chromatography, then the molécules were identified by NMR (Nuclear Magnetic Résonance) and Mass Spectrometry.
The results show a prédominant ie majority presence in each of these samples AsC4.5 and AsC4.6, of the said molécule A and of the said molécule B, in addition to a sériés of other molécules with a minority presence.
Thus, the molécule A and the molécule B are envisaged as being responsible for the “significant” biological activity for each of the samples AsC4.5 and Asc4.6.
In order to confirm this opinion, other analyses were performed.
In particular, ail of the fractions AsC4.5, AsC4.6, AsC4.2 and AsC4.7 exhibiting anti-HIV biological activity were mixed and separated on silica gel SiO2 column chromatography on a repeated basis (at least six times ), with use as eluent, of toluene/ CHCI3 creating a polarity gradient in order to obtain the sub - fractions referred to as AsC4.5.1 to AsC4.5.7.
Each of the sub - fractions AsC4.5.1 to AsC4.5.7 was then subjected to an in vitro assay as described here above on the viruses HIV 1 and HIV 2. The results of the tests showed a significant inhibitory activity against these two strains for each of the sub - fractions tested.
Each of the abovementioned sub - fractions Asc4.5.1, AsC4.5.2, AsC4.5.3, AsC4.5.4, AsC4.5.5, AsC4.5.6, and AsC4.5.7 was separated and purified on préparative thin layer chromatography by making use of different eluents as the mobile phase.
The eluents used as mobile phase are as follows :
- a mobile phase of CH2C12 / toluene in a volume to volume ratio of 1: 2 ;
- a mobile phase of toluene/ CHCI3 in a volume to volume ratio of 4: 1 ;
- a mobile phase of toluene/ CHCI3 in a volume to volume ratio of 1: 1 ;
- a mobile phase of 100% CHCI3.
More than ten different compounds were obtained for ail of the different mobile phases used. Each compound was individually tested against HIV-1 and HIV-2 according to the in vitro method mentioned above.
In a spécifie manner, among ail the compounds, only the compounds cited here below hâve shown a “significant” biological activity pursuant to the meaning of the invention against the prolifération of HIV -1 or HIV-2.
- The compounds AsC4.5.1.1 and R.3.6.6.1, obtained with a mobile phase of CH2CI2/toluene in a volume to volume ratio of 1: 2 ;
- The compound AsC4.5.2.1, obtained with a mobile phase of toluene /CHCI3 in a volume to volume ratio of 4: 1 ;
- The compound AsC4.5.4.1, obtained with a mobile phase of toluene/CHCI3 in a volume to volume ratio of 1: 1 ;
- The compounds AsC4.5.5.1, AsC4.5.6.1, AsC4.5.7.1 and AsC4.5.7.2, obtained with a mobile phase of 100% CHCI3.
In an advantageous manner, the compounds AsC4.5.1.1 and AsC4.5.7.2 exhibit an inhibitory activity that is at least two times greater against HIV - 1 and HIV - 2 than the other components identified as having a “significant” biological activity.
The identification of ail of the compounds with significant biological activity as mentioned here above was performed by means of nuclear magnetic résonance and mass spectrometry.
After identification, it tums out that the compounds identified which possess a biological activity against HIV - 1 and HIV - 2 are constituted of the molécules A to J cited above.
In a more précisé manner, it was noted that the compounds AsC4.5.1.1 and AsC4.5.7.2 correspond respectively to the molécule A and the molécule B claimed in the invention. These two molécules A and B are, as a conséquence, indeed responsible for significant inhibitory effect on the prolifération of HIV -1 and HIV - 2.
In addition, in the fractions AsC4.5 and AsC4.6 that are most active against HIV - 1, it was noted that the two molécules A and B are predominantly represented and in high concentrations.
In similar fashion, and still against HIV-1, in the active fraction AsC4.2 the molécule A is predominantly found (majority presence) but not the molécule B, and in the active fraction AsC4.7 the molécule B is predominantly found (majority presence) but not the molécule A.
