WO1997032577A1

WO1997032577A1 - Antidiabetic composition containing (2s, 3r, 4s)4-hydroxyisoleucine

Info

Publication number: WO1997032577A1
Application number: PCT/FR1997/000420
Authority: WO
Inventors: Yves Sauvaire; Gérard Ribes
Original assignee: Societe Civile De Gestion Jouvenet
Priority date: 1996-03-08
Filing date: 1997-03-07
Publication date: 1997-09-12
Also published as: AU2031997A; FR2745718B1; FR2745718A1

Abstract

An antidiabetic composition capable of stimulating insulin secretion and particularly suitable for treating non-insulin-dependent diabetes. The composition contains isomer (2S, 3R, 4S) of 4-hydroxyisoleucine having formula (I), and/or the lactone form thereof, and is substantially free of any other stereoisomer of this compound.

Description

ANTIDIABETIC COMPOSITION BASED ON (2S, 3R, 4S) 4-HYDR0XYIS0LEUCINE

The present invention relates to an anti-diabetic composition capable of stimulating the secretion of insulin and specially intended for the treatment of type II diabetes or non-insulin dependent diabetes.

It is known that diabetes affects more than 30 million people worldwide today, taking on the dimension of a major phenomenon in terms of public health; for example, we consider that in European countries, diabetes reaches between 2 and 5% of the population, and that in France, approximately 3 to 4% of residents suffer from non-insulin dependent diabetes which is by far the most frequent and in particular between 5 and 10% of subjects aged 60 to 70 years suffer from this disease.

In addition, and for various reasons related in particular to food richness, obesity, smoking or even a decrease in physical activity, the number of diabetics would have doubled in France in about twenty years, essentially due to an increase in non-insulin dependent diabetes.

This condition is characterized by an alteration in the secretion of insulin associated or not with an inino-resistance of the peripheral tissues. The alteration of the functioning of the pancreatic B cells which synthesize insulin, occurs from the initial phase of diabetes not insulin dependent. It results in a very marked reduction in insulin secretion in response to a glucose stimulation.

To treat this disease, specialists have therefore naturally been led to look for products capable of stimulating the secretion of insulin; among these, only the sulfonamides (sulfonylureas) have initially revealed an effectiveness. However, despite their advantages, sulfony¬ ureas have a certain number of drawbacks above all linked to the difficulties encountered in determining the appropriate dosage; this results in risks of overdose which can frequently cause hypoglycaemia, with the risk of a hypoglycaemic coma, especially in the elderly.

To remedy these drawbacks, we have in sequence sought to propose a drug capable of replacing the sulfonylureas to stimulate the secretion of insulin.

Thus, in accordance with document EP-0 587 476, it has been possible to highlight the anti¬ diabetic properties of certain mono or polyhydroxy amino acids among which the most active has been found to be 4-hydroxyisoleucine. formula :

5 4 3 2 1

OH CH ₃ NH ₂

and / or its lactonic form.

In accordance with this publication, a process for extracting the 4- hydroxyisoleucine from seeds of a particular species of trigonella (Trigonella sp: fenugreek Tricronel- foenum-graecum L) which is a legume easily cultivable in the Mediterranean regions. This process consists in implementing the following steps:

- fenugreek seeds are subjected to grinding and to a prior extraction with hexane, at ambient temperature so as to obtain a defatted cake, - this cake is subjected to several successive hydro¬ alcoholic extractions with ethanol at 70 % at room temperature,

- the extract obtained is concentrated under reduced pressure,

the concentrate is passed over a column containing a cation exchange resin in H form so as to retain the 4-hydroxyisoleucine sought in this column,

- this is eluted using an N or 2N ammonia solution, - after concentration and recovery with 70% ethanol, the mixture obtained is subjected to adsorption chromatography on silica gel, and

this is eluted with 70% ethanol before concentrating it under vacuum and purifying it by crystallization with the addition of diethyl ether so as to obtain a 4-hydroxyisoleucine titrating 99% purity.

To enable patients to benefit from the very specific anti-diabetic properties of 4-hydroxyisoleucine, it was decided to continue research on this product: in fact, the marketing of a new drug requires knowledge and precise description of the active ingredient; however, the skeleton of 4-hydroxyisoleucine contains three successive asymmetric carbons and consequently comprises eight stereoisomers which can be defined by the standardized notations {2S, 3R, 4S); (2S, 3R, 4R); (2R, 3S, 4R); (2R, 3S, 4S); (2S, 3S, 4S); (2S, 3S, 4R); (2R, 3R, 4R); (2R, 3R, 4S).

