EP2086586A2

EP2086586A2 - Anti-microorganism terpenic composition

Info

Publication number: EP2086586A2
Application number: EP07870301A
Authority: EP
Inventors: Aurèle Henri MANNARINI
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-11-17
Filing date: 2007-11-16
Publication date: 2009-08-12
Also published as: US8198321B2; US20100286413A1; FR2908660A1; CN101583378A; CA2669871A1; AP2009004854A0; WO2008068427A2; JP2010510199A; JP5616065B2; BRPI0721551A2; WO2008068427A3; AP2767A; FR2908660B1; WO2008068427A8

Abstract

The invention relates to a terpenic composition for use as an anti-micro-organism or anti-viral agent comprising a cyclic terpenic compound obtained from a terpene of the general formula (C5 H8)n, n being in the range of 2 to 5; and to a vector-forming organic substance, associated to the said cyclic terpenic compound.<0}

Description

Terpene anti-microorganism composition

The present invention relates to a terpene composition anti-microorganism and its use including against HIV. Terpenes and in particular camphor and its derivatives have been widely used in medicine and they were reputedly active against all ills. Camphor was generally used in local external application as an antiseptic or anesthetic. Camphor is also used internally as a tonicardiac. However, it is very slightly soluble in water which limits its use.

Camphor is generally obtained from alpha-pinene in turpentine, which is obtained from the coniferous sap that flows from the maritime pine. Thus, alpha-pinene is converted into camphor through various intermediates, including bornyl chloride, camphene and the organic ester of isoborneol.

On the other hand, camphor substitution products associated with mercury have also been widely used in the past as diuretics. However, given their low solubility in water, the use of terpenes and in particular derivatives of camphor or its synthetic intermediates, remains limited in human or animal medicine.

In addition, a number of its derivatives is found to be cytotoxic, including ketone derivatives.

However, the terpenes chemistry is old and well known now and therefore, the costs of obtaining these biologically active molecules and therapeutic virtues are relatively low. Also, a problem that arises and that aims to solve the present invention is to provide a terpene composition useful as an anti-microorganism agent and that can be administered in humans easily. Another goal is to provide a terpene composition whose effectiveness of the active ingredient is improved.

In order to solve this problem, the present invention provides, in a first aspect, a terpenic composition for use as an anti-microorganism agent. According to the invention, the terpene composition comprises: a cyclic terpene compound obtained from a terpene of generic formula (C ₅ H ₈ ) _n , n being between 2 and 5; a vector-forming organic substance associated with said cyclic terpene compound and said cyclic terpene compound associated with said organic substance is bonded to a metal cation.

Thus, a feature of the invention lies in the combination of a cyclic terpene compound which has anti-microorganism properties, with an organic substance for transporting the active terpene compound to the microorganisms; said cyclic terpene compound associated with said organic substance being bonded to a metal cation which then promotes the anti-microorganism properties of said composition. In addition, the organic substance optionally makes it possible to transfer the terpenic compound through the microorganism itself to reach a specific target, for example its genetic material, where the terpenic compound can cause its inhibition.

Advantageously, the cyclic terpene compound is obtained from a monoterpene, for which n = 2, and whose anti-microorganism activity is greater than that of the other terpenes. Preferentially, the monoterpene leads to a terpene compound having a single five-carbon ring.

For example, the terpene compound is obtained by oxidation of camphor, itself derived from the transformation of alpha-pinene as indicated above. Camphene, an isomer of pinene, and especially its acid derivatives and alcohols such as camphols: borneol, camphoric acid, camphoric hydroxide or camphoric diol, are terpene compounds quite suitable. They are synthetically obtained from camphene. With a peracid, an acid-alcohol is obtained by the reaction of Boeyer-Villiger. Or, an aldehyde by a treatment with potash. According to a particularly advantageous embodiment of the invention, the terpenic compound has at least one carboxylic acid function and preferably two. In addition, said terpenic compound forms ester bonds with said organic substance. Advantageously, the terpenic compound is camphoric acid. In addition, the metal cation associated with the terpenic compound is selected from the metals of the third and fourth period of the elements of the periodic table of the elements of Mendeleyev, also referred to as the periodic table of elements. Preferably, the metal cation is chosen from the elements of the group comprising zinc, copper, nickel and magnesium or even manganese.