As a conséquence, the technical effect of inhibiting prolifération of HIV 1 or HIV 2 in respect of the fractions having significant inhibitory activity is due to the spécifie presence and action of the molécules A and/or B.
The molécule A as well as the molécule B possess an activity serving to inhibit the prolifération of HIV -1 or HIV -2, each of the molécules A or B being capable of stimulating the immune activity of an infected organism or body.
In the light of these results, the molécules A and B alone or in combination hâve been specially selected and chosen by the applicant, from among the molécules A to J for which an inhibitory activity against HIV -1 and HIV - 2 has also been observed, in order to include them in the Chemical composition of the invention that biologically stimulâtes immune activity.
It follows from these studies that each of the molécules A to J of the invention isolated by means of the above noted techniques exhibit a synergistic inhibitory activity against the prolifération of HIV type 1 and HIV type 2 within cells infected therewith. The biological activity of each of these molécules taken individually or in combination against HIV -1 or HIV - 2 has been demonstrated through these experiments.
More specifically, the molécules A and B being represented predominantly (majority presence) in the fractions having inhibitory activity against the prolifération of HIV -1 and HIV - 2, are considered to be responsible for the principal and majority biological activity against viruses, in particular HIV 1 or HIV 2.
Further studies of the biological activity of the molécules A and B, taken alone or in combination hâve shown that the biological activity against HIV is mainly due to the presence of these two molécules. In particular, the presence of the molécule A and/or B in a Chemical composition thus has the effect of inhibiting the viral prolifération of HIV -1 and HIV - 2 and therefore of biologically stimulating the immune System.
That is why, in implementing an inventive approach, the applicant has specifically selected the molécules A and/or B from the group of molécules A to J having a biological activity, since these two molécules A and B exhibit the significant effect expected against the prolifération of HIV - 1 and HIV - 2 and provide the ability to stimulate immune activity.
The results of experiments hâve shown that both the molécule A alone, the molécule B alone, or the combination of the two molécules A and B, has/hâve a significant biological activity against HIV -1 or HIV - 2.
In addition, the significant biological activity is enhanced in the presence of the two molécules A and B. As a resuit, there is indeed synergy between the two molécules in respect of the activity and the effect thereof against HIV -1 or HIV - 2. In other words, a synergy exists between these two molécules so as to enhance their respective inhibitory effect against the prolifération of HIV - 1 and HIV - 2.
In an advantageous manner, the Chemical composition of the invention provides an effective alternative, one that is less expensive, and accessible to developing 5 countries, is easy to produce where it is derived from natural plant origin, which is biologically active, and stimulâtes the immune activity.
In particular, the Chemical composition of the invention may be used in pharmaceuticals domain in the treatment of infection by the HIV virus, as well as AIDS and the clinical manifestations that accompany it.
In addition, the Chemical composition of the invention may also be used as a food/dietary supplément that provides the means to stimulate immune activity and the natural immune defences of humans who may be immunocompromised.
Claims (16)
1. A Chemical composition characterised in that it comprises :
- the molécule A or one of the physiologically acceptable salts thereof ; and/or;
- the molécule B or one of the physiologically acceptable salts thereof, the said molécule A having the following formula :
and the said molécule B having the following formula
the molécule A or one of the physiologically acceptable salts thereof is biologically active and is a stimulator of immune activity ;
the molécule B or one of the physiologically acceptable salts thereof is biologically active and is a stimulator of immune activity.
2. A Chemical composition according to the preceding claim, characterised in that the said molécule A présents :
- at position 16, as a substitute for the carbonyl function =0, either an OH, or an aldéhyde carbonyl group -RCOH, or a ketone -RI -CO-R2, or an ester -R-COO-R', or an amide of the type -R-C(=O)NH2; and/or
- at position 3, a glycosylation or estérification in the form of - OR, where R is either a feruloyl or caffeoyl group, or a sugar or an amide.
3. A Chemical composition according to any one of the preceding claims, characterised in that the said molécule A is conjugated with amino acids, quatemary ammonium salts, glycosides, hemiphtalates or even carbamates.
