Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 334576
ANTIVIRAL AND IMMUNOSTIMULATING PHARMACEUTICAL COMPOSITIONS
ANO PROCESS FOR PREPARING SAME
This invention relates to antiviral and immuno-
stimulant pharmaceutical compositions.
According to an other aspect of the invention, there
is provided a process for the preparation of these composi-
tions.
The advantageous antiviral action of ~s-3 polyun-
saturated fatty acids / 5,8 5 11 ~14,17-eicosapentaenoic acid
(hereinafter: EPA) and 4,7,10,13,16,19-docosahexaenoic acid
(hereinafter: oHA)7 called a considerable attention. Szads
/~Antimicrobial Agents and Chemoterapy 12, 523 (1977)7
showed by in vitro experiments that polyunsaturated fatty
acids, e.g. both EPA and OHA,are capable to inhibit virus
replication. This fact was supported by Reinhardt et al.
/ J. of Virology 25, 479 (1977)7 investigating the inhibi-
tion of the replication on PR 4 bacteriophage.
The antiviral effect of polyunsaturated fatty acids,
among them that of EPA and OHA, is described in detail in
the United States patent specification No. 4,513,008. The
effect of EPA and OHA was compared in animal experiments
on mice and guinea pigs to Acyclovir / 9-(2-hydroxyethoxy-
methyl)guaninel, being the most frequently used antiviral
agent at present. It has been stated that the compositions
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particularly containing DHA showed a more preferable
action against herpes virus than that of acyclovir.
A number of articles have been devoted in the
literature to the antiviral effect of DHA and EPA, such
as: Whitaker et al. / Proc. Natl. Acad. Sci. USA 76, 5919
(1979)7 as well as Goodnight et al. / Arteriosclerosis,
2, 87 (1982)7 and Yoshiaki / Biochimica et Biophysica
Acta 793, 80 (1984)7.
Prickett et al. / Immunology 46, 819 ~1982)7 showed
that the humoral immune response was stimulated by eicosa-
pentaenoic acid as an arachidonic acid analogue. Under an
EPA-rich diet, the specific IgG and IgE production as a
response given to egg albumin was increased in inbred
Sprague-Dawley rats to a 4 to 8-fold value in comparison
to the control. According to this paper, an enhanced anti-
body response was induced by the EPA-rich diet. Further on,
it wasshown by the investigations that EPA acts through
an inhibition of the suppressive prostaglandin system where-
by it is capab]e to inhibit or correct, respectively, the
immune deficiency accompanying the aging and other
pathological processes (autoimmune processes, tumourogenesis).
These statements were supported by the articles of Kelley
et al. / J. of Immunol. 134, 1914 (1985)7 as well as Homey
et al. L Clin. Exp. Immunol. 65, 473 (1986)7.
It is long known on the basis of in vivo investiga-
tions of Pearson et al. / Proc. Soc. Exp. Biol. Med. 79,
409 (1952)7 that L-lysine has an effect inhibiting the ence-
phalomyelitis virus. Later on, Tankersley / J. Bact. 87,
- 3 - 1334576
609 (1964)7 called the attention to the fact that the
replication of herpes simplex virus (hereinafter: HSV) is
inhibited by lysine in human cells under in vitro conditions.
Based on human examinations, Kagan published L The Lancet, 1,
137 (1974)7 that both the oral and genital HSV-induced
laesions very rapidly disappeared on effect of a treatment
with L-lysine.
Griffith et al. L~Dermatologica, 156, 257 (1978)7
studied the therapeutic action of L-lysine on 45 patients
infected by HSV I and HSV II in various doses, with varied
treatment periods. The age of the patients (predominantly
women) varied between 8 and 60 years. It was stated that
L-lysine exerted only a suppressive but not healing effect
on HSV.
The aim of the present invention is to provide a
novel pharmaceutical composition combining the advantageous
therapeutical effects of ~-3 unsaturated fatty acids
with those of amino acids and certain essential amino acids,
particularly lysine, ornithine and histidine, and showing
a stronger action than that of any antiviral agent known
till now.
The invention is based on the recognition that the
advantageous antiviral effects separately known in se of
~ -3 unsaturated fatty acids, particularly EPA and OHA,
as well as of amino acids, respectively, particularly of
lysine, are synergistically increased in a mixture formed
therefrom and, in addition, the mixture shows an immuno-
stimulating action, too.