However, other metals of the fifth and sixth period of the aforementioned periodic table, such as tin or mercury, can also form a stable compound.

In addition, the organic substance forming a carrier and which makes it possible to convey the terpenic compound through the tissues and to lead it to its potential target, the microorganism to be inactivated, is chosen from among the carbohydrates, preferably the monosaccharides and osides, and gluconates, amino acid compounds, vitamins, nucleic acids or even benzene compounds. Included in these benzene compounds are salicylic or para-amino-salicylic acid and cinnamic acid.

Among the vitamins, folic acid, para-aminobenzoic acid or even ascorbic acid are particularly suitable. For example, zinc ascorbate-camphorate or bornyl folate of zinc or even born manganese gluconate will be formed. It is also anticipated that acetic acid will be used to form zinc aceto-camphorate. The metal ion of any of the above compounds can be replaced by one or other of the metal ions mentioned above, zinc or manganese.

According to another aspect, the invention proposes the use of a terpene composition as described above, for the production of a drug intended to overcome HIV or as an antimicrobial agent for external use. Thus, such a drug is likely to be produced at a very advantageous cost, since the aforementioned terpenic composition can itself be produced at low cost.

The terpene composition for the preparation of a medicament may be diluted in an aqueous solution or in a water / alcohol mixture and be packaged with a physiologically acceptable carrier, in the form of a gel for external application or under the form of an injectable solution. However, the terpenic composition is also capable of being packaged into powder for oral administration.

In general, the abovementioned terpenic composition can be used for the preparation of antiseptic, microbicidal or disinfectant agents, whether for the treatment of human and animal or plant pathologies or even in sanitary applications. According to another aspect, the invention relates to a process for the preparation of a terpene composition according to which an organic substance forming a vector is associated with a cyclic terpene compound obtained from a terpene of generic formula (C ₅ H ₈ ) _n , n being between 2 and 5, and said cyclic terpene compound being bonded to a metal cation.

According to yet another aspect, the invention relates to the use of a terpene composition as described above, for obtaining a medicament for combating a viral or retroviral infection, for example of the HIV type. Other features and advantages of the invention will become apparent on reading the description given below of particular embodiments of the invention, given for information but not limiting. A peculiarity of the invention lies in the use of a cyclic terpene compound derived from a terpene of general formula (C ₅ H ₈ ) n, where n is between 2 and 5 with a metal cation and a substance organic forming a vector. In the examples below, the terpene compositions according to the invention have been prepared proportionally with experimental amounts which make it possible to show their effects and their effectiveness. However, the extrapolation of these experimental quantities to industrial quantities raises no difficulty. Example 1

A first preparation containing ascorbic acid, camphoric acid and zinc was prepared under ambient conditions of temperature and pressure, is substantially ⁰ 298.15 K and 10 ⁵ Pa.

Nevertheless, in some phases the mixtures are slightly heated and stirred.

According to this first example, camphor is prepared from a well-known process using as raw material pinene, the camphoric acid of formula (I):

and this, by oxidation of camphor.

Thus, 200 mg of camphoric acid are solubilized in 1 g of ethanol at 90 ^e . Then, 0.5 g of ultra pure water is added to the mixture.

Then, the mixture of camphoric acid is associated with a zinc oxide in solution. For this, 81.3 mg of zinc oxide are mixed with 1 g of ultra pure water which after stirring form a milky solution. The aforementioned camphoric acid mixture is slowly poured into the solution milky. The whole is agitated moderately and intermittently. In addition, between each stirring phase, it is slightly heated, for example in an oven, to accelerate the reaction.

A precipitate is formed, the zinc camphorate, which is recovered by extraction of the solvent, either by heating or freeze-drying.

