Note: Descriptions are shown in the official language in which they were submitted.
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.
.
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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809rO
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
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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 _
: ~ .
: 5 - ::
~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
.
determination was also carried out.
Resul-ts were as follows;
~` :
~ 6 ~
~L~g~
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-
-- 7 --
] 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
:`
.
dissolving wlth 2 ml o~ 0.9~ injectable
sallne.
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