Note: Descriptions are shown in the official language in which they were submitted.
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Case_4-12418/+
Nasal preparations and processes for their production
The present invention relates to novel nasal
preparations for the prevention of infections caused by
viruses of the respiratory tract, especially by influenza
viruses A and B, processes for the production of these
preparations and the use thereof.
Surprisingly, it has been found that nitrogen-
containing polysaccharides obtained from fungi, in particular
the nitrogen-containing polysaccharide krestin discussed in
more detail below, have a prophylactic action lasting several
days against viruses of the respiratory tract, especially
influenza A and B viruses, when applied intranasally, and,
when mixed with viruses of the respiratory tract, neutralises
the infectivity of the viruses in question without impairing
their capacity to stimulate antibody formation.
Accordingly, it is an object of the present invention
to provide nasal preparations for the prevention of infections
caused by viruses of the respiratory tract, said preparations
containing, as active ingredient, one or more nitrogen-
containing polysaccharides obtained from fungi. The preparations
of this invention are in particular conventional medicinal
formulations for intranasal application.
The nitrogen-containing polysaccharide krestin, also
known as PSK, was obtained by research workers of the Kureha
Chemical Co. Ltd., Tokyo, by extraction from myzelia of fungi
of the genus Coriolus, in particular from Coriolus versicolor
(Fr.) Qel (Basidiomycetes, family of the Polyporaceae), at
elevated temperature with water, after there were grounds for
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supposing that myzelia contained tumour-inhibiting substances.
The extraction of a nitrogen-containing polysaccharide
mixture has been described in Japanese patent publication
75/36322 (filed on 3.10.68), and the fractionation of the
extracts with barium hydroxide to isolate the components
active againstSarcoma 180 is described in Japanese patent
publication 73/8489 (filed on 29.9.70). Further patent
applications claiming the Japanese priority of 18.12.75,
such as German Offenlegungsschrift 2 655 844, relate to a
modified method of extraction. Detailed particulars on the
fractionation of the crude extracts and isolation of the
tumour-inhibiting fractions, as well as on the nature and
amount of the sugar and amino acids obtained by hydrolysis
of the different fractions, are provided by Susumi Hirase
et al. in Yakugi Zasshi (J. Pharm. Soc. Japan) 96, 413-418
(1976). According to the subsequent publication [ibid. 96,
419-424 (1976)], the same authors investigated the
constitution of the ~-D-glucane components of the fractionated
polysaccharides. Shigeru Tsukagoshi et al. have reported
on the growth-inhibiting action of PSK on the sarcoma 180
in mice and the ascites-hepatome AH-13 in oral administration
in the periodical Gann 1974, 65(6) 557-8 ICA. 82, 119037a
(1975)], and in Prog. Chemother., Proc. 8th Int. Congr.
Chemother., 1973 [CA. 84, 54002e (1976)]. Chemical, and
especially pharmacological and clinical, publications and
reports on the tumour-inhibiting, nitrogen-containing
polysaccharide referred to herein as PSK, are summarised and
discussed in the in-house publication "An Outline of PSK",
1977, of the Kureha Chemical Industries Co. Ltd., Tokyo.
According to this publication, PSK is a brown or brownish
tas~less powder with a faint specific odour, sparingly soluble
in methanol, pyridine, chloroform, benzene and hexane, but
soluble in water. A 1% aqueous solution is brownish with
slight turbidity, and its pH value is around 6.6 to 7.2. PSK
. .
-- 3 --
(krestin) decomposes when heated to temperatures above 120C.
The prophylactic action against viruses of the respiratory
tract can vary within certain limits according to the
different lots of krestin produced and sold by the Kureha
Chemical Co. Ltd. for use as tumour-inhibiting substance.
For this reason it is advisable to test in advance samples
of the lots to be used by means of assays on mice for
affording protection against infection and, if necessary,
to refrain from using individual lots with lower activity,
e.g. an activity which is evidently lower than that of the
lots employed for the assays described hereinbelow.
The prophylactic action of krestin against infections
caused by influenza viruses A and B are apparent from e.g.
the assays described hereinbelow. The krestin used for all
assays was taken from the lots designated and sold by the
Kureha Chemical Co. Ltd. as Lo~s 1228 and 1214 J both of
which are recognised as having good prophylactic action
against influenza viruses.
