NL8900573A - NEW SALT TYPE COMPOUNDS, PHARMACEUTICAL PREPARATIONS WITH SUCH COMPOUNDS IN IT AND METHOD FOR THE PREPARATION THEREOF. - Google Patents

Description

«I

NL 35.804-Kp / ab x I

* r I

New salt-type compounds, pharmaceutical preparations with I.

such compounds therein and method of preparation I.

of it I

The present invention relates to I.

new antiviral and immunosystem stimulating salts and I.

pharmaceutical preparations containing such salts. I

According to another aspect of the invention, I

5 for a process for the preparation of the new salts. I

The advantageous antiviral effect of u -3-multiple-I

digly unsaturated fatty acids [5,8,11,14,17-eicosapentane carbon- I

acid (hereinafter: EPA) and 4,7,10,13,16,19-docasahexane carboxylic acid I.

(hereinafter: DHA)] have received special attention. Szads I

10 [Antimicrobial Agents and Chemoterapy 12, 523 (1977)] have I

in in vitro experiments demonstrated that polyunsaturated I

fatty acids, for example both EPA and DHA, are capable of I.

inhibit virus multiplication. The same fact became I.

supported by Reinhardt and employees [J. of Virology 25, I.

15 479 (1978)] which inhibits the multiplication of PR 4 I.

bacteriophage. I

The antiviral effect of polyunsaturated I.

fatty acids, below that of EPA and DHA, is Embodiment I.

described in U.S. Patent No. 4,513,008. I

The influence of EPA and DHA was compared in animal experiments with I.

mice and guinea pigs, with Acyclovir [9- (2-hydroxy-I

ethoxy-methyl) guanine] currently the most widely used antiviral I.

agent. It has been stated that preparations containing mainly I

DHA, have a more beneficial effect against herpes virus than I.

Acyclovir. I

A number of articles have been devoted to literature I.

to the antiviral activity of DHA and EPA, such as Whitaker and I.

employees [Proc. Natl. Acad. Sci. USA 76, 5919 (1979)] as well as Goodnight and co-workers [Arterioschlerosis, 2: 87 I

30 (1982)] and Yoshiaki [Biochimica et Biophysica Acta 793,80 I

(1984)].

Prickett and co-workers [Immunology 46, 819 (1982)] I

have shown that the humoral immune response was stimulated by eicosapentanecarboxylic acid as an arachidonic acid 89 00573. " «I - 2 - analog. In an EPA-rich diet, the specific IgG and IgE production as a response to egg albumin was increased in inbreed Sprague-Dawley rats from 4 to 8-fold values compared to the control. According to this article, an increased antibody response was induced by the EPA rich diet. Furthermore, studies have shown that EPA acts through inhibition of the suppressive prostaglendin system, in which it is able to inhibit the immune deficiency that occurs with aging and other pathological processes (autoimmuno processes, tumourogenesis). correct respectively. These findings were supported by articles by Kelley and co-workers [J. or Immunol. 134, 1914 (1985)] as well as Homey et al. [Clin. Exp. Immunol. 65, 473 (1986)]. It is based on the in vivo studies of

Pearson and co-workers [Proc. Soc. Exp. Biol. Med. 79, 409 (1952)] have long been known to L-lysine inhibit encephalomyelitis virus. Later Tankersley [J.

Bact. 87, 609 (1964)] draw attention to the fact that the multiplication of the herpes simplex virus (hereinafter: HSV) in human cells is inhibited by lysine under in vitro conditions. Based on human studies, Kagan has published [The Lancet 1, 137 (1974)) that both the oral and genital HSV-induced lesions resolved very rapidly under the influence of L-lysine treatment.

Griffith and co-workers [Dermatologies 156, 257 (1978)] studied the therapeutic effect of L-lysine on 45 patients infected with HSV I and HSV II at different dosages over various treatment periods. The age of the patients (mainly women) varied between 8 and 60 years. It was observed that L-lysine had only a suppressive but not a curative effect on HSV.

