HK1113910A - Antiviral and immunostimulating marine fish oil composition - Google Patents

Description

Antiviral and immunostimulating marine fish oil composition

Technical Field

The present invention relates to a novel antiviral and immunostimulating pharmaceutical composition.

Background

Omega-3-polyunsaturated fatty acids, including 5, 8, 11, 14, 17-eicosapentaenoic acid (hereinafter EPA) and 4, 7, 10, 13, 16, 19-docosahexaenoic acid (hereinafter DHA), are known to have antiviral effects. The conclusions of the initial in vitro experiments (see, e.g., Antimicrobial Agents and chemotherapeutics 12, 523(1977)) have been confirmed by in vivo animal tests (see, e.g., U.S. patent No. 4513008) and clinical data (see, e.g., j.of immunology 134, 1914(1985) or clin.exp.immunol.65, 473 (1986)).

L-lysine is also known to inhibit the replication of Herpes Simplex Virus (HSV) in human cells under in vitro conditions (see j. vact.87, 609 (1964)). However, clinical studies have found that L-lysine has only minimal therapeutic effect on HSV infection (see Dermatologica, 156, 257 (1978)).

It is widely accepted in the literature that HSV replication and other viral infections are associated with a lack of immunity in the immune system. It is also known that EPA and DHA and derivatives thereof can act on the immune system by inhibiting the prostaglandin system. This means that these agents are capable of inhibiting and/or correcting immunodeficiency, certain autoimmune processes and tumour production triggered by age and/or adverse environmental effects (see j. Immunology 134, 1914(1985) or Immunology 46, 819(1982) or Eur j. clin. nutr.56 suppl.3, 14-19 (2002)).

Hungarian patent 199, 775 discloses an interesting finding: according to the description, salts of 18 to 24-carbon fatty acids containing at least two double bonds with amino acids, preferably L-lysine, L-tyrosine, L-histidine, L-alanine or L-ornithine, are suitable as active ingredients in antiviral compositions. This information is supported by an in vitro test of the effectiveness of the compositions in inhibiting viral proliferation. The most significant effect reported was the tyrosine salt of polyunsaturated fatty acids. The disadvantages of said invention are: salt formation often results in a paste-like product that is difficult to purify and characterize (see, e.g., examples 10-14 of the cited patent specification). Even more readily crystallizable salts do not have a defined melting point. Therefore, the products obtained by the process disclosed in said patent specification often show a color change, which indicates that the active ingredient contains impurities and is of uncertain quality.

The authors of the hungarian patent 209,973 try to eliminate the drawbacks of the above-mentioned method. In this process, the authors do not use salts of fatty acids with the amino acids L-lysine, L-tyrosine or derivatives thereof, but rather are mixed with the omega-3-polyunsaturated fatty acids or their salts in a molar ratio of from 1: 4 to 4: 1. The mixture obtained by this route is used as active ingredient and converted into a pharmaceutical composition using standard pharmaceutical formulation procedures. The compositions described in the specification have immunostimulatory utility, but still have the disadvantages of: even the two most effective components (L-tyrosine-fatty acid and L-lysine monohydrate-fatty acid mixtures) require antioxidants for stabilization. As our own experiments show, despite the use of stabilizers, the mixtures prepared according to the above patent specification are still not suitable as ingredients for sufficiently stable pharmaceutical compositions.

Data show that HSV viruses isolated from clinical specimens can be inactivated to some extent by in vitro zinc salt treatment. The degree of inactivation depends on the HSV strain, the concentration of zinc salt and the duration of treatment (Max Ares and Sharon Travis: J. of Clinical Microbiology, 38, 1758-.

In addition to playing a key role in the activation of glutathione peroxidase and thus in oxidative stress conditions, selenium may play a role in viral replication in the form of selenium-containing proteins. The in vitro introduction of selenium can inhibit HIV viral replication, which has been demonstrated with chronically infected T lymphocytes (Hori et al: AIDS Res. human Retroviruses 13: 1325-32 (1997)).

