APPLIED MICROBIOLOGY, Feb. 1968,p.370-392 Vol. 16, No.2 Copyright © 1968 AmericanSocietyforMicrobiology Printedin U.S.A. In Vivo Antiviral Properties ,of Biologically Active Compounds Studies with Influenza and Vaccinia Viruses II. ROBERTW.SIDWELL,GLENJ. DIXON,SARAM.SELLERS,ANDFRANKM.SCHABEL,JR. VirusSection, Southern Research Institute, Birmingham, Alabama 35205 Received for publication 16 November 1967 D o w The in vivo anti-influenza virus andantivaccinia virus activity of 156 biologically n active compounds was determined. One oftwo criteria was used for evaluating ac- lo tivityagainst the influenza virus. The criteria were increase in survivor number and a d mean survival time, and reduction in virus-induced lung consolidation in treated, e infected Swiss mice. Increase in survivor number and mean survival time were the d criteria for evaluation ofantivaccinia virus activity. Several drug doses were tested fr againsttwovirusconcentrations to demonstrateantiviralactivitymoreclearly. Two om compounds were considered significantly active against the influenza virus: DL- noformicin (NSC72942) andamantadinehydrochloride (NSC 83653). Elevencom- ht pounds had reproducible activity againstvaccinia virus: isatin-,B-thiosemicarbazone tp (NSC 721), 6-azauracil (NSC3425), 9-a-fluoro-2a-methylhydrocortisone 21-acetate :/ / (NSC 12601), 5-[bis(2-chloroethyl)amino]uracil (NSC 34462), 5-iodo-2'-deoxyuri- a e dine (NSC 39661), streptonigrin (NSC 45383), N-methylisatin ,B-thiosemicarbazone m (NSC69811),cytovirin (NSC91770),9-f3-D-arabinofuranosyladenine (NSC404241), . a and 5-(mercaptomethyl)uracil (NSC 529351). s m . o Effective viral disease chemotherapy has be- MATERIALS AND METHODS rg come increasingly feasible in recent years as a Inifluentza virus. The strains ofinfluenza virus used o/ result of extensive screening studies and the ap- for primary screening experiments were the PR8n parent success of clinical trials with a few select strain of influenza A virus (PR8) or the Japan 305 M compounds. The chemotherapyapproach to viral strain of Asian influenza virus (Asian). In certaian disease control is ofparticular interest, since the follow-up studies, the Lee strain ofinfluenza B virurcs cmionsattiodne,siirsabwlheolallyterefnfaetcitvievetoonlthyisifctohnterovla,ccivnaecs- ((LPeRe3)01a)ndwertheealPsRo30e1mplsotyreadin. oTfheinPflRu8enzaandAPRv3ir0u1h 2s viruseswereobtainedinmousebrainsuspensionsfrom9 are used by the majority of a specific human Bernice Eddy, Laboratory ofVirology and Rickettsi, - population, and isineffective if the disease to be ology, National Institutes ofHealth, Bethesda, Md.;20 controlled is caused by a broad spectrum of the Asian agent was obtained in monkey kidney cel1l agents, such as is the case for the common cold. suspensionfromWiltonA. RightselofParke, Davis&9 Because of the potential of antiviral chemo- Co., Detroit, Mich.; and the Lee virus was receivebd y etvhaelraupayt,e wtheehianvveivcoararniteidvioruatlaactsievriietsyooffsatnuduimebsetro iRnobchionrsioonalloafntotihcemNeamtbironaanleCsuosmpmeunnsiiocnabflreomDRiosselaysne gu ofcompounds.Thecompoundsusedwereselected Ctehrneteer,timAetslantthar,ouGgah.mWiceepbaysseidntreaancahsalviriunsocautlalteiaosnest especially for their known biological activity in t priortouseinthesestudies.Virusstockwasprepared one or more in vivo tumor systems or their from homogenized, infected mouse lung suspended activityinvitroorinvivoinanimal-virus systems, in Hank's balanced salt solution. Dihydrostrepto- or becauseoftheirapparentchemicalrelationship mycin in a concentration of 10 mg/ml of virus was to biologically active compounds. Viral agents added before virus was used to infect animals. The used in these studies included Friend leukemia LD5ovirustiterswerecalculated (17) fromthenumbers virus, Rous sarcoma virus, mouse salivary gland of intranasally inoculated mice that died within 12 virus, influenza virus, vaccinia virus, and rabbit- days ofinoculation. Vaccinia and rabbitpox viruses. The International rpeosxulvtisruosf.sItnudaiepsrewviitohusthceomFmruiennidcalteiuoknem(i2a2),vitrhues Health Division (IHD) ortheWestern Reserve (WR) strains of vaccinia virus were used in all primary were described. The present report concerns the screening experiments. When antiviral activity was results of investigations with influenza, vaccinia, suggested in at least one screening experiment, the and rabbitpox viruses. Utrecht (Ut) strain ofrabbitpox virus wasoften used 370 VOL. 16, 1968 ANTIVIRAL EFFECT OF BIOLOGICALLY ACTIVE COMPOUNDS 371 in a follow-up study. All three viruses were obtained ofmean survival and in the number of survivors 21 in chorioallantoic membrane or mouse brain suspen- daysaftervirusinoculation. Comparativeexperiments sion from Arthur Brown, Fort Detrick, Frederick, carried out during these studies indicated that the Md. When received in our laboratory, the viruses reduction of virus-induced lung consolidation of were passed intracerebrally through ICR Swiss mice, drug-treatedanimalsonthe10thdayaftervirusinocu- and homogenized, infected mouse brains were sus- lation wasanequally sensitive procedure andhad the pended in Hank's balance salt solution. The LD5o advantage of requiring less time. The former proce- virus titers were determined from the 12-day mor- dure was therefore used in approximately two-thirds tality of intracerebrally inoculated mice. All viruses of the experiments and the latter method was used were stored in sealed ampoules at about -70C until intheremainderofthestudies. used. The results of the experiments