Mersalyl: a Diuretic with Antiviral Properties

Transcription

1 ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, Sept. 1975, p Vol. 8, No. 3 Copyright American Society for Microbiology Printed in U.S.A. Mersalyl: a Diuretic with Antiviral Properties M. J. KRAMER,* R. CLEELAND, AND E. GRUNBERG Department of Chemotherapy, Hoffmann-LaRoche Inc., Nutley, New Jersey Received for publication 15 May 1975 Mersalyl (Salyrgan), an organic mercurial diuretic, was tested against human and animal viruses with in vivo model infections in mice and tissue culture systems. Mersalyl was active against coxsackieviruses A21 and Bi in mice if administered intraperitoneally immediately after infection. No effect was observed if intraperitoneal treatment was delayed 1 or 2 h postinfection, or if treatment was administered either subcutaneously or per os. Topical treatment with a 5% aqueous solution of mersalyl produced a statistically significant effect against herpes simplex dermatitis in mice but the substance was inactive against systemic infections in mice with herpes simplex as well as Columbia SK, influenza, Semliki Forest, and Sendai viruses. Contact inactivation of coxsackieviruses A21 and Bi and herpes simplex virus was observed, but mersalyl was inactive in tissue culture against coxackieviruses A21 and B1, herpes simplex, influenza, rhinovirus, Semliki Forest, Sendai, and vaccinia viruses. Mersalyl (Salyrgan), an organic mercurial described by Brunn (2) in 1924 as an effective diuretic, has been shown by Dorne and Hirth (3, 4) to both degrade tomato bushy stunt virus and form a complex with tomato bushy stunt virus ribonucleic acid (RNA). The sulfhydryl-inhibitory effect of mersalyl (7) and the report by Billard and Peets (1) indicating that influenza virus polymerase was inhibited by antiviral compounds possessing sulfhydryl reactivity suggested that mersalyl might exert an antiviral effect against animal and human viruses. The present study was undertaken to answer this question. MATERIALS AND METHODS Compound description. Mersalyl, 2- [(3-hydroxymercuri-2- methoxypropyl) carbamyl ]phenoxyacetic acid, is a white, odorless, slightly hygroscopic powder which is slightly soluble in water and has the structural formula shown in Fig. 1. The acute toxicity of mersalyl was determined in 18- to 20-g mice at 72 h after a single administration of the substance by the intraperitoneal (i.p.), subcutaneous (s.c.), or per os (p.o.) routes, and the calculated mean lethal dose (LD,0) values were 400, 500, and 1,400 mg/kg, respectively. Virus infections in mice. Swiss albino mice (Royalhart) weighing 9 to 12 g (weanling) were infected i.p. with coxsackievirus A21 and herpes simplex viruses while intranasal instillation under light ether anesthesia was used to infect mice with influenza A2/Asian/J305 and Sendai (parainfluenza type 1) viruses. Mice (Royalhart) weighing 18 to 20 g were infected i.p. with Columbia SK, coxsackievirus Bi, and Semliki Forest viruses. Hairless mice (Jackson Laboratory) were lightly 295 F OCH2COOH OCONHCH2CH(OCH3)CH2HgOH FIG. 1. The structural formula of mersalyl. scratched on the flank with a 27-gauge needle and the HF C1-5 strain of herpes simplex virus was applied to the scarified area with a cotton swab. All animals received approximately 10 LD,0 of the appropriate virus. Treatment. Mersalyl, in aqueous suspension, was administered i.p., s.c., or p.o. (1.0 ml in adult mice and 0.5 ml in weanling mice), 24 h before virus infection, immediately after virus infection, and 24 h after virus infection for all virus infections in mice except influenza, Sendai, and herpes dermatitis. Mice infected with influenza and Sendai viruses received 7 i.p. treatments (at 0, 1, 5, 24, 30, 48, and 72 h relative to virus infection), while topical application, be.ginning 2 h after virus infection and then twice daily for the next 5 days, was used to treat herpes dermatitis in hairless mice. In vitro tube dilution assay. Monolayers of WI-38 and rhesus monkey kidney cells were infected with serial 10-fold dilutions of coxsackievirus (A21, Bi), herpes simplex, influenza (Asian), rhinovirus 42, Semliki Forest, Sendai, and vaccinia viruses. The mean tissue culture infective dose (TCIOD5) of the virus-infected cultures in the presence and absence of 5 sg of mersalyl per ml (the maximum tolerated noncytotoxic dose for tissue culture cells) was determined on the basis of cytopathogenic effect after 7 days of incubation for all viruses except influenza which was assayed by the hemadsorption technique 4 days after virus infection. Contact inactivation. Several RNA and deoxyri-

