Vidarabine (Vira-A®): Pharmacology and clinical experience

Pbarm,.. Ther,VoL g, pp 143 171

0163-7258180i0101-0143505,00;0

C~ Pergamon Press Lld 19~0. Printed in Great Britain

Specialist Subject Editor: DAVID SHUGAR

VIDARABINE (VIRA-A®): PHARMACOLOGY AND CLINICAL EXPERIENCE ROBERT A. BUCHANAN

Director of Clinical Research

and FRANK

HESS

Clinical Editor Warner-Lambert/Park~Davis Pharmaceutical Research Division, Ann Arbor, Michigan, USA

1. INTRODUCTION For the first time in the history of antiviral therapy there exists a drug that can be used to treat a systemic viral infection. Of course, the history of antiviral therapy, in contrast to that of antiviral prophylaxis, is not very long. It was only about 15 years ago that idoxuridine was introduced for the treatment of herpes simplex keratitis (Kaufman et al., 1962). Before that there were no effective drugs to treat viral infections. Now vidarabine (Vira-A ®) (Fig. 1) is available and is effective not only for herpes simplex keratitis, but also for herpes simplex encephalitis. As is the case with many antiviral candidates, vidarabine was first synthesized as a potential anticancer drug (Lee et al., 1960). It is not surprising that an intimate connection exists between the study of viruses and the study of cancer and that the results obtained in one field can often be used profitably in the other. The attempt to alter or prevent the abnormal growth of cells is the same sort of enterprise as the attempt to prevent the replication of viruses. The target is the same, the mechanisms of cellular reproduction. A drug that is effective for one might well be effective for the other. Early studies revealed that vidarabine effectively inhibits the growth of certain viruses and that it does so selectively, that is, with much less effect on the growth of the host cell. These discoveries supplied the incentive for the full study of vidarabine as a chemotherapeutic antiviral drug. For the past ten years the efforts of many investigators have been lent to this study. From this work has come a drug which is effective in treating herpes simplex keratitis and which has just recently been approved by the U.S. Food and Drug Administration for the systemic treatment of herpes simplex encephalitis, which, untreated, has one of the highest mortality rates of any viral encephalitis. N

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2. PRECLINICAL STUDIES OF VIDARABINE 2.1 In vitro ACTIVITY 2.1.1. D N A Viruses Vidarabine inhibits the replication of many DNA viruses. It was first reported active against herpes simplex virus (HSV) (reducing virus yield 45 per cent at 5 #g/ml and 99 per cent at 50 pg/ml) and vaccinia virus (VV) (reducing virus yield 90 per cent at 5 #g/ml and 99 per cent at 50/~g/ml) in HeLa cell culture (Privat de Garilhe and DeRudder, 1964). Further studies showed its activity against cytomegalovirus (CMV) in human embryonic lung (WI-35) ceils (Sidwell et al., 1967) and against varicella-zoster virus (VZV) in human WI-38 cells (Schabel, 1968). These are the clinically important DNA viruses against which vidarabine is active. Vidarabine was as effective as cytosine arabinoside (ara-C) in some studies with HSV in HEp-2 cell culture; both compounds reduced plaque formation at 3.1 #g/mi concentrations (Miller et al., 1969). But in other studies comparing vidarabine with ara-C and idoxuridine (IDU) against HSV type 1 (HSV~) and HSV type 2 (HSVz), VZV, and CMV in WI-38 cell cultures, ara-C was more effective than vidarabine, which was more effective than IDU. In these studies the minimal inhibitory concentrations (MIC) [based on the inhibition of 1015 to 10 2.5 cell culture infectious doses, 50 per cent (CCIDso)] of ara-C were only about 1/20 those of vidarabine; those of vidarabine ranged from 1/6 to 4/5 those of IDU (Fiala et al., 1974). According to some reports, HSV~ and HSV2 are not equally sensitive to vidarabine and IDU. When both viruses were tested in rabbit kidney cell culture against both antiviral drugs, HSV~ was more sensitive than HSV2 (Lowry and Rawls, 1969). But this does not apply to all variants of HSV~ ; one variant forms plaques in chick cells, as does HSV2, and is not as sensitive as the HSV1 variant that does not form such plaques (Lowry and Rawls, 1969; Lowry et al., 1971). The comparative sensitivity of the two types of HSV to antiviral drugs was also reported to be affected by the type of cell culture. In primary chick embryo fibroblast cultures, HSV1 was more sensitive than HSV2; in WI-38 cell cultures, the two types were equally sensitive; and in HeLa cell cultures, HSV2 appeared to be somewhat more sensitive than HSV~ (Person et al., 1970). Marks (1974) has explored other variables that affect the in vitro susceptibility of HSV to vidarabine. He determined the MIC, the concentration completely inhibiting cytopathogenic effects (CPE), using multiples of the mean tissue culture dose-50 per cent (TCD50). In comparing the effects of tissue cultures, he found that with inocula of HSV at 100 TCDs0, the MIC for vidarabine in primary rabbit kidney cells (PRK) was 66 pg/ml; in WI 38 cell cultures it was 40 #g/ml; in rat brain (RB) cell cultures it was 36 #g/ml. The particular HSV isolate tested also affected results; in RB with inocula of 10 TCDso, one HSV isolate was susceptible to an MIC of 0.3 pg/ml, another to an MIC of 8 #g/ml. As would be expected, the MIC was positively correlated with the strength ot inoculum. In PRK cells at an inoculum of 10TCD50, the MIC of vidarabine was 40/tg/ml; at 100TCD5o, it was 66pg/ml; at 1000TCD50, it was 200#g/ml; at 2000 TCDso, it was 400 ~tg/ml. Perhaps the most important influence on the in vitro antiviral activity of vidarabine is adenosine deaminase, which rapidly metabolizes vidarabine to its much less active metabolite, hypoxanthine arabinoside (ara-Hx). To measure the susceptibility of herpesvirus to unmetabolized vidarabine, Bryson et al. (1974) designed a system using an adenosine deaminase inhibitor, pentostatin ((R)-3-(2-deoxy-fl-D-erythropentofuranosyl)-3,6,7,8tetrahydroimidazol[4,5-d][1,3]diazepin-8-ol) (Woo et al., 1974) that prevented deamination of vidarabine. When vidarabine was tested in this system, it inhibited replication of HSV at concentrations much below those reported in tests without the enzyme inhibitor. Connor et al. (1975) used this system to measure the susceptibility of VV, HSVI, HSV2, and VZV to vidarabine, ara-Hx, vidarabine plus pentostatin, and ara-Hx plus pentostatin. Various lots of vidarabine and ara-Hx were prepared, and analyzed and purified by

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liquid chromatography. Prior to addition of vidarabine, ara-Hx, and viruses, 0.025 ml of a pentostatin solution was added to the appropriate wells. The tissue culture used by Connor et al. (1975) for the VV experiments was a mycoplasma-free stable cell line, LIC-MK2, a continuous passage monkey kidney cell line. Lyophilized standard VV in a dilution containing 101"5 to 101"7 plaque forming units was used. When vidarabine and ara-Hx were compared without pentostatin, their antiviral potencies did not differ; at concentrations of 5 pg/ml, they each reduced plaque development by approximately 80 per cent. Adding pentostatin had no effect on the antiviral potency of ara-Hx, but increased that of vidarabine approximately fifty-fold. Thus vidarabine, when not deaminated, was about fifty times as potent as ara-Hx. For the experiments with HSV, Connor et al. (1975) used human skin diploid fibroblastic cells. They studied 10 HSV1 and 10 HSV2 strains. Viral stocks were inoculated at dilutions giving 3(~50 plaques per well. The presence of pentostatin (0.8/~g/ml) increased the antiviral potency of vidarabine against both HSV1 and HSV2: without enzyme inhibitor, complete prevention of plaque development required 10 #g/ml of vidarabine; with inhibitor it required only 3 #g/ml. Ara-Hx had significant antiviral effect only at concentrations of 160/~g/ml, about fifty times the concentration of vidarabine with pentostatin. The VZV isolates used by Connor et al. (1975) were also cultured in human skin diploid fibroblastic tissue cell cultures. The inoculates of VZV used contained 30-60 plaque forming units. Results with VZV were similar to those with VV and HSV. When pentostatin was not present, the concentration of vidarabine necessary to inhibit 90 per cent of the plaque formation was 10/~g/ml. When pentostatin was present 90 per cent inhibition occurred at 0.5/~g/ml, 100 per cent inhibition at 1.0/~g/ml. In a different set of experiments, these investigators found that vidarabine with pentostatin completely inhibited plaque formation of VZV at 2/tg/ml; ara-Hx did so at 100/~g/ml (Bryson and Connor, 1976). Thus the potency ratio of nondeaminated vidarabine to ara-Hx in VZV cultures was also 50:1. The effects of pentostatin in these cultures clearly indicate that in vitro measurements of the antiviral activity of vidarabine are affected by any adenosine deaminase in the cell culture. Without an adenosine deaminase inhibitor to prevent the conversion of vidarabine to ara-Hx, the appraisal of vidarabine activity is only approximate. The results also have clinical implications; if the deamination of vidarabine could be prevented in treated viral infections, the dose might be profoundly reduced. Vidarabine showed in vitro effectiveness against some clinically important DNA viruses. It was also tested for activity against RNA viruses. 2.1.2. R N A Viruses Vidarabine has little activity in vitro against the non-oncogenic RNA viruses (e.g. polio virus, respiratory syncytial virus, influenza virus). No in vitro activity was observed against Coxsackie virus B1 as measured by plaque reduction in HEp-2 cell culture, nor against echovirus type 9, or rhinovirus 1B in primary cynomolgus monkey kidney cell culture (Miller et al., 1969). Nor has activity been observed against human influenza virus (Az/Aichi/2/68 and B/Lee/40) as measured by hemagglutinin titers in primary chick embryo fibroblast culture (Huffman et al., 1973). Vidarabine was also ineffective when tested against parainfluenza virus type 3 (Miller et al., 1969) and against measles virus in HEp-2 cell culture (Schabel, 1968). At 200 #g/ml vidarabine was reported to show in vitro activity against 3100 plaque forming units of rabies virus in BHK 21/135 cell culture by Janis and Harmon (1974), but the investigators found no in vivo effect in street virusinfected mice. Vidarabine has potentially important activity against several RNA tumor viruses. Miller et al. (1969), reported that vidarabine inhibited the growth of Rous sarcoma virus (RSV) in primary chick embryo fibroblasts. This activity against RSV was expected since the virus requires DNA for replication (Bader, 1964, 1966a, b). RNA tumor viruses generJ.P.T.