Then, 265.3 mg of zinc camphorate thus obtained are mixed with a solution of 10 g of pure water containing 176 mg of ascorbic acid of formula II:

Preferably, the mixing takes place at a temperature less than ⁰ 298.15 K. The preparation thus obtained is mildly agitated and is kept away from light to prevent oxidation of ascorbic acid. During stirring, zinc camphorate dissolves to form zinc ascorbate-camphorate in solution. However, only% of the zinc camphorate of the preparation dissolves, while the remaining ^3% of the remaining zinc camphorate remains undissolved in the mixture. In contrast, all of the ascorbic acid disappears and thus reacts with zinc camphorate. This preparation, then diluted to the 1/200 or the 200 ^th , therefore includes molecular compounds comprising mole% of zinc ascorbate-camphorate molecules, and in this case% of 1/1000 mole of molecules, for ³ A mole of zinc camphorate molecules and in this case for ³ A of 1/1000 mole of molecules. As will be explained below, this powder in solution, allows a terpene composition to inactivate microorganisms and viruses, including HIV.

A second preparation is carried out according to an example 2, with the same basic products, zinc camphorate and ascorbic acid, and according to the same protocol except the amount of ascorbic acid. Indeed, in this example 352 mg of ascorbic acid are dissolved in 10 g of ultrapure water with zinc camphorate. Thus, the second composition comprises not one but two moles of ascorbic acid for one mole of camphoric acid. On the other hand, according to this second preparation, it is now ^Λ A, half, of the zinc camphorate which dissolves, while the ascorbic acid reacts completely. Accordingly, the preparation includes molecular compounds comprising ^Λ A mole of zinc ascorbate-camphorate molecules for ^Λ A mole of zinc camphorate molecules.

A third preparation is carried out according to Example 3, also with the same basic products and according to the same protocol, except for the amount of ascorbic acid which is then 704 mg. The third composition then comprises four moles of ascorbic acid for one mole of camphoric acid. And according to this third preparation, all of the zinc camphorate dissolves and reacts with ascorbic acid. Thus, it is concluded that four moles of ascorbic acid react with one mole of zinc camphorate, from which it will be deduced that four molecules of ascorbic acid are attached to a molecule of zinc camphorate; and the molecule thus formed can be written: Zn [(C ₁₀ H ₄ O ₄ ) (CeH ₄ Oe) ₄ ]. This molecule can also form a number of hydrates that have not been described here.

It will be observed that the dissolved zinc ascorbate-camphorate can be recovered as a powder by lyophilization. Moreover, a fourth preparation, according to an example 4, is no longer prepared with ascorbic acid but from cinnamic acid of formula:

Example 4 148 mg of cinnamic acid are dissolved in 1 g of ethanol and 500 mg of ultrapure water are added thereto. 132.65 mg of zinc camphorate obtained according to the process above are incorporated in the solution thus produced. After dissolution, a precipitate is formed corresponding to cinnamo-camphorate zinc, soluble in 1/1000 water, denoted 1000 ^e (one part by weight per 1000 parts of water by weight) and which is recovered .

Thus, in Table I below, the molar proportions of the basic constituents of the above four preparations obtained in the four corresponding examples and on the basis of which terpene compositions according to the invention are produced are summarized.

Table I

In order to perform tests, cytotoxicity and inhibitory activity of infection, three cell types and two viral strains were used. This is a strain of HIV-1 _N DK (X4-tropic) laboratories that has been amplified by lymphocytes from healthy donors and a primary strain HIV-1 _Ba ι (R5-tropic) that has been amplified by macrophages.

For cell types, macrophages and dendritic cells were obtained from peripheral blood mononuclear blood cells. In addition lymphocytes were provided.

The cytotoxicity of the terpene compositions is controlled by the MTT test of Sigma. The above-mentioned macrophages or dendritic cells are then cultured and then treated with each of the compositions, and the MTT reagent is then added. The latter then forms crystals with highly metabolic living cells. The absorbance at 490 nm of the dissolved crystals then corresponds to the number of living cells. Also, in comparison with macrophages and cultured but untreated dendritic cells, a survival percentage is obtained revealing the toxicity of the terpene compositions.

The results show that the dilutions 1/10000 denoted 10 000 ^e, the 500 ^th and 100 ^th of the above four compounds are not toxic for the cells studied. Regarding the inhibitory activity, macrophages or dendritic cells are incubated with viral particles of the aforementioned strains, HIV-1 _N DK (X4-tropic) or HIV-1 _Ba ι (R5-tropic), in the presence and in the absence of terpene compositions. The cells are then washed, then put back into culture and finally centrifuged. The supernatants are then recovered and the viral particles are lysed in order to measure the concentration of a viral protein using the ELISA test. In this way, the inhibitory activity of the terpene compositions is evaluated by comparison between the untreated infected cells and the infected cells treated. In addition, virus transfer tests, from dendritic cells to autologous T cells, that is to say from the same stem cells, were carried out.

