CA1098040A - Antiviral agent - Google Patents

Abstract

ANTIVIRAL AGENT

ABSTRACT OF THE DISCLOSURE
Viral infections caused by viruses belonging to Herpes and Influenza groups can be controlled by administering an effective amount of 3-amino-4-homoisotwistane of the formula,

Description

1 This invention relates to an antiviral agent which comprises 3--amino-4-homoisotwistane or its salt as an active ingredient and a pharmaceutically acceptable carrier.
3-Amino-4-homoisotwistane hydrochloride (hereinafter referred to as compound A) was reported by Aigami et al. to show antiviral effects on Newcastle disease virus (J. Med. ~hem. 19, 536, 1976), but has never been so far reported to show the other an-tiviral activities.
The present inventors have studied in detai]
the antiviral activities of the compound A, and proved -tha-t the compound possesses the very strong antiviral activities on herpes and influenza viruses.
The compound A can be synthesized, for example, according to the methods described in "The ~ournal of Medicinal Chemistry", Vol. 19, p. 536 (1976).
It is well known that the so-called "caged compounds" such as amantadine often show the anti RNA viral activites but rarely exhibi-t the anti DNA
viral activltles. Among the caged compounds, only tromantadine is known to show an anti DNA viral ~; activities. ~ `
The antlvlral ef~fects of the compound A on herpes virus and so on are much superior to those of ~ ~ .
tromantadine. Thus, it can be said that the~compound is a very effectivç antiviral agent.
In the following, the antiviral activities, effective dosages, toxlcities of the compound ~ are . .

1 describcd.
Ex. 1. Effects of the compound A on the growth of herpes virus in tissue cultures Antiviral activities were determined by tube dilution method. The used cells for the assay were HeBa cells and KB cells. He~a cells were cultured in YBE medium and KB cells were cultured in Eagle MEM medium. Medium was supplemented with 10~ fetal calf serum. The monolayer of cells grown in tube was exchanged to the fresh medium supplemented with

2~o fetal calf serum, then 1000 TCD50 of herpes simplex ~ type 1 (H~ strain) and the -test compound were added.
; After 72 hr. incubation at 37C virus induced cytopathic effect (CPE) and the cytotoxicity of the compound were determined by microscopic examination.
Min:imum virus growth inhibition concentration (MIC) ànd minimum cytotoxic concentration (MCC) were shown in Table. I.

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Table I.

The effect of the compound A on growth of Herpes simplex virus Dl~gs Host cells ~ (~
. . . ~
Compound A He~a 5 5o ~ _ _ _ KB 5 5o ; HCl He~a 50 > 50 KB 50 > 50 Tromantadine KB 25 > 50 . _.

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l Ex. 2. Therapeutic effects of the compound A on experimental herpes vlrus infectlon Therapeutic effects were determined by using two experlmental infections as follows.
i) Effects on herpeskera-titis The corneal epithelium of each eye of rabblt was anesthetized and scratched, then each eye was ; infected with herpes simplex virus type l (HF strain).
One of each infected ey-es was used for treatment with lO the compound A and the other was used for viral con- -trol. One half % eye lotion of the compound A in 1.4% polyvinyl alcohol was applied every two hours, -five times a day during 7 days 12 hr. after virus infection. Each eye was daily examined and scored the leslon on the conjunctiva, cornea and the irls for 7 days. In this score O means normal and 4 means maximal severity. The eyes scratched but not infected w;th virus~were similarly treated with compound A in parallel as~toxicity~ control. The results were shown 20 ~ m Fle~
In ~ig. I, the numbers on the axis of abscissa~
indlcate the day after the vlral infectlon and those on the axis of~ordlnat~e lndlcate the score ~O(normal)~
4(maximal aeverity)~, and the mark -o- indlcates~the - `
score of an eye lotion whlch contalns 0.5~o~compound A~
; ~ ~ and the mark ~ ndlcates that of;;oontrol One half per;cent eye lotion of the compound A did not~prolong the;cure period- as com~pared with control, nor showed any other~toxicities.

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1 ii) Effects on herpesencephalitis Mice were anestheti~ed with ether and were infected intracerabrall~ c-) with 30 LD50 of herpes simplex virus type 1 HF strain. Infected mice were treated with various therapeutic schedules.
The antiviral effec-ts of the compound A was determined by comparing the number of survivors at 3 weeks after viral infection and the mean survival days of the drug-treated and placebo-treated animals. The results were shown in Table 2.

Table II.

The effects of the compound A on herpesencephalitis.

_ . Survi.vald ) Dosea) Administrationb) MeanC) ratio ~(mg/kg) rou-te survlval days no. of total mice) 7.2 7/10 i.c. 5.6 0/10 100 7.8 3/10 ; 0 P.o. 5.9 0/10 100 7.6 5/10 ' ~ O s.c. _ ~ ~ 0/10 ;

8.0 2/10 0 i.v. 6.2 0/10 - .-1 a). I)ose of administration b). Schedule of adm:inistration of each route was as follows, i.c. (intracerebrally): single administration simultaneously with vlrus infec-tion p.o. (per os): twice administration per day during 8.5 days from 4 hr. after virus infection s.c. (subcutaneously): twice administration per day during 8.5 days from 4 hr. after virus infection i.v. (intravenously): single administration at

3 hr. after vlrus infection c). The animals were examined for 21 days and deaths occuring were recorded.
d). Survivors at 21 st day.
Ex. 3. Acute toxicity of the compound A
Acute toxicities of the compound A against mice were compared with Amantad me (Symmetrel ~ ).
~; 20 The results were described in T~ble III.
:

Table III.
Acute toxicities of the compound A against mice ,~ ~: : ~ :, .
~D50 (mg/kg) Drugs _ : p.o. i.v.
. _ Compound ~ 300 32 Amantadine 400 _ : ~ .