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ally replicate through a DNA intermediate made possible by an RNA-directed DNA polymerase reaction (Baltimore, 1970; Temin and Mizutani, 1970). Studies of vidarabine activity against other RNA oncornaviruses, conducted because of this known DNA dependence, have been carried out. Shannon et al. (1974) showed that vidarabine inhibits Gross leukemia virus as measured by plaque reduction in rat XC sarcoma cell cultures. Significant reduction occurred at a vidarabine concentration of 0.4 #g/ml, complete inhibition at 40 #g/ml. At the higher concentration, vidarabine had some cytotoxic effects, reducing the number of viable Swiss mouse embryo cells upon 4 days treatment at 37°C. On the basis of the foregoing studies, it does not appear that vidarabine will ever be an effective drug against serious clinical RNA virus infections. 2.2. MECHANISM OF ANTIVIRAL ACTIVITY

The mechanism of action of vidarabine has not yet been fully elucidated. It does not appear to inactivate virus by direct contact; when concentrations of vidarabine up to 2000/~g/ml were combined with 2 × l04 plaque forming units of HSV, and dilutions absorbed on human epidermoid carcinoma cells, the plaque forming ability of the virus was not reduced when the dilutions were incubated for 3 days at 37°C (Miller et al., 1969). Nor does vidarabine appear to prevent the virus from penetrating the cell; when the drug was added to a human epidermoid carcinoma cell culture before virus and then removed, subsequently added HSV produced plaque at virtually the same rate as it did in untreated cultures, indicating that vidarabine did not alter the cells to prevent viral attachment or penetration (Miller et al., 1969). Circumstantial evidence indicates that vidarabine acts by inhibiting viral DNA synthesis. In cell culture studies with HSV1, viral replication was maximally inhibited when vidarabine was added to the culture up to 2 hr after the virus; if vidarabine was not added until 4 hr after the virus, viral replication was inhibited less (Shannon, 1975). These findings indicate that vidarabine affects some event in HSV replication that occurs between 2 and 4hr after virus is added to the culture; this time just precedes, and corresponds with, the beginning of viral DNA synthesis. Shannon (1975), in discussing this subject, said: 'From the data currently available, the three most likely explanations for the selective antiviral activity of ara-A (vidarabine) are that it is metabolized to ara-AMP or to the nucleoside triphosphate (ara-ATP), which causes either (1) the preferential inhibition of viral DNA polymerase, (2) the inhibition of virus-induced ribonucleotide reductase, or (3) the inhibition of some other virus-specific enzyme on the pathway of DNA synthesis that is more sensitive to the inhibitor than is the corresponding host-cell enzyme.' Several studies have been reported indicating that vidarabine is phosphorylated intracellularly to form ara-ATP. In exponentially growing mouse fibroblasts in suspension culture, an 0.1 mM concentration of vidarabine-3H resulted in a concentration of cellular ara-ATP calculated to be 2.0 x 10-SM after being incubated for 4 hr (Plunkett and Cohen, 1975). In cultures of human epidermoid carcinoma cells (KB cells) (containing 1 ~g/ml coformycin to prevent the deamination of vidarabine), both uninfected and infected with 10 PFU/cell of HSV1, vidarabine-3H exposure for 1 hr resulted in the formation of 3H-ara-ATP (but larger amounts of 3H-ATP), more in the infected cells than in the uninfected cells (Schwartz et al., 1974). In another study in KB cell cultures, which contained 3.5 x 10-6 M coformycin, both uninfected cells and cells infected with 3 PFU/ cell of HSV1 exposed to selected concentrations of vidarabine-3H showed 3H-ara-ATP. The amount of ara-ATP in the cells increased up to 12 hr of exposure and achieved levels as high as 1.5 x 10 .4 M (Schwartz et al., 1975). That the antiviral activity results from the ara-ATP rather than from vidarabine itself is suggested by the results of Bennett et al. (1975). They reported that DNA polymerases

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from HEp-2 cells, both uninfected and infected with HSV at 20 CCIDso/cell, were not inhibited by vidarabine or ara-Hx. In both the uninfected and infected cells, however, ara-ATP, at concentrations of 1 × 10 -5 M and 5 × 10 -5 M, gave a concentration-related inhibition of DNA synthesis. Further evidence that the antiviral activity of vidarabine results from its metabolite, ara-ATP, was given by Schwartz et al. (1975). In uninfected and HSV-infected KB cells exposed to various concentrations of vidarabine-3H, the amount of ara-ATP formed increased up to 12 hr. The rate of DNA synthesis declined with increasing levels of ara-ATP. When vidarabine was removed after a 6-hr exposure, the ara-ATP levels decreased and DNA synthesis recovered. The selective effect of vidarabine (presumably as ara-ATP) on viral DNA synthesis has also been demonstrated. In KB cell cultures, uninfected and infected with 10 PFU/cell of HSV1, to which coformycin (1 #g/ml) had been added, DNA synthesis with 1.0 × 10 -6 M of vidarabine was inhibited 20 per cent in uninfected cells and 25 per cent in infected cells. This inhibition increased to 85 per cent in uninfected cells and 90 per cent in infected cells at 5.0 x 10-5 M of vidarabine. In virus-infected cells without drug, 50 per cent of the total DNA synthesized was viral. In the presence of the, 5.0 × 10- 5 M concentration of vidarabine, however, only 25 per cent of the limited DNA synthesized was viral, indicating that the viral DNA synthesis was inhibited to a greater extent than the cellular DNA synthesis (Schwartz et al., 1974). In similar studies with KB cells, cultures containing 3.5 × 10 -6 M of coformycin and uninfected cells or cells infected with 3 PFU/ cell of HSV1, a similar selective activity of vidarabine was demonstrated. When the HSV infected cells had been exposed to 1.0 × 10-6M of vidarabine and the labeled DNA separated by isopycnic centrifugation, it was found that viral DNA synthesis had been inhibited by 58 per cent, but cellular DNA synthesis by only 20 per cent (Schwartz et al., 1975). Sidwell et al. (1970) reported that vidarabine did not appear to act as a competitive inhibitor of adenosine, deoxyadenosine, thymidine, guanosine, or deoxyguanosine, since addition of these substances did not reduce vidarabine's ability to inhibit the cytopathic effects of pseudorabies and rabbit myxoma viruses in rabbit kidney cell culture. More recently, however, Smith et al. (1976) reported that, in suspensions of KB cells infected with HSVI, 1.0 x 10-5 M of vidarabine inhibited HSV replication by 80 per cent, but this antiviral activity was reduced to 40 per cent by the concurrent addition of 5.0 x 10-5 M of deoxyadenosine (addition of adenosine to the culture had no such antagonistic effect.) These results suggest that some of the antiviral activity of vidarabine may result from a competitive inhibition of deoxyadenosine. At effective antiviral concentrations, vidarabine is not toxic for host cells, indicating it inhibits virus-coded enzymes of DNA synthesis more than the corresponding enzymes of the host cell. DNA polymerase activity increases in cells infected with HSV (Keir and Gold, 1963), and the enzyme appears to differ from the corresponding host cell enzyme (Keir et al., 1966). Therefore, the susceptibility of this enzyme to inhibition by vidarabine was tested. Although polymerase activity in herpesvirus-infected cells was inhibited by vidarabine, so was that in noninfected cells, indicating that inhibition of polymerase activity alone does not account for the selective activity of vidarabine (Shannon, 1975). It seems likely that other enzymes involved in viral DNA synthesis are also selectively inhibited by vidarabine. Recently, Champney et al. (1978) carried out experiments that may bear upon vidarabine's mechanism of action. They reported that vidarabine and ara-Hx had a synergistic effect. In one study in a rabbit kidney cell (RK-13) culture infected with HSV1, they found that when vidarabine at a concentration of 0.13 of its MIC and ara-Hx at a concentration of 0.25 of its MIC were combined, they produced antiviral activity equivalent to the MIC of either drug alone. By their method, they add these fractions of the MICs (0.13 + 0.25 = 0.38) and subtract the sum from 1.0 to arrive at a figure of 0.62, which is identified as the sparing effect. Any sparing effect equal to or greater than 0.5 is considered synergism. Six repetitions of the experiment gave a mean sparing effect of 0.54. The investigators do not yet have .any explanation for their findings.

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On the basis of the evidence to date, the mechanism of vidarabine's antiviral activity cannot be fully explained. But some distinctions can be made between more and less likely explanations. It does not appear that vidarabine inactivates virus by direct contact or prevents virus from penetrating the cell. Nor does it appear that competitive inhibition is prominently involved. Vidarabine does selectively inhibit viral DNA synthesis, and it appears that the active metabolite is ara-ATP. By what means vidarabine inhibits DNA synthesis is unclear; it may interfere with DNA polymerase activity, but this does not appear to be the entire mechanism. 3. E F F E C T S O F VIDARABINE O N I M M U N E M E C H A N I S M S Effective antiviral therapy with a virostatic agent requires that the host have at least a partially intact immune system. Therefore, the effects of any antiviral drug on immune mechanisms must be determined. Studies were done of vidarabine's effects on both the cellular and humoral immune mechanisms. 3.1. CELLULAR IMMUNE MECHANISMS The effects of vidarabine on cellular immune mechanisms were studied through evaluation of its effects on lymphocyte blastogenic response to mitogens, and on cell cultures infected with HSV1, HSV2, or VZV (Steele et al., 1975). A blastogenic index was calculated from the 3H-thymidine uptake for lymphocytes incubated with infected cells (or those incubated with mitogens), divided by the uptake following incubation with uninfected cells (or with medium alone). Vidarabine caused no significant effect on the blastogenic activation resulting from the use of the mitogens phytohemagglutinin, pokeweed, or concanavalin A. Neither a concentration of 3/tg/ml of vidarabine, nor an equivalent amount of phosphate buffer saline (the vehicle of the vidarabine preparation), altered the results of a 72-hr incubation. When cell cultures infected with HSV1 HSV2, or VZV were used in place of those incubated with mitogens, the results were similar, i.e. there was no significant difference in the blastogenic index whether the infected cell culture was used with vidarabine, placebo, or control. In addition to the in vitro studies, studies were conducted with blood samples taken from patients infected with HSV1, HSV2, or VZV and treated with vidarabine. Blastogenic indices were calculated with these blood samples when incubated with phytohemagglutinin, pokeweed, and concanavalin A, as well as with the infected blood samples alone. No significant difference was observed in the blastogenic index obtained before, during, or after treatment with vidarabine. In the same series of studies, lymphocyte cytotoxicity was also assayed. The target cells used were those infected with HSV1, HSV2, or VZV and labeled with 51Cr ' The quantitative release of 51Cr from the target cells was used as an index of lymphocyte-mediated cytotoxicity against the infected cells. Using this technique, no significant difference was found between the cultures with vidarabine and those without vidarabine in the infected cells. Nor were there any significant differences between the cytotoxicity found before, during, or after treatment with vidarabine among the infected patients. 3.2 EFFECT OF VIDARABINE ON HUMORAL IMMUNE RESPONSE The effect of vidarabine on humoral immune response was studied in rabbits infected with HSV (Zam et al., 1974). The rabbits were treated daily for 14 days with subcutaneous injections of vidarabine. The results were compared with those in a similarly infected group not treated with vidarabine. Antibody formation, measured by neutralizing antibody titers, was delayed in the treated group but reached levels comparable with the untreated group (1:32) by the second week. The results indicate that vidarabine has no immunosuppressive effect on humoral immune mechanisms.

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Not only does vidarabine not adversely affect the humoral immune response, but there actually appears to be a synergistic antiviral effect of vidarabine and humoral antibodies. Cho et al. (1976) reported that when human immune globulin (HIG) and vidarabine were administered concurrently s.c. to 3-week-old mice with experimental HSV encephalitis, the survival rate was increased to more than that when either of the agents was administered separately. The 20-day mortality rate for untreated control mice was 100 per cent, for mice treated with HIG alone it was 75 per cent, for mice treated with vidarabine alone it was 70 per cent, and for mice treated with vidarabine and HIG it was 25 per cent. They found that concurrent administration of vidarabine and rabbit immune globulin had a similar synergistic effect. 4. ANIMAL STUDIES OF ANTIVIRAL ACTIVITY In animal studies, vidarabine showed the same broad spectrum of anti-DNA virus activity as in vitro. It was active against HSV and VV in ophthalmic, cutaneous, and central nervous system infections. And, where the route was appropriate, it was effective intravenously, intraperitoneally, subcutaneously, subconjunctivally, intracerebrally, and topically. 4.1. OPHTHALMIC USE The earliest animal studies reported tested vidarabine against HSV ocular infections. In one of the first studies (Johnson and Jervey, 1964), the drug did not appear to be effective. These investigators reported that topical application of a 0.06 per cent saturated solution of vidarabine was ineffective in treating experimental ocular herpes infection in rabbits, but similar application of a 0.1 per cent solution of IDU was effective. The investigators thought the failure of vidarabine was due to its poor solubility; more likely it was due to the low concentration of vidarabine employed. Sidwell et al. (1969) observed significant therapeutic activity of topically applied vidarabine in an HSV keratitis in hamsters. Drug was applied directly to the infected corneas in ointments of varying concentrations. Twice daily administration for 15 days produced dose-related significant prevention of clinical disease and increase in survivor rates. Further, it prolonged survival time among animals that ultimately died of encephalitis. In these studies, the investigators found that a 0.3 per cent concentration of vidarabine was effective, indicating a therapeutic index (highest nontoxic dose to lowest effective dose) greater than sixty. Under the same conditions, IDU and ara-C had therapeutic indices of only 2-4. Miyai et al. (1974) also demonstrated vidarabine's topical efficacy in treating HSV keratitis in hamsters. Other studies compared vidarabine with IDU, both applied topically, for the treatment of HSV keratitis. Kaufman et al. (1970) found that 3.3 per cent vidarabine and 0.5 per cent IDU showed equivalent therapeutic effects in superficial HSV ocular infections in rabbits. As compared with untreated controls, treated animals had significantly higher survival rates, and their lesions were less severe. Okumoto et al. (1970) obtained therapeutically equivalent results with the topical application of 5 per cent vidarabine and 0.1 per cent IDU for HSV ocular infections in rabbits, as did Nesburn et al. (1970), using a 10 per cent suspension of vidarabine and a 0.5 per cent suspension of IDU, topically applied. These investigators also observed less corneal clouding and edema (conditions that indicate stromal involvement) among vidarabine-treated animals, and the dendritic lesions in the vidarabine-treated eyes generally remained dendritic until healing occurred, whereas the lesions in IDU-treated eyes tended to progress to geographic lesions before recovery began. In none of the three studies (Kaufman et al., 1970; Okumoto et al., 1970; Nesburn et al., 1970) did either vidarabine or IDU reduce the incidence of recurrences. The most likely reason these recurrences are not prevented is that the virus remains in the geniculate ganglion, protected from the topically applied antiviral drug. From there the virus can reinvade the cornea.

