The acute toxicity of BIOLF-143 in the rat

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

lo,3

13-320 ( 1988)

The Acute Toxicity of BIOLF-143 D. J. ECOBICHON,*

K. M. MEKHAEL,~

P. MAJOR,+

in the Rat AND K. K. OGILVIE~

*Department of Pharmacology and Therapeutics, @k-Gill Cancer Centre, and TDepartment of Chemistry, McGill University, Montreal, Quebec H3G 1 Y6, Canada ReceivedMay II, 1987:acceptedSeptember

16, 1987

The Acute Toxicity of BIOLF-143 in the Rat. ECOBICHON, D. J., MEKHAEL, K. M., MAJOR, OCSLVIE, K. K. (1988). Fundam. Appl. Toxicol 10, 3 13-320. BIOLF- 143, an experimental, purine-based acyclic nucleoside, was administered by iv or ip injection to young, adult, male and female Sprague-Dawiey rats in order to determine the (1) pharmacokinetic disposition, (2) route and rate of excretion, and (3) acute toxicity. HPLC analysis of plasma, hepatic, and renal tissue levels was conducted following iv injections of 50 and 100 mg& and ip injeo tions of 500 mg/kg. Metabolism/excretion studies were conducted following ip injections of BIOLF-143 (100 mg/kg). The assessment of acute toxicity was done following the ip injection of agent (250 mg/kg/hr for 8 consecutive hr). BIOLF-143 was rapidly distributed in the body, the estimated half-life in blood plasma being 18-23 min. The molecule was essentially unbound to plasma proteins (99% free) and was excreted unchanged in the urine. The recovery of the parent compound was 74.3 f 5.9% and 88.5 & 15.9% for male and female rats, respectively, with no metabolites or unidentifiable peaks being detected in HPLC chromatograms. No overt toxicity or untoward signs of latent toxicity were observed in the animals receiving doses up to 2000 mg/kg ip. No residues were detected in tissues at 24 hr post-treatment. A potential target organ in subchronic studies might be the kidney. High residue levels of BIOLF-143 were detected 1.0 hr post-treatment; however, the organ had cleared all residues by 24 hr after administration. 0 1988 Society ofToxicology. P., AND

The growing number of patients receiving immunosuppressive treatment represents a population particularly susceptible to severe, life-threatening viral infections with herpes simplex and cytomegalovirus (Meyers et al., 1980; Wade et al., 1982; Strauss et al., 1982). A number of nucleoside analogs have been synthesized and evaluated for human antiviral chemotherapy. The systemic toxicities of some of these experimental agents have excluded them from consideration for clinical use. Other factors such as low aqueous solubility and rapid detoxification have restricted their usefulness to dermal application or intravenous administration. Acyclovir (9[2-hydroxyethoxymethyl] guanine), ARA-A (arabinoside-A), and DHPG (9-[[2-hydroxyl-(hydroxymethyl)ethoxy]methyl] guanine, BIOLF-62) are three nucleoside analogs that have found a place in the antiviral armamen-

tarium for treating human cytomegalovirus and herpes simplex infections (Crumpacker et al., 1979; Corey et al., 1982; Whitley et al., 1982; Corey and Holmes, 1983; Shepp et al., 1985a,b, Felsenstein et al., 1985; Fletcher et al., 1986). The rapid inactivation of such agents as Acyclovir, ribavirin, and BIOLF-62 in vivo is the consequence of the oxidation of the free amino group by ubiquitous amine oxidases in body tissues. In an attempt to reduce biological inactivation, BIOLF- 143 (N-(dimethylamino)methylene - 9 - [[2 - hydroxy - l(hydroxymethyl)ethoxy]methyl] guanine), an analog of BIOLF-62 (Fig. I), was synthesized, introducing the dimethylaminomethylene substituent as a protective group on the guanine amino group. BIOLF-143 has shown a high level of potency against a spectrum of viruses and has moderate water solubility

313

0272-0590/88 $3.00 Com’isbt 8 1988 by the Society of Toxi.mlogy. All rights of reprcduction in any form resaved.