In view of this situation, we had the idea of seeking, in accordance with the invention, to which one (s) of these isomers should be attributed the antidiabetic characteristics or at least the essential of these characteristics. .

To this end, the work of Fowden et al. (Phytochemistry, 1973, vol.13, pp. 1707 to 1711) and d'Alcock et al (Phytochemistry, 1989, vol.28, pp. 1835-1841) according to which it has been established that in their natural state, Fenugreek contains only two isomers of 4-hydroxyisoleucine (diastereoisomers on carbon 2), namely the following isomers:

COOH COOH

H, N - C H H - C - NH-

H ₃ C - H H-.C - C - H

HO - C - H HO - C - H

CH, CH,

Isomer (2S, 3R, 4S) Isomer (2R, 3R, 4S)

More precisely, it has been proved that the Greek Fenu¬ contains approximately 97% of the isomer (2S, 3R, 4S) which must therefore be considered as the major isomer and approximately 3% of the isomer (2R, 3R, 4S) which should be considered as the minor isomer.

In accordance with the invention, it has been found experimentally that when Greek Fenu¬ seeds are extracted by the above process, described in the document EP-A-0 587 476, on a small scale, 4-hydroxyisoleucine is obtained by essentially retaining the above-mentioned proportions of major isomer and minor isomer; characteristics . physicochemical of the major isomer have been determined and correspond to the structure (2S, 3R, 4S).

'CH.

OH

This configuration could be attributed thanks to the following measurements and by analogy with previous work:

- Rotatory power (Perkin-Elmer) [α] ² ₎ ⁰ 31.3 (c = 1.2, H ₂ 0).

- SSppeeccttrroommééttrriiee iinnffrraarroouuggee ((PPeerrkkiinn - EEllmmeerr ,, FT-IR, Paragon

-i 1000) whose resonances are as follows in cm:

V _maχ 3500-2000, 1634, 1490, 1415, 1356, 1312, 1270, 1180,

1104, 1053, 1017, 962, 931, 899, 858, 817, 794.

- Mass spectrometry (JEOL, JMS-SX 102A) according to the “fast atom bombardment” technique in positive and negative modes with a glycerol-thioglycerol matrix:

FAB +: 148 (M + H) ⁺ 100; FAB-: 146 (MH) ⁺ 100

- H and C NMR spectrometry (Bruker AC 250 MHz) whose chemical shifts are as follows (in ppm: δ) ^λ E NMR δ: 1 (d, 3H, J = 7, CH ₃ C-3); 1.29 (d, 3H, J = 6.3, CH ₃ C-4): 1.97 (qdd, 1H, J = 7/4/8, CH-3); 3.88 ((qd, 1H, J) = 8 / 6.3, CH-4); 3.94 (d, 1H, J = 4, CH-2). ¹³ C NMR δ: 38.6 (C ₆ ), 47.2 (C ₅ ), 67.8 (C ₃ ), 83.5 (C ₂ ), 96.4 (C ₄ ), 200 (C _! ) .

Figures la to lf established the physicochemical characteristics of the (2S, 3R, 4S) isomer of 4-hydroxyisoleucine.

More precisely, FIG. 1 a corresponds to HPLC chromatography while FIGS. 1b to 1f represent respectively the IR spectrum, the mass spectrum "FAB" positive mode, the mass spectrum "FAB" mode ne-

1 13 gative, the H NMR spectrum and the C NMR spectrum.

On the other hand, we have realized that if we transpose the above-mentioned process to a larger scale for which the different stages require longer times, we obtain a product comprising approximately 85 to 90% of major isomer and approximately 10 to 15% of minimal isomer.

On the basis of these observations, an attempt was made to separate the two diastereoisomers by chromatography using a semi-preparative ultrabase C ₁₈ column which is characterized by a high hydrophobicity and a very low residual silanol index, therefore a high retention capacity; it is known that such columns are suitable for carrying out the separation of compounds having very polar functional groups, such as acidic or basic substances.

2 grams of an obte¬ naked mixture were thus purified following the implementation of the above-mentioned process under the following conditions: - Support: ultrabase C ₁₈ semi-preparative column,

5μM (10 mm x 15 cm)

- Detector: UV (190 to 220 nm) or refractometer,

- Eluent: pure water, - Flow rate: 2 ml / min.

