Nasal preparation and processes for their production

The present invention relates to nasal preparations which contain, as active ingredient, one or more nitrogen-containing polysaccharides obtained from fungi, in particular the polysaccharide krestin, and which exert a prophylactic action lasting several days against infections caused by viruses of the respiratory tract, especially influenza A and B viruses, as well as vaccine nasal preparations which contain the above mentioned active ingredient together with viruses of the respiratory tract of at least one strain and which impart a long-term protective action on single administration. The invention is also concerned with the production and use of these nasal preparations.

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 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 against Sarcoma 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 .beta.-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 [CA. 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 tasteless 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 (krestin) decomposes when heated to temperatures above 
120.degree. C. 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 Lots 1228 and 1214, 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 (H.sub.3 N.sub.2) 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 of 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 
observation period of 15 days in comparison to the control groups. The 
results are reported in Table 1. 
__________________________________________________________________________ 
Protective action against influenza viruses 
average average 
Application time 
A Hong Kong 1/68 
survival 
B/Ann Arbor 
survival 
in days (D) or mins. (M) 
survivors (%), 
time (%) 
survivors (%) 
time (%) 
C.sup.1 
before infection 
test/contr. 
test/contr. 
test/contr. 
test/contr. 
__________________________________________________________________________ 
40 
D5,D3,M30 80/10** 13.3/8.8** 
80/10** 
13.6/8.7*** 
10 
D5,D3,M30 80/10** 13.3/8.8** 
70/10* 12.9/8.7** 
10 
D3,M30 89/10** 14.4/8.8*** 
70/10* 13.5/10.5*** 
10 
M30 50/10 11.5/9.8 
20/10 10.4/10.5 
10 
D1 40/10 11.9/9.8 
20/10 11.1/10.5 
10 
D2 70/10 12.6/9.7 
10 
D3 80/10** 13.7/9.8*** 
50/10 12.5/10.5** 
10 
D4 80/10** 13.7/9.7*** 
10 
D7 80/10** 14.0/9.7*** 
10 
D9 70/10* 13.2/9.7*** 
10 
D11,D9,D7,D4,D2 
90/10** 14.2/9.7** 
4 
D3,M30 30/10 10.9/9.8 
60/10 13.1/10.5** 
1 
D3,M30 20/10 9.4/9.8 
50/10 12.7/10.5 
__________________________________________________________________________ 
*significant P&lt;0.05 
**significant P&lt;0.05 
***significant P&lt;0.001 
.sup.1 concentration of the solution in mg/ml 
contingency test for survivors and Cox test for average survival time 
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. 
Macroscopic and microscopic investigation of the lungs of mice to which a 
1% solution of krestin was administered nasally 7 days before 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 (H.sub.3 N.sub.2) and B/AA 4/56 influenza viruses has also been 
observed against infections caused by other A (H.sub.3 N.sub.2) strains, 
e.g. A/Vict. 3/75 and A/Port Chalmers 1/73 and especially A/Texas 1/77, by 
A (H.sub.2 N.sub.2) strains, e.g. A/Singapore 1/57, by A (H.sub.1 N.sub.1) 
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 1/Sendai. 
This action of krestin when applied nasally is superior to the prophylactic 
action of known antiviral substances, such as amantadine, against 
influenza viruses. Furthermore, krestin surprisingly affords also a very 
desirable protection in actual practice not only against influenza 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 (H.sub.3 N.sub.2) virus have shown. 
As corresponding assays have demonstrated, the prevention 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 suspension of A/HK 1/68 
(10.times.LD.sub.90 per 0.05 ml) with a 1% solution of krestin in vitro 
and allowing the mixture to stand for 30 minutes at 23.degree. C., 
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 resistant 
to a re-infection with the LD.sub.90 of the same virus. 
Mice to which a vaccine against A/Vict. 3/75 (Begrivac.RTM., 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 against infection by the same virus. 
The local 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 serum antibodies. 
Krestin has no cytotoxic or cytostatic activity and during its clinical 
trials as tumour-inhibitor was also well tolerated when administered 
orally in higher doses of usually 1 g and more per day. 
On the basis of the tumour-inhibiting action of krestin on oral 
administration, it was not to be expected that nasal application 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 protection 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 prophylactically 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), correponding 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 suitable for the intranasal 
application of 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 activity 
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 10 mg, 
preferably 5 mg, corresponding to a concentration of 0.5 to 2%, preferably 
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 to body temperature, especially at 20.degree. to 25.degree. C., and, 
if desired, with conventional excipients, e.g. preservatives. 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.degree.-25.degree. C., most preferably at 23.degree. C. The 
duration of action for A/Hong Kong 1/68 (H.sub.3 N.sub.2) 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)-benzenesulfonate], 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. 
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 liter of distilled water and, if desired 
after the addition of 10 mg of sodium timerfonate [sodium 
p-(ethylmercurithio)-benzenesulfonate], 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 liter 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 with vaccine action which contains a 1% 
aqueous solution of krestin containing originally 700 infectious 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 23.degree. C. 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 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.