FR2531338A1 - New therapeutic compositions intended for the treatment of contact allergies - Google Patents

Abstract

The invention relates to new therapeutic compositions intended for the treatment and prevention of allergies, containing as active principles at least one combination of catechol 2,3-oxygenase and an albumin.

Description

 The present invention relates to novel therapeutic compositions for the treatment and prevention of contact allergies produced by anacardiaceae and products derived therefrom.

 Approximately 80% of Americans (USA) are highly susceptible to "poison ivy (Toxicodendron radicants) in Dungeon oak" (T. diversllobum) or "poison sumad" (T vernix) and 50% of them have a clinical reaction. (contact allergy) (WL, Epstein, Cutis, 13, 544-548 (1974).

 The agent responsible for this "Rhus dermatitis" (AM Kligman, Arch Derm .., 77, 149-180 (1958)), is a mixture of catechols having a straight chain with 15 or 17 carbon atoms, more or less unsaturated.

The ivyl poison contains mostly derivatives in
C15: pentadecyl 3 catechol (PDC) pentadecene-8, yl-3 catechol, pentadecadiene-8, 11 yl-3 catechol and pentadecatrate-8,11,14-yl-3 catechol, PDC (saturated compound) constituting only 2 at 3% of the total called URUSHIOL.

The "poison oak" contains mainly catechols having a 3-membered chain of 17 carbon atoms 3-heptadecyl catechol (HDC), heptadecene 8-yl-3 catechol, heptadecadiene-8, 11 -3 catechol, heptadecatriene-8, 11, 14 -3 catechol (WF Symes, CR Dawson, J. Amer, Chem.

Soc., 76, 2959-2963, (1954), M. Gross, H-. Baer, H.M.

Fales, Phytochemistry, 14, 2263-2266, (1975), MD. Corbett,
S. Tickets, J. Pharm. Sci., 64, 175-1718 (1975). The oleoresins extracted from "poison oak" and "ivy" give cross-allergic reactions in patients who are sensitive to one or the other (WC Spain, JM Newell, MG Meeker,
J. Allergy, 5, 571-574 (1934). Dienes and trienes are more sensitizing than PDC or HDC saturates (RA Johnson, H. Baer, CH Kirkpatrick, QC

Dawson, R. G. Khurana, J. Allergy Clin. Immunol, 49, 2735 (1972).

This "urushiol" is also present in a Japanese tree from which is extracted the lacquer of China (or Japan),
Rhus verniciflua (lacquer tree) from where it was first isolated by Maxima (the name "urushiol" is derived from the Japanese name of this tree kiurushi). All the plants mentioned are part of the Anacardiaceae family, which also includes mango (Mangifera indica), bhilawa (Semecarpus anacardium, Indian marking nut tree), cashew nut tree (Cashew nut tree) and many more.

 Contact dermatitis with urushiol therefore concerns not only the plants themselves ('poison ivy', 'oak poison sumad') but also their derivatives: fruits (mango, cashew nut), lacquer, "ink to be marked" (S.

anacardium) etc. Cross allergies between all these plants or substances have been widely described in the medical literature (JC Mitchell, A. Rook,
Botanical Dermatology, Greengrass, Vancouver, 1979).

 US Pat. No. 4,002,737 describes a method for the treatment and prevention of this type of allergies by applying to the surface of the skin capable of coming into contact with allergenic compounds an effective amount of catechol 1,2-oxygenase. This patent also mentions the possibility of using in this type of treatment catechol 2,3-oxygenase prepared from P. avila. However, according to this patent, given the low stability of this enzyme, it does not seem particularly appropriate and has therefore not been the subject of any particular test.