It should be noted that the use of pure water as a diluting solvent has proved to be essential because the alcohol disturbs the separation. A preliminary analytical study carried out on

10 μg of mixture made it possible to verify that the retention time was 9.6 min. for the minor isomer and 10.2 min. for the major isomer and that it was therefore possible to separate the two isomers present. This possibility has been confirmed by experience and, the implementation of the separation process has effectively made it possible to obtain, from the initial two grams, and after purification, on the one hand, more than 400 mg of minor isomer. practically pure and on the other hand, approximately 1.5 g of major isomer with a purity of 97 to 98% identical to the compound isolated from document EP-A-0 587 476.

In accordance with the invention, pharmacological tests were then carried out to verify the antidiabetic activity of each of the isomers thus purified and it was found that the most powerful (eutomer) containing the major part of the antidiabetic activity is the major isomer while the minor isomer is clearly less powerful (distomer).

The object of the present invention is to take advantage of these experimental observations by proposing a new antidiabetic composition capable of stimulating insulin secretion and specially intended for the treatment of non-insulin dependent diabetes.

According to the invention, this anti-diabetic composition is characterized in that it contains, in as an active substance, the (2S, 3R, 4S) isomer of 4-hydroxyisoleucine of formula:

5 4 3 2 1 CH ₃ - CH - CH - CH - COOH i! I

OH CH ₃ NH ₂ 6

and / or its lactonic form, and in that it is practically free from any other stereoisomer of this compound.

According to a preferred characteristic of the invention, this active substance is of plant origin, and is in particular obtained from trigonella (Triαonella sp.) And more particularly extracted from fenugreek seeds (Triαonella fœnum-αraecum L).

The very specific anti-diabetic activity of the (2S, 3R, 4S) isomer of 4-hydroxyisoleucine will be brought to light by the following example, relating to an investigation at the level of islets of Langherans isolated from rat pancreas .

After digestion of the pancreas with collagenase, the islets of Langerhans which have B cells secreting insulin as well were separated from the other elements of the digest, taken under a binocular microscope, then placed in incubation tubes. This method, which has the advantage of requiring only a small amount of substance, allows a direct study of the effects of the composition on pancreatic endocrine cells, in particular insulin-secreting B cells, excluding ¬ sion of any interference with exocrine and anxious tissues.

On islets isolated from Wistar rats, incubated in the presence of 8.3 mM of glucose (1.5 g / l) for one hour, the effect of 200 μM of major isomer of 4- was sought. hydroxyisoleucine, on the one hand, and 500 μM and 1 mM of minor isomer on the other hand, on the secretion of insulin. The results obtained are shown in FIG. 2, on which the histograms represent for each dose of 4-hydroxyisoleucine isomer the insulin secretion measured over 60 minutes. The values given correspond each time to the average of 16 to 20 determinations.

On this diagram, it can be noted that for the major isomer, a significant stimulating effect on secretion appears for a concentration of 200 μM, while in the case of the minor isomer, this effect is observed only for a concentration 1 mM.

This pharmacological study therefore makes it possible to highlight the superior activity of the major isomer.

These results were confirmed by experiments carried out on the isolated pancreas and perfused in the presence of 8.3 mM of glucose.

In accordance with these experiments, the results of which are given in FIG. 3, the major isomer and the minor isomer of 4-hydroxyisoleucine were introduced at a concentration of 200 μM into the perfusion medium for 10 minutes.

The values given correspond each time to the average of 4 to 6 experiments.

This diagram clearly shows that, in the case of the major isomer, the stimulation is immediate and biphasic and persists throughout the duration of the infusion, whereas on the contrary, no notable effect is observed in the case of the 'minor isomer.

Claims

CLAIMS 1) Antidiabetic composition capable of stimulating the secretion of insulin and specially intended for the treatment of non-insulin dependent diabetes, characterized in that it contains, as active substance, the isomer (2S, 3R, 4S ) 4-hydroxyisoleucine of formula:

5 4 3 2 1

OH CH ₃ NH ₂

6 and / or its lactonic form, and in that it is practically free from any other stereoisomer of this compound. 2) Anti-diabetic composition according to Reven¬ dication 1, characterized in that the active substance is of plant origin.

3 °) Anti-diabetic composition according to Reven¬ dication 2, characterized in that the active substance is obtained from trigonella (Tricronella SP).

4) Antidiabetic composition according to Reven¬ dication 3, characterized in that the active substance is extracted from fenugreek seeds (Tricronella fœnum-graecum L).