FR2877576A1 - Composition containing eucalyptol, linalool, itaconic or methylsuccinic anhydride, terpinen-4-ol, alpha-terpineol, camphor, bornyl acetate, geranyl formate, geraniol and coumarin as mast cell proliferation stimulant e.g. to treat infections - Google Patents

Abstract

Composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic or methylsuccinic anhydride, terpinen-4-ol, alpha -terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin and diethyl phthalate is used to produce a pharmaceutical composition for preventing or treating bacterial, parasitic, viral or fungal infections or diseases involving mast cell deficiency or inactivation, other than cancer and alopecia. An independent claim is also included for use of a composition as above in a test for detecting allergies. ACTIVITY : Antibacterial; Antiparasitic; Virucide; Fungicide. MECHANISM OF ACTION : Mast cell proliferation stimulant. A composition comprising ethanol (20%), ethyl acetate (1%), eucalyptol (1%), dipropylene glycol (20%), linalool (13%), linalool acetate (2%), itaconic or methylsuccinic anhydride (1%), terpinen-4-ol (5%), alpha -terpineol (5%), camphor (1%), bornyl acetate (4%), geranyl formate (2%), geraniol or geranyl acetate (5%), coumarin (1%) and diethyl phthalate (20%) was applied to the shaved skin of mice twice a day. The mast cell density in skin samples after 7 days was 77.7 cells/mm2>, compared with 46.8 cells/mm2> for controls.

Description

The present invention relates to a pharmaceutical composition used

  to stimulate the proliferation and activation of mast cells.

  More particularly, it relates to pharmaceutical compositions used to enhance the immune response following the introduction of infectious agents into the human or animal body. It also relates to compositions used in or for performing tests for detecting allergies.

  The immune system can eliminate and neutralize foreign substances that can enter the body and especially infectious agents such as bacteria, parasites, viruses or fungi. Specific cells involved in the immune response include lymphocytes, phagocytic cells, basophils, platelets and mast cells.

  Mast cells are characteristic cells of connective tissue and account for about 5% of skin cells. They are found more particularly in the tissues of the face and the scalp, near the blood vessels and nerves. Mast cells exert their functions via mediators (histamine, heparin, angiogenic proteins) present in their granules.

  Mast cells play a role in the immune response, in the control of parasitic and bacterial infections and in the stimulation of angiogenesis (Metcalfe DD et al., Mast et al., Physiol Rev 1997, 77: 1033-70).

  Mast cells have the ability to produce proinflammatory and immunoregulatory cytokines (Okayama Y et al., Cytokine: Production by Human Mast, Human Basophils and Mast, C: Marone Ed., Chem Immunol Base / Karger 1995; 34) a property that brings them closer to T helper T cells. It has been suggested that mast cells play a role in the orientation of the immune response (Collective Book, Mastocytes, Basophils and Clinical Biology, Biotem Edition, 1996).

  It has also been shown that mast cells play a fundamental role in tissue reconstruction and fibrous tissue formation (fibrosis) (Garbuzenko E et al., Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis, Clin Exp AII 32: 237-246, 2002. LeviShaffer F. hast cell / fibroblast interactions in health and disease, Chem Immunol 61: 161-185, 1995). It has been demonstrated that the activation of mast cells increases the proliferation of fibroblasts and the synthesis of collagen. Indeed, some mediators released by the mast cells are at the base of fibroblast growth factor (Artuc M et al.) Mast ce / 1-fibroblast interactions: human mast cells and inducers of fibroblast and epithelial growth factors J Inv Dermatol 118: 391-395,: 2002).

  Mast cells also act in hair growth: they are found in greater numbers in the anagen phase of the hair cycle (Li et al., Flair shaft elongation, fol / ide growth, and spontaneous regression in long-term, gelatin sponge-supported Proc Natl Acad Sci 1992 Sep 15; 89 (18): 8764-8) histoculture of human scalp skin.

  Deficiency and / or deactivation of mast cells can therefore adversely affect a wide range of biological processes, such as immune function, vascularization, fibrous tissue formation, tissue regeneration or wound healing. A composition that increases and / or stimulates mast cells is therefore quite useful in the cosmetic and medical environment.

  The mast cell is the main cell involved in the initiation and amplification of the inflammatory reaction in the case of allergies. The allergic phenomenon is due to an overproduction of immunoglobulin E (IgE) which binds abundantly to the mast cells of tissues to cause their degranulation in response to the binding of specific antigens to the antibodies. This degranulation causes the release of various inflammatory substances including histamine.

  To determine the allergens involved skin tests, provocation tests, IgE assays can be performed. The skin test involves applying small quantities of products suspected of being allergenic to the skin. A local reaction gives a reliable and rapid response to the responsible substances.