4 1 334576
According to the an aspect of the present
invention, there is provided an antiviral and
immunostimulating pharmaceutical composition comprising a
naturally occurring amino acid and/or a derivative thereof
wherein the carboxyl group is substituted by a C14 alkyl
group or amino group, or by an alkali metal, an alkali
earth metal or an ammonium cation; or a hydrate and/or a
salt of the amino acid and/or the derivative thereof; and
at least one ~3 C1824 fatty acid containing at least two
double bonds, and/or a derivative thereof, wherein the
molar ratio of the amino acid to the fatty acid is from 1:4
to 4:1.
According to another aspect of the present
invention, there is provided a process for the preparation
of an antiviral and immunostimulating pharmaceutical
composition comprising the steps of mixing a naturally
occurring amino acid and/or a derivative thereof wherein
the carboxyl group is substituted by a C~ 4 alkyl group or
amino group, or by an alkali metal, alkali earth metal or
ammonium cation; or a hydrate and/or a salt of the amino
acid and/or the derivative thereof; and at least one ~3
C18z4 fatty acid containing at least two double bonds,
and/or a derivative thereof, and transforming the mixture
into a pharmaceutical composition, wherein the molar ratio
of the amino acid to the fatty acid is from 1:4 to 4:1.
Thus, the present invention relates to an
antiviral and/or immunostimulating pharmaceutical
composition, which comprises, as active ingredients,
naturally occurring amino acids (such as taurine,
cycloserine, homoserine, homocysteine, canavanine,
sarcosine, hydroxyproline, hydroxylysine, 4-
hydroxyphenylalanine, essential amino acids) and/or their
derivatives wherein the carboxyl group is substituted by a
C~ 4 alkyl group or amino group, or by an alkali metal, an
alkali earth metal or an ammonium cation, preferably a
sodium or a potassium cation; or the hydrates and/or salts
of these compounds together with one or more C1824 fatty
acid(s) containing an alkyl group bearing at least two
5 1 334576
double bonds, optionally in admixture with carriers and/or
additives and antioxidants commonly used in the
pharmaceutical industry, wherein the molar ratio of the
amino acid and the fatty acid(s) is from 1:4 to 4:1.
C1824 ~-3 fatty acids containing at least two
double bonds and amino acids which are basic in character
are preferably used as components of the compositions
according to the invention. It is suitable to use lysine,
histidine or ornithine, or their derivatives as the amino
acids.
Substances usually employed in the pharmaceutical
industry such as lactose, starch or magnesium stearate may
be used as carriers and additives in the compositions
according to the invention.
The use of ~-tocopherol (vitamin E), glutathione
or traditional antioxidants, such as butylhydroxytoluene,
is suitable to inhibit the oxidation of the composition.
Among the compositions according to the
invention, the activity and eventual toxicity of mixtures
containing an ~-3 polyunsaturated fatty acid mixture
(containing 27.6% of EPA and 44.6% of DHA) and L-lysine in
molar ratios of 1:1, 1:4 and 4:1 were studied on the basis
of their effect exerted on the propagation and morphology
of Hep 2 cells (human epithelial tumour cell line). These
experiments were carried out on a plastic tissue-
cultivating sheet containing 6x4 wells (each well having an
area 1.9 cm2).
A stock solution containing the test composition
in a concentration of 10 mg/ml was prepared in Eagle MEM
medium (manufactured by Serva GmbH Co., Heidelberg, German
Federal Republic). An aliquot of the stock solution was
diluted 10-fold. The solution obtained was diluted 2-fold
and then, by twice repeating the 2-fold dilution of the
latter solution, solutions with successively decreasing
concentrations of the test composition (solutions Nos. 1 to
5) were prepared. The cells in each well were treated with
1 ml of a solution containing the active ingredient in the
following concentrations:
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Solution Concentration
No. /ug/ml
10000
2 1000
3 500
4 250
125
The treatment was carried out for 1 hour, then the
cultures were washed twice with buffered sodium chloride
solution (phosphate-buffered saline, hereinafter: P8S),
whereafter anutritive medium was added to the cultures.
After 24 hours and fixation by methanol, the cultures were
dyed by ethanolic Giemsa solution (manufactured by Reanal,
Budapest, Hungary) and the morphology of the cells was
evaluated under light microscope. It could be stated that
the test compound proved to be toxic only above a concentra-
tion of 1000/ug/ml.
The virus replication-inhibiting effect was examined
on Hep 2 cells as described above. Type I of HSV was used
for infection. The concentration of the virus amounted to
about 1000 PFU (plaque forming unit). Solutions containing
the test composition in a concentration of lnO0, 500 and
250 /ug/ml, respectively, were used and for the treatment
n . 1 ml of solution was added to the undiluted virus suspension.
