Drug for treating affections provoked by a too high histamine level, of the gastroduodenal mucosa and allergic affections

Drug for treating affections provoked by a too high histamine level in the body, for treating affections of the gastroduodenal mucosa and allergic affections, comprising the reaction product of (+)-catechin with at least one basic amino-acid, which is more particularly selected from the group consisting of L-lysine, L-arginine and L-ornithine.

This invention relates to a drug and a method for treating affections 
provoked by a too high histamine level in the body, affections of the 
gastroduodenal mucosa and allergic affections. 
According to the invention the drug comprises the reaction product of 
(+)-catechin with at least one basic amino-acid. 
According to a particular embodiment of the invention, said drug comprises 
the reaction product of (+)-catechin with said basic amino-acid and 
further at least one other organic or inorganic acid. 
The amino-acid may be natural or not, such as, for example, lysine, 
arginine, ornithine, L-lysine, L-arginine and L-ornithine. 
The inorganic acids may be, for example, hydrochloric acid, sulfuric acid 
or phosphoric acid and the organic acids may be aliphatic, cycloaliphatic, 
aromatic, araliphatic or heterocyclic carboxylic or sulphonic acids, such 
as, for example, acetic, propionic, glycolic, gluconic, lactic, tartaric, 
citric, ascorbic, glucuronic, glutamic, methanesulfonic, toluenesulfonic, 
malonic, galactaric, galacturonic, maleic, fumaric acids. 
Based on new biochemical and pharmacological observations, it has been 
found that said reaction products of (+)-catechin and at least one basic 
amino-acid, and of (+)-catechin, one basic amino-acid and at least another 
organic or inorganic acid, are able to lower the histamine level in 
various tissues and cells, including gastric mucosa mast cells, peritoneal 
mast cells, leucocytes and lung tissue, and to inhibit the histamine 
release from mast cells. 
It is known that the histamine present in human gastric mucosa is 
exclusively localized in the mastocyte cells and that said histamine has 
an important effect on the secretion of gastric acid and on the origin of 
gastroduodenal ulcers (K. Mohri, et.al., Agents and Actions, 8, 372, 
1978). 
Furthermore, it is well known that histamin release caused by allergens 
plays a major role in allergic affections. 
Consequently, the above mentioned reaction products of (+)-catechin present 
great pharmaceutical interest for the treatment of gastroduodenal ulcers 
and affections of the gastric mucosa, of gastro-intestinal allergy and of 
allergic affections of the skin of the respiratory system. 
Of particular interest for said treatments are reaction products of 
(+)-catechin and L-lysine or L-arginine and of (+)-catechin and L-lysine 
or L-arginine and a mineral acid, such as hydrochloric acid, or an organic 
acid, such as acetic acid, citric acid or ascorbic acid. 
Examples of (+)-catechin derivatives which are particularly interesting 
are: 
(+)-catechin hydrochlorolysinate 
(+)-catechin ascorbolysinate 
(+)-catechin lysinate. 
The newly discovered pharmaceutical properties of said (+)-catechin 
derivatives have been proved by several studies in vitro, on animal models 
and on human beings, which are summarized hereafter. 
Biochemical studies in vitro have indicated that (+)-catechin 
hydrochlorolysinate inhibits histidine decarboxylase extracted from rabbit 
stomach with an IC.sub.50 of 10.sup.-4 M. 
Further studies, carried out in vitro, have shown that the release of 
histamine from rat peritoneal mastocytes, stimulated by the release 
inducer 48/80, is inhibited by (+)-catechin hydrochlorolysinate by 27% at 
a concentration of 10.sup.-50 M. 
The amount of released histamine has been measured after incubation of the 
cells at 37.degree. C. during 10 minutes with either an amount of compound 
48/80 determinated to obtain a release of 50%, either with such quantity 
of compound 48/80 in combination with the test substance. 
The inhibiting activity of said (+)-catechin derivatives on histamine 
release from rat peritoneal mastocytes has been confirmed by in vivo 
studies. In these experiments (+)-catechin hydrochlorolysinate was 
administered per os to Sprague-Dawley rats, 3 times a day during 3 days, 
at a dose of 30 mg/kg. 
