Use Of A Fatty Acid For Preparing A Topical Composition For Allaying Inflammatory Reaction Due To Vennemous Hymenoptera Strings

Abstract

The invention relates to the use of a fatty acid for preparing a composition for allaying inflammatory reactions due to hypenoptera stings.

Description

The invention will be better understood upon consideration of the examples below which refer to the following figures:

FIG. 1: Percentage of inhibition of bee venom PLA2 activity as a function of acid concentration as follows:

FIG. 1A: Linoleic acid (example 1)

FIG. 1B: Oleic acid (example 1)

FIG. 2: Effect of oleic acid and linoleic acid on rat-paw volume increase in an inflammation model as a function of time following bee venom injection (example 2, experiment 1).

FIG. 3: Effect of oleic acid and linoleic acid on rat-paw volume increase in an inflammation model as a function of time following bee venom injection (example 2, experiment 2).

FIG. 4: Effect of oleic acid and linoleic acid on rat-paw volume increase in an inflammation model as a function of time following bee venom injection (example 2, experiment 3).

EXAMPLE I

In vitro inhibiting effects of linoleic acid and oleic acid on bee venom phospholipase A2 (PLA2) activity, a major inflammatory component of said venom.

The effect of linoleic acid and oleic acid on PLA2 activity is measured according to the hexane extraction method described by M. Katsumata, G. Gupta, and A. S. Goldman, (1986), Anal. Biochem. 154 (2), 676-681. PLA2 activity is determined by using a radioactive substrate, L-a-dipalmitoyl-[2,9,10(N)-3H-palmitoyl]-phosphatidylcholine. The reaction is carried out in 1 ml of glycine/NaOH buffer, pH 9, containing 2.2 mM deoxycholate, 0.11 μCi of dipalmitoyl-PC and 32 mU/ml of bee venom PLA2. After 20 minutes of incubation, the reaction is quenched by adding 0.2 ml of a Triton X-100/EDTA solution, and then the reaction product, radiolabeled palmitic acid, is extracted by a hexane solution containing 0.1% acetic acid and 0.7 g/ml Na₂SO₄. The radioactivity of the extract is determined using a liquid scintillation counter. The results represent the mean±SEM of the CPM values obtained in two independent experiments.

FIGS. 1A and 1B illustrate the percentage of inhibition of PLA2 activity (vertical axis) as a function of the concentration of the fatty acid used (horizontal axis).

These results show that linoleic acid and oleic acid inhibit the enzymatic activity of bee venom PLA2 in a dose-dependent manner.

EXAMPLE II

Activity of oleic acid and linoleic acid in a rat model of inflammatory edema caused by bee venom (G. A. Rabinovtch, E. C. Sotomayor, C. M. Riera, I. Bianco and S. G. Correa (2000), Eur. J. Immunol. 30, 1331-1339; J. Chen, C. Luo, H. L. Li, and H. S. Chen, (1999), Pain, 83, 67-76).

Experiment 1:

Bee venom (20 μg/ml in 0.9% NaCl) is incubated at room temperature in the presence or absence of linoleic acid (32 μM). Thirty minutes after incubation begins, the solutions (25 μl) are injected subcutaneously in the upper surface of the foot of Sprague-Dawley rats (110-140 g). Edema is measured using a plethysmometer at 5, 10, 15, 20, 30, 60 and 90 minutes following injection. The results represent the mean±SEM of 5 animals. Statistical comparisons are performed using a t-test, for which the control group is comprised of animals having received venom alone. (*p<0.05, ***p<0.005) The results are represented in FIG. 2.

Experiment 2:

At 120 and 30 minutes before the injection of bee venom (20 μ0.9% NaCl), the Sprague-Dawley rats (110-140 g) either receive or do not receive, on the top of the foot, an application of topical linoleic acid. Edema is measured using a plethysmometer at 5, 10, 15, 20 and 30 minutes following subcutaneous injection of bee venom in the upper surface of the animal's foot. The results (FIG. 3) represent the mean+SEM of 20 animals. Statistical comparisons are performed using a t-test, for which the control group is comprised of animals treated with the carrier (acetone).(*p<0.05, **p<0.01, ***p<0.005)

Experiment 3:

At 120 and 30 minutes before the injection of bee venom (20 μg/ml in 0.9% NaCl), the Sprague-Dawley rats (110-140 g) either receive or do not receive, on the top of the foot, an application of topical oleic acid. Edema is measured using a plethysmometer at 5, 10, 15 and 20 minutes following subcutaneous injection of bee venom in the upper surface of the animal's foot. The results (FIG. 4) represent the mean±SEM of 5 animals. Statistical comparisons are performed using a t-test, for which the control group is comprised of animals treated with the carrier (acetone). (*p<0.05)

Claims

1-6. (canceled)

7. A method for allaying an inflammatory reaction due to a hymenoptera sting, comprising: administering a composition comprising a fatty acid to a subject,wherein the composition is administered at least one of topically or by injection.

8. The method according to claim 7, wherein the fatty acid comprises a C12-C24 acid.

9. The method according to claim 7, wherein the fatty acid comprises at least one of lauric (n-dodecanoic) acid, myristic (n-tetradecanoic) acid, palmitic (n-hexadecanoic) acid, stearic (n-octadecanoic) acid, arachidic (n-eicosanoic) acid, behenic (n-docosanoic) acid, lignoceric (n-tetracosanoic) acid, palmitoleic (cis-9-hexadecenoic) acid, oleic (cis-Δ9-octadecenoic) acid, linoleic (cis,cis-Δ9-,Δ12-octadecadienoic) acid, linolenic (all-cis-Δ9-,Δ12-,Δ15-octadecatrienoic) acid, or arachidonic (all-cis-Δ5-,Δ8-,Δ11-,Δ14-eicosatetraenoic) acid.

10. The method according to claim 7, wherein the fatty acid is selected from the group consisting of lauric (n-dodecanoic) acid, myristic (n-tetradecanoic) acid, palmitic (n-hexadecanoic) acid, stearic (n-octadecanoic) acid, arachidic (n-eicosanoic) acid, behenic (n-docosanoic) acid, lignoceric (n-tetracosanoic) acid, palmitoleic (cis-Δ9-hexadecenoic) acid, oleic (cis-Δ9-octadecenoic) acid, linoleic (cis,cis-Δ9-,Δ12-octadecadienoic) acid, linolenic (all-cis-Δ9-,Δ12-, Δ15-octadecatrienoic) acid, and arachidonic (all-cis-Δ5-,Δ8 ,Δ11-,Δ14-eicosatetraenoic) acid.

11. The method according to claim 7, wherein the fatty acid comprises at least one of linoleic acid or oleic acid.

12. The method according to claim 7, wherein the fatty acid is selected from the group consisting of linoleic acid and oleic acid.

13. The method according to claim 7, wherein the composition has a form of at least one of an ointment, a cream, a gel, a patch, a powder, a spray, a lotion or a stick.

14. The method according to claim 7, wherein the composition is provided in an injectable form.

15. The method according to claim 7, wherein the composition is applied to a region of tissue close to the sting.