2 296 KRAMER, CLEELAND, AND GRUNBERG bonucleic acid (DNA) viruses (coxsackieviruses A21 and Bi, herpes simplex, influenza A2/Asian, Semliki Forest and Sendai) were incubated in the presence of 1,000 ug of mersalyl per ml for 1 h in a 37 C water bath, and viral infectivity was determined in tissue culture by a tube dilution assay in comparison to a nontreated virus control. RESULTS In vivo antiviral effects of mersalyl. The effects of i.p. treatment with mersalyl against virus infections in mice are shown in Table 1. Mersalyl, at doses of 200, 100, and 50 mg/kg, protected more than 50% of the mice infected with coxsackievirus A21 while 48 to 50% of coxsackievirus Bl-infected mice survived when treated with mersalyl at these doses. The protective effect of lower doses of mersalyl, i.e., 12.5 and/or 25 mg/kg, was also statistically significant (P < 0.05) against these two strains of coxsackievirus. No effect (P > 0.05) was seen against Columbia SK, herpes simplex, influenza, Semliki Forest, and Sendai viruses at any of the doses tested. Administration of mersalyl by the p.o. or s.c. routes was found to be without effect even at TABLE mg/kg against coxsackievirus A21 and Bi infections in mice (Table 2). Since activity was observed after i.p. but not p.o. or s.c. administration of mersalyl by the -24, 0, and +24 h schedule, the time of i.p. treatment was studied to determine if the 0-h treatment was critical for activity, as Grunberg and Prince (5) had reported in their study of the antiviral effect of 3,4-dihydro-1-isoquinolineacetamide hydrochloride. Therefore, the protective effect afforded by i.p. treatment with mersalyl against coxsackievirus A21 and Bi infections in mice was investigated in a series of comparative experiments wherein the treatment normally given immediately after virus infection (0 h) was delayed 1 or 2 h postinfection while retaining the treatments administered at -24 h and +24 h. Treatment with mersalyl at -24, 0, and +24 h produced a significant protective effect against coxsackievirus A21 (Table 3) (18 of 23 infected, drug-treated mice survived, whereas only 3 of 24 infected, control mice survived). If, however, treatment was administered at -24, +1, and +24 or -24, +2, and +24 h, no protective effect of mersalyl was observed. Sim- Effect of mersalyl against virus infections in mice ANTIMICROB. AGENTS CHEMOTHER. Dose No. of mice surviving/ % Virusna (mg/kg no. treated at 21 days Corrected Pd Treated Control uval Coxsackievirus A /63 4/64 73 < /60 3/64 74 < /101 7/ < /63 4/64 43 < /56 4/ Coxsackievirus Bi /31 2/30 48 < /64 1/61 50 < /55 1/55 49 < /55 1/55 29 <0.001 Columbia SK 200 0/7 2/7 0 > /31 2/32 13 >0.05 Herpes simplex 200 0/8 0/7 0 > /40 5/40 2 > 0.05 Influenza A2/ 100 0/16 0/14 0 >0.05 Asian/J /16 0/14 0 > 0.05 Semliki Forest 200 3/21 4/23 0 > /16 2/15 12 > 0.05 Sendai 100 0/14 0/16 0 > /15 0/16 0 > 0.05 a Weanling (9- to 12-g) mice were infected i.p. (coxsackievirus A21, herpes simples virus) or intranasally (influenza, Sendai) with approximately 10 LD50; 18- to 20-g mice were infected i.p. (coxsackievirus Bi, Columbia SK, Semliki Forest) with approximately 10 LD,0. btreatment: 0.5 ml for 9- to 12-g animals, 1.0 ml for 18- to 20-g animals. Treatment schedule: i.p. three times at -24 h, 0 h, and +24 h relative to infection at 0 h. In the case of influenza and Sendai viruses, i.p. seven times at 0, 1, 5, 24, 30, 48, and 72 h relative to infection. c Percentage of survival in treated mice less percentage of survival in control. d Fisher exact test.