The mixture was incubated at 37 C for 1 hour. Then, the
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cells were treated by the virus pre-incubated with the test
composition and the development of cytopathologic (CP)
alterations was observed for 7 days. It could be stated
that the virus replication was directly inhibited by a
500 /ug/ml or 250 /ug!ml dose of the test composition: the
infective titre value of the cytopathologic dose (neg.
log. CPD50) was 1.75 to 2.04 calculated for 0.1 ml, in
comparison to the neg. log. CPD50 value of 4.5 of the un-
treated control virus culture. The value of the virus inhibi-
tion exceeds by two orders of magnitude that of the control.
Under the same conditions, EPA and DHA did not show any
valuable virus inhibition; and lysine in itself inhibited
the virus replication to an extent being only by about one
order of magnitude higher than that of the control.
Vaccine virus was also treated in an in vitro
experiment carried out in the above manner. The neg. log.
CPD50 value of the test composition measured on vaccine
virus proved to be 1.75 to 2.15 in comparison to the 5.5
value of the untreated control virus, i.e. the test composi-
tion showed a virus-inhibiting effect which was by three
orders of magnitude stronger than that of the control.
The in vitro action on the immune system of the
compositions according to the invention was studied on the
activation of lymphocytes by polyclonal mitogens. The
blastic transformation of the lymphocytes was investigated
in such a way that a lymphocyte cell population obtained
on a Fico lJromiro gradient / Scand. J. Clin. Lab. Invest.
1 334576
2i, 97 (1968)7 was pipetted into the hollow holes of
flat-bottom sheets, 25 /ug/ml of Concanavaline A (herein-
after: Con A) (manufactured by Pharmacia, Uppsala, Sweden)
and then a solution containino the compositions according
to the invention in a concentration of 0.1, 1.0 or 10 /ug/ml,
respectively, were added to each of the parallel cultures.
A culture containing 25 /ug/ml of Con A without test composi-
tion was used as control. The sheets were maintained under
an air atmosphere containing 5% of carbon dioxide at 37 C
for 72 hours and 0.4 /uci of 3H-thymidine was added to
each sample after 64 hours, before ending the cultivation.
After 72 hours, the cultures were filtered through a glass
filter, the filters were put into a scintillation cuvet
and the radioactivity was measured in 5 ml of toluene solu-
tion each by using a beta-counter device. The results are
summarized in the foliowing Table.
1 334576
No. Compound Concentration Counts/minute Eva].uation of
name of the active cpm the effect
ingredient
/'~9/9
1 Control 25 8969 + 2984
(Con A)
2 L-tyrosine 0.1 12915 + 2045 weakly significant
growth
3 L-lysine 0.1 11063 + 1528 not significant
1.0 11833 + 1823 weakly significant
growth
4 Na salt of an ~i-3 0.1 12332 + 2235 not significant
fatty acid mixture
(see the composition 1.0 12039 + 1640 not significant
in Example 1)
Control 25 8396 + 2233
(Con A)
6 Product of Example 5 0.1 14605 + 1747 highly significant
growth
1.0 12010 + 2285 not signifi.cant
7 Product of Example 1 0.1 16085 + 2063 highly significant
growth
1.0 14681 + 1769 significant growth
8 Product of Example 3 0.1 126kl + 1548 significant growth
1.0 12420 + 1452 significant growth
9 Product of Example 4 0.1 11920 + 2005 significant grnwth
1.0 12240 + 1950 significant growth
- lo 1 334 5 76
It is obvious from these examinations that the
compositions according to the invention, particularly the
polyunsaturated fatty acid mixture used together with
lysine and tyrosine, gave a significant test result, i.e.
a strong immunostimulating effect, while the separate
components of the test materials according to the invention
in themselves proved to have no or only a weak biological
action.
It is suitable to use as starting material of C18 24
10 r~r_3 unsaturated fatty acids, representing one compnnent
of the composition, first of all oils which can be obtained
from fishes of the northern seas such as salmon, codfish,
sardine or from their liver but oils arising from fresh-
-water fishes may also be utilized. The c~r-3 polyunsaturated
fatty acids may be obtained from the above oils by using a
known method / J. Am. Chem. Soc. 59, 117 (1982)7. These
fatty acids are liable to oxidation. For the inhibition of
oxidation, it is suitable to use antioxidants such as e.g.
vitamin E.
The active ingredients mentioned above can be trans-
formed to capsules, tablets, dragées or other pharmaceutical
compositions formulated in a manner known per se.