314

ECOBICHON OH

f? HN ’

N

HN

\

“,, H.~‘N Lb HO

N 0

3 ‘N

(cbi,&-cH=N HO

lJ

Y

0 Y

OH BIOLF-

62

J

OH

BIOLF-

143

FIG. 1. The chemical structure of BIOLF-143 (N(dimethylamino)methylene-9-[[2-hydroxy-I-(hydroxymethyl)ethoxy]methyl] guanine) and the antiviral analog BIOLF-62 (9-[[2-hydroxy-l-(hydroxymethyl)ethoxy]methyl] guanine).

(Smith el al., 1982). In this report, we present the results on the acute toxicity, the tissue distribution, and the pharmacokinetics of BIOLF-143. METHODS Synthesis. The synthesis of BIOLF-143 has been described (Ogilvie et al., 1985). The chemical was purified by recrystallization from hot methanol to a state of microanalytical purity (99.9%). Samplepreparation. BIOLF- 143 was dissolved in 0.9% sodium chloride solution with the aid of sonication and the addition of drops of 1.O N sodium hydroxide until a solution was obtained. A maximum concentration of 40 mg BIOLF-143 could be dissolved in an aqueous medium but the alkalinity of the solution was approximately pH 12.0. Routinely, a stock solution of 25 mg/ml was prepared in the same manner, the pH being 10.2. In vitro binding. Aliquots (1 .O ml) of a pooled sample of fresh heparinized rat plasma were treated with BIOLF143 concentrations of 0.5, 1.0, 2.5, 5.0, 10, 25, and 50 pmol added in a volume of 0.1 ml. These samples were incubated at 37°C for 10 min in an oscillating water bath and then were pipetted into Amicon Centrifree micropartition tubes (Amicon Canada Ltd., Oakville, Ontario, Canada) and were centrifuged for 15 min at 3000gto separate the free (unbound) fraction of the drug into a protein-free ultrafiltrate suitable for analysis. Animal treatment. Young adult male (65 days) and female (80 days) Sprague-Dawley rats (CD-CrlCD (SD)BR strain) of250-275 g body weight were purchased (Charles River Canada, St. Constant, Quebec, Canada) and acclimatized for 7 days in the animal care facility in a room with a 12-hr 1ight:dark cycle, housed five rats per

ET AL. plastic shoebox cage and provided water and standard Purina rat chow ad libitum. For studies of the pharmacokinetic disposition of BIOLF-143 following intravenous administration, the rats were lightly anesthetized with diethyl ether and a small incision was made in the ventral surface of the neck to expose the jugular vein for injection through the overlying muscle layer. The animals received intravenous injections of suitable volume of a 25 mg/ml stock solution of agent to yield dosages of 50 or 100 mg/kg body wt. Following injection, the incision was closed with a metal surgical clamp for the duration of the experiment. Subgroups (n = 5) of rats were anesthetized with chloroform and exsanguinated by cardiac puncture into heparanized syringes at post-treatment intervals of 15,30,60,90, 120, 150. 180, and 240 min. Samples of liver and kidney were removed at necropsy and were stored at -20°C along with blood plasma samples to await analysis. Preliminary studies showed that BIOLF- 143 was stable in tissues frozen at -2o’C for longer than 2 weeks. All analyses were done within that time period. Due to the limited aqueous solubility of BIOLF-143 which would necessitate the intravenous administration of a large volume of drug solution, the highest dosage, 500 mg/kg body wt, was administered intraperitoneally. Only male rats were used in this experiment. The results demonstrated the need for a study of the equivalence of the ip versus the iv routes if relatively high doses of BIOLF- I43 were to be administered. Groups of male and female Sprague-Dawley rats of 250-275 g body wt received ip injections of BIOLF- 143 at dosages of 100 mg/ kg body wt and subgroups (n = 4 or 5) were euthanized as described above at similar time intervals after treatment, blood samples being collected for analysis of plasma residues of agent. Male and female Sprague-Dawley rats received ip injections of BIOLF-143 at a level of 100 mg/kg body wt and were placed in Nalgene metabolic cages overnight, being provided with powdered rat chow and drinking water ad libitum. Urine and feces were collected overnight (18 hr), the receptacles being immersed in an ice bath to minimize degradation of the agent. Ahquots of excreta were stored at -20°C for analysis of BIOLF- 143 residues and to detect any metabolites and/or degradation products. High dose exposure to BIOLF-143 posed a problem because of the limited water solubility and the volume of drug solution that would be administered. To ascertain the maximum tolerated dose and/or that eliciting minimal toxicity, repeated doses of BIOLF- 143 were administered to attain a final concentration of 2000 mg/kg body wt. For this experiment, male Sprague-Dawley rats of 250-300 g body wt received an intraperitoneal injection of BIOLF- I43 to achieve a dosage of 250 mg/kg body wt every 60 min for 8 consecutive hr. A subgroup (n = 6) of the rats was euthanized 60 min following the last dose