The object of the present invention is to prepare pharmaceutical compositions with enhanced effect which are not only completely free of the problems with large scale technology, composition and stability inherent to previous methods, but also have more valuable advantages.

Disclosure of Invention

The present invention is based on the following findings: the above object can be achieved if the following conditions are met: if instead of using free omega-3-polyunsaturated fatty acids (i.e. EPA and DHA) esters thereof are used, the effect of said esters can be enhanced in a synergistic manner by the addition of L-lysine or salts thereof, optionally in addition of zinc salts or selenium compounds. Unexpectedly, the following steps are carried out: the compositions obtained in this way have lower toxicity and greater efficacy than the known compositions, in other words the therapeutic index of the compositions prepared on the basis of the process of the invention disclosed above is superior to that of all the known similar compositions.

Detailed Description

The invention therefore relates to novel antiviral and immunostimulating pharmaceutical compositions containing 20 to 85% by mass of an omega-3-polyunsaturated fatty acid ester as active ingredient, in particular 20 to 70% by mass of a fish oil concentrate (containing esters of 5, 8, 11, 14, 17-eicosapentaenoic acid and 4, 7, 10, 13, 16, 19-docosahexaenoic acid) and L-lysine or salts thereof, optionally containing a zinc salt, selenium or a selenium compound, and also additives and carriers.

In a preferred embodiment, according to the invention, the product contains the omega-3 polyunsaturated fatty acids in the form of esters with primary, secondary or tertiary alcohols, preferably ethyl or glycerol esters, and also the L-lysine salt L-lysine hydrochloride.

The amount of the concentrate of fish oil containing an omega-3-polyunsaturated fatty acid ester is preferably from 30 to 70% by mass, more preferably from 40 to 60% by mass, most preferably from 55 to 60% by mass, and in particular it contains from 20 to 70% by mass, preferably from 25 to 45% by mass, more preferably from 30 to 40% by mass, most preferably from 31 to 35% by mass, of 5, 8, 11, 14, 17-eicosapentaenoic acid, while the amount of 4, 7, 10, 13, 16, 19-docosahexaenoic acid is from 20 to 70% by mass, preferably from 25 to 45% by mass, more preferably from 30 to 40% by mass, most preferably from 31 to 35% by mass.

As lysine salts, not only lysine hydrochloride but also all pharmaceutically acceptable lysine salts are within the scope of discussion. Non-limiting examples are lysine fumarate, maleate and oxalate. The amount of lysine salt may range from one-fourth to four-times the equimolar amount of the omega-3-polyunsaturated fatty acid ester.

The concentration of the zinc salt is 1 to 10% by mass, preferably 2 to 6% by mass.

In another embodiment of the invention, the composition contains a zinc salt that is zinc gluconate or zinc lactate and also contains a selenium compound that is one or more natural selenium compounds incorporated into natural yeast.

The omega-3-polyunsaturated fatty acid esters specified for use as components of the composition in the present invention may be present predominantly in oils obtained from fish in the north sea. Fish oil concentrates which can be prepared from the fish oils described contain 50 to 65% by mass of esters of omega-3-polyunsaturated fatty acids, 20 to 70% by mass of which are esters of 5, 8, 11, 14, 17-eicosapentaenoic acid and esters of 4, 7, 10, 13, 16, 19-docosahexaenoic acid, are prepared in a manner known per se (see, for example, j.am. chem. soc, 59, 117 (1982)).

Another essential ingredient of the compositions described herein is L-lysine or certain L-lysine salts, preferably acetate or hydrochloride salts (see, e.g., the United states Pharmacopeia 27-NF 22Supplement 2.).

The third component of the composition is a zinc salt, preferably zinc gluconate or zinc lactate (see, e.g., U.S. pharmacopoeia 27-NF 22Supplement 2.).