The present invention relates to new therapeutic active salts and to the use of such salts for the preparation of pharmaceutical preparations, in which the beneficial therapeutic effect of the ω-3 saturated fatty acids is combined with that of the amino acids, such as in particular lycin , ornithine and histidine, which preparations have a stronger effect than the so far 8900573.

% I

known antivirals. I

The invention is based on the recognition that the I

salts obtained by the salt formation of a ω-3 unsaturated I.

the fatty acids, in particular EPA and DHA, with amino acids or I.

5 their derivatives, especially with lysine, a strong anti-I

have viral and immunostimulatory action. I

Accordingly, the invention relates to I.

the new antiviral and immunostimulating salts with the I.

general formula I 1

10 I

R-C00 “AH + (1)

in which I

R is a C 1-8-24 "alkYllop with at least two double I.

15 bindings? while I

WA "represents an amino acid found in living organ. I

ganisms and / or a derivative thereof with a carboxyl-I

group which is substituted by a C 1-4 alkyl group I.

or an amino group or by an alkali metal cation. I

According to the present invention, these new I

salts prepared by conversion of an I as the basic component

amino acid (A), as represented by general formula 1, I.

wherein the substituents have the meanings defined above I

with an acid of the general formula (2) I

25 R-COOH (2)

where R has the above meanings, in a polar I

solution. I

The compounds of the general formula (1) are at I.

preferred salts formed from ω-3 unsaturated fatty acids with I.

therein at least two double bonds with basic I.

amino acids or their derivatives. I

The potential toxicity to a cell culture of the I

Salt compound of the invention was studied with an I.

salt, formed from a ω-3 polyunsaturated fatty acid I

mixture (containing 27.6% EPA and 44.6 DHA) with L-lysine at I.

based on its action on the multiplication and morphology of I.

Hep 2 cells (human epithelial tumor cell line). This experiment I

8900573.

2-4 vaccinations were performed on a plastic tissue culture plate containing 6x4 cavities (cavities with a bottom area of 1.9 cm2 each).

A stock solution containing the test compound at a concentration of 10 mg / ml was prepared in an Eagle HEM medium (manufactured by Serva GmbH Co., Heidelberg, Federal Republic of Germany). A certain part of the stock solution was diluted 10 times, after which the resulting solution was diluted 2 times and then by repeating 2 times the 10-fold dilution of the last solution, one has solutions with successively decreasing concentrations of the compound to be tested (solutions nos. 1 to 5) prepared. The cells were treated with 1 ml of a solution, each of which contained the active ingredient in the following concentrations:

Solution Concentration of the active ingredient n ° __ u q / ml_ 1 10,000 20 2 1,000 3 500 4 250 5 125 25 The treatment was carried out for 1 hour, after which the cultures were washed twice with buffered sodium chloride solution (phosphate buffered saline, hereinafter : PBS), after which a nutrient medium was added to the cultures. After 24 hours and fixation by methanol, the 30 cultures were stained with an ethanolic Giemsa solution (produced by Reanal, Budapests, Hungary) and the morphology of the cells was evaluated under light microscope. It could be established that the test compound was only toxic above a concentration of 1000 µg / ml. The virus multiplication inhibition activity was tested on Hep 2 cells as described above. HSV type I was used for infection. The virus concentration was about 1000 PFU (plaque forming unit). Solutions containing the test compound at a concentration of 8900573.

rates of 1000, 500 and 250 µg / ml, respectively, were I.

used, while for the treatment to the undiluted I

virus suspension 0.1 ml of the solution was added. The I

mixture was incubated at 37 ° C for 1 hour. Then I

5 cells treated with the virus pre-incubated with the I.

connection to be investigated while the development of the I

cytopathalogical (hereinafter: CP) changes during 7 I.

days were observed. I

It could be established that the virus multiplication I

10 g is immediately inhibited by a 500 µg / ml or 250 I.

µg / ml dose to the test compound, while the I

infective titre value of the cytopathalogical dose (neg. I

log. CPD50) was 1.75 calculated for 0.1 ml compared to the I.

neg. log. CPD50 value of 4.5 from the untreated control I.