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In rabbits with corneal HSV infections, vidarabine, as a 5 per cent suspension, by subconjunctival administration and at 250mg/kg by s.c. administration showed some therapeutic effect (Kaufman et al., 1970). Vidarabine in oral doses up to 500 mg/kg had no therapeutic effect in HSV corneal infections in hamsters (Sloan, 1975), probably because it is rapidly deaminated in the gut. Although vaccinial keratitis is not common among humans and does not usually recur, it can be serious if it occurs as a complication of vaccination. In rabbit studies, in which both drugs were applied topically, vidarabine (5 per cent) reduced the severity of vaccinial keratitis lesions significantly more than did IDU (0.1 per cent) (Hyndiuk et al., 1976). 4.2. USE IN CUTANEOUS VIRAL INFECTIONS

Vidarabine was also evaluated in animals for the treatment of cutaneous HSV diseases. Unfortunately, the disease in animals does not closely follow that in man. Lesions in animals are usually atypical, inconsistent, and variably persistent. Nevertheless, some relevant knowledge can be gained by animal tests. Lieberman et al. (1973) reported that a zosteriform cutaneous infection induced in hairless mice by herpesvirus usually resulted in neurological involvement and death if left untreated. Tested in this animal model, vidarabine (125 mg/kg) given intradermally at the time of inoculation and twice daily for 14 days, reduced mortality and the severity of cutaneous lesions. In this study, ara-C (12.5 mg/kg), administered by the same route and for the same duration, neither inhibited lesion development nor prevented fatal sequelae. Klein et al. (1974), also using the hairless mouse with a cutaneous infection, found that vidarabine at 300 mg/kg per day given i.p. from the time of viral inoculation and for 11 days (total dose: 3300 mg/kg) resulted in mean lesion scores 25 per cent lower than those in controls and survival of 90 per cent of the animals (40 per cent survival in controls). Both differences were statistically significant (P < 0.03). At 300 mg/kg given only every 48 hr through day 10 (total dose: 1800 mg/kg), lesion scores and survival rates did not differ significantly from those of controls. At 600 mg/kg per day from 24 hr after viral inoculation and for 6 days (total dose: 3600 mg/kg), mean lesion scores were lowest and all the animals survived (20 per cent survival in controls), both differences highly significant (P < 0.001). They concluded that the total dose of vidarabine administered was more important in determining outcome than was the route of administration or the schedule of doses. IDU at doses of 100 mg/kg per day administered i.p. from the day of, or from 24 hr after, viral inoculation and continued to day twelve resulted in significantly lower mean lesion scores and higher survival rates than found in controls (P < 0.01), but administration of the dose beginning on the third day after inoculation had no therapeutic effect. With both vidarabine and IDU, the investigators observed a greater antiviral effect when effective doses of the drugs were begun 24 hr after viral inoculation. The investigators considered the results with vidarabine somewhat superior to those with IDU. Topical application twice daily for 4 days of 3.3 per cent vidarabine in dimethylsulfoxide (DMSO) and 30 per cent vidarabine in Jelen ® had therapeutic effects in hairless mice inoculated intradermally with HSV2; lesion size and severity were lower and per cent survival higher in treated animals than in controls (Sloan, 1975). Although not as successful as those reported by Lieberman et al. (1973) and Klein et al. (1974) with parenteral administration, the Sloan results do indicate that topical application can be effective.

4.3. USE IN NEUROLOGICAL INFECTIONS Untreated HSV encephalitis in humans is fatal in 70-80 per cent of proven cases (Lerner et al., 1976; Olson et al., 1967). Vidarabine has been studied extensively in animals with intracerebral HSV infections.

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Early results were summarized by Schabel (1968). In one of the studies, mice were inoculated intracerebrally with thirty-two times the LO50 of HSV. Vidarabine at 250 to 1000 mg/kg per day, administered i.p., produced significantly more 21-day survivors and significantly longer survival times among the mice that died than occurred among the untreated control mice. In the same study, IDU, at 500-2000 mg/kg per day, failed to provide any protection. Schmidt-Ruppin (1971) reported that single 500 mg/kg i.p. injections of vidarabine also provided protection against intracerebral HSV (LD90) infections in mice; among the animals receiving vidarabine, 50 per cent survived; among untreated controls, only 10 per cent survived. The effectiveness of vidarabine against intracerebral VV infections was also tested. Sidwell et al. (1968) reported significant antiviral activity by i.p. administered vidarabine in mice with VV (10 and 32 LO50 ) intracerebral infections; 31-250 mg/kg per day for 9 days after inoculation produced 75 per cent survival at the lower infective dose and 50 per cent survival at the higher. In studies by Dixon et al. (1969), vidarabine was compared with N-methylisatin-3-thiosemicarbazone in mice infected intracerebrally with VV (10 and 32 LD50 ). Both drugs, given i.p. once a day for 9 days at 1/2 the LOlo, provided significantly greater survival rates than were found among controls. When survivors were rechallenged with 1000 LOso VV 21 days after the first inoculation, 91 per cent of the rechallenged animals survived. The survival of the challenged animals indicated that vidarabine had been therapeutic rather than simply prophylactic; its effects were obtained after the infection had been present long enough to stimulate immunity in the animal. This therapeutic activity has been observed by Sidwell et al. 0968) and Sloan et al. (1969). In oral doses of 1000 mg/kg, vidarabine had significant therapeutic effect in mice with intracerebral infections of HSV or VV (Dixon et al., 1969; Sloan et al., 1969). Since neither oral nor i.p. administration of vidarabine, in the large doses required, would be practical for treating viral encephalitis in man, s.c. administration was also tested in mice intracerebrally-inoculated with 32 LO5o HSV. Sloan et al. (1969) found that 2000 and 3000 mg/kg administered s.c. in four divided doses within 1 day resulted in 25 per cent survival rates, but when administered every hr for 8 hr, or in two doses 4 hr apart, 3000 mg/kg resulted in a higher per cent of survivors than did 2000 mg/kg. Miller et al. (1970) also found that s.c. vidarabine offered protection to mice which had received intracerebral inoculations of 32 LDs0 of HSV. They further observed that survival rate increases varied inversely with the extent of the delay in treatment. When 1000 mg/kg of drug was given three times per day 24 hr after inoculation, 57 per cent of the mice survived; when given 48 hr after inoculation, 41 per cent survived; when given 72 hr after inoculation, 22 per cent survived; and when given 96 hr after inoculation ll per cent survived. For each of these schedules, however, the survival rate was significantly greater than for untreated controls. When these investigators (Miller et al., 1970) used brain tissue from the inoculated mice to prepare a virus seed to challenge other mice intracerebrally, they found the HSV was still susceptible to the action of vidarabine. This provided in vivo evidence that HSV in one passage through the mouse did not develop resistance to vidarabine. 4.4 COMPARISONOF SENSITIVITYOF INTRACEREBRALLYINOCULATED HSV 1 AND HSV 2 TO VIDARABINE The effectiveness of vidarabine in HSV1 and HSV 2 intracerebral infections was compared in mice. Although reports of in vitro studies indicated that H S V 2 was less susceptible to vidarabine than HSV1 (Lowry and Rawls, 1969), Sloan et al. (1973) found that in some circumstances vidarabine was equally effective against both types. When vidarabine, in buffered aqueous solution, was given s.c. in three divided doses for one day begining 24 hr after inoculation at a total dose of 3000 mg/kg, it provided equivalent significant protection (increased survival rate and survival time compared with controls) against inoculations of 32 LDso of either HSV1 o r H S V 2. The authors did find that

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increasing the size of the infecting HSVz inoculum to 320 or 1000 LDso decreased the survival rate, but the mean survival times were still significantly greater than those among the controls. Generally survivors were resistant to rechallenge with 1000 LDs0 doses of homologous virus, indicating that vidarabine had been effective after the infection had been sumciently established to induce immunity. Based on the whole study, the authors concluded that vidarabine is as effective against the genital type HSV (type 2) as against the oral type HSV (type 1). 5. TOXICOLOGY Early toxicological information about vidarabine was obtained in studies meant to elucidate the cytotoxic and biochemical actions of the drug. Brink and LePage (1964) found that i.p. administration to mice of 240 mg/kg per day of vidarabine for 6 days caused moderate weight loss but did not alter circulating elements of the blood. Vidarabine was clearly a potent DNA antagonist but did not produce the usual toxic signs. The toxicity of vidarabine was studied most systematically in the research laboratories of Parke-Davis. These studies, reported by Kurtz et al. (1969) and Kurtz (1975), will be summarized. The earliest toxicity results of vidarabine were those found in control animals in pharmacology studies which indicated that animals tolerated single parenteral doses in excess of 1000 mg/kg. Following these preliminary observations, formal acute toxicity and irritation studies were conducted. The oral LD50 of vidarabine was greater than 5 g/kg in both rats and mice. Installation of vidarabine in the eyes of rabbits for 7-28 days produced no more irritation than did vehicle alone and, when vidarabine was applied to rabbit skin for the same period, few signs of irritation were observed. Autopsy and histological study of the treated animals showed no signs of toxicity. Constant dose oral tolerance tests of vidarabine were conducted in mice. Doses ranging from 170 to 3000 mg/kg per day were administered in the diet. At the end of two weeks, the highest dose was reduced to 1400 mg/kg per day and all doses were continued for another 2 weeks. The high dose was reduced because the animals on it lost weight and became debilitated. The animals at 170 mg/kg remained clinically normal for the entire 28 days. Those at the higher doses had depressed weight gain. Clinical laboratory tests indicated a tendency toward neutrophilia and lymphopenia and variable doserelated malfunction of the liver. Histopathological examination showed poorly defined liver cell abnormality and variable gonadal atrophy among the more severely affected animals. When another 28-day study was conducted with mice receiving 1600 mg/kg per day orally and then 6 weeks of standard diet, gonadal recovery was complete, but enlarged liver cells were observed. Further study showed that these enlarged cells were only found in mice and rats and only after large single i.p. doses or after 1 month of daily oral toxic doses in mice or 5 months of regular i.m. injection in rats and mice. By 1 year after withdrawal of drug, the livers were normal. Twenty-eight-day constant dose parenteral studies of vidarabine were conducted in rats, dogs, and rhesus monkeys. Among the rats given i.m. doses of a 20 per cent suspension up to 15 mg/kg per day, some suppression of weight gain and tenderness at the site of injection were observed at the highest dose. No clinical laboratory abnormalities or enlarged liver cells were observed. Among dogs given i.m. doses up to 50 mg/kg per day, the only evidence in the living dogs or at autopsy of any toxicity was inflammation and necrosis at the sites of injection. Although i.m. vidarabine was well tolerated systemically, the local findings indicate poor tolerance by this route, perhaps due to vidarabine's poor solubility. The rhesus monkeys proved to be the most sensitive animals tested in the constant dose parenteral studies. At 15 mg/kg per day of vidarabine, administered i.m., the animals completed the 28-day dosing period with nothing more than tenderness at the injection sites. But at 25, 37.5, and 75 mg/kg per day, animals had tremors, weakness, incoordination, and somnolence, and convulsions could be triggered easily in some of them. Sensi-