4. A Chemical composition according to any one of the preceding claims, characterised in that the said molécule B présents, at position 28, an estérification of an amide or a sugar.
5. A Chemical composition according to any one of the preceding claims, characterised in that it comprises, in addition, at least one of the following molécules or one of the physiologically acceptable salts thereof, or one of the possible combinations of the said following molécules, the said one or more molecule(s) being selected from the following list :
- The molécule C having the formula :
wherein n is comprised between 8 and 26 ;
- The molécule D having the formula :
- The molécule E having the formula :
- The molécule F having the formula:
- The molécule G having the formula :
- The molécule H having the formula :
- The molécule I having the formula :
With n comprised between 14 and 26 ;
- The molécule J having the formula:
6. A Chemical composition according to any one of the preceding claims, characterised in that each of the said molécules A, B, and each of the physiologically acceptable salts thereof is of synthetic origin and derived from a Chemical synthesis in the laboratory or from a hemi - synthesis.
7. A Chemical composition according to any one of claims 1 to 5, characterised in that each of the said molécules A, B and each of the physiologically acceptable salts thereof is of natural origin and derived from a plant extract of Anthostema senegalense.
8. A Chemical composition according to the preceding claim, characterised in that the plant extract of Anthostema senegalense consists of a polar extract, that is aqueous, alcoholic or hydro - alcoholic or an apolar (or non - polar) extract or a mother tincture of Anthostema senegalense with an éthanol content of 70% V / V.
9. A Chemical composition according to the preceding claim, characterised in that the said plant extract is obtained from the trunk / stem bark, in particular dried bark, of Anthostema senegalense.
10. A Chemical composition according to any one of the preceding claims, intended to be used as a médicament or as a food - dietary supplément.
11. A pharmaceutical composition, characterised in that it comprises of the Chemical composition according to any one of claims 1 to 10, intended to be used in the treatment of HIV infection type 1 or type 2, AIDS and the clinical manifestations that accompany it.
12. A pharmaceutical composition according to the preceding claim, characterised in that it is in an oral galenic form.
13. A pharmaceutical composition according to the preceding claim, characterised in that it is in an oral galenic form of microspheres produced by a method of extrusion and spheronisation, of tablets, soft capsules, granules, gel capsules, powders, ampoules, syrups or even décoctions.
14. An isolation method for isolating by means of extraction, identification and sélection of the one or more biologically active molecule(s) of the Chemical composition of the invention according to any one of claims 1 to 10 in which :
- A part of the Anthostema senegalense plant is dried ;
- A plant extract is prepared, from the dried part of the plant, by extraction with an apolar or polar solvent or by macération, infusion, décoction, percolation, digestion or leaching ;
- The said plant extract is concentrated under vacuum ;
- The molécules of the plant extract are separated by chromatography ;
- In vitro testing is carried out on the biological activity of the said molécules against an HIV - type viral pathogen according to the method of E de Clercq ;
- The molécules having a selectivity index against the said pathogen > 50 and a 50% cytotoxic concentration CC50 > 45 pg/mL are selected and isolat ed ;
- It is then necessary to identify the one or more molécules that hâve been separated, selected and isolated by means of nuclear magnetic résonance and mass spectrometry.
15. A method according to the preceding claim, characterised in that :
- The part of the Anthostema senegalense plant which is dried consists of the bark of the trunk/stem, or the bark of the roots, or the leaves or the fruits ;
- A plant extract is prepared by percolation with CHC13 in order to obtain an apolar plant extract ;
- After concentration under vacuum, the molécules of the plant extract are separated by means of repeated column chromatography and thin layer chromatography.
16. A method, according to the preceding claim, characterised in that it is necessary to separate the molécules of the said apolar plant extract by means of repeated column chromatography, using as the stationary phase a polar silica gel and, as the mobile phase, an apolar eluent constituted of a gradient of CHC13 in hexane.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1660230 | 2016-10-21 | ||
| FR1750080 | 2017-01-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA19672A true OA19672A (en) | 2020-12-31 |
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