3 VOL. 8, 1975 ANTIVIRAL EFFECT OF MERSALYL 297 TABLE 2. Effect of p.o. or subcutaneous administration of mersalyl against coxsackievirus A21 and Bl infections in mice No. of mice surviving/ Dose Virus (mg/kg, no. treated at 21 days Corrected P 3 times)' Treated Control survival Coxsackievirus A p.o. 3/24 3/24 0 > p.o. 3/16 1/16 12 > P.O 3/24 3/24 0 > s.c. 3/19 3/24 3 > s.c. 2/16 1/16 6 > S.C. 3/24 3/24 0 > 0.05 Coxsackievirus Bi 400 p.o. 0/20 1/21 0 > p.o. 1/16 1/15 0 > P.O. 0/8 0/7 0 > s.c. 1/13 0/13 7 > s.c. 0/23 0/21 0 > S.C. 0/16 1/15 0 > 0.05 a Virus infection, see Table 1. b Treatment: 0.5 ml for coxsackievirus A21, 1.0 ml for coxsackievirus B1; all treatments given - 24 h, O h, +24 h relative to infection at 0 h. TABLE 3. Effect of different regimens of mersalyl treatment on infection of mice with coxsackieviruses A21 and BJ infectiona Dose Treatment schedule (no. of mice surviving/no. treated at 21 daysb) (mg/kg, i.p. x 3-24, 0, -24, +1, -24, +2, Control +24 h +24 h +24 h Coxsackievirus A /23 2/24 5/24 3/24 Coxsackievirus Bi /24 1/23 1/24 0/22 avirus infections, see Table 1. Relative to virus infection at 0 h. ilar results were obtained with coxsackievirus Bi infection in mice. The requirement for an i.p. treatment with mersalyl at 0 h suggested that the antiviral effect against coxsackieviruses A21 and Bi might be due to a contact-inactivation of the viruses by mersalyl. To test this possibility, several RNA and DNA viruses were first incubated for 1 h at 37 C in the presence of mersalyl and then assayed for viral infectivity in tissue culture. The infectivity of coxsackieviruses A21 and Bi was reduced by at least 4.2 logarithms while the titers of herpes simplex viruses (Sabin and HF Cl-5 strains) were decreased by at least 5 logarithms (Table 4). Contact with mersalyl, however, had no appreciable effect on the infectivity of influenza A2/Asian/J305, Semliki Forest, and Sendai viruses. Inactivation of the two strains of herpes virus after contact with mersalyl suggested that topical application of the substance might be effective. Therefore, hairless mice were infected dermally with the HF Cl-5 strain of herpes simplex and treated topically with mersalyl. Application of a 5% aqueous suspension of mersalyl protected 38% of the infected mice from an otherwise lethal infection (P = 0.001) (Table 5). Effect of mersalyl on growth of viruses in tissue culture. The in vitro antiviral effect of mersalyl was tested in tissue culture against several RNA and DNA viruses. From the results presented in Table 6 it is apparent that mersalyl did not significantly (A log > 2) inhibit the in vitro growth of any of the viruses tested. DISCUSSION Previous reports of the antimicrobial effects of mersalyl have indicated that the substance is without effect against pathogenic fungi such as Trychophyton interdigitale and Achorion schoenleinii (8) but possesses marked activity against typhoid bacilli in the bile 2 h after intravenous injection into human typhoid carriers (6).