TOXICITY

OF BIOLF-143

and additional subgroups (n = 6) were euthanized at 24 and 72 hr and at 14 days post-treatment. At necropsy, samples of blood, liver, and kidney were obtained for residue analysis and samples of liver and kidney were fixed in buffered formalin for sectioning, staining, and examination by light microscopy. Analysis. Aliquots of the stored blood plasma, peritoneal fluid, and urine were pipetted into Amicon Centrifree micropartition tubes and were centrifuged at 3000s for 15 min to obtain protein-free ultrafiltrates for analysis. For liver and kidney samples, 10% w/v homogenates were prepared in 0.067 M phosphate buffer, pH 7.4, and were centrifuged at 4’C for 15 min at 9OOOg. Aliquots (0.5 ml) of the resulting supematants were pipetted into Centrifree tubes to recover the free (unbound) fraction of BIOLF-143. Frozen fecal samples were homogenized as described for other tissues above, and aliquots (0.5 ml) of the 10% w/v homogenate were transferred to the Centrifree tubes to obtain protein-free ultratiltrates for analysis by HPLC. Samples of liver and kidney were removed at necropsy and fixed in 4.0% neutral-buffered formalin, later to be embedded in paraffin with standard thickness sections being stained with hematoxylin and eosin for light microscopy. Analyses of BIOLF- 143 residues were carried out on a Waters HPLC system (Model 441 detector, Model 740 data module, Model 680 controller, and Model 510 pump). Separation was effected on a Shandon Spherisorb C 18 column (0.46 X 15 cm, 5-pm particle size) (Chromatography SciencesCo., Inc., Montreal, Quebec, Canada), using a mixture (85:15) of 10 mM KH2P04 buffer, pH 4.9, and methanol as the mobile phase at a flow rate of 1.0 ml/min. The residues were detected spectrophotometrically at 254 nm, the change in optical density was recorded on a strip chart, and the areas under the peak(s) were integrated. The levels of residues were quantitated using a standard curve prepared with known amounts of BIOLF-143 and plotting the areas under the peaks eluted.

RESULTS The pharmacokinetic disposition of BIOLF- 143 in the blood plasma of male and female rats receiving the agent by iv (50 and 100 mg/kg) or ip (500 mg/kg) injection revealed a dose-dependent, rapid disappearance of the agent from the bloodstream (Fig. 2). The drug was absorbed rapidly from the abdominal cavity following ip injection, peak levels being attained 15-30 min after administration. There were no significant differ-