Virus culture. The value of the virus inhibition increases by an I.

factor of 2 relative to the control. Under the same I

circumstances ΕΡΆ and DHA had no valuable virus-inhibiting I

ming, and lysine alone inhibited virus multiplication by I.

a factor of only about once. I

The vaccine virus was also treated in an in vitro I.

test carried out in the manner described above. The I

infective titer value (neg. log. CPD50) of the test I

a compound measured by the vaccine virus was found to be I.

To be 1.75 compared to the 5.5 value of the untreated I.

25 control virus, that is, the compound to be tested I

thing had a virus-inhibiting effect three times stronger I

than that of the control. I

The in vitro action on the immune system of the I

bonds according to the invention were studied with reference to I.

30 of the activation of the lymphocytes by polyclonal mitogenic

nen. The blastic transformation of the lymphocytes was assessed at I.

examined a way that a lyphocyte cell population obtained on a Fico Uromiro gradient [Scand. J. Clin. Lab.

Invest. 21, 97 (1968)] was pipetted into the cavities of flat plates 35 µg / ml Concanavalin A (hereinafter: Con A) I

(manufactured by Pharmacia, Uppsala, Sweden) and then a solution containing a new compound of the invention I.

at a concentration of 0.1, 1.0 or 10 µg / ml, respectively, was added to each of the parallel cultures. I

8900573.

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As a control, a culture containing 25 µg / ml Con A without the test compound was used therein. The plates are kept under an air atmosphere containing 5% carbon dioxide at 37 ° C for 72 hours, with 0.4 Ci 3 H-thymidine added to each sample after 64 hours prior to termination of the culture. After 72 hours, the cultures were filtered through glass filter, then the filters were transferred to a scintillation cuvette, measuring the radioactivity in 5 ml of toluene solution using a beta counter. The results are shown in the following table.

No. Compound Concentration of Count / min Evaluation name the active constituent of the 15 parts uq / g activity 1. Control 25 8969 + 2984 (Con A) 20 2 L-tyrosine 0.1 12915 + 2045 weak significant growth 1.0 12313 + 2003 3. L-lysine 0.1 11063 + 1528 not significant 1.0 11833 + 1823 weakly significant growth 4. Na salt of 0.1 12332 + 2235 not significant 30 ω-3 fatty acid mixture (see 1 , 0 12039 + 1640 non-signifi- able preparation in cant example I) 35 5. control (Con A) 25 8396 + 2233 6. Product of 0.1 14965 + 2143 strong signature example XVI cant 1.0 12517 + 2195 not signifi- cant 40 cant 7. Product of 0.1 15985 + 2102 strong signature example Instant growth 1.0 14068 + 1665 significant 45 growth

These studies clearly showed that the compounds according to the invention, in particular the salt of 50 lysine or tyrosine, formed with a mixture of multiple 8900573,

-Λ I.

unsaturated fatty acids, significant research results I

that is, a strong immunostimulatory action, I

while the individual salt-forming components as such I

were found to have no or only a weak biological effect

5 have. I

As a starting material of CIG-24 ω ~ 3 unsaturated I.

fatty acids being one component of the salts, it is suitable I.

first, to use all the oils that I

can be obtained from North Sea fish, such as I.

10 salmon, cod, sardine or oils from their livers, I.

however, also oils from freshwater fish I

are also suitable. The ω-3 polyunsaturated I.

fatty acids were obtained from the above oils under I

application of a known method [J. Am. Chem. Soc. 59, 117 I.

15 (1982)].

The active compounds of the general formula I I.

can be transferred into capsules, tablets, dragees and I.

other pharmaceutical preparations which are known per se

assembled in a manner using carriers I.

And / or additives, such as lactose, starch, magnesium stearate I.

and the like, which are commonly used in the pharmaceutical industry I.

are applied. I

For inhibiting the oxidation of the preparation I.

is the application of ω-tocopherol (vitamin E), glutathione or I.