These two enzymes have, indeed, the property of cutting the catechol cycles to turn them into non-allergenic acyclic derivatives, according to the following scheme for catechol 2,3-oxygenase (hereinafter referred to as C 2,3-O):

Urushiol being a mixture of compounds of formula

<tb><SEP> C <SEP> 15 <SEP> H31
<tb><SEP> - (CH) 7 <SEP> - <SEP> CH <SEP> = <SEP> CH <SEP> - <SEP> (CH2) 5 <SEP> - <SEP><SEP> CH3
<tb><SEP> 27 <SEP>
<tb> - <SEP> (CH2) 7 <SEP> - <SEP> CH <SEP> = <SEP> CH <SEP> CH2 <SEP> - <SEP> CH <SEP> = <SEP> CH <SEP> - <SEP> (CH2) 2 <SEP> - <SEP> CH3
<tb><SEP>(<SEP> - <SEP> (CH2) 7 <SEP> - <SEP> CH <SEP> = <SEP> CH <SEP> - <SEP> CH2 <SEP> - <SEP> CH <SEP> = <SEP> CH <SEP> - <SEP> CH2 <SEP> - <SEP> CH- = <SEP> CH2 <SEP>
<Tb>
this can be detoxified by the previous enzymes which cut the catechol cycles.

 However, this theoretical activity proves very difficult to demonstrate in vivo, in fact urushiol being a very poorly soluble product the activity of the enzyme is greatly reduced.

 Even using surfactants, the observed degradation results prove to be very insufficient.

 However, the Applicant has discovered that it is possible to achieve almost complete detoxification of urushiol by using, in combination with catecol 2,3-oxygenase, an albumin.

 This is why the present invention relates to novel compositions, in particular therapeutic compositions useful in the treatment and prevention of contact allergies caused by plants of the family Anacardiaceae and their products derived, comprising as active principle the least a combination of catechol 2,3-oxygenase and albumin.

The characteristics of the catechol 2,3-oxygenase enzyme and processes for its preparation are described in the French patent application No. 82 01574 filed on February 1, 1982 in the name of the Applicant.
The United States also mentions other methods of preparation of C 2,3-O, especially from P. avila.

Although as part of this description
albumin has been used essentially as human bovine serum, it is possible to use a
any albumin for example an original albumin
possibly to limit the risk of irritation
local level.

The use of this protein presents in
the framework of therapeutic compositions for the u
wise a lot of advantages besides the acti
detoxifying life.

Indeed, it is a product presenting
an emulsifier common to all proteins
and which lends itself particularly well to the realization
creams or laibs
But it is also a very product
soluble in water that can be easily removed by
ethanol and is also suitable for preparing
in the form of spray or lotion.

 The compositions according to the present invention can also be used for the decontamination of objects, in particular clothes, for example by washing.

The concentrations of active principle can vary over a wide range and depend in particular on the action envisaged, namely preventive or curative action, but also on the amount of allergenic agent to which
the subject may or may be exposed.

In general, it is possible to use
compositions according to the present invention which are saturated
albumin, but it is possible to use quantities
much lower albumin up to
0.01% by total weight of the composition.

 With regard to the concentration of catechol 2,3-oxygenase, this concentration can vary widely; for example between 20 and 0.01% by weight of the composition, although it is generally preferable to use smaller amounts of the order of 5 to 0.01% taking into account the cost of this enzyme.

 In terms of its activity, it seems that lgalbumine can play a role vis-à-vis the lipophilic compounds, and block the catechol alkyls when they are oxidized.

In addition, it should be noted that this is a product with low toxicity applied to the skin because it does not penetrate the skin barrier and believed it is a cheap product
The compositions according to the present invention can be used both for preventive and curative purposes.

The compositions according to the present invention may be in any form that can be used topically, whether it is cream, lotion or
spray, which will not pose a problem for their shaping since the principles actis used are so-
lubles in the water. It is also possible to make milks or powders. In some cases, it can be
interesting to provide the preparations in extempora form
born in which the active ingredients will be present
in a form ensuring their preservation; lyophilic form
for example, which will not be put in contact with
the substrate intended for their application only a short time
before use.

Although the process for the preparation of catechol 2,3-oxygenase is not one of the elements
However, in the aforementioned French patent application, said enzyme can be prepared from Bacillus subtilis, whereas until now this enzyme was only prepared from Gram-negative bacteria such as Psendomonas and Escherichia coli which are pathogenic for humans.