Method
Male NMRI mice having a body weight of 16-18 g and
divided into groups of 10 are slightly anaesthetised with
ether and infected intranasally with a suspension of
influenza A/Hong Kong 1/68 virus (H3N2) and influenza B/Ann
Arbor virus in 0.05 ml of water. The suspension is fatal to
90% of the animals. The mice in the different groups, except
those in the two control groups, are treated beforehand once
or repeatedly by intranasal application or 0.05 ml of
solutions of krestin in different concentrations in distilled
water and at the times indicated in Table 1. The effect of
the prophylactic treatments is determined from the percentage
of mice surviving after 15 days in comparison to the control
groups, and from the prolongation of the average survival time
within an observaticn period of 15 days in comparison to the
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control groups. The results are reported in Table 1.
~ Protective action aqainst influenza viruses
C ) Application time A Hong Kong 1/68 3/Ann Arbor average
in days (D) or m1ns. (M) survivors (d)~ average survivors (~) survival
before infection test/contr. survival test/contr. time (d)
time (I) test/contr.
_ test/contr .
D5,D3,hl30 80/10** 13.3/8.8** 80/10** 13.6/8.7***
D5~D3,il30 80/10~* 13.3/8.8*~ 70/10* 12.9/8.7**
D3,M30 89/10** 14.4/8.8*** 70/10* 13.5/10.5***
M30 50/10 11 . 5/9 . 8 20/10 10 . 4/10 . 5
Dl 40/10 11.9/9.8 20/10 11.1/10.5
02 70/10 12.6/9.7
D3 80/10~* 13 . 7/9 . 8*** 50/10 12 . 5/10 . 5~ *
04 80/10** 13.7/9.7***
07 80/10~* 14.0/9.7***
09 ' 70/10* 13.2/9,7***
Dl 1 ,D9,D7,04,D2 90/10** 14.2/9.7**
4 03,M30 30/10 10.9/9.a 60/10 13.1/10.5*~
1 03,M30 20/10 9.4/9.8 50/10 12.7/10.5
* significant P<0.05 contingency test for survivors
** significant P<O.OS and Cox test for
**~ significant P~0.001 average survival time
1) concentration of the solution in mg/ml
It is evident from the results reported in Table 1
that krestin at a concentration of 10 mg/ml = 1% ensures a
strong protective action against influenza A and B viruses
even on single administration if this is made at least 2-3
days up to 7 days before infection. Lower concentrations,
e.g. 0.4%, are somewhat less effective.
i l 4 3
-- 5 --
~ acroscopic and microscopic investigation of the
lungs of mice to which a 1% solution of krestin was administered
nasally 7 days be,ore infection with A/HK 1/68, revealed
much less pronounced pneumonic findings than in control animals
treated with placebo.
A protective action similar to that obtained against
the above A/Hong Kong 1/68 (H3N2) and B/AA 4/56 influenza
viruses has also been observed against infections caused by
other A (H3N2) strains, e.g. A/Vict. 3/75 and A/Port Chalmers
1/73 and especially A/Texas 1/77, by A (H2N2) strains, e.g.
A/Singapore 1/57, by A (HlNl) strains, e.g. A/USSR 92/77, as
well as against infections caused by influenza B viruses
such as influenza B/Lee 40, and parainfluenza viruses such
as parainfluenza l/Sendai.
This action of krestin when applied nasally is
superior to the prophylactic action of known antiviral sub-
stances, such as amantadine, against influenza viruses.
Furthermore, krestin surprisingly affords also a very desirable
protection in actual practice not only againstinfluenza A
viruses, but also against infections caused by influenza B
viruses and other respiratory viruses. As against this, the
oral or subcutaneous administration of krestin does not
effect any significant protective action, as assays with
mice infected in the manner indicated above with influenza
A/Hong Kong 1/68 (H3N2) virus have shown.