  An increase in mast cells in the area where these tests are carried out is therefore quite useful for locally amplifying the allergic reaction in order to better identify the allergenic substances.

  Most factors stimulating the activation and proliferation of mast cells are large proteins that can only be obtained through complex and expensive synthetic pathways. 15

  The applicant has demonstrated that a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic acid, terpinen-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, stimulates surprising proliferation and activation of mast cells.

  The toxicological profile of these different compounds is not likely to induce a risk to human health and the formulation does not present an innovation likely to cause adverse reactions to humans. Moreover, it is not known to date of harmful interaction between these compounds.

  According to the invention, this composition is used in the manufacture of a pharmaceutical composition intended to enhance the immune response and more particularly for the manufacture of a pharmaceutical composition intended to prevent or treat bacterial infections, parasitic infections, viral infections or fungal infections.

  According to another characteristic of the invention, this composition is used for the manufacture of a pharmaceutical composition intended to prevent or treat pathologies in which deficiency or inactivation of mast cells is involved, other than cancers or alopecia.

  According to another characteristic of the invention, this composition is used in or for performing a test for detecting allergies. This composition is particularly suitable for use in human medicine but can also be used in veterinary medicine.

  Preferably, the compounds are assayed in proportions close to the following values: ethanol 20%; ethyl acetate 1% eucalyptol 1%; dipropylene glycol 20%; linalool 13%; linalool acetate 2%; itaconic anhydride or methyl succinic 1%; terpinene-4-ol 5% a-terpineol 5%; camphor 1%; bornyl acetate 4%; Geranyl formate 2%; geraniol or geranyl acetate 5%; coumarin 1% diethyl phthalate 20%.

  The usable quantity depends on the desired effect and the composition may be used pure or diluted with distilled water or any other equivalent solvent, for example between 0.3 and 1%, advantageously at 0.5%.

  Most of the compounds at the base of the composition can be distilled off from natural products, for example: ethanol (fermented grape), euca / ypto / (eucalyptus radiata), linalool (occimum basilicum), linalool acetate (citrus aurantium) , terpinene-4-ol (malaleuca alternifolia), a-terpineol (citrus aurantifolia), camphor (rosmarinus officinalis), bornyl acetate (cistus ladaniferus), geranyl formate (pelargonium graveolens), geraniol (citrus lemonum), geranyl acetate (cymbopogon nardus), coumarin (Tonka bean). The other compounds can be obtained by simple and inexpensive manufacturing processes, for example, ethyl acetate (WO 01/46117A1), dipropylene glycol (US 3574772), itaconic anhydride (GB 854999), methyl succinic anhydride (FR 2735775). ), diethyl phthalate (US 2618651).

  According to one characteristic of the invention, these compounds are advantageously extracted by distillation from a mixture of iodized salt, lemon juice, fresh onion, citric acid and alcohol, assayed in the proportions of: % iodized salt, 15% lemon juice, 50% fresh onion, 5% citric acid, 20% alcohol.

  These different characteristics make it possible to obtain a composition containing only natural compounds of very low cost, easy to prepare and easy to use.

  The compounds of the invention may be used alone or conventionally associated with vehicles, carriers or excipients conventionally used in the medical and pharmaceutically acceptable medium: preservatives, antioxidants, solvents, stabilizers ....

  The routes of administration of the composition are carried out in an appropriate medicinal form, established according to the pathology to be treated, for example by the oral route, locally by topical application to the skin and mucous membranes or by intravenous injection. , intramuscular, intraperitoneal or subcutaneous. These compositions may be in any pharmaceutical form customarily used in human or veterinary medicine: tablets, capsules, drops, granules, injectable preparations, serums, ointments, creams, lotions, gels or patches.

  As regards the administration concentrations, it will be necessary to adapt the concentration to the targeted pathology and possibly to the patient. The concentrations can be between 0.3 and 1% and preferably about 0.5%. The composition can also be used pure.

  The dosage will be adapted according to the concentration of the basic mixture, the pathology to be treated and the profile of the patient. For example, to treat parasitic infections (eg leishmaniasis) the applicant's composition can be used pure and prepared in such a way as to administer daily from 0.01 to 1000 mg by topical application to the area to be treated.

  The various compounds used in the invention may optionally be used with other agents stimulating proliferation and / or activation of mast cells, or agents blocking the degranulation of certain mediators (in particular histamine) responsible for inflammatory reactions or other adverse reactions to the body.

  In addition, the composition may be used in combination with other active ingredients when the stimulation of the proliferation and / or the activation of the mast cells will constitute only an adjunct therapeutic treatment to a main treatment where proliferation and / or Mast cell activation is beneficial.