For this purpose, dendritic cells are transferred and incubated with each of the terpene compositions and the virus. Then, after washing, autologous T lymphocytes are added in a ratio of one dendritic cell to five T lymphocytes. Then, the concentration of viral proteins of the aforementioned type is evaluated by the ELISA test.

Then, inhibition of macrophage infection was evaluated. The latter being potential reservoirs of HIV, it is a question of measuring the ability of the terpene composition to prevent the infection of macrophages. Table II: Inhibition test for macrophage infection

It turns out here that the terpene composition obtained from the third preparation, and diluted to 1/200 noted 200 ^e , inhibits more than 95% infection of macrophages by strains of tropical group X4 and R5. Moreover, for the third and fourth compositions respectively obtained from the third and fourth preparations, and diluted, no longer in the 200 ^th but in the 2000 ^th , we obtain the same results

In addition, tests have been conducted on the inhibition of dendritic cell infection, as these cells are involved in the transmission of HIV in the mucosa.

Table III: Inhibition test for dendritic cell infection

Again, the terpene composition obtained from the third compound, and diluted at 200 ^e more than 95% inhibits the infection of dendritic cells by strains X4 and R5. It will be noted here that the third composition obtained from the third preparation and diluted in 2000 ^e also makes it possible to obtain the same results. Also, diluted ^100th the same third preparation is able to make non-infectious X4 strains.

The dendritic cells express on their surface a DC-SIGN adhesion protein forming a viral receptor capable of sensing HIV and facilitating the infection of permissive cells by a so-called trans-infection mechanism. Indeed, the DC-SIGN receptor can interact with the HIV envelope and retain the virion in an infectious state and then transmit it to the permissive T lymphocytes at the adjacent lymph nodes.

Table IV: Inhibition test of dendritic cell transfer to T lymphocytes

Thus, a 200 ^th dilution of the terpene composition from the third compound then causes an inhibition of the infection of the dendritic cells by the tropical R5 and X4 strains by more than 95%. Again, the third composition obtained from the third preparation and diluted in the ^2000th leads to the same results. On the other hand, another terpene composition, in accordance with the invention, zinc acetosalicylo-borneolate, has been prepared in organic solution. This fifth preparation, diluted to 50%, was tested in the inhibition of macrophage infection; and it inhibits by more than 50% the infection of macrophages by strains of tropical group R5. It is thus shown that other organic substances, and in this case, according to this fifth preparation, acetic acid and salicylic acid, can be adopted to form vector. Consequently, terpene compositions based on the abovementioned compositions allow the preparation of drugs for internal or external use to fight against HIV. It will be noted that the most effective terpene composition with the highest activity in all of the above tests is the composition from the third preparation. Also, the role of ascorbic acid as a vector is critical.

Note also that this terpene composition from the third preparation, has a detergent activity vis-à-vis the X4 strains, when diluted to one hundredth.

With respect to external applications, the terpene compositions are formulated with a physiologically acceptable excipient in the form of a gel or ointment.

Also, in order to use the terpene composition from the third preparation in a microbicidal application for the control of HIV, vaginal toxicity tests were conducted on the rabbit model.

The New Zealand white rabbit model was used to show whether repeated applications of the terpene composition from the third preparation led to vaginal irritation. For this, the animals were treated with doses of 1 ml of 2 compositions, one containing 2.1 μg / ml of the composition and the other 4200 μg / ml. Control animals were treated only with PBS buffer under the same conditions.

All animals, 9 in total, received daily intravaginal doses of the above compositions for 10 consecutive days.

24 hours after the last application of compositions, the vaginal apparatus of all animals was excised and histopathological evaluation was performed. As expected, control animals treated with PBS buffer exhibited normal tissues. The vaginal tissues of the other two groups of animals, treated with the two different dilution compositions, had infiltrations of polymorphic core cells in the epithelial connective tissues and subepithelial. However, the observed infiltration and vascular congestion were considered "minimal" and no edema was observed. The vaginal epithelium remained entirely intact and only minor morphological changes were noted. Thus, the composition according to the two dilutions mentioned above, caused only "minimal" vaginal irritation. Also, this irritation rate is acceptable for vaginal use. Therefore, these tests made it possible to verify the safety of the composition.