As corresponding assays have demonstrated, the
preventive treatment with krestin does not inhibit the
formation of hemagglutinative serum antibodies against the
virus. In assays with influenza virus infections of tissue
cultures, krestin exhibits no inhibition of plaque formation
and thus does not have antiviral properties analogous to
those of e.g. amantadine. On the other hand, after mixing a
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suspension of A/HK 1/6~ (10 x LD90 per 0.05 ml) with a 1%
solution of krestin in vitro and allowing the mixture to
stand for 30 minutes at 23C, infectivity in mice on nasal
application was neutralised, but there was found to be an
increase in the hemagglutination inhibitory titre in the
serum of mice treated with this mixture,and these animals
were reslstant to a re-infection with the LD90 of the same virus.
Mice to which a vaccine against A/Vict. 3/75
(Begrivac ~ , registered trade mark of Behringwerke, Marburg
a.d. Lahn, Federal Republic of Germany) was administered
subcutaneously with simultaneous nasal application of a
1% solution of krestin, were protected in the antibody-free
latency period agains~ infection by the same virus.
Thelocal tolerance of aqueous solutions of krestin
in low concentration is good. The twice daily application of
the 1% aqueous solution to rabbits' eyes over 4 days resulted
in no irritation - a fact which is all the more remarkable,
as treatment intervals of several days are possible in
practical application.
The nasal application of 0.1 ml of 1% or 3%
solutions to guinea pigs three or five times weekly for 4
weeks resulted neither in allergic symptoms nor in the
formation of serum antibodies, whereas animals treated in
the same way with the known allergen ovalbumin died of
anaphylaxis during or after the first 4-week treatment and
had seru~. antibodies.
Krestin has no cytotoxic or cytostatic activity and
during its clinical trials as tumour-inhibitor was also well
tolerated when adminis~ered orally in higher doses of usually
1 g and more per day.
3 ~ 4 3
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On the basis of the tumour-inhibiting action of
krestin on oral administration, it was not to be expected
that nasal applica~ion of krestin would exert the protective
action observed in the practice of this invention against
virus infections. The known oral administration of krestin
in relatively high doses as tumour inhibitor in no way made
obvious its nasal application for the same purpose and
the production of appropriate preparations.
The nasal preparations of this invention are, in
particular, nasal drops, sprays, gels and ointments. These
preparations contain the active ingredient in a prophylacti-
cally effective amount and concentration, i.e. one ensuring
a protective action against virus infections of the above
mentioned kind. Accordingly, the nasal preparations of the
invention contain the active ingredient in a concentration
suitable for administration of at least 2 mg of active
ingredient each time. The upper limit is 4% (w/v) and the
lower limit - because single administration can be made not
only by once only nasal application, but also by repeated
application once or more than once after the preparation
has dried - is about 0.2% (w/v), corresponding to 1 ml of a
ready-for -use nasal preparation for the above minimum dose.
Nasal drops have in particular a concentration between 1%
and 2% and sprays a concentration between 0.2 and 1% of
active ingredient. In both formulations the solvent is
preferably water. The aqueous solutions optionally contain
conventional pharmaceutically acceptable excipients for
stabilising the active ingredient, as well as for buffering,
preserving and/or lowering the surface tension, and they
can be made isotonic in conventional manner, e.g. with
sodium chloride or buffer solutions.
The invention relates in particular to spray bottles
filled with the above solutions and to similar containers
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suitable for the intranasal application o~ such solutions.
The preparations of the present invention for the
prevention of influenza A, influenza B, and other infections
caused by other viruses of the respiratory tract, are preferably
administered at intervals of 2 to 3 days or twice weekly.
Instead of using the preparations of the invention for
prolonged preventive treatment, e.g. right through the whole
cold season, they can also be used only during part of the
cold season when there is an obviously increased risk of
infection. In addition, the preparations of the invention can
also be used for bridging the latency period of vaccinations
against influenza, i.e. simultaneously with or directly after
vaccination, as the simultaneous nasal application with
parenterally injected vaccine ensures protection in the
antibody-free latency period without impairment of the
antibody formation. The risk of catching an infection within
the first two weeks after vaccination can thereby be reduced.