in which the first Mice. Random-bred ICRSwiss mice weighing 8 to procedure was used were statistically evaluated by 10 g were used in the majority ofthe influenza virus comparing (bymeans ofthe ttest) themeansurvival experiments. In certain follow-up experiments, 18- to time of drug-treated, virus-infected animals dying D 21-gmicewereemployed. Forthevacciniaandrabbit- on or before day 21 with the mean survival time of o pox virus experiments, 18- to 21-g mice were used. theviruscontrolanimals. Ifthe Pvalueobtainedwas w All animals were obtained from Southern Animal <0.05 but >0.001, the antiviral activity was con- n Farms, Prattville, Ala., and mice of the same sex sidered questionable. Ifthe Pvalue was <0.001, the lo a were used in individual experiments. They were drug was considered to have possible antiviral activ- d housedfivetoacage. ity.Thedatawerequestionableifasignificantincrease e Compounds tested. A total of 156 compounds were in mean survival time occurred in animals treated d tested for antiviral activity. The Cancer Chemo- with low dosages of the drug, but did not occur in fr therapy National Service Center (Washington, D.C.) animals treated with higher nontoxic concentrations. o m supplied the majority of these compounds; hence, The same consideration applied if activity was ob- when each is described, the CCNSC (NSC) number served in mice infected with a high level ofvirus but h is included. Among the compounds tested were not in those infected with lower virus doses. The tt p antimetabolites, alkylating agents, antibiotics, hor- number of surviving infected, treated animals was : / mone and hormonelike compounds, guanidines, compared with the number of virus control mice /a thiosemicarbazones, semicarbazones, terephthalani- surviving (ifany) by meansofthechi-square analysis e lides,andothermiscellaneousagents. Eachcompound technique. Ifthe P values calculated by this method m was dissolved or suspended in the most appropriate were <0.3 but >0.05 (approximate standard error), .a medium, which was either sterile water, physiological theantiviralactivity wasquestionable, whereasvalues s m saline, diluent E (a steroid-suspending vehicle con- of <0.05 wereindicativeofpossibleantiviralactivity. taining 9% sodium chloride, 5% sodium carboxy- The evaluation ofantiviral activity on the basis of .o methylcellulose 7LP, 0.4% polysorbate 80, and 0.9% reduction of virus-induced lung consolidation was rg benzyl alcohol in water), 1% NaHCO3, 5%o gum carried out by scoring each lung on a "blind" basis, / acacia in saline, or 0.4% carboxymethylcellulose in i.e.,thescorersdidnotknowthehistoryofthedonors o n phosphate-buffered saline. ofthe lungs. The lungs were graded accordingto the M Influenza virus testing. Mice were inoculated intra- following scale: 5 = death with consolidation, 4 = nasallywith0.06mlofvirus (10or32LD5o) suspended 100% consolidation, 3 = -75% consolidation, a r in Hank's balanced salt solution. After virus inocula- 2 = -50% consolidation, 1 = -25% consolidation, c h tion, the animals were randomized and divided into 0 = no consolidation. An averagelungconsolidation groups of 10 for each drug dosage. Twenty animals score was calculated by dividing the total grade of 2 9 wereusedasviruscontrols. consolidation by the number of lungs graded. The , One oftwo drug treatment schedules was used in data werestatistically evaluated using White's modifi- 2 0 the primary screening evaluations. Schedule 1 con- cation ofthe Wilcoxon test (28). A Pvalue of <0.05 1 sisted oftwice daily drug injections beginning 1 day calculated by this method indicated questionable 9 prior to virus inoculation and continuing for 9 days, antiviral activity, and a P value of <0.01 indicated b andschedule 2consisted ofonce dailydruginjections possible antiviral activity. The same considerations, y starting 1 day after virus inoculation and continuing of dose response and virus concentrations described g for 9 days. Drug doses used were the approximate above were also used in this method. u e LDIO, LD1o/2, LD,o/4, and LD1o/8 as based on previous Any compound having questionable or possible s toxicitytestsinnormalanimals held 21 daysfromthe antiviral activity when tested by either of the above t beginning of treatment. The compounds were ad- procedures was retested for confirmation. If possible ministered intraperitoneally on a milligram per kilo- activity was again seen, additional experiments with gram basis, with each animal being weighed daily. other virus strains, different treatment schedules, or Other treatment schedules were utilized in additional differentmethodsforevaluationwerethencarriedout. experimentswithcertaindrugs;thesetreatmentsched- Mice which had beenexposed only to virus diluent uleswerechoseninattemptstoobtainamorepositive were treated with identical drug dosages at the same demonstration of antiviral activity for compounds time as the test animals. These animals were held 30 that appeared active in the screening studies and to days after the end of treatment and served as drug elucidate prophylactic or therapeutic activity. These toxicitycontrols. various schedules are indicated in the appropriate Vaccinia virus testing. Mice were inoculated intra- tables. cerebrally with 0.03 ml containing 10 or 32 LD5o of The initial criteria for evaluation of anti-influenza the virus suspended in Hank's balanced salt solution virus activity ofeach drug were increases in the time and were treated as described above for influenza 372 SIDWELL ET AL. APPL. 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