Furthermore, an optional ingredient in the compositions described herein is a selenium compound, which may be a selenium compound incorporated into natural yeast or any other selenium compound. The concentration of selenium is 0.05-0.30 mass%, preferably 0.1-0.2 mass%, but not more than 75 μ g.

The active ingredients described above can be formulated using generally known pharmaceutical composition formulation methods, whereby compositions known per se are obtained, preferably encapsulated in soft gelatin capsules. As additives and/or adjuvants, preference is given to using silica gel, glycerol, dyes and other substances.

The antiviral and immunostimulatory effect of the compositions described in the present invention was demonstrated as follows:

A. test substance and test method description

I. Test substance composition coding

SIN-E1: salts of omega-3-polyunsaturated fatty acids with L-lysine monohydrate (see example 1 of Hungarian patent 209,973).

SIN-E2: omega-3-polyunsaturated fatty acid ester + L-lysine hydrochloride (see example 3 of the present application).

SIN-E3: omega-3-polyunsaturated fatty acid ester + L-lysine hydrochloride + calcium gluconate (see example 1 of the present application).

Test methods

1. Toxicity testing of Primary monkey Kidney cell cultures

Primary monkey kidney cells were treated with different dilutions (1: 3, 1: 10, 1: 30, 1: 100) of the test substances SIN-E1, SIN-E2 and SIN-E3. The final toxic effect of the substances on the tissues was examined after 3 hours of incubation (see Arens, M.and Travis, S.: J.of Clinical Microbiology, 38, 1758-.

2. Study on antiviral Effect of the test substance

The test substances SIN-E1, SIN-E2 and SIN-E3 were used to pretreat the virus, which was then cultured with the second generation monkey kidney cells to investigate the infection.

Different dilutions of virus and different dilutions of test substance were incubated for 1 hour at a ratio of 1: 1, corresponding to a dilution of 0.5% (see table 1), followed by infection of the second generation monkey kidney cell cultures with the pretreated virus. Microscopic examination was used on day seven to determine the pathogenic effects of untreated Herpes Simplex Virus (HSV) and herpes simplex virus pretreated with test substances SIN-E1, SIN-E2 and SIN-E3 on monkey kidney cells. The aim of the test is to determine the direct Antiviral effect of the test substance on the cells (see Lawetz, C, Liuzzi, M.: Antiviral Res.39(1), 35-46 (1998)).

3. Studies on the influence of serum proteins on the viral inactivating effect of test substances SIN-E1, SIN-E2 and SIN-E3.

The experiment was repeated in medium containing 10% calf serum with the dilution in the experiment at point 2 showing complete inactivation and the next higher dilution in the experiment, with no serum added as a blank, and the experiment was evaluated on day 7 as described at point 2.

4. Evaluation of

In toxicity studies after 3 hours of incubation and antiviral effect studies after 7 days, cells were examined using an inverted microscope. Morphological changes, vacuole generation, separation and cell wall disruption were recorded.

B. Study of efficacy

1. Toxicity study of test substances in tissue culture

Organizing: second generation monkey kidney cell culture, cell number 5X 10⁶。

-all three test substances were diluted 2-fold in series, added to the tissue and subsequently incubated for 3 hours at 37 ℃.

-the blank contains only the tissue in question,

after 3 hours the material was poured off, 100. mu.l of Parker's medium containing 2% calf serum was added to the whole plate, and the morphological changes were observed with a microscope,

based on the examination after 3 hours, it was demonstrated that the substance SIN-E1 was not toxic at a dilution of 1: 32768, whereas the substances SIN-E2 and SIN-E3 were not toxic at a dilution of 1: 2048,

-the same results were obtained the next day by repeated microscopic examination,

hereinafter, for SIN-E1, the dilution at 1: 32768 is marked "concentrated", whereas for SIN-E2 and SIN-E3, the dilution is 1: 2048. (results report is shown in Table 1.)