IN RATS

315

ences between sexes in the rate of elimination of BIOLF- 143 from the plasma over the 3&r time interval following administration. Estimated half-lives from best-fit lines of the initial portions of the curves for male rats gave values of 18.5,20.0, and 23.0 min for dosages of 50, 100, and 500 mg/kg body wt, respectively. No distinct beta (&phase was ob served above the limit of detection and/or over the time interval studied. The analyses of BIOLF- 143 residues in livers and kidneys of the rats euthanized at each time interval in the pharmacokinetic study are shown in Table 1. The results demonstrated proportionally higher levels of BIOLF- 143 in the kidney compared to the residue concentrations measured in the liver, suggesting that renal excretion may be the primary route of elimination from the body. The rate of disappearance of BIOLF- 143 from the livers and kidneys of these animals was somewhat slower than that measured in the blood. After observance of the levels detectable in the bloodstream and the rapid disappearance of the drug, an in vitro experiment was conducted to ascertain the extent of proteinbinding in rat plasma. At a concentration range of 0.5-50 pg BIOLF-143/ml plasma, treated, pooled rat plasma was incubated with agent and centrifuged in micropartition tubes, and the protein-free ultrafiltrate was analyzed for free drug. The agent was not bound to plasma proteins to any extent over the concentration range tested, the recovery being 96.8% at the lowest and 101.6% at the highest concentration used. An experiment was conducted to study the equivalence of ip versus iv administration, the former route being necessary at high dose levels. Single doses of BIOLF- 143 (100 mg/ kg) were administered by either route to male and female rats, and subgroups (n = 3 or 4) of animals were euthanized at suitable time intervals after treatment and blood samples were taken for analysis. The results, shown in Fig. 3, revealed that, after the initial 30-min

316

ECOBICHON

ET AL. ,000 1

1000 ma

500

MALE

RATS FEMALE

100

100

5Q

50

.

10

10

-

I

RATS

-

5.

500 I.(

l.O-

O.!

0.5.

\E 1

l \ 100 1

5.

60

180

(20 TIME

240

0

TIME

(min.)

180

120

60

240

(min.)

FIG. 2. The elimination of BIOLF-143 from the blood plasma of male and female Sprague-Dawley rats following the administration of 50 and 100 mg/kg body wt by iv injection or 500 mg/kg body wt by ip injection. The values presented are the average plasma concentrations SD of residue levels measured in five individual animals euthanized at each time interval indicated.

time interval where large variation in plasma level was observed with ip administration, there was a good correlation between the plasma levels of BIOLF- 143 administered by either route. No significant differences in

equivalence were observed, suggesting that the ip route would satisfactorily reflect the direct iv administration of the drug. Sample chromatograms of diluted urine from a female rat, collected during an 1%hr

TABLE 1 HEPATICANDRENAL

RESIDUES OF BIOLF-143 INMALERATSATVARIOUSTIMEINTERVALS FOLLOWINGINTRAVENOUSADMINISTRATION

BIOLF- 143 (50 mg/kg)” Time post-treatment (min) 30 60 120 180

BIOLF- 143 ( 100 mg/kg)

Liver bplg)

Kidney Wg)

Liver bJg)

Kidney Wg)

6.1 f 5.3 ND ND ND

138 *68 102 k81 20.3 f 13.9 ND

65.6 f 14.0 19.2* 11.6 5.2 f 2.9 ND

605 +401 74+- 15 302 7.1 ND

a The values shown are average residue concentrations f SD for the tissues of six animals euthanized at the posttreatment intervals shown. ND indicates not detectable.

TOXICITY IP 20

F

+

MALE

’

FEMALE

r

15

OF BIOLF-143

l

z 10 :: s

5

i :lf/I

0 5 PLASMA

10 CONC.

15 ( W/ml

20

IV

)

FIG. 3. The correlation between ip and iv administration of BIOLF-143 to male (+) and female (0) SpragueDawley rats at a dosage of 100 mg/kg body wt. The values shown are mean blood plasma concentrations of agent, the bars being the standard deviations determined for n = 3 rats per point. The line represents the equivalence correlation if there was no difference in the routes of administration.