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tivity tended to be dose-related. Although such pronounced clinical reactions were observed, the hematologic and blood biochemical changes were only those reflecting inflammation and muscle necrosis. Gross and microscopic study at autopsy showed no anatomic changes to explain the clinical reactions. Shannon (cited in Kurtz, 1975) reported that New World owl monkeys tolerated daily doses of 300 mg/kg of vidarabine for 1 month. A muscle irritation study was done in rabbits. They received 0.5 ml of a 20 per cent suspension of vidarabine i.m. and animals were sacrificed 1, 7, 14, and 28 days after administration and the injection sites examined. Unabsorbed crystals of vidarabine were found in sections taken up to 7 days. Moderate necrosis and acute inflammation were present early, and at 7 and 14 days granulomatous changes with foreign body and regenerating muscle giant cells were observed. The lesions had usually resolved by day 28. In the dog and monkey i.m. studies described above, scar, regenerating muscle fibers, and foreign body cells remained longer than 1 month. Therefore i.m. administration would not be appropriate. In rhesus monkeys i.v. administration of 15 mg/kg per day for 28 days was accomplished with no clinical signs of toxicity. Clinical laboratory and pathologic examinations showed no abnormalities attributable to treatment with vidarabine. Vidarabine, administered i.m., had teratogenic effects in rats, rabbits, and rhesus monkeys. Rabbits were most sensitive; doses over 3-5-mg/kg, given each day during the period of organogenesis in the fetus, produced teratogenic effects, including severely deformed offspring. Of five pregnant rhesus monkeys given 15 mg/kg per day i.m. during the period of organogenesis in the fetus, three delivered completely normal offspring, one aborted early, and one had a macerated fetus. Teratogenesis was also observed in rabbits when a 10 per cent vidarabine ointment was applied topically to more than 5 per cent of the body surface of pregnant females during the period of organogenesis in the fetus. However, intravaginal instillation of 10 per cent vidarabine in gel in pregnant rats from day 15 of gestation to the day of parturition produced no effects on offspring at birth or up through weaning. The mutagenic potential of vidarabine was tested by both the dominant lethal test and by the host-mediated assay. In the dominant lethal test, male animals were treated with vidarabine, placebo as negative control, or triethylene melamine as positive control. They were then mated with untreated females to see if defective chromosomes were formed which resulted in aborted fetal implantations. In the host-mediated assay (similar to the Ames test), mice were treated with vidarabine, placebo as negative control, or N-methylN'-nitro-N-nitrosoguanidine as positive control; the mice were injected with strains of Salmonella typhimurium with particularly labile genetic traits associated with nutritional requirements; the bacteria were recovered and plated on media of various nutrient characteristics to see if any genetic changes had occurred that were demonstrable by growth characteristics. In both of these assays, the positive controls were mutagenically active, but vidarabine was not. Wilkerson et al. (1973) studied blood from two human patients who had received vidarabine i.v. (12 hr/day) for severe viral infections. In vitro lymphocyte preparations showed a significant increase in chromosome breaks and gaps. Samples taken within 30 min of starting therapy showed an increased incidence (13 per cent vs 3 per cent) of metaphase plates having one or more chromosome breaks. Between 18 and 24 hr, the peak incidence of 25 per cent occurred. Gradually the incidence declined during the rest of treatment, but 4 weeks after treatment was stopped, the incidence was still 9-10 per cent. Kurtz (1975) conducted tumorigenesis studies with mice and rats given 30 or 50 mg/kg vidarabine three times a week for 5 months. The study was terminated at 24 months. By termination, almost all control and treated rats had developed tumors and the incidence, types, and latency periods were similar. Among mice, the incidence and types of tumors were also similar between controls and treated animals. These studies are presently being repeated using the National Cancer Institute guidelines. Subacute toxicity studies are

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performed at different dose levels to determine a well-tolerated dose. Then this dose i~ administered for 21-24 months. Fishaut et al. (1974) compared vidarabine, ara-Hx, and ara-C for their effects on th~ postnatal development of the rat, paying particular attention to the cerebellum. At i.p doses up to 50 mg/kg given on days 2, 3, 4, and 5 of life, vidarabine had no adverse effect,. on survival, weight gain, vigor, or developmental motor behavior. No effects wer~ observed grossly or histologically on cerebellar development. Ara-Hx at 20 mg/kg als~ had no effects on rat development. Ara-C at 3 mg/kg caused retarded growth and disor ganization of the cerebellar cytoarchitecture. But repair occurred so that mature animal: had no behavioral or developmental abnormalities. At doses of 5 mg/kg or more, how ever, ara-C caused profound disruption of cerebellar cytoarchitecture, and, in survivin1 adult animals, gross neurologic abnormalities and growth retardation. The results sug gest that vidarabine, but not ara-C, should be acceptable for use in newborns.

6. METABOLIC DISPOSITION Extensive metabolic studies were conducted using vidarabine tagged with tritium (3H' These have been reported by Glazko et al. (1975), whose report serves as the basis for thq following summary. In the dog, i.v. administration of 10 mg/kg of vidarabine produced peak plasma level (approx. 10 #g/ml) of nonvolatile 3 H (NV-3H) (total drug) within 5 min and the plasm', half-life was 30 min. The total plasma 3H (T-all), as opposed to the NV-3H, remaine~ high over a 5-day period, however, indicating rapid incorporation of 3H into body watel NV-3H levels in the erythrocytes were close to those in the plasma initially, fell fi parallel with those in the plasma, then rose slowly for the remainder of the experimenl Thin layer chromatographic examination of the plasma and erythrocytes showed that th major metabolite was ara-Hx. Following i.m. administration of 37.5 mg/kg of vidarabin to the dog, the peak plasma level of NV-aH (approx. 0.5 pg/ml) was lower than tha following i.v. administration and the peak appeared several hours later. The T - 3 I " reached a plateau in about 3 days indicating slow absorption of the drug from th, injection site, due to the low solubility. The erythrocyte levels of NV-3H rose slowly, h the dog, urinary excretion accounted for about 20 per cent of the administered 3H mostly in the form of NV-3H, whether vidarabine was administered i.v. or i.m. Th maximum excretion rate occurred within 8 hr after i.v. administration, but 2448 hr afte i.m. administration. In the rhesus monkey the differences between i.v. and i.m. administration of vidarabin were similar to those in the dog. After the i.v. administration of 4 mg/kg, the peak plasm, level (5 pg/ml)of NV-3H occurred in about 15 min, the half-life was 2-4 hr, the T-3H fel with the NV-3H for 4-8 hr then remained constant, the erythrocyte NV-aH level followe~ that of the plasma level closely, and the major metabolite was ara-Hx, which was identi fled within 5 min of administration by high pressure liquid chromatography. After i.rr administration of 15 mg/kg, the peak plasma level of 1 to 2 #g/ml occurred in about 4 hi the half-life was in the range of 24-72 hr, the elevated T-all lasted for about 8 days, anq the erythrocyte NV-aH level followed that of the plasma closely. The main differences in metabolism between the dog and the rhesus monkey were th~ the half-life was shorter in the dog (30 min vs. 2-4 hr), the tritiated water level in th plasma of the rhesus monkey was lower, and the level of NV-aH in the erythrocytes ( the rhesus monkey was higher. In contrast to the dog, in which urinary excretio accounted for only 20 per cent of the dose, in the rhesus monkey it accounted for abot 90 per cent of the dose; about 85 per cent of the T-all was in the form of NV-3H. As i the dog, the major part of the dose had been excreted within 8 hr after i.v. administratio but somewhat later after i.m. administration (12-24 hr). Five and l0 mg/kg per day i.v. doses of vidarabine were administered to rhesus mor keys for 3 days. The plasma levels 5 min after administration on the first and last da3

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were similar, indicating that vidarabine did not accumulate in the blood with continued administration up to 3 days. In mice that received large i.m. doses of vidarabine, the largest concentration of NV-3H was found in the kidney. In rats receiving large oral doses, the largest concentration was found in the liver, where drug was first carried by portal circulation. In both species, however, and in the rhesus monkey, the largest concentrations of drug following i.v. administration were found in the liver, spleen, and kidney. Appreciable levels of radioactivity were found in the brain in all the animals, indicating that vidarabine or its metabolites pass easily into the central nervous system. 7. CLINICAL STUDIES OF VIDARABINE 7.1. PROBLEMS ENCOUNTERED Before describing the clinical applications of vidarabine, some of the problems encountered in its development may be described with profit. Nearly all of these problems will be faced whenever an antiviral drug is under development. When human trials with vidarabine began, the effects of the drug on metabolism were only poorly understood. Therefore, because of possible risk, studies could only be done in patients for whom the drug was indicated, that is, very sick patients with serious DNA viral infections. Such patients were available for study only at irregular, often inconvenient, times. Most of them were immunosuppressed with other cytotoxic drugs, which made the evaluation of vidarabine's side effects difficult. Analytical methods for the separation of the drug from these other agents had to be developed. Study of the clinical use of vidarabine was also seriously hampered by incomplete knowledge of the natural histories of viral diseases and of the limitations of antiviral therapy. Consequently, results of the clinical studies were often unexpected. For example, vidarabine was tested in immunosuppressed patients with varicella infections; most had varicella zoster, but some had chicken pox. Since the immunological systems were compromised in these patients, it was thought that this would be a severe test of vidarabine. In the patients receiving vidarabine, the skin viral cultures cleared and the patients healed. But 60 per cent of the control patients receiving placebo also had their skin cultures clear and their lesions heal. And only a small number of the control patients developed hepatitis, pneumonitis, or encephalitis. Thus, even without antiviral therapy, varicella infections in the immunosuppressed can be self-limiting. On the other hand, when vidarabine was tested in a population with HSV encephalitis, there was a significant difference in mortality between the group on placebo and the one that received vidarabine. Among the patients receiving placebo, the mortality was 70 per cent; among those receiving vidarabine it was 28 per cent. The rate on placebo is similar to that reported for the disease when it is untreated. The controlled studies of HSV encephalitis also showed that the diagnosis can only be certified by a brain biopsy. Clinical suspicion, accompanied by the expected physical and laboratory findings, typical encephalographic abnormalities, arteriography indicating inflammation, focal computerized axial tomography, and radionuclide brain scan changes are all helpful. But in this study, when the results of these tests pointed to HSV encephalitis and brain biopsy was done, the diagnosis was confirmed in only 50 per cent of the suspected cases. Clearly our knowledge of the diseases being treated was deficient. Diagnostic methods were often crude. 7.2 HUMAN PHARMACOLOGY The earliest studies of vidarabine in humans used tritium-labeled drug. This was necessary because a chromatographic method was not available at the time the data were needed. Glazko et al. (1975) reported that, following the i.v. administration of vidarabine labeled with l mCi/mg of 3H, the plasma level of NV-aH 30min after dosing was