TABLE 1

Comparison of the toxicity of the substances SIN-E1, SIN-E2 and SIN-E3

	1∶128	1∶256	1∶512	1∶1024	1∶2048	1∶4096	1∶8192	1∶16384	1∶32768	1∶65536	1∶131072	1∶262144
													SIN1						+	+	+	-	-	-	-
SIN1						+	+	+	-	-	-	-
													SIN1						+	+	+	-	-	-	-
SIN2	+	+	+	+	-	-	-	-	-	-	-	-
													SIN2	+	+	+	+	-	-	-	-	-	-	-	-
SIN2	+	+	+	+	-	-	-	-	-	-	-	-
													SIN3	+	+	+	+	-	-	-	-	-	-	-	-
SIN3	+	+	+	+	-	-	-	-	-	-	-	-
													SIN3	+	+	+	+	-	-	-	-	-	-	-	-
Negative control	-	-	-	-	-	-	-	-	-	-	-	-
													Negative control	-	-	-	-	-	-	-	-	-	-	-	-

Symbols used in Table 1

+ ═ cytotoxic dose

Non-cytotoxic dose

And (3) test evaluation:

toxicity studies carried out clearly show that the toxicity of the substance SIN-E1 is at least an order of magnitude greater than that of the substances SIN-E2 and SIN-E3. It is evident that the toxicity of the test substance SIN-E1 disappeared at the final dilution of 1: 32768 of the standard solution, whereas the substances SIN-E2 and SIN-E3 disappeared at the final dilution of 1: 2048. In the following studies, the above final dilution was taken as a basis.

2. Test substance antiviral Effect detection

-organization: second generation monkey kidney cell culture, cell number 5X 10⁶。

-preparation of dilution 10^-1-10^-8Detecting the infectious titer of said virus. Entered in table 2 are the negative log values of infectious titer per 0.1 ml.

-preparing test substance SIN-E1 at a dilution of 1: 32768 and test substances SIN-E2 and SIN-E3 at a dilution of 1: 2048, respectively. These dilutions were labeled "concentrated" respectively.

The "concentrated" dilutions were tested after 1: 3, 1: 10, 1: 30 and 1: 100 fold dilutions.

-mixing the virus and the substance in a ratio of 1: 1, respectively diluted.

Followed by incubation for 1 hour.

After this the medium on the tissue was decanted and 100. mu.l of the mixed virus and test substance dilution was added to the appropriate row.

Followed by incubation at 37 ℃ for 1 hour.

-pour off material and add 100 μ l Parker's medium containing 2% calf serum.

The samples were placed at 37 ℃, evaluated microscopically over a period of 7 days and compared to untreated virus.

The results are shown in table 2.

TABLE 2

Antiviral Activity assay

Test substance dilution mg/mL	1∶31.5	1∶100.5	1∶300.15	1∶1000.05	Viral control
						SIN-E1	0*	0*p＜0.01	4.0	5.15	5.15
SIN-E2	0*	0*p＜0.01	3.2	4.8	5.15
						SIN-E3	0*	0*p＜0.01	0*p < 0.01vs control p < 0.05vs SIN-E2	3.5	5.15

Symbols used in Table 2

0-total inhibition

Evaluation of results

The study performed showed that all three test substances completely inhibited viral proliferation at dilutions of 1: 3(1.5mg/mL) and 1: 10(0.5 mg/mL). Partial inactivation was observed within 1 hour for the substances SIN-E1 and SIN-E2 even at a dilution of 1: 30(0.15mg/mL), while complete inhibition was observed for SIN-E3 even at this dilution, which was significant compared to SIN-E2 and the control. Partial inactivation was found at a dilution of 1: 100(0.05mg/mL) for SIN-E3, but this result was not statistically significant.

3. Effect of serum proteins on the inactivation Effect of SIN-E1, SIN-E2 and SIN-E3 viruses the experiments were carried out as described in point 2.