After sacrifice the peritoneal mastocytes were incubated during 10 minutes 
at 37.degree. C. in the presence of 0.5 .mu.g/ml of compound 48/80, which 
induced 50% histamine release in the control group (6 rats per group). 
Compared to the controls the histamine release of the mastocytes of the 
treated group was decreased by 22%. 
Further studies, made in vivo, indicated that intraperitoneal injection of 
(+)-catechin hydrochlorolysinate at 30 mg/kg, induces a decrease of 33% of 
the histamine content in rat stomach mucosa and of 38% in the lungs. 
The effect of the (+)-catechin derivatives on the histamine content of 
stomach mucosa has been confirmed by clinical studies on human beings. 
In this study, 4 groups of 10 healthy persons received per os in double 
blind either placebo, either 500 mg, 1000 mg or 1500 mg of (+)-catechin 
hydrochlorolysinate per day during 3 days. Biopsies were taken before and 
after the treatment. In the treated groups, a decrease of the histamine 
level of the mucosa of the fundus, of the corpus and of the antrum has 
been observed which is dose dependent and which reaches a 30% decrease at 
the highest dose. The number of mastocytes has decreased in the same way. 
In the placebo group, the histamine level remained constant in the three 
areas of the stomach. 
Taking into account the histamine release inhibiting properties of said 
reaction products of (+)-catechin and a basic amino-acid and the known 
effect of histamine on the genesis of gastric ulcers, it was expected that 
the amino-acid derivatives of (+)-catechin present beneficial effects for 
the treatment of affections of the gastroduodenal mucosa.

The preventive and curative effect of said (+)-catechin derivatives has 
clearly been proved by studies on animal models, which are summarized here 
after. 
The preventive effect of (+)-catechin hydrochlorolysinate and of 
(+)-catechin ascorbolysinate has been studied as follows: 
(a) on ulcers induced by stress caused by immobilization: After one week 
adaptation to the environment, female rats, (30 animals per group, 5 per 
cage), were immobilised during 12 hours by wrapping them, under narcosis 
with ether, in a plaster bandage. 
After sacrifice of the animals, the dissected stomach was visually 
inspected and the lesions of the mucosa were scored according to their 
number and their intensity. 
(b) on ulcers induced by stress caused by cold: The experimental conditions 
are the same as the ones used in the study of ulcers induced by 
immobilisation, but in this model, stress was induced by placing the rats, 
one per cage, in a cold room at -10.degree. C. for 5 hours. 
The products to be tested were administered, immediately before the test, 
by intraperitoneal injection of 2 ml of a freshly prepared saline 
solution. The doses tested varied from 0.1 to 50 mg/kg. The controls 
received 2 ml of saline. 
The results obtained showed that treatment with amino-acid derivatives of 
(+)-catechin induced a significant and dose dependant decrease in the 
number of lesions of the stomach of stressed rats. 
When expressed in terms of dry (+)-catechin, the ED.sub.50 in the gastric 
ulcers induced in rats by immobilisation is: 
for (+)-catechin ascorbolysinate: 1.5 mg/kg 
for (+)-catechin hydrochlorolysinate: 5 mg/kg. 
Similar results were obtained against cold induced ulcers in rats and 
guinea-pigs. 
Furthermore, the preventive action of the catechin derivatives against 
stress induced gastric ulcers has been shown also after their 
administration by oral route. 
The technique used consisted of oral administration to rats of 30 mg/kg of 
(+)-catechin hydrochlorolysinate, given as a suspension in a 1% aqueous 
solution of carboxymethylcellulose. The tested product was given 3 times a 
day during the 3 days preceding the experiment. The last administration 
was given 1 hour before immobilisation of the rats during 12 hours. 
After sacrifice, inspection of the gastric mucosa revealed a significant 
decrease of the proportion of rats with severe lesions: from 38% for the 
control group to 15% for the treated group. 
The curvative activity on gastro-duodenal mucosa of the amino-acid 
derivatives of (+)-catechin has been established on animal models by means 
of the following technique: 
In rats fasted for 24 hours, necrosis of the gastric mucosa was induced by 
intra-gastric administration of acetic acid (1 ml at 20%) one hour before 
treatment. 
As a suspension in an aqueous solution of 1% carboxymethylcellulose, 
(+)-catechin hydrochlorolysinate was administered orally twice a day 
during 5 days. 