control period (Fig. 4A) and during the 18-hr period following treatment with BIOLF-143 (Fig. 4B), demonstrate that the agent was excreted unchanged with no breakdown products, metabolites, or unidentified peaks being detected by HPLC analysis. The major fraction of the agent was excreted via the urine. The average recovery values (m f SD), ascertained from 6 rats of each sex were 74.3 + 5.9%formaleand88.5+ 15.9%forfemale rats, respectively. Repeating the experiment with male rats and holding the animals in the metabolic cages for 48 hr did not improve the recovery of BIOLF- 143, the average recovery (m + SD) for 5 male rats was 70.3 f 8.7%. No BIOLF-143 was detected in the feces within the limits of detection (0.2 ,ug/g). The administration of a total dose of 2000 mg BIOLF- 143/kg body wt in eight ip injections of 250 me/kg body wt at hourly intervals resulted in no overt toxicity or adverse signs in any of the animals other than slight discomfort and irritability. No mortality occurred up to 14 days post-treatment. The results of the tissue analyses at 1.O, 24, and 72

317

IN RATS

hr and 14 days post-treatment are shown in Table 2. At 1.0 hr after the eighth and final dose, the plasma levels of BIOLF-143 were not excessively high considering the fact that these animals had just received a cumulated total of 2000 mg/kg body wt of the agent. A considerable amount of drug was detected in the peritoneal fluid. High levels of BIOLF143 were measured in the livers at 1.Ohr posttreatment and extremely elevated concentrations were measured in the kidneys. The general appearance of these two tissues at necropsy was normal. An observation made while handling the animals for repeated injection was that a clear, almost colorless urine was eliminated which, when the liquid evaporated, left a white paste which was ascertained to be predominantly BIOLF- 143. By 24 hr post-treatment, BIOLF- 143 was not detected in the blood plasma. No peritoneal fluid remained and the drug levels in the liv-

si ii

WOLF-143

0

t

2.5

5.0

7.5

RETENTION

10.0

12.5

TIME

15.0

17.5

bin)

FIG. 4. A representative HPLC chromatogram of (A) a dilution ( 1:1000) of urine collected over 18 hr from a control, untreated, female Sprague-Dawley rat and(B) a dilution ( 1: 100) of the 18-hr urine sample collected from the same animal following treatment with BIOLF-143 (1CJowhg~yW

318

ECOBICHON

ET AL.

TABLE 2 RESIDUES OF BIOLF-

143 DETECTED

IN TISSUES OF MALE

RATS RECEIVING

EIGHT

CONSECUTIVE DOSES (250 mg/kg) AT HOURLY INTERVALS” Time post-treatment (hr) 1 24 12

Peritoneal fluid WmUb 116.la

92.5

Plasma Wml) 42.1 f 7.3

ND ND ND

14 Days

ND ND ND

Liver WP) 111.7 f 10.8 ND ND ND

Kidney b&g) 265.8

+ 54.9

ND ND ND

a The animals received BIOLF-143 by ip injection at a concentration of 250 mg/kg body wt at 60-min intervals until a total of eight doses had been administered, attaining a total concentration of 2000 mg/kg of drug administered. * The values shown are average residue concentrations f SD for the tissues of six animals euthanized at the posttreatment time intervals shown. ND indicates not detectable.

ers and kidneys had decreased below the level of detection. No drug could be detected in any tissues at subsequent time intervals. No untoward effects or latent toxicity was noted in any animals held for a 14-day post-treatment observation period. Light microscopic examination of stained sections of hepatic and renal tissue showed no evidence of morphological changes. DISCUSSION BIOLF- 143 is a member of a class of antiviral agents composed of pyrimidine- and putine-based acyclic nucleosides which includes such agents as Acyclovir and BIOLF-62 (BW B759U, 2-NDG, or DHPG), all of which exhibit potent inhibitory effects on human cytomegalovirus and herpes simplex viruses (Smith et al., 1982; Bach et al., 1985; Felsenstein et al., 1985; Gauntt et al., 1985; Shepp et al., 1985a,b). All of these agents are characterized by a relatively low order of toxicity accompanied by rapid distribution and elimination from both animals and man (deMiranda et al., 198 1, 1982; Blum et al., 1982; Fletcher et al., 1986). The results of the acute studies with BIOLF-143 demonstrated that this agent at-