TABLE 3

Test substance for antiviral Activity in the Presence of bovine serum

Test substance dilution mg/mL	1∶31.5	1∶100.5	1∶300.15	1∶1000.05	Viral control
						SIN-E1	0*	0*p＜0.01	4.0	5.15	5.15
SIN-E2	0*	0*p＜0.01	3.2	4.8	5.15
						SIN-E3	0*	0*p＜0.01	0*p < 0.01vs control p < 0.05vs SIN-E2	3.5	5.15

Symbols used in Table 3

0-total inhibition

TABLE 4

Test substance for antiviral Activity in bovine serum free Medium

Test substance dilution mg/mL	1∶31.5	1∶100.5	1∶300.15	1∶1000.05	Viral control
						SIN-E1	0*	0*p＜0.01	4.2	5.15	5.15
SIN-E2	0*	0*p＜0.01	3.5	4.9	5.15
						SIN-E3	0*	0*p＜0.01	0*p < 0.01vs control p < 0.05vs SIN-E2	3.7	5.15

Symbols used in Table 4

0-total inhibition

Evaluation of the test

The presence of the protein did not affect the inactivation activity of the test compound against herpes virus, as this has been demonstrated in serum-free medium (see table 4) and in medium containing 10% fetal bovine serum (see table 3).

In summary, based on studies on the influence of serum proteins, it can be shown that the composition described in this patent shows significant additional activity compared to the data available in the literature (see, for example, U.S. Pat. No.4,513,008, inventor e.recivii et al.) showing that it is capable of destroying or completely eliminating the infectivity of viruses with capsid, since various unsaturated fatty acid compositions can cleave the surface structure of the virus. According to other literature data (see, for example, Vollenbrooch, D.et al: biologicals.Sept.; 25 (3): 289-97(1997)), the virus-inactivating activity of unsaturated fatty acids is destroyed by small amounts of serum proteins and is therefore lost in therapy. In contrast, our own experiments in primary monkey kidney tissue did not inhibit the inactivation effect of our composition on herpes virus even in the presence of 10% fetal bovine serum.

The advantages of the novel pharmaceutical composition claimed in the present application are summarized as follows:

by the present invention, it is possible to prepare, in contrast to the prior art, a stable pharmaceutical composition with a long shelf life, which has the advantage of effectively protecting omega-3-polyunsaturated fatty acids from oxidation during storage,

the use of the process detailed in the present invention provides a simple and economical process for the preparation of other compositions containing other and/or new antiviral agents,

-omega-3-polyunsaturated fatty acid esters and compositions containing such esters are less toxic than the parent omega-3-polyunsaturated fatty acid or compositions containing such fatty acids.

The compositions prepared according to the invention provide a more versatile and flexible means of antiviral treatment in a biological sense, while also providing more effective inhibition of viral replication,

the process described in the present invention eliminates the technical problems associated with the preparation of salts of omega-3-polyunsaturated fatty acids with basic components and the costs caused by the difficulties mentioned.

The compositions prepared according to the invention are illustrated by the following examples:

example 1

Capsule form preparation (coding in the table is SIN-E3)

A mixture of esters of omega-3-polyunsaturated fatty acids (362 g) obtained from concentrated marine fish oil, containing 35 mass% of ethyl 5, 8, 11, 14, 17-eicosapentaenoic acid (EPA ethyl ester) and 25 mass% of ethyl 4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA ethyl ester), was mixed with L-lysine hydrochloride (203 g) and zinc gluconate (30 g) at room temperature. In this way a homogeneous mixture was obtained, and then colloidal silica gel (25 g) and lecithin (1 g) were added. After further homogenization, the material is filled into 1000 soft capsules in a manner known per se.

Example 2

Preparation in capsule form

The procedure was followed in all respects as described in example 1, with the difference that the composition contained a mixture comprising: contains 32.8% by mass of ethyl 5, 8, 11, 14, 17-eicosapentaenoic acid (EPA ethyl ester) and 22.2% by mass of an omega-3-polyunsaturated fatty acid ester of ethyl 4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA ethyl ester) (362 g), and also the active ingredients and additives described in example 1, and furthermore selenium (1 g) incorporated in natural yeast is added.