Accordingly the rats were sacrified, the stomach mucosa was photographed 
and the lesions were scored. 
In these conditions, at a dose of 150 mg/kg, a significant curative effect 
was noted as a reduction of 50% of the number of animals with severe 
lesions. 
The lowering effect on the histamine level of the gastric mucosa and the 
curvative activity on gastroduodenal ulcers of (+)-catechin 
hydrochlorolysinate have been confirmed in human beings in a double blind 
study, in which the aminoacid derivative of (+)-catechin has been compared 
to cimetidine. 
Patients suffering from duodenal ulcer were treated daily either with 1500 
mg (+)-catechin hydrochlorolysinate (11 patients) either with 800 mg 
cimetidine (10 patients) during 4 weeks. Photographs of the ulcer and 
biopsies of the stomach mucosa were taken before and after each week of 
treatment. 
The results obtained with (+)-catechin hydrochlorolysinate confirmed the 
activity observed previously, since a decrease of the histamine levels and 
a decrease of the number of mastocytes in the fundus, corpus and antrum 
mucosa by .+-.20% has been measured. Also a decrease of the mean histamine 
level of the plasma from 0.7 (.+-.0.3) ng/ml to 0.3 (.+-.0.1) ng/ml has 
been observed. The decrease was observed after one week treatment and 
remained constant during the following 3 weeks of the treatment. 
(+)-catechin hydrochlorolysinate has no effect on the intragastric pH. 
In the other group cimetidine showed no notable effect or induced an 
increase, in some cases of more than 10% of the histamine levels in the 
stomach mucosa and in the plasma. 
Further results, based on the measurements of the ulcer diameter, confirmed 
the curative properties of (+)-catechin hydrochlorolysinate on human 
duodenal ulcer: 7 patients out of 11 had fully recovered after 1 month of 
treatment. The ulcer diameter of the other patients had decreased 
significantly. 
In further biochemical studies carried out in vitro it was found that 
amino-acid derivatives of (+)-catechin are effective inhibitors of antigen 
induced histamin release from leucocytes of persons who are sensitive to 
allergic affections provoked by pollen or house dust. 
This activity of the catechin derivatives has clearly been established by 
the following technique: the leucocytes, preincubated during 10 minutes at 
37.degree. C. with hydrochlorolysinate of (+)-catechin, were treated with 
the allergin, pollen or house dust, during 30 minutes at 37.degree. C. to 
activate the histamine release. 
Then the histamin level was measured in the leucocyte suspension and the 
supernatant liquid and compared to the values obtained from the controls. 
Preincubation of the leucocytes by 10.sup.-5 M or 10.sup.-6 M 
hydrochlorolysinate of (+)-catechin resulted in a release inhibition of 
45%. 
The activity of amino-acid derivatives of (+)-catechin on the histamine 
level of lung tissue is shown by the following experiment: two groups of 
10 female Sprague-Dawley rats received either placebo either 30 mg/kg 
(+)-catechin hydrochlorolysinate during 3 days, 3 times a day. The drug 
was administered per os as a suspension in carboxymethyl cellulose (1% in 
water). Five hours after the last treatment the rats were sacrified and 
the histamine level in the homogenized lung tissue has been determined. In 
the treated group a decrease of the histamine level of 38% compared to the 
control group has been measured. 
Toxicological studies have shown that the considered amino-acid derivatives 
of (+)-catechin have extremely low toxicity. 
The acute toxicity of (+)-catechin hydrochlorolysinate, administered per os 
in rat is over 6 g/kg in both sexes. No mortality was found up to this 
dose, which did not cause behavioural changes. 
Subacute toxicity studies in rat and in Cynomolgus monkeys indicated that 
(+)-catechin hydrochlorolysinate is well tolerated at doses up to 1000 
mg/kg administered daily and per os during 5 weeks to monkeys and during 6 
weeks to rats. 
Besides, (+)-catechin hydrochlorolysinate is free of mutagenic properties, 
as is proven by the results of the Ames test and by the absence of 
chromosomic aberrations on human lymphocytes in vitro. 
The experimental results given above clearly demonstrate that the 
amino-acid derivatives of (+)-catechin, specified previously, exhibit 
important protective and curative activities on lesions of gastroduodenal 
mucosa and anti-allergic properties since they are able to lower the 
histamine content in various tissues and to decrease the histamine release 
from various cells. 