tamed a rapid distribution in the body following either iv or ip administration (Fig. 2) and had a very short plasma half-life of 18-23 min for dosages up to 500 mg/kg body wt. There was a somewhat slower rate of disappearance of the agent from the liver and kidney (Table 1). Such a rapid rate of elimination is characteristic of an agent that is not extensively protein-bound, a fact confirmed by our in vitro plasma protein-binding studies. The major route of elimination of BIOLF- 143 is via the kidney and is characteristic of this class of agents. Both Acyclovir and BIOLF-62 are recovered unchanged in the urine in excess of 90% of the administered dose (deMiranda et al., 1982; Fletcher et al.. 1986). While no untoward signs of overt toxicity or latent secondary toxicity were observed in the rats receiving single or repeated doses of BIOLF- 143 up to 2000 mg/kg body wt, a potential target organ would appear to be the kidney because of the high levels of agent detected in this organ (Tables 1 and 2). Acyclovir, given by daily iv injection in subchronic tests, caused obstructive nephropathy in rats and dogs receiving high doses (Tucker, 1983). The rapid injection of such high levels caused precipitation of crystals in the renal tubules. In the present acute studies,

TOXICITY

OF BIOLF-143

BIOLF-143, administered iv at a dosage of 100 mg/kg body wt, was eliminated from the kidney within 3 hr (Table 1). The solubility of BIOLF- 143 in urine was such that the voided urine from animals receiving eight consecutive ip injections of 250 mg/kg body wt evaporated to a white paste which, on analysis, was shown to be BIOLF- 143. No drug was detectable in the renal tissue at 24 hr after treatment. The experiences with Acyclovir suggest that renal toxicity may be an important aspect of daily, single-dose, subchronic studies with BIOLF- 143. The administration of fractionated doses should minimize renal toxicity related to solubility and still permit the detection of chronic or cumulative toxicity in other tissues. The initial studies with BIOLF143 indicate that this antiviral drug should have limited toxicity if care is given to the rate of administration of the drug. Plasma levels attained by repeated injection should be above those concentrations needed in vitro for antiviral activity while decreasing the risk of renal toxicity. Our initial rodent studies indicate that BIOLF-143 has minimal toxicity and warrants further toxicity evaluation. In the present acute studies, there was no evidence of biotransformation of BIOLF143. Unidentified compounds in blood plasma, tissues, urine, and feces were not resolved by HPLC analysis, BIOLF- 143 being the only agent detected (Fig. 4). These results suggest that chemical modification by the addition of the N-dimethylaminomethylene substituent protected the nucleoside from enzymatic degradation. While 74-88% of the parent chemical could be accounted for in the urine collected overnight, the balance may be distributed throughout the body, necessitating the use of radiolabeled BIOLF- 143 to measure organ sequestration of low levels of the agent and/or metabolites formed. ACKNOWLEDGMENTS These studies were funded by the Medical Research Council of Canada (Grant SP-17). The technical assistance of Anne M. Comeau is gratefully appreciated.

319

IN RATS

REFERENCES BACH, M. C., BAGWELL, S. P., KNAPP, N. P., DAVIS, K. M., AND HEDSTROM, P. S. (1985). 9-( 1,3dihydro2-propoxy-methyl)guanine for cytomegalovhus infections in patients with the acquired immunodeficiency syndrome. Ann. Intern. Med. 103,381-382. BLUM, R. M., LIAD, S. H. T., AND DE MIRANDA, P. (1982). Overview of acyclovir pharmacokinetic clisposition in adults and children. Amer. J. Med. 73, 186192. COREY, L., NAHMAIS, A. J., GUINAN, M. E., BENEDETTI, J. K., CRITCHLOW, C. W., AND HOLMES, K. K. (1982). A trial of topical acyclovir in genital herpes simplex virus infections. N. Engl. J. Med. 306, 13 131319. COREY, L., AND HOLMES, K. K. (1983). Genital herpes simplex virus infections: Current concepts in diagnosis, therapy, and prevention. Ann. Intern. Med. 98, 973-983.

CRUMPACKER, C. S., SCHNIPPER,L. E., ZAIA, J. A., AND LEVIN, M. J. (1979). Growth inhibition by acycloguanosine of herpes viruses isolated from human infections. Antimicrob. Agents Chemother. 15, 642645.