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1-2 #g/ml, about 70 per cent of the T-3H. The NV-3H plasma half-life was 3-5 hr. T-3I as in animals, fell more slowly and remained at a relatively high level for several da3 Shortly after dosing, the erythrocytes had slightly higher levels of NV-3H than did tl plasma; the erythrocyte levels fell to a minimum about 24 hr after dosing, then ro slowly during the next 5 days. Among infants and children receiving unlabeled i.v. dos of vidarabine, the maximal plasma level was on the order of 1-2 gg/ml and the half-lJ was about 1.5-2.5 hr, shorter than in the adult. In adults receiving i.m. vidarabine, t'. maximal plasma level was about 0.24).3 ~tg/ml and the plasma half-life about 10-16t The delays and poor tolerance observed with i.m. administration in animals were al observed in humans. Therefore further study of the i.m. route was discontinued. With i.v. administration of vidarabine to humans, the maximum urinary excretion r~ of 3H occurred in the first 4 hr after dosing with mean recovery of about 45 per ce within 24 hr. The excretion was mainly of NV-3H, indicating little release of 3H. Agai as in animals, maximums were delayed after intramuscular administration. Maximu excretion rate occurred in the 4-8 hr period. Most of the recovered product was NV -3 Urinary excretion of 3H in man accounted for only 50 per cent of the dose, whereas the rhesus monkey it accounted for 90 per cent. About 95 per cent of the NV-3H excret by humans was as the metabolite ara-Hx. LePage e t al. (1973), studied the excretion of 3H-labeled vidarabine and ara-Hx human patients receiving rapid intravenous infusions (1/2-2 hr), continuous intraveno drips (24 hr), or intramuscular injections. They reported that in the two patients receivi rapid infusions, 90 per cent of the drug was recovered in the urine in 24 hr. In the t~ patients receiving continuous drips, about 35 per cent was recovered in 24 hr. In the t~ receiving i.m. injections, about 5 per cent was recovered in 24 hr. Kinkel and Buchanan (1975) studied the plasma, cerebrospinal fluid, erythrocyte, a urine levels of vidarabine and ara-Hx following i.v. infusion in seven patients. Vidarabi was administered by either i.v. drip or by a constant rate infusion pump over a 12 period. The daily dose ranged from 10-20 mg/kg and was administered over a range 4-10 days. In the collection tubes for whole blood, approximately 20/~g/ml of deamin~ inhibitor was present. This inhibited the conversion of vidarabine to ara-Hx e x vivo. T plasma was immediately separated from the erythrocytes and snap-frozen to prevent a further conversion of vidarabine to ara-Hx. The assay was conducted by high presse liquid chromatography. Standard curves were determined from known samples in appI priate blanks and were alternately assayed with the unknowns. The assay sensitivity approximately 0.1/~g/ml in plasma and cerebrospinal fluid, 0.6/~g/ml in erythrocytes, a 5 #g/ml in urine for both compounds. The levels of vidarabine appearing in the plasma were just barely above the limits assay detectability. The levels of ara-Hx, however, rose promptly during the infusi period and then declined promptly when infusion was stopped. The elimination half-li' in the patients observed were between 3 1/2 and 4 hr. The levels found on day 1 and d 5 were similar, indicating no accumulation or change in metabolism, distribution, excretion over that period. In one patient, renal function was impaired following a re~ transplant. The plasma levels of ara-Hx in this patient were much higher than in 1 patients with normal renal function. These findings indicate that vidarabine should administered with extra care to patients with impaired renal function. About one-half of the dose was eliminated through the urinary excretion of ara-t Only small amounts of vidarabine were detected. In one patient, weight 36 kg, receivin l dose of 15 mg/kg per day by i.v. infusion, the urinary excretion on the first day was 4 per cent of the dose, on the second day, 52.8 per cent of the dose, on the fifth day, 5, per cent of the dose, and on the tenth day, 41.6 per cent of the dose. The excretion rz was highest during the 12 hr following the infusion. Steady state daily excretion appeal to be attained by the third day. The average renal clearance for ara-Hx, calculated fr, the 2-hr plasma levels, and the O 4 h r urinary excretion for days 1, 3, and 10, v 38.1 ml/min. The distribution of ara-Hx between erythrocytes and plasma indicated that erythroc

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levels parallel plasma levels. The mean ratio of erythrocyte content to plasma content was 0.98. The distribution of ara-Hx between plasma and cerebrospinal fluid was variable, but in the cerebrospinal fluid it was about 1/3 of that in the plasma. This degree of penetration suggested that vidarabine would be effective in managing HSV encephalitis. The results of this study indicate that vidarabine is rapidly distributed into the tissues and is rapidly metabolized to ara-Hx. The deamination to ara-Hx probably occurs through the action of the erythrocytes. Drug accumulation was not found, since the elimination half-life is short. 7.3. OPHTHALMICSTUDIES 7.3.1. Comparison of Vidarabine with IDU for Treatin9 HSV Keratitis A double-blind multicenter controlled study was conducted to evaluate the efficacy and safety of vidarabine in treating HSV keratitis (Pavan-Langston and Buchanan, 1976). IDU was used as the control medication, since it was the only approved drug for this disease. The patients in this study presented the typical dendritic or geographic ulcers that are pathognomonic for HSV keratitis. No viral cultures were made because of technical difficulties and because the lesions are so typical. The lesions were identified and measured by Haag-Streit slit lamp examination. Patients were randomly placed in groups, one to receive a 3 per cent ointment of vidarabine, one a 0.5 per cent ointment of IDU. The vidarabine ointment was formulated in a base containing approximately two parts liquid and three parts solid petrolatum, U.S.P. Drugs were self-administered five times a day during the waking hours. The first application was made under the instruction of the participating ophthalmologist so that patients learned the proper method. Administration consisted of instillation of approximately a half-inch strip of ointment into the lower conjunctival sac. This delivered approximately 0.9 mg of vidarabine. Medication was to be continued for approximately 7-10 days after the epithelium had healed. If the ulcer had not improved, or if it was larger by day 14, or if it had not healed by day 28, the treatment was considered a failure. Treatment was considered a success when the epithelium did not become stained when fluorescein dye was applied after re-epithelialization had occurred. Drug tolerance and adverse reactions were recorded. If staining with the fluorescein dye increased or if a serious adverse reaction occurred, the double-blind administration of medication was stopped and open label administration or other treatment was begun. The results were analyzed for eighty-seven patients who had received vidarabine and for eighty-two who had received IDU. By the end of 2 weeks of treatment, the majority of patients in each treatment group had healed. Among those who had not healed by that time, most had shown improvement and were kept on medication for 2 more weeks. By the end of 4 weeks of treatment, approximately 90 per cent of the patients in each group had healed. The mean time to heal for the group that received vidarabine was 6.9 days; for the group that received IDU it was 7.2 days, virtually the same. The proportion of failures in the group that received IDU was twice that of the group that received vidarabine (18.8 per cent vs 9.5 per cent), but the difference was not statistically significant. However, the proportion of patients whose distant visual acuity improved was significantly greater (P < 0.05, chi-square test) in the group receiving vidarabine than in the group receiving IDU (66.2 per cent vs 43.5 per cent) suggesting a better quality of corneal healing among the patients receiving vidarabine. In addition, open-label studies were conducted with 146 patients. Of these, 118 (81 per cent) had healed within 4 weeks of treatment with vidarabine. Among the patients entering the open study, 115 (79 per cent) had been unsuccessfully treated with IDU for previous episodes of keratitis so they could not be included in a double-blind trial in which they might receive IDU again. In most of these failures, the disease had been refractory to IDU, but some of the patients had had allergic or toxic reactions to the drug. Of these 115 patients, 79 per cent had clearing of the cornea within 4 weeks of

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treatment with vidarabine, showing that vidarabine can be used in the IDU-refractory c IDU-allergic patient. In the double-blind studies, ten patients (10.9 per cent) receiving vidarabine and six (6. per cent) receiving IDU had adverse reactions. Among those receiving vidarabine tl~ reactions were tearing, conjunctival injection, burning, irritation, anterior uveitis, eder~ of the stroma, superficial punctate keratitis, punctal occlusion, redness, secondary glat coma, sensitivity, and trophic epithelial defect. Among those receiving IDU, the reactiot were irritation, blurred vision, corneal edema, and stinging. Many of the complain could be considered symptoms associated with the underlying disease. On the basis of the results of the double-blind studies, it appears that vidarabine is least as effective as IDU in the treatment of herpetic keratitis. The results of the opt studies show that vidarabine is indicated in cases of herpetic keratitis that have not bec successfully treated with IDU. 7.3.2. Comparison with Trifluorothymidine for Treating H S V Keratitis Clinical studies comparing vidarabine to trifluorothymidine (F3T) in the treatment, herpes simplex keratitis have also been conducted (Coster et al., 1976). Each of 1( patients presenting dendritic or ameboid ulcers was assigned to either a group to recei' 3.3 per cent vidarabine ointment or a group to receive 1 per cent FaT drops five tim daily, until the ulcers healed. The healing rate for the dendritic ulcers was 5.13 days c vidarabine and 5.75 days on F3T. The ameboid ulcers healed at a daily rate of 0.85 m on vidarabine and 1.00 mm on F3 T. Among forty-eight patients who received vidarabiJ there were four treatment failures; among fifty-four patients who received F3 T there w one treatment failure. The results suggest that FaT is also an effective agent in tl treatment of herpetic keratitis, comparable to vidarabine. 7.3.3. Intraocular Penetration To explore the possibility of using vidarabine to treat HSV infections of the stroma uveal tract, both animal and human studies were done to determine the degree to whk vidarabine penetrates the deeper structures of the eye. Vidarabine was compared wi ara-Hx and IDU. In the animal studies (Pavan-Langston et al., 1973), albino rabbits were divided in eight groups. Two groups received 3 per cent vidarabine, one in a petrolatum ointme~ the other in a water-soluble cream base; two groups received 3 per cent ara-Hx, one ir petrolatum ointment, the other in a cream base; one group received 25 mg of vidarabi subconjunctivally; one group received 100 mg of vidarabine subconjunctivally; the 1~ two groups received 0.5 per cent IDU, one in a petrolatum ointment, one in a crea base. With each animal, active medication was administered to one eye, placebo to tl other. Animals were sacrificed 1, 2, 4, 6, and 24 hr after drug application. No vidarabi: was detected in the aqueous humor from eyes of the animals receiving vidarabine. In animals, whether they received vidarabine or ara-Hx, some level of ara-Hx was fou~ higher levels in the aqueous humor from eyes to which ara-Hx had been applied. Su conjunctival injection of vidarabine also produced high levels of ara-Hx. The high~ concentrations were found in the aqueous humor taken from eyes of rabbits sacrific, 2 hr after administration. Intraocular penetration studies were also conducted in humans (Poirier et al., 197 Twenty-one patients undergoing routine cataract surgery were studied. Each patient w given 3 per cent vidarabine ointment preoperatively every 6 hr for a total of eight dos The planned intracapsular cataract surgery was done under local anesthesia using 2 I~ cent Xylocaine. The conjunctival sac was irrigated with balanced salt solution and a residual vidarabine ointment cleaned off before the anterior chamber was enter~ Aqueous humor was aspirated into a syringe and the entire contents immediately sna frozen in liquid nitrogen and stored at - 1 9 6 ° C until assayed. Aqueous samples wc obtained from twenty-five eyes of twenty-one patients. In none of the aqueous samp

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was vidarabine detected. Varying concentrations of ara-Hx from 0.02 #g/ml, the lower limit of sensitivity of the assay, to 0.28 #g/ml were detected. The results of the assays of the aqueous humor from eyes to which vidarabine had been applied in both rabbits and humans indicate that the cornea is capable of deaminating vidarabine to the hypoxanthine, although the exact site of deamination is unknown. These results indicate that topical vidarabine would not be effective for treating deeper ocular HSV infections. But they also indicate that topical application of vidarabine should not be toxic to the deeper ocular structures because only the relatively inactive ara-Hx penetrates. 7.3.4. Corneal Wound Healing As vidarabine was studied, the impression grew among various investigators that it was less toxic to the corneal epithelium than was IDU. Specifically, these investigators felt that the use of vidarabine resulted in less superficial punctate staining and epithelial edema and a lower incidence of persistent epithelial defects. This was of interest because problems of epithelial healing in chronically inflamed eyes or corneal transplants are frequently encountered and many require treatment with antiviral agents. To test this impression, a study was conducted in mature male albino rabbits (Langston et al., 1974). In each eye of the rabbits a central defect, 5-10 mm in diameter, was made by removing epithelium under slit-lamp observation. A 0.3 per cent gentamicin solution and 0.5 per cent IDU, 3 per cent vidarabine, or placebo were then instilled in the eye. The active medications were prepared in a petrolatum base; the base acted as the placebo. The antibiotic was instilled once a day and the ointment four times a day throughout the experiments. No animal received the same drug in both eyes. The fluorescein-stained epithelial defects were photographed daily. The closure of the 5-mm lesion was studied in thirty eyes, ten treated with IDU, ten with vidarabine, and ten with placebo. The closure of the 10-mm lesion was studied in eighteen eyes, six in each group. There was no significant difference in the rate of healing between the groups. By the end of 60 hr for the 5-mm lesions, and the end of 8 days for the 10-mm lesions, treatment had produced virtually complete healing. In the eyes in which the 5-mm lesions had been produced, the quality of the regenerating epithelium was evaluated with the slit lamp and histologically. A 0-4 scoring system was developed in which 0 indicated no visible abnormality under the slit lamp and 4 indicated epithelium thickened and cloudy with a rough and irregular surface. The eyes were examined daily and two independent observers, who did not know which medication was being used, scored the eyes on days 2, 4, and 7. Their observations correlated with a coefficient of 0.81. The results in the IDU treated eyes were consistently worse than those in the vidarabine and placebo treated eyes. The difference was statistically significant (P < 0.05, 2-way Analysis of Variance) on days 4 and 7 but not on day 2. Eyes from each treatment group were prepared for histological examination immediately after the animals were sacrificed. After paraffin processing, 6 mm sections were cut and stained for microscopic examination. The histologic examination showed no changes specific for any of the treatment groups, but the severity of the abnormalities present correlated with the slit lamp examination, i.e. the more severe histologic abnormalities were more frequently seen in the IDU treated group. These investigators (Langston et al., 1974) also conducted another study in rabbits, comparing the effects of vidarabine and IDU on the strength of healed stromal wounds. In anesthetized animals, a full thickness 2 mm central corneal button was trephined. When the plasmoid aqueous of the rabbit had clotted, gentamicin solution was instilled. IDU, vidarabine, and a placebo of Ringer's ointment were applied in the same manner as in the previous experiment. The ointments were not applied until the third day, by which time slit lamp observations with fluorescein dye showed that all the wounds had a firm plug of fibrin and were well covered by epithelium. Three weeks after the wounds were made, during which time they were observed daily, the fibrin plug had been replaced by scar tissue in all the wounds. The animals were sacrificed and the wound strength was