Consequently, said compounds have great therapeutical value for the 
treatment of gastroduodenal ulcers and for the treatment of allergic 
affections of the respiratory system, such as asthma and rhinitis, of 
allergic contact dermatitis and urticaria, of gastro-intestinal allergy 
and of allied allergic diseases. 
The catechin derivatives useful for this invention may be administered, in 
view of the intended therapeutical application, orally, parenterally, 
topically, rectally or as an aerosol in various galenic forms. 
This comprises pharmaceutical compositions containing as active ingredient 
at least a minimal effective amount of at least one of said reaction 
products of (+)-catechin and L-lysine, L-arginine or L-ornithine and which 
may contain also diluents, carriers or excipients and/or other 
pharmaceutical active ingredients. 
Thus for example the compositions to be administered orally can be liquids 
or solids and exist as tablets, sugar-coated pills, coated tablets, 
capsules, granules, powders, syrups or suspensions. The dry oral 
formulations comprise additives and excipients usually used in galenic 
pharmacy, inert diluents, desintegration agents, binders and lubricants, 
such as lactose, starch, talc, gelatin, stearic acid, cellulose and 
derivatives thereof, silicic acid, magnesium stearate, 
polyvinylpyrrolidone, calcium phosphate, calcium carbonate and the like. 
The aqueous suspensions, the emulsions and the oily solutions are prepared 
in the presence of sweetening agents, such as dextrose or glycerol, 
flavouring agents, such as vanillin for example, and can also contain 
thickening agents, wetting agents, preservation agents. 
The oily emulsions and solutions are prepared in an oil of vegetal or 
animal origin and can contain emulsifiers, flavouring, dispersing, 
sweetening and antioxidant agents. For parenteral administration, sterile 
water, an aqueous polyvinylpyrrolidone solution, peanut oil, ethyl oleate 
and the like are used as a vehicle. These aqueous or oily injectable 
solutions can contain thickening, wetting, dispersing and gelling agents. 
For rectal administration, several galenic forms can be used as 
suppositories or rectal capsules or gels. 
The catechin derivatives useful for treating and preventing gastroduodenal 
affections can be used alone or in combination with other pharmaceutical 
active compounds exerting a similar or a different activity. 
Usual daily doses for oral and rectal administration of the (+)-catechin 
derivatives, may vary from 200 mg to 5 g, and are preferentially 1500 mg. 
Usual daily doses for parenteral administration of the (+)-catechin 
derivatives vary from 50 mg to 5 g and are preferentially 1500 mg. 
For topical administration galenic forms such as, for example, creams, 
pastes, ointments, solutions, suspensions, emulsions or gels can be used 
and the usual concentration of active compound in said galenic forms may 
vary from 0.1% to 30%. 
Hereafter, a few galenic formulations are described as non limitative 
examples in which the active compound is represented by A and in chosen 
from the following compounds: 
(+)-catechin hydrochlorolysinate 
(+)-catechin ascorbolysinate 
(+)-catechin lysinate. 
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Tablets 
A 500 mg 
Ac-Di-Sol 90 mg 
Aerosil 200 20 mg 
Polyvinylpyrrolidone 30 mg 
Talc 30 mg 
A 200 mg 
Aerosil 200 12 mg 
Talc 12 mg 
Ac-Di-Sol 35 mg 
Magnesium stearate 1 mg 
Suppositories 
A 500 mg 
Witepsol H 15 2500 mg 
Injectable 
A 20 mg 
Benzylalcohol 20 mg 
Lysine ad pH = 7.4 
aqua purificata ad 1 ml. 
Cream 
A 10 mg 
Glycerin 2 g 
Perhydrosqualene 8 g 
Liquid paraffin 8 g 
Solid paraffin 6 g 
Cetylstearyl alcohol 4.5 g 
Sodium cetylstearylsulfate 
0.5 g 
Emulgine B-3 2 g 
Aluminium stearate 0.3 g 
Citric acid 0.1 g 
Nipasept 0.2 g 
Distilled water ad 100 g 
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It has to be noted that the studies and tests referred to hereinabove are 
also valid for drugs comprising the reaction products of (+)-catechin with 
L-arginine or L-ornithine and possibly with another acid as defined 
hereinabove.