Proc. Nati. Acad. Sci. USA Vol. 86, pp. 332-336, January 1989 Medical Sciences Marked in vivo antiretrovirus activity of 9-(2-phosphonylmethoxy- ethyl)adenine, a selective anti-human immunodeficiency virus agent (acquired immunodeficiency syndrome/antiviral chemotherapy) JAN BALZARINI*t, LIEVE NAESENS*, PIET HERDEWUN*, IVAN ROSENBERGt, ANTONIN HOLYt, RUDI PAUWELS*, MASANORI BABA*, DAVID G. JOHNS§, AND ERIK DE CLERCQ* *Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium; tInstitute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 16610 Prague, Czechoslovakia; and §Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by R. C. Gallo, October 6, 1988 (receivedfor review July 1, 1988) ABSTRACT 9-(2Phophonylmethoxyethyl)adenne (PMEA) in vivo. No animal model is known in which HIV infection leads is a potent and selective inhibitor of the replication of human to severe immunodeficiency and animal death. Recently, a immunodeficiency virus (HIV) in vitro in human T-Iymphocyte variety of murine, feline, and simian retrovirus test systems MT4, H9, and ATH8 cells. PMEA also inhibits Moloney munne have been proposed for the in vivo evaluation of promising sarcoma virus (Mo-MSV)-induced transformation ofmurine C3H anti-HIV compounds (17-22); in several of these models the embryo fibroblasts. Moreover, PMEA causes a dose-dependent antiretrovirus activity ofAZT has been assessed. Tavares et al. suppression of tumor formation and assocated mortality in mice (17) found that AZT prevents Rickard feline leukemia virus inoculated with Mo-MSV. At a dose of 50 or 20 mg/kg per day infection in 6-week-old kittens ifadministered immediately after PMEA effected a 90-100% protection of the mice against Mo- infection. Ruprecht et al. (19) found that in mice infected with MSV-induced tumor formation and mortality. Even with a PMEA Rauscher murine leukemia virus AZT prevented development dose as low as 1 to 5 mg/kg per day, tumor formation was of splenomegaly and suppressed viremia when treatment was signifiantly delayed and the survival rate was siantly en- started soon after virus inoculation. In this study, AZT was hanced. In parallel experiments, azidothymidine exhibited a com- given i.p. at daily doses of60, 120, and 240 mg/kg (or 0.5 to 1.0 parable inhibitory effect on Mo-MSV-induced tumor formation mg/ml in drinking water). and associated death only at a 25-fold higher dose than PMEA. Our group reported that AZT was endowed with remarkable Because PMEA has stronger in vivo andretrovirus potency and antiretrovirus activity when given i.p. at daily doses of 125, 25, selectivity than azidothymidine and various other compounds and 5 mg/kg ofbody weight to newborn NMRI (Naval Medical currently being subjected to clinical trials, PMEA studies should be Research Institute) mice infected with Moloney murine sar- pursued to assess the potential of this compound in the treatment coma virus (Mo-MSV) (22). This murine retrovirus, when of acqured immunodeficiency syndrome (AIIDS) and other retro- injected s.c. in the left hind leg of 2- to 3-day-old NMRI mice, virus infections in humans. causes tumor formation within 4 to 5 days and death within 10 to 12 days after infection. Both tumor appearance and the Acquired immunodeficiency syndrome (AIDS) is caused by associated mortality are functions of the virus inoculum size. the retrovirus, human immunodeficiency virus (HIV) (1, 2). Both Mo-MSV-induced tumor formation in vivo and Mo- Because an urgent need exists to find an effective chemo- MSV-induced cell transformation in vitro depend on the viral therapeutic drug against AIDS, many substances have been replicative power, as does the cytopathogenicity of HIV for evaluated for antiretrovirus activity in vitro, and some human T4 lymphocytes in vitro. We therefore considered compounds reported to be effective against HIV replication Mo-MSV-induced tumor formation in vivo and associated in vitro are currently being clinically investigated for AIDS mortality an appropriate model to assess the in vivo antiretro- treatment (3-15). So far, only 3'-azido-2',3'-dideoxythy- virus activity of selected anti-HIV agents. midine (N3ddThd, AZT, Zidovudine) has unambiguously Recently, we described a series ofacyclic purine derivatives demonstrated clinical benefit (7-9). In a double-blind, place- endowed with antiretrovirus and broad-spectrum anti-DNA bo-controlled trial, 282 patients with AIDS or AIDS-related virus activity (23, 24). Among them, we found that the acyclic complex (ARC) were randomly assigned to receive either adenosine derivative PMEA [9-(2-phosphonylmethoxyeth- AZT (250 mg) or placebo by mouth every 4 hr for a total yl)adenine] (Fig. 1) inhibits HIV-induced cytopathogenicity in period of 24 weeks. When the study was completed, 19 MT4 cells and HIV antigen expression in H9 cells at concen- placebo recipients (of 137) and one AZT recipient (of 