Thus, tests show that terpene derivatives have high anti-retroviral activity, and are capable of inhibiting viral replication within mucosal target cells, such as macrophages, dendritic cells, and lymphocytes. Some of them effectively inhibit the transfer of HIV from dendritic cells to CD4 cells, which is one of the major hypothetical mechanisms involved in the mucosal crossing of the virus and in the amplification of its dissemination within the mucosa.

Since terpene derivatives are a class of antiretroviral compounds distinct from those that already exist and they inhibit the entry and replication of HIV-1 by novel mechanisms, these compounds become attractive sources of anti-viral molecules in a perspective that is both preventive and therapeutic.

For internal applications, the terpene compositions are obtained in solid form, for example powdery, and can then be tabletted for oral administration or solubilized in a physiologically acceptable excipient for parenteral administration.

Moreover, the terpene compositions were tested in toxicological terms in the mouse and revealed an LD 50 (lethal dose from which 50% of the individuals die) at 48 hours after administration, 5.5 g more or less 0.5 g per kilogram. Thus, such compositions are considered to be non-toxic. In addition, the subacute toxicity tests have also shown that the terpene compositions according to the invention are suitable for the treatment of human pathologies.

In human therapeutics, the above terpene compositions are formulated from 1 to 300 mg by absorption for fractional or non-fractional doses and for a dosage ranging from 1 to 3000 mg per day in an adult.

Of course, the dosages could be higher if other embodiments of the invention were used, and in particular if the terpene compound was derived from bornyl.

Claims

Terpene composition for use as an anti-microorganism or anti-viral agent, characterized in that it comprises: a cyclic terpene compound obtained from a terpene of generic formula (C ₅ H ₈ ) _n , n being included between 2 and 5; and,

an organic substance forming a vector, associated with said cyclic terpene compound; and in that said cyclic terpene compound associated with said organic substance is bonded to a metal cation.

Terpene composition according to claim 1, characterized in that said terpene is a monoterpene for which n = 2.

3. terpene composition according to claim 2, characterized in that said terpene compound has a single ring with five carbon atoms.

4. terpene composition according to any one of claims 1 to 3, characterized in that said terpenic compound has a carboxylic acid function.

5. terpene composition according to claim 4, characterized in that said terpenic compound forms ester bonds with said organic substance.

6. Terpene composition according to claim 4 or 5, characterized in that said terpene compound is camphoric acid.

7. terpene composition according to any one of claims 1 to 6, characterized in that said metal cation is selected from the metals of the third and fourth period of the elements of the table of the periodic table of elements.

8. terpene composition according to claim 7, characterized in that said metal cation is selected from the elements of the assembly comprising zinc, copper, manganese and magnesium.

Terpene composition according to any one of claims 1 to 8, characterized in that said organic substance The carrier is selected from the group consisting of carbohydrates, amino acid compounds, vitamins and benzene compounds.

Terpene composition according to claim 9, characterized in that said organic substance is ascorbic acid.

11. Terpene composition according to claim 9, characterized in that said organic substance is cinnamic acid.

12. A method of manufacturing a terpene composition according to any one of claims 1 to 11, characterized in that associates an organic substance forming a vector to a cyclic terpene compound obtained from a terpene of generic formula (C ₅ H ₈₎ _n, n being between 2 and 5, said cyclic terpene compound being bound to a metal cation.

13. Use of a terpenic composition according to any one of claims 1 to 12 for the preparation of an antimicrobial agent.

14. Use of a terpene composition according to any one of claims 1 to 11, for obtaining a medicament for lute against a viral infection.

15. Use of a terpenic composition according to any one of claims 1 to 11 for the preparation of a terpene composition for external use.

16. Use of a terpene composition according to any one of claims 1 to 11 for the preparation of a terpenic composition that can be administered parenterally or orally.

17. Use of a terpenic composition according to claim 14 or 16 for the preparation of a medicament capable of being administered in a dosage of between 1 and 3000 mg per day for an adult.