The traditional antioxidants, such as butylhydroxytoluene, I.

desirable. I

The main advantages of the dilution I

according to the invention and the pharmaceutical preparations, I

prepared therefrom can be summarized as follows. I

30 1. They can be used against acute virus I.

infections; they are particularly suitable in the herpus virus infection for suppressing the infection at an early stage. I

2. Due to their immunostimulatory action, they can preferably be used against retroviruses, in particular against the immunodeficiency syndromes (eg.

AIDS) induced by HTLV III and HTLV IV type viruses.

3. They contain only natural active agents, which are essential from a biological point of view. They are 8900573.

*, - 8 - therefore suitable for prophylactic, long-term, healing-promoting treatment in the event of a risk of viral infection and in diseases of the immune system.

4. She also explores internally. Thus, the inconvenient external treatment commonly used in antiviral therapy can be omitted.

The compounds and preparations according to the invention and their process for their preparation are further illustrated by the following non-limiting examples.

EXAMPLE I

To 164 g (1 mol) of L-lysine monohydrate, dissolved in 500 ml of water at room temperature, was added 320 g (ca 1 mol) of 15 a 15-3 polyunsaturated fatty acid mixture (consisting of 27.6% EPA, 44.6% DHA and 0.1% vitamin E) was added dropwise.

The mixture was stirred under gentle heating (at 40 ° C) and under nitrogen atmosphere for 3 hours and then evaporated under reduced pressure (4 to 5.3 kPa) to yield 465 g of a crystalline salt, mp 188-195 ° C

(under decomposition), wherein the unsaturated fatty acid composition is identical to that of the starting mixture.

EXAMPLE II

155 g (1 mole) of L-histidine was dissolved in 450 ml of water at room temperature, giving 320 g (about 1 mole) of a ω-3 polyunsaturated fatty acid mixture (consisting of 27.6% EPA and 44.6% DHA ) was added dropwise to the solution over 5 min. Furthermore, the procedure of Example I was followed to yield 471 g of a crystalline substance, melting point: 192-200 ° C (with decomposition).

EXAMPLE III

The procedure described in Example II was followed except that 133 g of L (+) ornithine was used in place of L-histidine to yield 449 g of a crystalline product, mp: 189-195 ° C (with decomposition).

EXAMPLE IV

After dissolving 1.64 g (0.01 mole) of L-lysine monohydrate in 5 ml of water at room temperature, 3.02 g (0.01 mole) of eicosapentanecarboxylic acid [solution 8900573] was added to the solution.

- 9 -

dazzled by Sigma Co. st. Louis, USA under catalog No. I

E-7006 (1987)] in Small portions. The mixture was I.

stored under nitrogen at 40 ° C for 3 hours, then I.

evaporated under a pressure of 4-5.3 kPa to yield I.

4.9 g of a yellow-brown crystalline salt, melting point I.

194-196 ° C (with decomposition). I

EXAMPLE V I

The method described in Example IV was I.

followed, except that instead of the eicosapentane carbon- I

Acid 3.28 g (0.01 mol) docohexacarboxylic acid [produced by I.

Sigma Co., St. Louis, USA, under catalog no. D-6508 (1987)] I

was used to yield 4.85 g of a crystalline I.

salt, melting point! 195-198eC (under decomposition). I

EXAMPLE VI

The method described in Example IV was I.

followed except that 1.55 g (0.01 mol) of L-histidine was I.

used in place of L-lysine monohydrate to form I.

4.8 g of crystalline salt, mp 196-200 ° C (with decomposition). I

EXAMPLE VII I

20! The method described in Example IV was I.

repeated, except that instead of L-lysine monohydrate 1.55 I.

g (0.01 mol) L-histidine was used and instead of EPA I.

3.28 g (0.01 mol) of DHA to yield 3.78 g of an I.

crystalline salt, melting point: 196-199 ° C (with decomposition). I

EXAMPLE VIII

The method described in Example IV was I.

repeated, except that instead of L-lysine monohydrate 1.32 I.

g (0.01 mol) L (+) ornithine was used under delivery I.

of 4.6 g of a crystalline salt, melting point! 190-193 ° C I.

30 (under decomposition). I

EXAMPLE IX

The method described in Example IV was I.

repeated except that in place L-lysine monohydrate 1.32 g I.