Example 3

Capsule form preparation (coding in the table is SIN-E2)

A mixture of esters of omega-3-polyunsaturated fatty acids (362 g) obtained from concentrated marine fish oil, containing 35 mass% of ethyl 5, 8, 11, 14, 17-eicosapentaenoic acid (EPA ethyl ester) and 25 mass% of ethyl 4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA ethyl ester) was mixed with L-lysine hydrochloride (203 g) at room temperature. In this way a homogeneous mixture was obtained, and then colloidal silica gel (25 g) and lecithin (1 g) were added. After further homogenization, the material is filled into 1000 soft capsules in a manner known per se.

Example 4

Preparation in capsule form

The procedure is carried out in each case as described in example 1, except that instead of the mixture of ethyl omega-3-polyunsaturated fatty acid esters, a mixture of 5, 8, 11, 14, 17-eicosapentaenoic acid triglyceride (EPA triglyceride) and 4, 7, 10, 13, 16, 19-docosahexaenoic acid triglyceride (DHA triglyceride) is used.

Claims (7)

1. An antiviral and immunostimulating pharmaceutical composition characterized by comprising 20-85 mass% of an omega-3-polyunsaturated fatty acid ester as an active ingredient, 20-70 mass% of 5, 8, 11, 14, 17-eicosapentaenoic acid ester and 4, 7, 10, 13, 16, 19-docosahexaenoic acid ester in a marine fish oil concentrate, and also L-lysine or a salt thereof, optionally 1-10 mass%, preferably 2-6 mass% of a zinc salt, 0.05-0.3 mass%, preferably 0.1-0.2 mass%, but not more than 75 μ g of selenium or a selenium compound, and further additives or carrier ingredients.

2. Pharmaceutical composition according to claim 1, characterized in that it comprises as active ingredient preferably from 30 to 70 mass%, more preferably from 40 to 60 mass%, most preferably from 55 to 60 mass% of a fish oil concentrate containing omega-3-polyunsaturated fatty acid esters, which fish oil concentrate comprises in particular from 20 to 70 mass%, preferably from 25 to 45 mass%, more preferably from 30 to 40 mass%, most preferably from 31 to 35 mass% of 5, 8, 11, 14, 17-eicosapentaenoic acid, and from 20 to 70 mass%, preferably from 25 to 45 mass%, more preferably from 30 to 40 mass%, most preferably from 31 to 35 mass% of 4, 7, 10, 13, 16, 19-docosahexaenoic acid.

3. Product according to claims 1 to 2, characterized by containing omega-3-polyunsaturated fatty acids, preferably omega-3-polyunsaturated fatty acid ethyl esters or omega-3-polyunsaturated fatty acid glycerides esterified with primary, secondary or tertiary alcohols, and also containing the L-lysine salt L-lysine hydrochloride, fumarate, maleate or oxalate, preferably hydrochloride.

4. A product according to claims 1 to 3, characterized by containing the zinc salt zinc gluconate or zinc lactate.

5. Product according to claims 1 to 4, characterized by containing selenium compounds natural selenium compounds or selenium compounds incorporated in natural yeasts.

6. A pharmaceutical composition according to claims 1 to 5 for use as a medicament.

Use of 20-85 mass% of an omega-3-polyunsaturated fatty acid ester, containing 20-70 mass% of 5, 8, 11, 14, 17-eicosapentaenoic acid ester and 4, 7, 10, 13, 16, 19-docosahexaenoic acid ester, and L-lysine or a salt thereof, optionally 1-10 mass%, preferably 2-6 mass% of a zinc salt, and 0.05-0.30 mass%, preferably 0.1-0.2 mass%, but not more than 75 μ g of selenium or a selenium compound in a marine fish oil concentrate, for the preparation of a pharmaceutical composition for the treatment of a viral infection.