The main advantages of the pharmaceutical composition
according to to the invention can be summarized as follows:
a) The antiviral therapeutic effect of the active
ingredients of the composition is synergistically increased.
11 - 1 334576
b) It can be used against acute virus infections,
particularly with herpes virus infections,for suppressing
the infection in an initial stage.
c) Owing to the immunostimulating effect, it can
preferably be used against retroviruses, especially against
the immunodeficiency syndromes (e.g. AIDS) induced by HTLV
III and IV type viruses.
d) It contains exclusively native active compounds
which are essential from a biological viewpoint; thus, it
is useful for a prophylactic, long-lasting, cure-like
treatment in case of a danger of virus infection as well
as in diseases of the immune system.
e) It acts also internally; thus, the inconvient
external treatment commonly used in the antiviral therapy
can be avoided.
The compositions according to the invention are
illustrated in detail by the following non limiting Examples.
Example 1
164 9 (1 mole) of L-lysine monohydrate and 320 9
(about 1 mole) of an ~-3 polyunsaturated fatty acid
mixture (containing 27.6 % of EPA and 44.6 % of DHA) are
admixed at room temperature. The homogeneous mixture thus
obtained is filled by using a known encapsulation process
into hard gelatine capsules capable to receive 500 mg of
active ingredient.
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Example 2
155 9 (1 mole) of L-histidine are mixed with 320 9
(about 1 mole) of an ~-3 polyunsaturated fatty acid
mixture (containing 27.6 % of EPA and 44.6 % of DHA).
Further on, the process described in Example 1 is followed.
Example 3
41.0 9 (0.25 mole) of L-lysine monohydrate are mixed
with 320 9 (about 1 mole) of an ~-3 polyunsaturated fatty
acid mixture (containing about 90.0 % of EPA and 0.1 % of
vitamin E), then the process described in Example 1 is
followed.
Example 4
The process described in Example 3 is followed,
except that 164 9 (1 mole) of L-lysine monohydrate and, as
a polyunsaturated fatty acid, 328 9 (1 mole) of docosa-
hexaenoic acid (DHA) / manufactured by Sigma, St. Louis,
USA under the catalogue number D-6508 (1987)7 are used.
Example 5
The process described in Example 1 is followed,
except that 181 9 (1.0 mole) of L-tyrosine are used, instead
of L-lysine.
Example 6
The process described in Example 1 is followed,
- 13 ~ 1 33 4 5 7 6
except that 120 9 (1.0 mole) of L-threonine are used,
instead of L-lysine.
Example 7
The process described in Example 3 is followed,
except that 164 9 (1 mole) of L-lysine monohydrate and
80 9 (about 0.25 mole) of an ~-3 polyunsaturated fatty
acid mixture (containing about 90.0 % of DHA and 0.1 % of
vitamin E) are used.
Example 8
The process described in Example 3 is followed,
except that 164 9 (1 mole) of L-lysine monohydrate and,
instead of a polyunsaturated fatty acid mixture, 302 9
(1 mole) of eicosapentaenoic acid (EPA) / manufactured by
Sigma, St. Louis, USA under the catalogue number E-7006
(1987)7 are used.
Example 9
The process described in Example 1 is followed,
except that 132 9 (1.0 mole) of L-ornithine are used,
instead of L-lysine.
Example 10
The process described in Example 3 is followed,
except that 33 9 (0.25 mole) of L-ornithine are used,
instead of L-lysine.
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1 334576
Example 11
The process described in Example 4 is followed,
except that 132 9 (1.0 mole) of L-ornithine are used,
instead of L-lysine.
Examp]e 12
The process described in Example 1 is followed,
except that 133 9 (1.0 mole) of L-aspartic acid are used,
instead of L-lysine.
Example 13
The process described in Example 1 is followed,
except that 211 9 (1.0 mole) of L-arginine hydrochloride
are used, instead of L-lysine.
Example 14
The process described in Example 1 is followed,
except that 105 9 (1.0 mole) of L-serine are used, instead
of L-lysine.
Example 15
The process dessribed in Example 1 is followed,
except that 145 9 (1.0 mole) of L-glutamine are used,
instead of L-lysine.
- 15 - 1 3 3 4 576
Example 16
Preparation of tablets
From the composition as described in Example 1
tablets are prepared each of which contains the following
components:
mg
Composition according to Example 1 500
Lactose 120
10 Starch 63
Poly~inylpyrrolidone 3.5
Magnesium stearate 3.5
If desired, the tablets are covered with a sugar
coat by using a panning machine.