  For use in or for performing an assay for allergies, the composition may be used pure or diluted. It may be in the form of a lotion, cream, patch or any other equivalent form, which is applied beforehand on the area of the body (for example on the hand or the arm) where a skin test will be performed. allergy.

  Other characteristics and advantages of the present invention will appear more clearly on reading the following example, given by way of nonlimiting example, and the appended drawings in which: FIG. 1 is a graph showing the density of mast cells counted on a group of animals treated (T) with the composition of the applicant and on a control group (C).

  - Figure 2 is a graph showing the amount of hair follicles in the anagen phase on the treated group (T) and on the control group (C).

  FIG. 3 is a graph showing the amount of hair follicles in the catagen phase on the treated group (T) and on the control group (C).

  EXAMPLE: Evaluation of the Effect of the Composition According to the Invention on the Proliferation and Activation of Mastocytes in an In Vivo Model Carried Out in Mice

  The studies were performed on young BAL / C male mice aged 7 weeks. 30 of these animals were shaved with a razor in the dorsal region in the morning and then divided into two groups: a control group (C) without treatment and a treated group (T) with the composition of the applicant used pure, twice by day, on the shaved area.

  The basic mixture of the composition which is the subject of the invention is measured in proportions close to the following values: base% mass ethanol 20 ethyl acetate 1 eucalyptol 1 dipropylene glycol linalool 13 linalool acetate 2 itaconic anhydride or methyl succinic 5 Terpinene-4-ol-terpineol 5 camphor 1 bornyl acetate 4 geranyl formiate 2 geraniol or geranyl acetate 5 coumarin 1 diethyl phthalate 20 After 2, 7, 14 and 21 days of treatment, the animals were sacrificed and skin samples removed and fixed in formalin to make histological sections to be tested by an aqueous solution 0.5% toluidine blue (Merck).

  Mast cell analysis is performed using an Image Analysis System (Kontrom300) on a dermal surface and the mast cell density is expressed as the number of cells per mm 2 of tissue.

  In parallel with this analysis, a histological study of a section of the tissues made it possible to count the hair follicles in the anagen growth phase and in the involution catagen phase.

  Statistical analysis: the differences between each group are estimated by the Student's T test and the correlation between the count and the days of treatment is evaluated by a simple regressive analysis. The data is evaluated with Sigma Stat Softwear (Jandel Sientific). P <0.05 is considered significant.

  The results of the mast cell analyzes are presented in the following table: Number of days 2 7 14 21 Number of Group 49.3 46. 8 48.7 46.2 control mast cells (C) per mm2 Group 75.3 77.7 49.6 48.1 treated (T) The comparison of the Mast cell density between the two groups of animals, using the Student's test, is shown in Figure 1 (p <0.001).

  The results of the counting of the hair follicles in the anagen phase and in the catagen phase, using the Student's test, are represented respectively in FIGS. 2 and 3 (p <0.001).

  The treated group (T) shows an increase in the mast cell density on days 2 to 7 and then a decrease in the density of days 14 to 21. On the control group (C), no variation in density is observed different measuring points. By comparing the mast cell density between the two groups studied (Figure 1), there is a higher density of mast cells in the treated group (T). These results show that the composition of the Applicant significantly stimulates the proliferation of mast cells in the skin.

  At the same time, a greater number of hair follicles in the anagenic phase are observed in the treated group (T) than in the control group (C) (FIG. 2) and conversely a smaller amount of hair follicles in the catagen phase in the treated group (T ) than in the control group (C) (Figure 3). However, the anagenic phase of the hair cycle is characterized by activation of mast cells (Maurer M et al., Actived skin mast is involved in murine hair follicle regression.Lab Invest 1997 Oct. 77 (4): 319). These results show that the Applicant's composition significantly activates the mast cells in the skin.

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Claims (10)

  1. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for preventing or treating bacterial infections.   2. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for preventing or treating parasitic infections.   3. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for preventing or treating viral infections.   4. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for preventing or treating fungal infections.   5. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for preventing or treating diseases in which mast cell deficiency or inactivation is implicated, other than cancer or alopecia.   6. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic acid , terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, in or for performing a test to detect allergies.   7. Use according to one of the preceding claims, characterized in that the various components are extracted from a composition comprising iodized salt, lemon juice, fresh onion, citric acid and water. alcohol.   8. Use according to claim 12, characterized in that the components are dosed in the proportions of: 10% iodized salt, 15% lemon juice, 50% fresh onion, 5% citric acid, 20 alcohol.   - 12 - 9. Use according to one of the preceding claims, characterized in that the composition is diluted with water between 0.3 and 1%.   10. Use according to claim 9, characterized in that the composition is diluted with water to about 0.5%.