(0.01 mol) L (+) ornithine was used and instead of EPA 35 3.28 g (0.01 mol) DHA yielding 4.55 g of a

crystalline salt, melting point: 191-195 ° C (with decomposition). I

EXAMPLE X

2g (22mmol) L-alanine was dissolved in the solution of 0.88g (22mmol) sodium hydroxide in 20ml 8900573.

♦ - 10 - water with stirring at room temperature, after which 7.5 g (22 mmol) of a ω-3 polyunsaturated fatty acid mixture (containing 27.6% EPA, 44.6% DHA and 0.1% vitamin E) the above solution was added dropwise at 5 40®C. The mixture was stirred at 40 ° C under nitrogen for 2 hours, then the solvent was distilled off under reduced pressure of 4 to 5 kPa to yield 10.4 g of a light brown pasty solid product, mp: 210-220 ° C.

In the following Examples XI-XIV, the processes described in Example X were followed, except that the corresponding amino acid was used instead of L-alanine.

EXAMPLE XI

15 L-proline 1.5 g (13.0 mmol) water 40.0 ml sodium hydroxide 0.52 g (13.0 mmol) ω-3 fatty acid mixture (according to 4.35 g (13.0 mmol) Example X) 20 A brown oily product was obtained in 6.3 g. Yield.

EXAMPLE XII

L-leucine 1.0 g (7.6 mmol) water 5.0 ml sodium hydroxide 0.3 g (7.6 mmol) 3-3 fatty acid 2.54 g (7.6 mmol)

A brown crystalline product was obtained in a yield of 3.70 g, which was liquefied in air.

EXAMPLE XIII

30 L-threonine 0.5 g (4.2 mmol) water 10.0 ml sodium hydroxide 0.16 g (4.2 mmol) ω-3 fatty acid 2.5 g (7.5 mmol)

A yellow crystalline product was obtained in a yield of 1.95 g, melting point: 204-213 ° C (with decomposition).

EXAMPLE XIV

L-aspartic acid 1.0 g (7.5 mmol) water 40.0 ml 8900573 · 4 - 11 - sodium hydroxide 0.6 g (15.0 mmol)] ω-3 fatty acid 2.5 g (7.5 mmol) .

A yellow crystalline salt was obtained in an I.

yield of 3.97 g, melting point: 200 ° C (with decomposition). I

EXAMPLE XV I

7.1 mmol of sodium metal was dissolved in 20 ml of I.

anhydrous ethanol, after which the solution was cooled to an I.

temperature of between 0 to 10 ° C, followed by addition I.

1.5 g (7.1 mmol) of L-arginine hydrochloride. After stirring at I

At room temperature for 20 minutes, the mixture was I.

filtered and the filtrate under reduced pressure I.

was evaporated. The evaporated residue was dissolved in the I

solution of 0.28 g (7.1 mmol) sodium hydroxide in 15 ml I.

water, followed by removing 2.37 g (7.1 mmol) of a ω-3 multiple I.

15 unsaturated fatty acid mixture (containing 27.6% EPA, 44.6% DHA I

and 0.1% Vitamin E) was added. I

The mixture was then stirred under I for 2 hours

nitrogen was stirred at 40 ° C, after which the solvent was removed

distilled under reduced pressure of 4-5.3 kPa under I.

Delivery of 4.05 g of a yellow crystalline salt, I.

melting point: 207-211 ° C (with decomposition). I

EXAMPLE XVI I

2.0 g (11.0 mmol) of L-tyrosine were dissolved in one I.

solution of 0.44 g (11.0 mmol) of sodium hydroxide in 20 ml I.

25 water and 20 ml of methanol at room temperature, after which 3.68 g of I.