145) had trations ('1.6-2 ,uM) significantly below the toxicity threshold died. A decreased frequency of opportunistic infections, for the host cells ("40-67 AM) (Table 1 and data not shown) increased base-line Karnofsky performance score, increased (24). PMEA also proved effective in inhibiting Mo-MSV- weight, and a significant increase in the number of CD4 cells induced transformation of murine C3H embryo fibroblasts in were noted in the AIDS or ARC subjects receiving AZT (9). vitro; it did so at a concentration similar to that found inhibitory However, serious adverse reactions-particularly bone mar- to HIV replication in MT4 and H9 cells. row suppression (megaloblastic anemia)-but including also We then evaluated PMEA for its antiretrovirus effect in headache, confusion, anxiety, nausea, and insomnia were Mo-MSV-infected newborn NMRI mice and included in this observed, indicating that AZT is not an innocuous drug (16). study a number of anti-HIV agents that are currently in These results further emphasize the need for more efficacious and selective antiretrovirus agents. Abbreviations: HIV, human immunodeficiency virus; Mo-MSV, A major problem in screening compounds proven active Moloney murine sarcoma virus; AIDS, acquired immunodeficiency against HIV in vitro is the lack ofany suitable HIV test system syndrome; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; AZT, 3'- azido-2' ,3'-dideoxythymidine; ddCyd, 2' ,3'-dideoxycytidine; ddAdo, 2',3'-dideoxyadenosine; ATA, aurintricarboxylic acid; The publication costs of this article were defrayed in part by page charge CCID50, cell culture 50%6 infective dose; ED50, 50%o effective dose; payment. This article must therefore be hereby marked "advertisement" CD50, 50%o cytotoxic dose. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 332 Downloaded by guest on September 29, 2021 Medical Sciences: Balzarini et al. Proc. Natl. Acad. Sci. USA 86 (1989) 333 NH2 Inhibitory Effects of Test Compounds on the Initiation of Mo-MSV-Induced Tumor Formation in NMRI Mice and on the Survival of Mo-MSV-Inoculated NMRI Mice. Two- to three- day-old NMRI mice (weighing -2 g) were inoculated s.c. in the as N left hind leg with 50 ,ul of Mo-MSV (100 focus-forming units measured by in vitro determination of the virus-induced trans- formation ofmurine C3H embryo fibroblast cells). At 4 to 5 days LM2I 0I l 11 after infection, tumors develop and rapidly increase in volume CH2OCH2- P- OH upon further aging of the mice. Within 10 to 12 days after I infection, mice (then weighing -5 to 6 g) die from the viral OH infection. Drug treatment was started 2 to 3 hr before injection of the and further administration was FIG. 1. Formula of PMEA. virus, compound given daily i.p. for an additional 9 days. The mean day of tumor clinical trial or considered for clinical studies-i.e., AZT, initiation (± SD) and the mean day of survival of the mice (± 2' ,3 '-dideoxycytidine (ddCyd), 2' ,3 '-dideoxyadenosine SD) were calculated, and the statistical significance of the (ddAdo), ribavirin, fusidic acid, and suramin. average delay oftumor formation and the mean day of survival in the treated groups versus the untreated (control) group was assessed the two-tailed Student's t test. MATERIALS AND METHODS by Inhibitory Effects of Test Compounds on HIV-Induced RESULTS Cytopathogenicity in MT-4 Cells. The procedure to determine Antiretrovirus Effect of PMEA and Other Test Compounds the anti-HIV activity in MT-4 cells has been described (25). in Vitro. Of the compounds listed in Table 1, AZT emerged Briefly, HIV-infected MT-4 cells were seeded at 5 x 104 cells as the most potent and selective inhibitor of HIV replication into 0.32-cm2 wells ofa 96-well microplate containing various in MT-4 cells (ED50, 0.004,.M), followed by ddCyd (ED50, concentrations of the test compounds. Virus input was 100 0.6 1LM) and Evans blue (ED50, 1.5 MtM). With an ED50 of 2 cell culture 50% infective doses (CCID50) per well. After a MtM, PMEA was slightly superior to ddAdo and aurintricar- 5-day incubation at 370C, the number of viable cells was boxylic acid (ATA) (ED50, 5-10 MM), whereas suramin determined in parallel for both HIV- and mock-infected cells showed a 50% inhibitory effect at 32 MLM. Ribavirin as well as in a blood cell-counting chamber using the trypan blue fusidic acid were inactive as anti-HIV agents at subtoxic dye-exclusion method. The 50% effective dose (ED50) was concentrations. These data agree with previously reported defined as the concentration of compound that protected values for ribavirin in HIV-infected ATH8 cells (26) and for HIV-infected cells by 50%, whereas the 50% cytotoxic dose fusidic acid in HIV-infected H9 cells (unpublished data). AZT (CD50) corresponded to the concentration of compound that and PMEA were equally potent in HIV-infected MT-4 cells reduced the number of viable mock-infected cells by 50%.