 It is obvious that the possibility of preparing such an enzyme from a non-pathogenic microorganism for subtilis Bacillus man is a considerable advantage in the application of this enzyme in the therapeutic field.

The catechol 2,3-oxygenase can be prepared from Escherichia coli according to the process described by Inouye Sachye, Nakazawa Atsuhi and Nakazawa Teruko, 1981,
Journal of Bacteriology 145, 1137-1143 Z "Molecular Cloning of TOL Genes XylB and XylE in E. coli". The preparation of the same enzyme from Bacillus subtilis has already been described in the French patent mentioned above.

 The following tests were conducted using a 2,3-0 C prepared from a strain of E. coli BZ18 containing the plasmid pTG203 which carries the catechol gene 2,3-oxygenase, this strain is described in the French patent mentioned above. It should be noted, however, that the process for the preparation of C 2.3-0 does not constitute a feature of the present invention and that it is possible to prepare this enzyme using other bacterial strains.

 The general properties of catechol 2,3-oxygenase have already been widely studied in the aforementioned French patent application.

 Essentially, it should be remembered that the enzyme is stable up to a temperature of about 600C, and that acetone shows a stabilizing effect.

In addition, the optimum pH of activity of 2,3-C
0 is 6.75, as to its optimum temperature of activity
it is close to 45 0C.

Storage trials of the enzyme at different
temperatures and under different conditions have shown that
it could be kept without difficulty during
durations of a few months.

The following experiments highlight the remarkable properties of the compounds
according to the present invention.

 The experiments are conducted on six Hartley albino guinea pigs which are sensitized respectively to pentylcatechol, pentadecylcatechol and urushiol.

 The guinea pigs were sensitized by the so-called "Freundis complete adjuvant test" technique.

 The protocol consists in making 3 intradermal injections of 0.1 ml of 1% sensitizing emulsion in the postnuqual region at days 1, 3 and 5, respectively. The emulsions are made of Freund's complete adjuvant and water. physiological 1/1 by volume.

The control group is treated in the same manner but with an emulsion containing no allergenic agent. With regard to sensitization to urushiol, it was carried out by the natural route that is to say the epicutaneous route by applying 0.1 ml of a 0.1% ethanolic solution of urushiol % on 8 cm2 of flank-shaved and shorn of the animal on days 1,3 and 5, these unimaux were boosted by injectior
10) Sensitivity tests apyres actïonde enzvme in vitro
In this first group of experiments the allergen is previously incubated with the enzyme in vitro, it is verified by spectroscopic methods or on chromatoplate that the substrate has been completely transformed. This medium is then applied to the flank of sensitive guinea pigs. The animals are tested simultaneously with an allergen solution containing no enzyme (positive controls).

The observed results are summarized in the table below.

<Tb>

-Substance <SEP> Reaction <SEP> 24 <SEP> h <SEP> Reaction <SEP> 48 <SEP> h
<tb> Pentylcatechol <SEP> 18 <SEP> mM <SEP> 1.9 <SEP> 1.5
<tb> Pentylcatechol / Enz /
<tb> BSA <SEP> O <SEP> O <SEP>
<tb> Urushiol <SEP> 0.03 <SEP>% <SEP> 1.7 <SEP> i <SEP> 1.5
<tb> Urushiol <SEP> 0.03-e / BSA <SEP> 0.6 <SEP> 0.4
<tb> Urushiol <SEP> 0.03% / BSA / <SEP>
<tb> Enzyme <SEP> O <SEP>
<Tb>
The figures represent the average degree of irritation observed in the animals according to the scale of zero = no irritation, to five = necrosis.

 The above results clearly demonstrate the remarkable activity of the compositions according to the present invention and in particular the quite remarkable activity of albumin alone used in the treatment of this type of dermatoses.

 By the implementation of the compositions according to the present invention no cutaneous reaction is visible either at 24 hours or 48 hours.

 Figures 1 to 5 attached show the photos of these different experiences.

 The experimental protocols are recalled below.

Figures 1 & 2: Action of 2,3 catechol oxygenase in the presence of bovine serum albumin (BSA) on the outward effect of pentylcatechol "PC"
Surface A: allergen alone
Surface area B allergen + BSA + C 2,3 0
Protocol: two two solutions are prepared
Solution 1: 18 mM PC in Ethanol,
Application of 25jul on the surface.

