Archives of Virology
Archives of Virology 55, 225--231 (1977)
© by Springer-Verlag 1977
Enhancement of Antiviral Protection Against Eneephalomyoearditis Virus by a Combination of Isoprinosine and Interferon By C. CHARY and I. CERUTTI Institut National de la Santd et de la Recherche lViddieale, I-I6pital St. Vincent de Paul, Paris, France, and
Institut de Recherehes sur le Cancer C.N.R.S., Villejuif, France With 1 Figure Accepted June 15, 1977
Summary The antiviral effect of interferon against encephalomyocarditis (EMC) virus infection in mice was enhanced b y isoprinosine. However, the enhancement was only obtained when both interferon and the virus were inoculated into the peritoneum; the inoculation route of isoprinosine did not modify significantly the final results. I n addition, the time sequence of injections was of great importance ; generally the injection of isoprinosine had to precede that of interferon b y a few hours.
Introduction The antiviral effect, of interferon in vitro and in vivo is presently well documented. The amount of interferon needed to obtain a therapeutic effee~ in vivo is relatively important and requires repeated injections (13). Among the possible explanations are a) a rapid degradation of interferon in the circulation or in the cell, b) a limited diffusion to the target area where high concentrations are necessary to trigger interferon receptor sites on the cell membrane (8), and c) the presence of interferon antagonists in the tissues (9). From a practical viewpoint, two possibilities are now available to overcome these difficulties: the administration of purified interferon at high concentrations and the use of adjuvants to improve its antiviral (or other) effect. Such adjuvants might potentiate interferon action b y a parallel but independent inhibitory effect on virus replication or/and through the ceil b y increasing the interferon-induced antiviral state. I n a series of articles we have recently shown that the structure of the cell membrane (5), as well as the integrity of the cytoskeletal components (4), play an
226
C. C~Am-Yand I. C E n ~ - ~ :
i m p o r t a n t role in t h e e s t a b l i s h m e u t of t h e interferon-induced a n t i v i r a l state. M a n y substances w i t h a f f i n i t y for gangliosides can m o d i f y t h e cellular response to interferon (1, 2, 3, 16). Likewise, m o d i f i c a t i o n of t h e celt m e m b r a n e due to cell t r a n s f o r m a t i o n (i. e., oncogenic viruses) can also change t h e i n t e r f e r o n s e n s i t i v i t y of t h e cells (7). Thus, in a search for drugs which m i g h t p o t e n t i a t e t h e a n t i v i r a l a c t i o n of interferon, we h a v e selected isoprinosine since this c o m p o u n d affects a p p a r e n t l y t h e m e m b r a n e of i m m u n o c o m p e t e n t or other somatic cells. I t has been r e p o r t e d , for instance, t h a t isoprinosine decreases the a m o u n t of P H A n e e d e d for t h e s t i m u l a t i o n of h u m a n p e r i p h e r a l blood l y m p h o e y t e s . I n t h e absence of P H A isoprinosine alone has no similar s t i m u l a t i n g a c t i o n (14). As shown in a p r e l i m i n a r y r e p o r t (6), c o m b i n e d t r e a t m e n t w i t h isoprinosine a n d interferon i m p r o v e s c o n s i d e r a b l y the p r o t e c t i v e effect of a single interferon dose a g a i n s t t h e l e t h a l effect of e n e e p h a l o m y o c a r d i t i s (EMC) virus in mice. E a c h of these two p r o d u c t s i n j e c t e d s e p a r a t e l y a t the same c o n c e n t r a t i o n is either ineffective or o n l y s l i g h t l y effective.
Materials and Methods
Drug Isoprinosine is the p-acetamidobenzoic acid salt of N, N-dimethylamino-2-propanol : inosine complex, 3 : 1 molar ratio, and was supplied b y Laboratories DelMande (Courbevoie, France). The white crystalline powder is stable at temperatures up to 150 ° C, its maximal UV absorption rate is at 260 nm, and it is water soluble. The physical and chemical properties have been described elsewhere (10).