The above mentioned property of polysaccharides
obtained from fungi, such as that of krestin, of neutralising
the infectivity of virus suspensions, but not -their ability
to induce antibody formation, affords in addition the
possibility of producing nasal preparations having vaccine
ac,ivity for the prevention of infections caused by viruses
of the respiratory tract. Such preparations for vaccination
contain in aqueous medium, per 0.5 ml, i.e. in the amount of
fluid which can comfortably be applied to each nostril by
the single application of 0.25 ml, 2.5 to lO mg, preferably
5 mg, corresponding to a concentration of 0.5 to 2%, prefer-
ably of 1%, of a nitrogen-containing polysaccharide obtained
from fungi, such as krestin, in admixture with viruses of the
respiratory tract of at least one strain, in an amount not
exceeding that which becomes non-infectious as a result of
the action of the nitrogen-containing polysaccharide at room
1 4 ~
~ 9 _
to body temperature, especially at 20 to 25C, and, if
desired, with conventional excipients, e.g. preserva~ives.
Such preparations are obtained by mixing an aqueous solution
of a nitrogen-containing polysaccharide derived from fungi,
especially krestin, of suitable concentration with an aqueous
suspension of at least one strain of viruses of the respiratory
tract. The concentrations of the aqueous solution of the
polysaccharide and of the virus suspension are chosen such
that the resultant mixture contains, per 0.5 ml, 2.5 to 10 mg
of the polysaccharide, e.g. of krestin,-and especially about
5 mg of krestin, and the viruses in an amount not exceeding
that which can become non-infectious as a result of the
action of the polysaccharide at room to body temperature.
The duration of action at given or also varying temperature
is determined by means of assays on mice for affording
protection against infection with a corresponding sample
mixture and is between about 15 minutes and about 120 minutes,
depending on the sensitivity of the virus in question, in the
preferred temperature range from 20-25C, most preferably
at 23C. The duration of action for A/Hong Kong 1/68 (H3N2)
is shorter e.g. than for A/Texas 1/77. Then, if desired,
excipients are added, e.g. preservatives such as sodium
timerfonate [sodium p-(ethylmercurithio)-benzenesulfonate3,
and the preparation is stored in a refrigerator until use.
The preparations of the invention are administered in a
manner similar to that of non-vaccine preparations, but
administration is made just once for a prolonged period of
time, e.g. at the start of the cold season.
The following Examples illustrate a number of
medicinal formulations, but without in any way restricting
the scope of the invention thereto.
1 1 5 ~
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Example 1: Nasal spray
A nasal spray with a 1% content of active ingredient is
prepared by dissolving 10.0 g of krestin in 1 litre of
distilled water and, if desired after the addition of 10 mg
of sodium timerfonate [sodium p-(ethylmercurithio)-benzene-
sulfonate], filling 10 ml bottles with the solution. For
prevention of influenza and other infections caused by viruses
of the respiratory tract, each nostril is sprayed 2-3 times
e.g. every second day or twice weekly from a full bottle, or,
if necessary, more often from a partially empty bottle.
Example 2: Nasal drops
Nasal drops containing 1% of active ingredient are prepared
by dissolving 10.0 g of krestin in 1 litre of distilled water
and, if desired after addition of 10 mg of sodium timerfonate,
filling dropper bottles of 5 or 10 ml content provided with a
stopper in the form of a pipette. In the same intervals as
indicated in Example 1, 4-6 drops, corresponding to about 0.2
or 0.3 ml containing about 2-3 mg of active ingredient, are
applied to each nostril.
Example 3: Nasal preparation with vaccine action
100 ml of a nasal preparation withvaccineaction which contains a 1%
aqueous solution of krestin containing originally 700 infect-
ious units (IU) of A/Texas influenza virus per 0.5 ml (usual
vaccination dose), are prepared by mixing 80 ml of 1.25%
solution of krestin in distilled water with 20 ml of a
suspension of A/Texas 1/77 virus with a content of 7000 IU
per ml and which has been purified in the usual manner. The
mixture is kept at 23C for a period of time that was
determined beforehand in assays on mice for affording
protection against infection and in which a corresponding
sample mixture proved to be non-infectious but induced a
high antibody titre and resistence to re-infection, and
11561~3
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which in the present instance was 60 minutes. Afterwards, if
desired, a preservative is added, e.g. 1.0 mg of sodium
timerfonate, and the preparation is stored in a refrigerator
until use.
For influenza prevention by vaccination, preferably at the
start of the cold season, 0.25 ml of the preparation is
dropped into each nostril with a pipette.