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~Q~Q~i 1 ~x. ~. 13ffects o~` the compound ~ on experlmental Influenza virus infection The antiviral activities were determined by the modified Horsfall's method (Tani et aL., ~ukuoka Igaku Zasshi, 58, 9 (1967)).
Drug preparation The compound A and amantadine hydrochloride as a control were dissolved in sterile physiological saline for injection.
Animals ddY male mice weighing about 12 g were used in this study. Ten a~limals were used at each experi-ment.
Virus Influenza AoPR/8 was used.
Drug evaLuation Five ~D50 of influenza AoPR/8 was used for infecting mice by the aerosol. Subcutaneous drug treatment using various dosages started at ~ hours ~ 20 prej 2, 69 ]8, 30, 42, 54, 665 789 90, 102, 114, 126, `~ 138 and 150 hours post infection in order to determine the efficacy of the compound A and amantadine hydro-chloride.
Lung lesion score (LLS) was determined 7 davvs after infection by sacrificing the animals. When ~- the mice were died within 7 days af-ter infection, ~LS
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determination was also carried out.
Resul-ts were as follows;
~` :

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I,ung ~es:ion 1 l~xp. No. Drug dose (mg/kg) Score 1 0 ~.8 2 amantadi.ne HCl (10 mg/kg) 4 3-x ~ " (25 mg/kg) 4.1*

4 " (50 mg/kg) 4.0*

compd. A (7.5 mg/kg) 4.6 6 " (15 mg/kg) ~.o-x- :

7 " (30 mg/kg) 4.2*

* P ~ 0.05 (Probability value, Student's t test) As mentioned above, the compound A shows a very strong antiviral activities in vivo as well as in vitro, and can be used for the therapy of human . .
herpes viral diseases, for example, herpes keratitis, herpesencephalitis and herpeslabialis, and human i.nfluenza infections in the pharmaceutical forms such as an ointmen-t, eye lotion, injection, tabl.et and so on.
: : The dose of the compound A used in the treat-ment for an adult is varied by administration routes.
.
; ~ When used in a formula of ointment~ or eye lotion, the dosage level of 0.1 - 0.5% concentration, preferably 0.2%, which is administered several times per day, is .
desirable.

When administered orally or subcutaneously, .
100 - 500 mg, preferably some 200 mg per day is de- :
: 25 sirable, and when administered intraveneously, 10 - 50 mg, preferably 10 mg per day is deslrable.
The said compound A can be formulated to ointmen-t, eye lotion, injection, ta~let, capsule? troche and so on in a manner well-known to pharmaceutical chemists refferin-] to the represerltative antiviral agents.
~[n the following, the pharmaceutical uses of the present invention are described.
Example 1. Eye lotion ; 5 l~ -Phenylethylalcohol 5 ml 3-Amino-4-homoisotwistane HCl5 g Saline water 995 ml Total volume 1000 ml The materials were dealt aseptically.
Example 2. Ointment One half per cent of 3-amino-4-homoiso-twistane HCl in liquid paraffin. The materials were dealt aseptically.
Example 3. Tablet 3-Amino-4-homoisotwistane HCl100 mg Sucrose 88 mg Kaolin 150 mg Potato starch 20 mg `~ ; Magnesium stearate 5 mg Tablets were prepared according to usual pharmaceutical methods. Easily soluble film coating tablets are, if necessary, :
able to be prepared by usual methods.
~xample 4. Injection Sterile 3-amino-4-homoisotwistane HC1 (10 mg) was asep-tLcally put into an ampoule and : : :
sealed to prevent from humidity and micro-biral contamination. Before use, it can be dlssolved~into 2 ml~of 5~0 injeGtable glucose solution. It can be also used by :`

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dissolving wlth 2 ml o~ 0.9~ injectable sallne.

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Claims (4)

1. THE EMBODIMENTS OF THE INVENTION IN which AN EXCLUSIVE
   PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
   
   l. A pharmaceutical composition useful as an antiviral agent for the treatment or prevention of infectious diseases caused by herpes or influenza viruses which comprises a pharmacologically effective amount of 3-amino-4-homoisotwistane of the formula, or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
2. 2. An eye lotion or ointment comprising the pharmaceutical composition of claim 1, said 3-amino-4-homoisotwistane or its pharmaceutically acceptable salts being present in the lotion in an amount of about 0.1 to 0.5%.
3. 3. A tablet comprising the pharmaceutical composition of claim l, said 3-amino-4-homoisotwistane or its pharmaceutically acceptable salts being present, in the tablet in an amount of about 100 mg.
4. 4. An ampoule comprising the pharmaceutical composition of claim l, said 3-amino-4-homoisotwistane or its pharmaceutically acceptable salts being present in the ampoule in an amount of about 10 mg.