Inter/eron Mouse interferon was prepared in C-243 cells (17) using standard procedures. The antiviral titer was estimated b y 50 per cent CPE inhibition using mouse L cells and vesicular stomatitis virus (VSV) as the challenge virus. The crude interferon preparation was purified b y two successive steps of ammonium sulfate precipitations as described b y K~IG~T (15). After dialysis against PBS, the tenfold concentrated preparation titered 400,000 reference units/ml. F o r all experiments this preparation was diluted in MEM to the final concentration of 20,000 reference units/0.5 ml. The specific activity of the diluted preparation was 4 × l0 s reference units/rag protein.
Viruses Vesicular stomatitis virus (VSV, Indiana strain) and encephalomyocarditis virus (EMC, Mengo strain) were propagated in L cells. The stock virus titer was around 10~ P F U / m l for VSV and 10l° P F U / m l for EMC. F o r the latter, one lethal dose (LD~0) in the mice equals 10a plaque-forming units (PFU).
Animals All experiments were performed using consanguine Swiss male mice, average weight of 20 g, routinely inbred in our laboratory.
Results
E//ect o/ Isoprinosine W e h a v e t e s t e d t h e effect of isoprinosine on a n t i v i r a l p r o t e c t i o n a g a i n s t the l e t h a l effect of EMC virus. U p to 50rag/mouse, no t o x i c i t y was observed after a single i n t r a p e r i t o n e a l (i.p.) injection, a n d half of this a m o u n t a d m i n i s t r a t e d b y
A n t i v i r a l Effects of I n t e r f e r o n a n d I s o p r i n o s i n e
227
the intravenous (i.v.) route was not found to be toxic. Thus, in all the following experiments, we used 25 rag/mouse for the i.p. and subcutaneous (s.c.) routes and 12.5 mg/mouse for the i.v. route. The amount of virus injected (100 LDs0) was sufficient to kill 85--90 per cent of the animals in the different treated or untreated control groups after 9--10 days. Isoprinosine was injected into groups of 15 mice 24 hours before, at the same time, or 24 hours after EMC virus (100 LDs0) ; both isoprinosine and virus were administrated b y the I P route. As shown in Table 1, no significant effect on the 50 per cent survival time and final survival rate was observed in these different groups. T a b l e t. E]/ect o / i s o p r i n o s i n e on the survival time a n d survival rate o / m i c e inoculated with E M C virus
E x p . No.
Iso 24 h o u r s before EMC
Iso+EMC simultaneously
I s o 24 h o u r s after EMC
Control
1
7a
7
7
7
5/15~
~/~5
2/15
3/~5
2
8~
ND
5
6
3
7• 2/15"
ND
ND
7 2/15
a 50 p e r c e n t s u r v i v a l t i m e (days) b Final survival rate
Association o/ Isoprinosine and Inter/eros I n the following experiments, both interferon and isoprinosine were injected and the mice were challenged with EMC virus. Both drugs as well as the virus were injected b y the i.p. route. The five chronological sequences b y which isoprinosine, interferon and the virus were administrated gre shown in Figure 1 (groups A E). CHRONOLOGICAL ORDER OF INJECTIONS
ISO
A
EMC IF
~
24h
~'lh~ EMC IF
B
~rlh~r ISO
C
~"
EMC IF
~r 3h ~'lh~ ISO
0
ISO
24h
*
IF
EMC
24h ISO EMC +IF
E Fig. l. Chronological s e q u e n c e s b y w h i c h isoprinosine, i n t e r f e r o n a n d t h e v i r u s were administered
228
C. CI4AN¥ and I. CEl~T~I:
As shown in Table 2, the survival rate was greatly dependent on the chronological order of the injections of isoprinosine and interferon. In groups A, C and D, isoprinosine was injected at 24 hours, 4 hours, and 3 hours before interferon respectively. In all experiments, interferon alone was ineffective or only slightly effective, whereas the association of interferon and isoprinosine was highly effective, since 60--70 per cent of the animals survived. No such results were obtained when isoprinosine was added 24 hours after interferon (group B) or when added together with interferon 1 hour after the inoculation of EMC virus (group E). Only in group C was interferon active alone, but even in this ease, the association of the two compounds was significantly more protective ( P =
Archives of Virology 55, 225--231 (1977)
© by Springer-Verlag 1977
Enhancement of Antiviral Protection Against Eneephalomyoearditis Virus by a Combination of Isoprinosine and Interferon By C. CHARY and I. CERUTTI Institut National de la Santd et de la Recherche lViddieale, I-I6pital St. Vincent de Paul, Paris, France, and
Institut de Recherehes sur le Cancer C.N.R.S., Villejuif, France With 1 Figure Accepted June 15, 1977
Summary The antiviral effect of interferon against encephalomyocarditis (EMC) virus infection in mice was enhanced b y isoprinosine. However, the enhancement was only obtained when both interferon and the virus were inoculated into the peritoneum; the inoculation route of isoprinosine did not modify significantly the final results. I n addition, the time sequence of injections was of great importance ; generally the injection of isoprinosine had to precede that of interferon b y a few hours.