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determined. To do this a 25-gauge needle was inserted into the anterior chamber and the intraocular pressure was increased by 5 psi at 5-sec intervals until the wound ruptured. Results were obtained from twenty-four eyes and showed that IDU and ara-A treatment both resulted in weaker wounds. The difference in wound strength was statistically significant between each active drug group and the placebo group (P < 0.02 for vidarabine). The difference between the IDU and vidarabine treated groups was not significant. The corneas of the eyes were excised and inverted and the button of scar tissue was punched out with a 2 mm trephine. The buttons were assayed for hydroxyproline as a measure of collagen content. The differences in collagen content between the active drug-treated wounds and the controls were statistically significant (P < 0.01 for IDU and P < 0.05 for vidarabine). The difference between the IDU and vidarabine treated groups was not significant. The results of the studies showed that neither vidarabine nor IDU ointment in clinical concentrations retards the closure of large or small corneal epithelial wounds in rabbits. But the quality of regenerated epithelium with IDU treatment appeared to be significantly worse than that with vidarabine treatment. Both drugs decreased the strength ol healing stromal wounds to the same degree. This decrease in strength is associated with a decrease in the collagen content of the wounds, as indicated by the hydroxyproline assay. The difference found in the quality of regenerated epithelium suggests that vidarabine would be more suitable than IDU when antiviral therapy is necessary following corneal surgery. It may also explain the significantly greater incidence of improved visual acuity among keratitis patients receiving vidarabine, as compared to those receiving IDU.

7.3.5. Intravenous Vidarabine for Herpes Eye Infections Since i.v. administration of vidarabine had been successful in treating systemic HSV disease, this method of administration was tested in the treatment of deeper herpes infections of the eye. Topical treatment of these infections had not been successful for reasons already explained. Both a double-blind and an open study were conducted. The patients selected for the double-blind study (Abel et al., 1975) met these criteria: they had clinically established HSV keratouveitis (usually with disciform corneal edema) or stromal keratitis combined with anterior chamber activity; they had had no previous systemic or topical vidarabine therapy, no antiviral therapy for 7 days prior to the study, and no topical or systemic corticosteroids for 7 days prior to the study; and they had nc evidence of severe hepatic, renal, or cardiac disease. Eighteen patients entered the study eight received vidarabine and ten received placebo. Patients who received active dru~ received 20 mg/kg per day infused over 8-12 hr for 7 days. As measured by changes ir discomfort, ciliary injection, visual acuity, corneal edema, and anterior chamber activity the results among the patients receiving vidarabine were significantly better than thos~ among the patients receiving placebo. Discomfort, injection, and anterior chamber ac. tivity, particularly, showed improvement. Visual acuity and corneal edema were im proved in 50 per cent of the patients on vidarabine but in only 10 per cent of those or placebo. In the open-label studies the dose of vidarabine was the same as in the double-blinc studies. There were twelve patients in the open-label studies, eight of whom had receivec placebo in the double-blind study. The results in this study showed the value of earl,. treatment with vidarabine. In the four patients who had not received placebo, discomfort injection, and anterior chamber activity improved in three, and visual acuity and cornea edema in two. Among the eight who had received placebo and then vidarabine, discom fort improved in four, corneal edema in three, while injection and visual acuity improve~ in only two of them. Only in anterior chamber activity did a majority of the patient show improvement. The results suggest that the delay, while the patients were in th. double-blind study on placebo, hindered the effectiveness of vidarabine. The results of this study indicate that the intravenous administration of 20 mg/kg pe day of vidarabine to patients with herpetic keratouveitis has a therapeutic effect. Thl

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results further demonstrate that the effect is greater when the drug is administered earlier in the course of the infection, as would be expected. 8. T O P I C A L A P P L I C A T I O N O F VIDARABINE FOR G E N I T A L HERPETIC I N F E C T I O N S Herpes progenitalis is one of the most widespread venereal diseases and is spreading (Yabrov, 1977). Generalized herpes of the newborn may result from direct inoculation from vulvar lesions at the time of parturition. Infected newborns have a mortality rate of over 70 per cent (Overall et al., 1975). Vidarabine was evaluated in the treatment of genital herpetic infections in two studies. One of these was conducted in fifty-five men and forty-two women who presented active disease and from whose lesions HSV was recovered (Adams et al., 1976). Each of the patients had formed new lesions within 2 days of entering the study. Patients were grouped as either having an initial, or recurrent, episode of genital herpetic infection. Patients were randomly assigned to treatment with 3 per cent vidarabine in petrolatum ointment base, treatment with petrolatum ointment placebo, or no treatment at all. At each visit one or more external lesions were cultured for HSV. If a crust was present, it was lifted and the surface under the crust was swabbed. Before the lesions were swabbed, they were cleaned of any residual vidarabine. Cultures were done in human fetal tonsil diploid fibroblasts, which were examined for CPE (cytopathic effects) after 7 and 14 days. Isolates were typed by an immunoperoxidase method. Patients were re-examined 3 and 8 days after the start of therapy and weekly thereafter until all lesions had cleared. The measures used to evaluate therapy were the persistence of pain, the number and type of herpetic lesions present, and a culture obtained from the lesion in the earliest stage of development. Only patients whose lesions yielded HSV were included in the final evaluation. Final evaluation was applied to 63 episodes in fifty-five men and 45 episodes in forty-two women. Because the clinical manifestations and the course of the disease were different for initial and recurrent episodes, the results were analyzed separately for the two groups of patients. The mean duration of lesions, new lesions, pain, and viral shedding did not differ significantly between the groups (P > 0.05, Student's t test). In the other study, 34 episodes of herpes progenitalis in thirty-two men were studied (Goodman et al., 1975). The study was conducted in the same manner as the one just described. The results were also similar; there was no significant difference between the drug and control groups in clinical or virological change. The results of these studies suggested that vidarabine is not effective in the treatment of genital herpes infections. The virus probably replicates in the deeper tissues, such as the sacral ganglia. Effective antiviral topical therapy of genital disease would require either a drug or delivery system capable of deeper tissue penetration. 9. PARENTERAL USE O F VIDARABINE Clinical studies have been conducted to evaluate the use of vidarabine, parenterally administered, in the treatment of HSV, VZV, and CMV infections. Many of these diseases start locally but can become progressive and debilitating, particularly in immunosuppressed patients. Others are life-threatening, even when the immune system is not compromised. 9.1. H S V Encephalitis A multi-center double-blind placebo-controlled study was conducted in patients with biopsy-proved HSV encephalitis to evaluate vidarabine (Whitley et al., 1977). Patients were entered into the study only if they showed the clinical features of HSV encephalitis. The diagnosis was considered confirmed only upon brain biopsy of the focal area with J.PA. 8/1

K

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HSV isolation. Following biopsy, brain tissue was minced in a standard cell culture medium and inoculated as a 10 per cent (weight by volume) homogenate into various, types of cell cultures sensitive for isolation of HSV. In addition to those from brain tissue cultures were also prepared from urine and throat swabs and, occasionally, frorr white blood cells and stools. All cultures were observed for at least 4 weeks before bein~ discarded as negative. The drug or placebo was given in a double-blind, randomized fashion. Vidarabine wa, administered i.v. at a dose of 15 mg/kg per day over 12 hr in concentrations not exceed. ing 0.7 mg/ml of standard i.v. solution. Biopsy-positive patients continued to receive dru~ or placebo for 10 days. For the patients who were entered because they exhibited clinica signs, but who were subsequently found to be biopsy-negative, treatment was stopped a 5 days to avoid possible drug toxicity. Toxicity was monitored on admission and a 5-day intervals for 25 days. It included complete blood counts (hemoglobin and hemato crit, WBC counts and differential, reticulocyte and platelet counts), serum glutamic oxa loacetic transaminase (SGOT), serum bilirubin, total protein, blood urea nitrogen, serurr creatinine, serum lactic dehydrogenase, serum alkaline phosphatase, and routine urinalvsis. Twenty-eight of the patients were found to be brain biopsy-positive. (All viral isolate: were typed as HSV~.) Eighteen received vidarabine and ten received placebo. Five (28 pe: cent) of the eighteen receiving vidarabine died. Seven (70 per cent) of the ten receivin~ placebo died. The difference was statistically significant (P < 0.03, chi-square test). A: soon as it became clear that there was a significantly lower mortality rate among th~ patients receiving vidarabine than among those receiving placebo, the double-blind stud~ was discontinued and all subsequent patients have been given vidarabine. (Another twenty-six patients were entered into the study but were found to be biopsy negative; that is, the HSV encephalitis diagnosis was not confirmed. Death occurred in ! per cent of those who received vidarabine and 27 per cent of those who received placebo This difference was not statistically significant. These results indicated that the clinica impression of the presence of HSV encephalitis is not accurate and that vidarabine doe not increase mortality.) Of the eighteen patients with biopsy-confirmed HSV encephalitis who received vidara bine, seven were able to perform at their previous level or were able to perform at gainful level, although hampered by varying degrees of neurologic deficit. This was truq for two of the ten placebo-treated patients. Among the patients with biopsy-prove~ encephalitis, the prognosis also depended upon the level of consciousness at the time c therapy. Among the patients treated with vidarabine, none died who were only lethargic 25 per cent died who were semicomatose, and about one-half died who were comatos~ Morbidity was similarly affected; six of the seven lethargic patients survived with onl' moderate or no sequelae. One of the four semicomatose patients had only moderat sequelae and none of the seven patients in a coma survived without severe sequela~ Among the patients treated with placebo the prognosis was similarly related to the levc of consciousness; three of four lethargic patients survived, but none of those who were i~ a semicoma or a coma. The two treatment groups of patients with biopsy-proved encephalitis differed in th frequency of seizures, which was higher in the group receiving vidarabine. The tw, groups were similar in mean duration of alteration of consciousness and in personalit change. Pleocytosis, however, was present longer before biopsy in the drug-treated reci pients; the mean duration among those receiving vidarabine was 4.8 days, among thos receiving placebo, 3.0 days. This difference suggests that, on the average, the encephaliti may have been more severe among the patients receiving vidarabine. Adverse reactions could not be satisfactorily monitored because of the altered state c consciousness. The investigators did not observe any serious adverse effects of vidarabin on bone marrow, liver, or renal function. The results of this study indicate the effectiveness of vidarabine, parenterally adminis tered, in the treatment of HSV encephalitis. Results also indicated the importance c