Solution 2: PC O 18 mN
C 2.3 0: 300 U / ml
BSA. half-saturation
Phosphate buffer 0 50 mM
pH 6.8
Incubation 1 h at 370C, storage during
night at room temperature (22 C)
Application of 25uI on surface B
Figures 3,4 & 5: Action of C2,3-0 and BSA on the allergenic effect of urushiol
Surface A Urushiol + C 2,3 0 + BSA
On face 3: Urushiol + BSA
On face C Urushiol
Protocol: 3 solutions are prepared.

Solution 1 0 Urushiol: 0.03%
C 2.3 0: 350 umites / ml
BSA: 1/3 saturated
TpShosphate: 50 mM
pH 6.8 Incubation Ih at 370C, storage during
night at room temperature (220C)
25 l deposited on the surface A
of each animal
Solution 2: Urushiol: 0.03%
BSA: 1/3 saturated
TpPhosphate: 50 mM
pH 6.8
Incubation 1 hr at 370C
Overnight storage on time
ambient temperature (22bC)
25 l deposited on the surface B
of each animal.

 Solution 3: Urushiol 0.03% in methanol.

20) Sensitivity test after direct action of
of the enzyme on the skin
In these experiments, the procedure is as follows: at time O, 25 l of an ethanolic solution of allergen is applied to 2 cm 2 of skin. When the ethanol is evaporated, the enzymatic medium is applied to the same surface in the form of an aqueous solution.

It is dried under a stream of warm air after a few minutes and a second application of 25 μl of enzymatic medium is made 1 hour later, which is dried under the same conditions. The results are read after 24 and 48 h. It should first be noted that during the first application of the enzymatic medium after a time of about 1 minute, a yellow coloration develops which indicates that the oxidation of the allergens has already started. During the second application no color reappears.

 The results observed in this experiment are summarized in the table below.

Enzymatic medium: C 2,3-O: 350 U / ml BSA.saturated at 1/3
TpPhosphate, pH-6.8 - 30 mM

<tb><SEP> Substance <SEP> Reaction <SEP> 48 <SEP> h
<tb> 1) <SEP> Pentylcatechol <SEP> 18 <SEP> mM <SEP> (0.3%) <SEP> 0.5
<tb> 2) <SEP> Pentylcatechol <SEP> 18 <SEP> mM / Enz / BSA <SEP> 0.1
<tb> 1) <SEP> Urushiol <SEP> 0.03% <SEP> i <SEP>1; 2 <SEP>
<tb> 2) <SEP> Urushiol <SEP> 0.03% <SEP> Enz.BSA <SEP> 0.8
<Tb>
The skin reactions, although positive, have very markedly decreased after application of the compositions according to the invention.

Claims (10)

 1) Composition useful in the treatment and prevention of contact allergies comprising as active ingredient at least one combination of catechol 2,3-oxygenase and albumin.  2) Composition according to claim 1, characterized in that it is a therapeutic composition applicable topically.  3) Composition according to claim 1, characterized in that it is a decontamination composition.  4) Composition according to one of claims 1 to 3, characterized in that it is a cream, a lotion, a milk, a powder or a "spray".  5) Composition according to one of claims i'à 4, characterized in that lacatechol 2,3-oxygenase is presented in freeze-dried form.  6) - Composition according to one of claims 1 to 5, characterized in that the concentration of catechol 2,3-oxygenase is between 20 and 0.01% by weight of the composition.  7) Composition according to claim 6, characterized in that the concentration of catechol 2,3-oxygenase is between 5 and 0.01% by weight of the composition.  8) Composition according to one of claims 1 to 7, characterized in that the albumin concentration is between 0.01% by weight of the composition and saturation.  9) Composition according to one of claims 1 to 8, characterized in that 11 albumin used is bovine serum albumin.  10) Composition according to one of claims 1 to 9, characterized in that the catechol 2,3-oxygenase is prepared from Bacillus subtilis.