Introduction The antiviral effect, of interferon in vitro and in vivo is presently well documented. The amount of interferon needed to obtain a therapeutic effee~ in vivo is relatively important and requires repeated injections (13). Among the possible explanations are a) a rapid degradation of interferon in the circulation or in the cell, b) a limited diffusion to the target area where high concentrations are necessary to trigger interferon receptor sites on the cell membrane (8), and c) the presence of interferon antagonists in the tissues (9). From a practical viewpoint, two possibilities are now available to overcome these difficulties: the administration of purified interferon at high concentrations and the use of adjuvants to improve its antiviral (or other) effect. Such adjuvants might potentiate interferon action b y a parallel but independent inhibitory effect on virus replication or/and through the ceil b y increasing the interferon-induced antiviral state. I n a series of articles we have recently shown that the structure of the cell membrane (5), as well as the integrity of the cytoskeletal components (4), play an
226
C. C~Am-Yand I. C E n ~ - ~ :
i m p o r t a n t role in t h e e s t a b l i s h m e u t of t h e interferon-induced a n t i v i r a l state. M a n y substances w i t h a f f i n i t y for gangliosides can m o d i f y t h e cellular response to interferon (1, 2, 3, 16). Likewise, m o d i f i c a t i o n of t h e celt m e m b r a n e due to cell t r a n s f o r m a t i o n (i. e., oncogenic viruses) can also change t h e i n t e r f e r o n s e n s i t i v i t y of t h e cells (7). Thus, in a search for drugs which m i g h t p o t e n t i a t e t h e a n t i v i r a l a c t i o n of interferon, we h a v e selected isoprinosine since this c o m p o u n d affects a p p a r e n t l y t h e m e m b r a n e of i m m u n o c o m p e t e n t or other somatic cells. I t has been r e p o r t e d , for instance, t h a t isoprinosine decreases the a m o u n t of P H A n e e d e d for t h e s t i m u l a t i o n of h u m a n p e r i p h e r a l blood l y m p h o e y t e s . I n t h e absence of P H A isoprinosine alone has no similar s t i m u l a t i n g a c t i o n (14). As shown in a p r e l i m i n a r y r e p o r t (6), c o m b i n e d t r e a t m e n t w i t h isoprinosine a n d interferon i m p r o v e s c o n s i d e r a b l y the p r o t e c t i v e effect of a single interferon dose a g a i n s t t h e l e t h a l effect of e n e e p h a l o m y o c a r d i t i s (EMC) virus in mice. E a c h of these two p r o d u c t s i n j e c t e d s e p a r a t e l y a t the same c o n c e n t r a t i o n is either ineffective or o n l y s l i g h t l y effective.
Materials and Methods
Drug Isoprinosine is the p-acetamidobenzoic acid salt of N, N-dimethylamino-2-propanol : inosine complex, 3 : 1 molar ratio, and was supplied b y Laboratories DelMande (Courbevoie, France). The white crystalline powder is stable at temperatures up to 150 ° C, its maximal UV absorption rate is at 260 nm, and it is water soluble. The physical and chemical properties have been described elsewhere (10).