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early administration of the drug during the course of the disease. Early treatment requires a high index of suspicion and prompt diagnostic work up. 9.2. HERPESZOSTER IN IMMUNOSUPPRESSEDPATIENTS In a multi-center study, parenterally administered vidarabine was evaluated in the treatment of herpes zoster (varicella zoster, shingles) in immunosuppressed patients (Whitley et al., 1976). Hospitalized patients with active herpes zoster, either localized or disseminated, who were immunosuppressed were entered into the study. None were receiving zoster immune globulin or any other experimental antiviral drugs. Vidarabine was administered i.v. over 12 hr at a dose of 10 ms/ks per day for 5 days. A cross-over design was employed in which one group of patients received active drug for 5 days and then placebo for 5 days; the other group received the treatments in reverse order. Although the value of a crossover design is questionable in diseases of short duration, it was employed so that no patient would be denied therapy. Clinical progress was monitored daily by qualitative assessment of pain, analgesic requirement, temperature, and evidence of cutaneous healing. Quantitative measures for healing were viral persistence, new vesicle formation, and time to pustulation and scabbing. For culturing, vesicles were aspirated daily. The aspirant was inoculated immediately into tubes containing monolayers of human fibroblast cells. Each series of cell cultures was examined for cytopathologic changes at least three times a week for 1 month before being considered negative. Toxicity was evaluated by clinical observations, by complete blood counts (hemoglobin, hematocrit, WBC and differential, and platelet and reticulocyte counts), and by clinical chemistry determinations (SGOT, serum bilirubin, total protein, blood urea nitrogen, creatinine, serum lactic dehydrogenase, serum alkaline phosphatase, and routine urinalysis). Forty-seven patients received vidarabine first and forty patients received placebo first. Because the zoster healed so rapidly, the cross-over information could not be used and evaluation was based on only the first period in which one group received placebo and the other active medication. Of the patients in the active drug group, twenty-six had reticuloendothelial malignancies and thirteen had other malignancies. Six had other diseases with immunosuppression. Of those in the placebo group, twenty-three had reticuloendothelial malignancies, thirteen had other malignancies. Six had other diseases with immunosuppression. Significantly more of the patients receiving vidarabine (34.83 per cent) had relief of acute pain than did those receiving placebo (17.46 per cent) (P = 0.001, chi-square test). Although the lesions among the placebo-treated patients healed rapidly, healing was significantly more rapid among those who received vidarabine. The differences favoring the group receiving vidarabine were significant for viral clearance (P = 0.01), cessation of new vesicle formation (P = 0.004), and time to total pustulation (P = 0.01) (Wilcoxon Analysis). These differences became even more pronounced after 48 hr of therapy. The time to total scabbing could not be assessed statistically, but it was shorter in the group receiving vidarabine. When therapy with vidarabine was begun within 6 days of onset of the herpes zoster rather than later, viral persistence was significantly shorter (P -- 0.001, Wilcoxon Analysis). The same was true to cessation of new vesicle formation (P = 0.001) and to total pustulation (P = 0.001). Differences between medication groups were not significant when the patients had disease for more than 6 days before treatment. As was true in the study of HSV encephalitis, antiviral chemotherapy must be started promptly to be effective. Nausea or vomiting was observed in 16 per cent of the patients, but was easily controlled medically and did not require discontinuation of treatment. Other adverse experiences that might be attributed to drug were diarrhea (5 per cent) and skin rashes (5 per cent). Hallucinations were experienced by 2 per cent of the patients but were difficult to evaluate because spinal fluid examinations were not performed to rule out varicella encephalitis. None of the changes in laboratory values indicated significant adverse

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effects of vidarabine on bone marrow, liver, or renal function. Comparison of the laboratory values from the treated and untreated groups indicated no significant difference in the changes between the two. It appears that the underlying diseases, rather than the vidarabine, were responsible for most of the laboratory aberrations detected. On the basis of this study, it appears that vidarabine has a therapeutic index suitable for treating herpes zoster in the immunosuppressed. The low level of solubility of the drug, which necessitates slow administration and hospitalization of the patients, means that vidarabine should not be administered for trivial infections. On the other hand, the need to hospitalize the patients helps assure appropriate use of the drug. 9.3. CYTOMEGALOV1RUS(CMV) INFECTIONS Since in vitro studies indicated that vidarabine is active against CMV, it is reasonable to suppose that it would be effective in treating CMV infections in man. CMV has the greatest clinical impact in two patient populations. One population is the newborn. The neonate with CMV infection presents fever, petechiae, thrombocytopenia, hepatomegaly and splenomegaly. If the central nervous system becomes involved, often evident chorioretinitis or intracerebral calcification, then permanent mental retardation becomes a real possibility. The other patients particularly subject to CMV infection are those who have had a renal transplant. Perhaps one month following surgery, while they are still immunosuppressed, the patients will develop a CMV pneumonia. Baublis et al. (1975) used vidarabine to treat both infants and adults with cytomegalovirus infections. Nine infants, aged 4 days to 4 weeks, with disseminated neonatal CMV infection, were given vidarabine. The lowest daily dose was 1 mg/kg, the highest was 20 mg/kg. The shortest duration of therapy was 5 days, the longest was 21 days. In all patients viral excretion in the urine or throat was suppressed, but three patients died and two others had no change in their clinical condition. Four patients experienced subjective clinical improvement, but in three of these, development was retarded and there occurred sequelae such as deafness, spasticity, and convulsions. Therapeutic results were not correlated with dose or duration of treatment. These investigators also gave 13 courses of vidarabine treatment to ten babies (ages l0 weeks-3 years) with cytomegalic inclusion disease. The lowest daily dose was 5 mg/kg, the highest was 20 mg/kg. The shortest duration of therapy was 8 days, the longest was 21 days. Viral excretion was suppressed in these patients, but one died and two did not improve. Six experienced subjective clinical improvement after one or more courses ot therapy. In three of the six who improved, development was retarded to some degree. These investigators also treated four infants (aged 7 to 28 months) who had CMV pneumonitis. Each patient received 20 mg/kg per day of vidarabine for l0 days. In all the patients viral excretion was suppressed and the respiratory disease resolved, but development was retarded in one. Six immunosuppressed adult patients, two with CMV mononucleosis and four with CMV infections following renal transplant, were each given 10 mg/kg per day of vidarabine for l0 or 14 days. In both mononucleosis patients, viral excretion was suppressed and clinical improvement occurred. Of the four renal transplant patients, two died and neither of the others improved. These studies indicated that vidarabine suppressed, but did not completely eliminate viral excretion. Some time after cessation of vidarabine therapy, an insidious viremia ol viruria developed. This suggests that a longer duration of therapy or a higher dose ma3 be necessary to successfully treat CMV infections. A major limitation to raising the dos~ is the large volume of fluid necessary to administer vidarabine. 9.4. VARIOLA VIRUS INFECTIONS (SMALLPOX)

Vidarabine is effective both in vitro and in vivo against vaccinia or cowpox virus. No laboratory or animal studies of its action against variola virus have been conducted because of the virulence of the virus. During a period when civil disobedience disrupted

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the vaccination program in Bangladesh, a field study was conducted comparing vidarabine with placebo in the treatment of patients with acute variola infections. Koplan et al. (1975) evaluated vidarabine for smallpox in a placebo-controlled study. Each of twenty smallpox patients was randomly assigned to either a group to receive vidarabine (nine patients) or one to receive placebo (eleven patients). The dose of i.v. administered vidarabine was 20 mg/kg per day, infused over 8 hr. The duration of treatment was scheduled for 7 days. The measures of efficacy were mortality, duration of fever, time to scabbing, and length of time that virus could be isolated. Four (36 per cent) of the eleven placebo patients and five (56 per cent) of the vidarabine patients died; the difference was not statistically significant. Among the surviving patients, the mean number of febrile days was 7.7 in the placebo group and 7.3 in the vidarabine group, a difference not statistically significant. Among the surviving patients, the mean time to scabbing was 11.3 days in the placebo group and 8.3 days in the vidarabine group; the time in the vidarabine group was significantly shorter than that in the placebo group. But if the mean number of days of rash before treatment was added into the calculation, the mean time to scabbing was 14.7 days among those on placebo and 13.0 days among those on vidarabine, a not significant difference. Variola virus was isolated from lesions in all patients throughout the entire study. Among patients in whom the blood levels of vidarabine were determined, the investigators found only ara-Hx, except in samples taken during the infusions, when small amounts of vidarabine were also detected. On the basis of this study, it appeared that vidarabine at the dose administered was not effective in the treatment of smallpox. With the apparent success of the World Health Organization program to eradicate smallpox (Henderson, 1976), the results of this study are probably of only historic interest. 9.5. EPSTEIN-BARR VIRUS (EBV) INFECTION (INFECTIOUS MONONUCLEOSIS)

Even though infectious monoucleosis is generally considered a benign disease, its treatment usually requires that the patient be hospitalized. Therefore, the disease often means a significant economic loss. The EBV is a herpesvirus and might be expected to be susceptible to vidarabine therapy. There is no animal model of infectious mononucleosis in which vidarabine could be tested, but in vitro studies have been conducted. The results are presented here rather than in the preclinical section because there have been no clinical studies, but the interest lies in the clinical possibilities. Coker-Vann and Dolin (1977) tested the activity of vidarabine against EBV (Burkitt's lymphoma virus) in two cell lines, P3HR-1, which spontaneously produces EBV, and Raji, which does not. Using indirect immunofluorescence, they found that vidarabine reduced the percentage of P3HR-1 cells that expressed viral capsid antigen (VCA), indicating effectiveness against the virus. This effect was observed at vidarabine concentrations of 5 and 10/~g/mi, well below the toxic concentrations, 25 and 50/~g/ml, that interfered with the growth of the cells. In this study, IDU was effective but toxic at 2.5/~g/ml. Further evidence of the low toxicity of vidarabine was that early antigen was neither induced nor did it accumulate in the VCA-free cells under the influence of vidarabine except at very high concentrations (50 pg/ml). Vidarabine at concentrations of 5 to 50/~g/ml did not inhibit the superinfection of Raji cells when EBV particles were suspended in a medium containing Raji cells. Although most infections with EBV are subclinical or self-limiting, on those occasions when they are more serious, vidarabine may be efficacious. 9.6. SAFETY EVALUATION OF INTRAVENOUSVIDARABINE

The most rigorous evaluation of the safety of i.v. administered vidarabine was during the Phase III double-blind studies done to obtain approval for marketing the drug. The patients treated in these studies had VZV (varicella and herpes zoster) and HSV encephalitis infections. Because their treatment was part of multicenter cooperative studies con-

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ducted jointly by the National Institute of Allergy and Infectious Diseases (NIAID) and Parke-Davis, these patients had the most complete examinations for adverse reactions and for laboratory abnormalities. Not all of the data collected have been published and those which have are scattered throughout the literature. They are briefly presented here as they were submitted in the New Drug Application for approval of the parenteral form of vidarabine. One hundred and fifty-nine patients participated in the three studies, 106 with herpes zoster or chicken pox and fifty-three suspected of having HSV encephalitis. Of those in the studies of VZV infections, all 106 patients received vidarabine and 100 received placebo. In the studies of encephalitis, thirty-one patients received vidarabine and twentytwo received placebo. Thus, a total of 137 patients received vidarabine and 122 received placebo. The major adverse reactions were gastrointestinal. In the studies of herpes zoster, nausea, vomiting, or both occurred in ll per cent of the patients while they were receiving vidarabine and in 6 per cent while they were receiving placebo. No adverse reactions occurred among the nineteen patients in the chicken pox studies. In the HSV encephalitis studies, no adverse reactions were reported, but many of the patients were lethargic or comatose and, therefore, unable to report their states accurately. Among the patients for whom accurate reports were available, the incidence of the gastrointestinal reactions did not differ appreciably between those receiving vidarabine and those receiving placebo. The only laboratory abnormalities encountered with any frequency in these studies were decreased hemoglobin, hematocrit, and white blood cell counts, and elevations in SGOT. The changes considered clinically significant were decreases of 2 g/100 ml or 15 per cent or more for hemoglobin, decreases of 15 per cent or more for hematocrit, decreases of 2000/mm 3 or more for WBC count, decreases of 75,000/mm 3 or more for platelet count, and increases of 50 international units or more for SGOT. By these criteria, 19 per cent of the patients receiving vidarabine and 16 per cent of those receiving placebo showed decreases in hemoglobin; 19 per cent of those receiving vidarabine and 17 per cent of those receiving placebo had decreases in hematocrit; 16 per cent of those receiving vidarabine and 13 per cent of those receiving placebo had decreases in platelet count; and 22 per cent of those receiving vidarabine and 16 per cent of those receiving placebo had elevations of SGOT. (For WBC count, 14 per cent of those receiving vidarabine and ll per cent of those receiving placebo had decreases, but this includes the patients in the HSV encephalitis studies for whom the criterion was a decrease of 5000/mm3.) The similarity of the incidences of the abnormal laboratory values between the groups that received vidarabine, and the groups that received placebo, suggests that these changes were more the result of the patients' conditions rather than the administration of vidarabine. 10. ANALOGUES OF VIDARABINE From the beginning of investigations of vidarabine, analogues were sought that would offer greater effectiveness, greater solubility, and less susceptibility to deamination in clinical use. Ara-C and ara-Hx have been described in the sections dealing with vidarabine. Many other candidates have been synthesized. This section describes a few of these that appear most promising. 10.1. 9-fl-D-ARABINOFURANOSYLADENINE-5'-PHOSPHATE (VIDARABINE MONOPHOSPHATE)

The analogue of vidarabine that has received the most attention is vidarabine monophosphate. It was originally synthesized as a potential anticancer agent (Furth and Cohen, 1967). It has formulation advantages over the parent nucleoside in that it has greater solubility (> 50 times as soluble at 37°C). It also is only slowly dephosphorylated and therefore provides sustained blood levels (LePage et al., 1972). It has been evaluated in vitro and in vivo for anti DNA virus activity.