4 298 KRAMER, CLEELAND, AND GRUNBERG TABLE 4. Effect of in vitro contact with mersalyl on the infectivity of RNA and DNA viruses in tissue cultures of mammalian cells Log,0 TCID,, Virus ~ ~ ~ ~ ~ ~ ~ ~~~AVirus control + mersalyla Virus Virus Virus Log inactivation" Coxsackievirus A <1.0 >4.2 + Coxsackievirus B Herpes simplex (Sabin) 6.5 <1.0 >5.5 + Herpes simplex (HF Cl-5) 7.0 <1.0 >6.0 + Influenza A2/Asian Semliki Forest Sendai a Virus control and virus + mersalyl (final concentration, 1,000 Ag/ml) were incubated for 1 h in a 37 C water bath before assay for infectivity in tissue culture. bvirus inactivation was defined as a difference of at least 2 logarithms between the TCID,O of the virus control and virus + mersalyl. TABLE 5. Effect of topical application of mersalyl against herpes simplex dermatitis in hairless mice a No. of mice Drug surviving/no. % Drug treated at 21 days corrected P concn' Treated Control survival 5% 11/26 1/ % 2/12 1/12 8 >0.05 a Hairless mice (18 to 20 g) were lightly scratched on the flank with a 27-gauge needle and the HF Cl-5 strain of herpes simplex virus was applied to the scarified area with a cotton swab. All animals received approximately 10 LD,,. b An aqueous suspension of the test substance was applied topically to the infected area 2 h postinfection and twice daily for the next 5 days thereafter. TABLE 6. In vitro antiviral effect of mersalyla ALog,, TCID,, Virus of control Activity" treated cells Coxsackievirus A Coxsackievirus Bi Herpes simplex nfluenza A2/Asian Rhinovirus Semliki Forest Sendai Vaccinia a Tested at 5 ug/ml. b Activity was defined as a difference of at least 2 logarithms between the TCID,O of the virus control and drug-treated cells. The results of the present study indicate that mersalyl is also active against coxsackievirus A21 and Bi infections in mice if treatment is administered by the i.p. route. It was further ANTIMICROB. AGENTS CI-IEMOTHER. demonstrated that the i.p. treatment administered at 0 h (time of virus infection) is essential for activity and that delaying treatment 1 or 2 h after virus infection results in a loss of activity. These findings, taken in conjunction with the in vitro contact-inhibitory effect of mersalyl against coxsackievirus A21 and Bi, suggest that the in vivo antiviral effect of mersalyl on these viruses might be due to contact inactivation of these two viruses. Although mersalyl inactivated two strains of herpesvirus on contact, there was no effect against a systemic infection in mice with herpes simplex virus (Sabin) when mersalyl was given i.p. by the same treatment schedule which protected mice against coxsackievirus infections. Topical application of mersalyl, however, was effective against a herpesvirus dermatitis caused by the second strain (HF Cl-5) and this protective effect may also be due to a direct effect of the compound on the virus. The contact inactivation studies indicate that mersalyl could significantly reduce the infectivity of both DNA and RNA viruses, but some degree of selectivity exists since not all viruses tested were similarly affected. Among the RNA viruses, the nonenveloped coxsackieviruses A21 and Bi were inactivated by mersalyl while the infectivity of three other viruses (influenza, Semliki Forest, and Sendai) with lipoprotein envelopes was not significantly reduced. It seems unlikely that the presence of an envelope per se protects these enveloped RNA viruses since mersalyl inactivated a DNA-containing enveloped virus (herpes simplex) on contact. LMRATURE CITED 1. Billard, W., and E. Peets Sulfhydryl reactivity: mechanism of action of several antiviral compounds

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