Inter/eron Mouse interferon was prepared in C-243 cells (17) using standard procedures. The antiviral titer was estimated b y 50 per cent CPE inhibition using mouse L cells and vesicular stomatitis virus (VSV) as the challenge virus. The crude interferon preparation was purified b y two successive steps of ammonium sulfate precipitations as described b y K~IG~T (15). After dialysis against PBS, the tenfold concentrated preparation titered 400,000 reference units/ml. F o r all experiments this preparation was diluted in MEM to the final concentration of 20,000 reference units/0.5 ml. The specific activity of the diluted preparation was 4 × l0 s reference units/rag protein.
Viruses Vesicular stomatitis virus (VSV, Indiana strain) and encephalomyocarditis virus (EMC, Mengo strain) were propagated in L cells. The stock virus titer was around 10~ P F U / m l for VSV and 10l° P F U / m l for EMC. F o r the latter, one lethal dose (LD~0) in the mice equals 10a plaque-forming units (PFU).
Animals All experiments were performed using consanguine Swiss male mice, average weight of 20 g, routinely inbred in our laboratory.
Results
E//ect o/ Isoprinosine W e h a v e t e s t e d t h e effect of isoprinosine on a n t i v i r a l p r o t e c t i o n a g a i n s t the l e t h a l effect of EMC virus. U p to 50rag/mouse, no t o x i c i t y was observed after a single i n t r a p e r i t o n e a l (i.p.) injection, a n d half of this a m o u n t a d m i n i s t r a t e d b y
A n t i v i r a l Effects of I n t e r f e r o n a n d I s o p r i n o s i n e
227
the intravenous (i.v.) route was not found to be toxic. Thus, in all the following experiments, we used 25 rag/mouse for the i.p. and subcutaneous (s.c.) routes and 12.5 mg/mouse for the i.v. route. The amount of virus injected (100 LDs0) was sufficient to kill 85--90 per cent of the animals in the different treated or untreated control groups after 9--10 days. Isoprinosine was injected into groups of 15 mice 24 hours before, at the same time, or 24 hours after EMC virus (100 LDs0) ; both isoprinosine and virus were administrated b y the I P route. As shown in Table 1, no significant effect on the 50 per cent survival time and final survival rate was observed in these different groups. T a b l e t. E]/ect o / i s o p r i n o s i n e on the survival time a n d survival rate o / m i c e inoculated with E M C virus
E x p . No.
Iso 24 h o u r s before EMC
Iso+EMC simultaneously
I s o 24 h o u r s after EMC
Control
1
7a
7
7
7
5/15~
~/~5
2/15
3/~5
2
8~
ND
5
6
3
7• 2/15"
ND
ND
7 2/15
a 50 p e r c e n t s u r v i v a l t i m e (days) b Final survival rate
Association o/ Isoprinosine and Inter/eros I n the following experiments, both interferon and isoprinosine were injected and the mice were challenged with EMC virus. Both drugs as well as the virus were injected b y the i.p. route. The five chronological sequences b y which isoprinosine, interferon and the virus were administrated gre shown in Figure 1 (groups A E). CHRONOLOGICAL ORDER OF INJECTIONS
ISO
A
EMC IF
~
24h
~'lh~ EMC IF
B
~rlh~r ISO
C
~"
EMC IF
~r 3h ~'lh~ ISO
0
ISO
24h
*
IF
EMC
24h ISO EMC +IF
E Fig. l. Chronological s e q u e n c e s b y w h i c h isoprinosine, i n t e r f e r o n a n d t h e v i r u s were administered
228
C. CI4AN¥ and I. CEl~T~I:
As shown in Table 2, the survival rate was greatly dependent on the chronological order of the injections of isoprinosine and interferon. In groups A, C and D, isoprinosine was injected at 24 hours, 4 hours, and 3 hours before interferon respectively. In all experiments, interferon alone was ineffective or only slightly effective, whereas the association of interferon and isoprinosine was highly effective, since 60--70 per cent of the animals survived. No such results were obtained when isoprinosine was added 24 hours after interferon (group B) or when added together with interferon 1 hour after the inoculation of EMC virus (group E). Only in group C was interferon active alone, but even in this ease, the association of the two compounds was significantly more protective ( P =