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Sidwell et al. (1973) reviewed much of the early experimental work on vidarabine monophosphate. It was compared with vidarabine in cell culture studies with human carcinoma cells of the nasopharynx, rabbit kidney cells (RK-13), human embryo lung fibroblasts (WI-38), and secondary mouse embryo cells. Vidarabine monophosphate was essentially equal to vidarabine in antiviral activity in cell cultures. By a virus rating scale based on the inhibition of cytopathic effect both compounds showed marked or moderate activity against several strains of HSVI, HSV2, CMV, and VV. In HSV keratitis experiments in New Zealand rabbits, vidarabine monophosphate was moderately more effective than vidarabine when used topically. In experiments with mice that had been infected intracerebrally with HSV1 and HSV2, vidarabine monophosphate was more effective than vidarabine when the drugs were administered intracerebrally, probably largely because the maximum tolerated dose of vidarabine monophosphate was approximately twenty times that of vidarabine. Kern et al. (1978) compared vidarabine monophosphate, vidarabine, and phosphonoacetic acid in mice with genital H S V 2 infections. Intravaginal administration of phosphonoacetic acid at 500 mg/kg or as a 5 per cent cream, when given 3 hr after infection, completely inhibited viral replication in the genital tract and prevented subsequent death; when given 24-72 hr after infection it reduced viral replication but did not protect from subsequent death. Neither vidarabine monophosphate nor vidarabine at 500 mg/kg or as 10 per cent cream affected viral replication or mortality. On i.p. administration, none of the drugs had any therapeutic effects. Hull et al. (1977) tested several antiviral solutions in eyes taken from freshly killed rabbits. The solutions tested were vidarabine monophosphate, 500 #g/ml, vidarabine monophosphate, 1000 #g/ml plus adenosine deaminase inhibitor, vidarabine, 500/~g/ml, vidarabine 250/~g/ml plus adenosine deaminase inhibitor, and ara-Hx, 1000 #g/ml. At the end of 3 hr of perfusion, none of the corneas showed any change in thickness significantly different from the controls, and electron microscopy showed preservation of the endothelium. At the concentrations and duration tested, none of the compounds had any effect on corneal endothelial cell function or ultrastructure. Foster and Pavan-Langston (1977) compared 0.1 per cent IDU, 1 per cent F3 T, and 3 per cent vidarabine monophosphate for their effects on corneal wound healing in New Zealand rabbits. They found that vidarabine phosphate retarded wound healing appreciably more than the other drugs; by day 4, healing in the eyes treated with IDU, FaT, and the control solution was complete, but by day 5, the mean defect remaining in the eyes treated with vidarabine monophosphate was > 25 per cent. The quality of regenerated epithelium in all eyes treated with drugs was worse than that in the control eyes, but that in the eyes treated with vidarabine monophosphate was significantly worse than that in the eyes of the rabbits treated with IDU or FaT. The eyes treated with vidarabine monophosphate had extensive networks of subepithelial neovascularization; the corneal epithelium was thickened, grossly cloudy, and marred by extensive punctate defects. The strength of the healed wound, however, was significantly greater in the eyes treated with vidarabine monophosphate than in the controls, which was somewhat stronger than that in the eyes treated with IDU or F3T. Preliminary studies of the use of vidarabine monophosphate for treating HSV keratitis in human patients have also shown adverse reactions. Three of four patients receiving a 3 per cent ophthalmic solution of vidarabine monophosphate for epithelial keratitis developed punctate epithelial granularity in the area of dendritic keratitis, which had been asymptomatic at the start of treatment. 10.2. 9-fl-D-ARABINOFURANOSYLADENINECYCLIC-3',5'-(HYDROGEN PHOSPHATE)(CYCLIC ARA-AMP) Cyclic ara-AMP was synthesized by Lee et al. (1971) and by Khwaja et al. (1972). Sidwell et al. (1973) reviewed the in vitro and in vivo studies of its antiviral activity (in the same article in which they described the experiments with vidarabine phosphate). I n

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vitro, cyclic ara-AMP was active against HSVI and HSV2, CMV, and VV. It was moderately more effective than vidarabine in treating HSV keratitis in rabbits. In mice infected intracerebrally with HSV, cyclic ara-AMP intracerebrally administered was more effective than vidarabine, based on the fraction of maximum tolerated dose.

10.3.

9-fl-D-ARAB1NOFURANOSYLADENINE-5'-PHOSPHATE-1-OXIDE (Ara-AOP)

Sidwell et al. (1972) studied ara-AOP in cell cultures of KB, RK-13, EBTr, Vero, and secondary mouse cells to which 100 CCIDso of virus had been added. Several concentrations of ara-AOP were added within 20 min of adding the virus. Using criteria such as inhibition of both viral cytopathic effect and of intra- and extracellular virus titers, the investigators found significant activity against HSV1, pseudorabies, infectious bovine rhinotracheitis virus, murine cytomegalovirus, VV, and myxoma virus. Against HSV2, reduction of virus titer was observed but not inhibition of cytopathic effect. The MIC ot ara-AOP ranged from 1-10 ~g/ml, depending on the virus. Survival increases up to 65 per cent were seen among mice infected intracerebrally with HSV1 and treated with 500, 250, or 125 mg/kg per day of ara-AOP. Significant inhibition of HSVl-induced tail lesions was observed when ara-AOP in a l0 per cent solution was applied topically. 10.4. CARBOCYCLICARAB|NOSYLADEN1NE(C-ara-A) In a search for analogues of vidarabine that would be resistant to the effects of adenosine deaminase, and thereby not be metabolized as quickly as vidarabine, Vince and Daluge (1977) synthesized C-ara-A. The drug was tested for antiviral activity in cell culture studies and found to have virus ratings well over 1 (indicating definite activity) against HSVa and VV. The approximate minimal effective dose (50 per cent) was about 9/~g/ml, a concentration not cytotoxic to host cells. Further investigations are underway with this potential antiviral drug. 10.5,

N'-(9-fl-D-ARABINOFURANOSYL-9H-PURIN-6-YL)-N,N-DIMETHYLFORAMIDINE

This derivative of vidarabine was synthesized by Hanessian (1973) in an attempt tc find an analogue that would maintain its antiviral activity longer than the parent compound. The acute i.p. toxicity of the derivative was approximately the same as that ot vidarabine and the in vitro antiviral activity against HSV and VV were also similar. But in mice infected intracerebrally with HSV, the derivative did not appear to have therapeutic activity equal to that of vidarabine. 11. C O N C L U S I O N Vidarabine is active in vitro and in animals against a variety of DNA viruses. In ar ophthalmic ointment it is indicated for topical therapy of HSV keratitis and in suspension for intravenous therapy of HSV encephalitis. It has the greatest practical value o: any of the presently approved antiviral drugs. But even beyond its therapeutic activity, just by its existence vidarabine contributes tc the development of antiviral drugs. It removes any doubts that effective systemic antivira agents are possible. Vidarabine also offers a substance on which to base future synthese~ to try to attain more effective antiviral drugs. And further, when we understand th~ method of action of vidarabine, we will probably understand more fully the action o antiviral drugs generally and the pathogenic action of viruses. REFERENCES ABEL, R., JR., KAUFMAN, H. E. and SUGAR,J. (1975)Effectof intravenous adenine arabinoside on herpes simple:

keratouveitis in humans. In Adenine Arabinoside: An Antiviral Agent, pp. 393~,00, PAVAN-LANGSTON,D. BUCHANAN,R. A. and ALFORD,C. A., JR. (eds). Raven Press, New York.

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ADAMS, H. G., BENSON, E. A., ALEXANDER,E. R., VONTVER, L. A., REMINGTON, M. A. and HOLMES, K. K. (1976) Genital herpetic infection in men and women: Clinical course and effect of topical application of adenine arabinoside. J. Infect. Dis. 133 (Suppl): A151-A159. BADER, J. P. (1964) The role of deoxyribonucleic acid in the synthesis of Rous sarcoma virus. Virology 22: 462-468. Bader, J. P. (1966a) Metabolic requirements for infection by Rous sarcoma virus--I. The transient requirement for DNA synthesis. Virology 29: 444-451. Bader, J. P. (1966b) Metabolic requirements for infection by Rous sarcoma virus--lI. The participation of cellular DNA. Virology 29: 452-461. Baltimore, D. (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226: 1209-1211. Baublis, J. V., Whitley, R. J., Ch'ien, L. T. and Alford, C. A., Jr. (1975) Treatment of cytomegalovirus infection in infants and adults. In Adenine Arabinoside: An Antiviral Agent, pp. 247 260, PAVAN-LANGSTON, D., BUCHANAN, R. A. and ALEORD, C. A., JR. (eds). Raven Press, New York. BENNETT, L. L., JR., SHANNON, W. M., ALLAN, P. W. and ARNEYr, G. (1975) Studies on the biochemical basis for the antiviral activities of some nucleoside analogs. Ann. N.Y. Acad. Sci. 255: 342-358. BRINK, J. J. and LEPAGE, G. A. (1964) Metabolism and distribution of 9-fl-D-arabinofuranosyladenine in mouse tissues. Cancer Res. 24: 1042-1049. BRYSON, Y. J. and CONNOR, J. D. (1976) In vitro susceptibility of varicella zoster virus to adenine arabinoside and hypoxanthine arabinoside. Antimicrob. Agents Chemother. 9: 540-543. BRYSON,Y., CONNOR,J. D., SWEETMAN,L., CAREY,S., STUCKEY,M. A. and BUCHANAN,R. (1974) Determination of plaque inhibitory activity of adenine arabinoside (9-fl-D-arabinofuranosyladenine) for herpesviruses using an adenosine deaminase inhibitor. Antimicrob. Agents Chemother. 6: 98-101. CHAMPNEY, K. J., LAUTER,C. B., BAILEY,E. J. and LERNER, A. M. (1978) Antiherpesvirus activity in human sera and urines after administration of adenine arabinoside, d. Clin. Invest. 62: 1142-1153. CHO, C. T., FENG, K. K. and BRAHMACUPTA,N. (1976) Synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to Herpesvirus hominis, d. Infect. Dis. 133:157 167. COKER-VANN,M. and DOLIN, R. (1977) Effect of Adenine arabinoside on Epstein-Barr virus in vitro. J. Infect. Dis. 135: 447-453. CONNOR, J. D., SWEETMAN, L., CAREY, S., STUCKEY, M. A. and BUCHANAN, R. (1975) Susceptibility in vitro of several large DNA viruses to the antiviral activity of adenine arabinoside and its metabolite, hypoxanthine arabinoside: Relation to human pharmacology. In Adenine Arabinoside: An Antiviral Agent, pp. 177 196, PAVAN-LANGSTON, D., BUCHANAN, R. A. and ALFORD, C. A., JR. (eds). Raven Press, New York. COSTER, D. J., McKINNON, J. R., McG1LL, J. I., JONES, B. R. and FRAUNFELDER, F. T. 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