(11.0 mmol) of a ω-3 polyunsaturated fatty acid mixture I

sel (with the same composition as in example I) to the I

the above solution at 40 ° C was added dropwise. After I

stirring the reaction mixture under nitrogen at 40 ° C

The solvents were reduced for 1 hour for 2 hours

pressure of 4-5.3 kPa evaporated to yield a yellow, I.

solid powdery crystalline salt residue in a yield I.

of 6.18 g, melting point: 150 ° C (with partial decomposition). I

In the following examples XVII and XVIII, the one described in I

35 Example XVI described procedure followed, except that I

salt is formed using the corresponding I.

amino acid instead of L-tyrosine. I

EXAMPLE XVII

L-serine 1.0 g (9.5 mmol) I.

8900573.

4 - 12 - methanol 10.00 ml water 15.0 ml sodium hydroxide 0.38 g (9.5 mmol) ω-3 fatty acid mixture (according to 3.17 g (9.5 mmol) 5 Example I)

After evaporation, a light brown crystalline product was obtained in a yield of 4/5 g, which was decomposed at a temperature above 175 ° C.

EXAMPLE XVIII

10 glutamine 2.0 g (13.77 mmol) methanol 20.0 ml water 105.0 ml sodium hydroxide 0.55 g (13.77 mmol) ω-3 fatty acid mixture 4.60 g (13.77 mmol) 15 A brown powdery crystalline salt

obtained in a yield 7.11 g, melting point; 181-190 ° C. EXAMPLE XIX

Preparation of a pharmaceutical formulation in capsule form. A salt mixture prepared according to Example I was transferred into hard gelatin capsules suitable for receiving 500 mg of active ingredient using a known encapsulation method.

EXAMPLE XX

25 Preparation of tablets

Tablets were prepared from the salt mixture obtained in Example I, each of which contained the following ingredients; salt mixture according to example I 500 mg lactose 120 mg starch 63 mg polyvinylpyrrolidone 3.5 mg magnesium stearate 3.5 mg

If desired, tablets can be coated with a sugar layer using an appropriate machine therefor.

8900573.

Claims (11)

1. New antiviral and immunostimulating salts I of the general formula (1) I R-COO-AH + (1) I 2. A salt according to claim 1, characterized in that, as the acid component, eicosapentacarboxylic acid (EPA) contains I, as the basic component L-lysine. I Salt according to claim 1, characterized in that as the acid component docosahexacarboxylic acid (DHA) contains I and as the basic component L-lysine. I A salt according to claim 1, characterized in that it is a mixture of docosahexacarboxylic acid and I eicosapentacarboxylic acid as the acid component and L-lysine as the basic I component. I 5. Antiviral and immunostimulatory pharmaceutical preparation, characterized in that it contains a salt I of general formula 1, wherein the substituents have the meaning defined in claim 1, mixed with I the carriers and / or additives and optionally antioxidants , which I are commonly used in the pharmaceutical industry. I 5 where I R represents a C 1-8 24 alkyl group at least two double I bonds? while I "A" represents an amino acid found in living organisms and / or a derivative thereof containing an I carboxyl group substituted by a C 1-4 alkyl group or an amino group or by an alkali metal ion. I 6. Process for the preparation of the new salts I of general formula (1) I R-COCTAH +, (1) wherein R and A have the meanings defined in claim 1, characterized in that the basic component is an amino acid, in which the substituents having the meanings stated in claim 1 8900573. are reacted with an acid of the general formula (2) R-COOH, (2) 5 wherein R has the meanings defined in claim 1, in a polar solvent. Process according to claim 6, characterized in that eicosapentacarboxylic acid is reacted with Process according to claim 6, characterized in that docosahexacarboxylic acid is reacted with L-lysine. 9. Process according to claim 6, characterized in that a mixture of docosahexacarboxylic acid and eicosapentacarboxylic acid is reacted with L-lysine. 10. Process for the preparation of pharmaceutical preparations with antiviral and immunostimulating action, characterized in that a salt of general formula (1), prepared according to the method of claim 6, is mixed with carriers and / or excipients and optionally with antioxidants commonly used in the pharmaceutical industry, formed by converting the mixture thus obtained in the form of tablets, dragees, capsules, suppositories, etc. into pharmaceutical preparations. 10 L-lysine. Use of salts of general formula (1) according to claim 1 for the preparation of pharmaceutical preparations and having antiviral and immunostimulating action. 8900573.