DE MIRANDA, P., GOOD, S. S., LASKIN, 0. L., KRASNY, H. C., CONNOR, J. D., AND LIETMAN, P. S. (198 1). Disposition of intravenous radioactive acyclovir. Clin. Pharmacol. Ther. 30,662-672. DE MIRANDA, P., KRASNY, H. C., PAGE, D. A., AND ELION, G. B. (1982). Species differences in the disposition of acyclovir. Amer. J. Med. 73,3 l-35. FELSENSTEIN,D., D’AMICO, D. J., HIRSCH, M. S., NEUMEYER, D. A., CEDERBERG, D. M., DE MIRANDA, P., AND SCHOOLEY, R. T. (1985). Treatment of cytomegalovirus retinitis with 9-[2-hydroxy-l-(hydroxymethyl)ethoxymethyl]guanine. Ann Intern. Med. 103, 377-380.

FLETCHER, C., SAWCHUK, R., CHINNOCK, B., DE MIRANDA, P., AND BALFOUR, H. H. (1986). Human pharmacokinetics of the antiviral drug DHPG. Clin. Pharmacol. Ther. 40,28 l-286. GAUNTT, C. J., ARIZPE, H. M., KUNG, J. T., OGILVIE, K. K., AND CHERIYAN, U. 0. (1985). Antimyocarditic activity of the guanine derivative BIOLF-70 in a Coxsackevirus B3 murine model. Antimicrob. Agents Chemother. 27, 184- 19 1. MEYERS, J. D., FLOURNOY, N., AND THOMAS, E. D. (1980). Infection with herpes simplex virus and cellmediated immunity after marrow transplant. J. Infect. Dis. 142,338-346. OGILVIE, K. K., HANNA, H. R., NGUYEN-BA, N., AND SMITH, K. 0. (1985). N-substituted acyclopurinenucleosides with antiviral activity. Nucleosides and Nucleotides 4,507-5 13.

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ECOBICHON

SHEPP,D. H., DANDLIKER, P. S., DE MIRANDA, P., BURNETTE, T. C., CEDERBERG, D. M., DIRK, E., AND MEYERS, J. D. (1985a). Activity of 9-[2-hydroxy-l(hydroxymethyl)ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia. Ann Intern. Med. 103,368-373. SHEPP, D. H., NEWTON, B. A., DANDLIKER, P. S., FLOURNOY, N., AND MEYERS, J. D. (1985b). Oral acyclovir therapy for muco-cutaneous herpes simplex virus infections in immunocompromised marrow transplant recipients. Ann. Znt. Med. 102,783-785. SMITH, K. O., GALLOWAY, K. S., KENNELL, W. L., OGILVIE, K. K., AND RADATUS, B. K. (1982). A new nucleoside analog, 9-[[2-hydroxy- I -(hydroxymethyl)ethoxylmethyllguanine, highly active in vitro against herpes simplex virus type 1 and 2. Antimicrob. Agents Chemother. 22,55-6 1.

ET AL.

STFUUSS, S. E., SMITH, H. A., BRICKMAN, C., DE MIRANDA, P., MCLAREN, C., AND KEENEY, R. E. ( 1982). Acyclovir for chronic mucocutaneous herpes simplex virus infection in immunosuppressed patients. Ann. Intern. Med. 96,270-277. TUCKER, W., JR. (1982). Preclinical toxicology of acyclovir: An overview. Amer. J. Med. 73,27-30. WADE, J. C., NEWTON, B., MCLAREN, C., FLOURNOY, N., KEENEY, R. E., AND MEYERS, J. D. (1982). Intravenous acyclovir to treat mucocutaneous herpes simplex virus infection after marrow transplantation: A double-blind trial. Ann. Intern. Med. 96,265-269. WHITLEY, R. J., BLUM, M. R., BARTON, N., AND DE MIRANDA, P. (I 982). Pharmacokinetics of acyclovir in humans following intravenous administration: A mode1 for the development of parenteral antivirals. Amer. J. Med. 73, 165 17 1.