The purine path to chemotherapy.

Research on antimetabolites of nucleic acid purines led to drugs for the treatment of acute leukemia (6-mercaptopurine and thioguanine), gout and hyperuricemia (allopurinol), and herpesvirus infections (acyclovir), and for the prevention of organ transplant rejection (azathioprine).

Increased melphalan activity in intracranial human medulloblastoma and glioma xenografts following buthionine sulfoximine-mediated glutathione depletion.

In previous studies we demonstrated that administration of buthionine sulfoximine (BSO) to athymic BALB/c mice bearing intracranial human glioma xenografts resulted in highly selective depletion of glutathione in neoplastic tissue with minimal effects on contralateral normal brain tissue. In the present study we treated athymic BALB/c mice bearing intracranial human glioma (D-54 MG) or medulloblastoma (TE-671) xenografts with melphalan alone or BSO followed by melphalan. Administration of BSO depleted intracellular glutathione to 7.5% of the control level. BSO plus melphalan resulted in a significant increase in median survival over that produced by melphalan alone: 45.3% versus 26.4% in TE-671 and 69% versus 27.6% in D-54 MG. These studies justify further efforts to modulate chemotherapeutic and radiotherapeutic interventions of primary malignant brain tumors by depletion of glutathione.

Combination chemotherapy in vitro exploiting glutamine metabolism of human glioma and medulloblastoma.

The human glioma-derived cell line D-54 MG and the human medulloblastoma-derived cell line TE-671 have been shown to be sensitive in culture to the pharmacological interference with glutamine metabolism by acivicin, 6-diazo-5-oxo-L-norleucine, and methionine sulfoximine. Using as a guide the multiple contributions of glutamine to the biosynthesis of proteins, purines, and pyrimidines, we now have identified six additional antimetabolites active against these lines in vitro at clinically relevant concentrations. The 50% growth-inhibitory levels of the drugs against D-54 MG in 6-day continuous exposure experiments were: L-asparaginase, 0.057 IU/ml; 5-fluorouracil, 0.5 micrograms/ml; 6-mercaptopurine, 0.8 micrograms/ml; actinomycin D, 0.0007 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 2.3 micrograms/ml; and 5-azacytidine, 0.2 micrograms/ml (3-day exposure. The corresponding 50% growth-inhibitory values in TE-671 were: L-asparaginase, 0.54 IU/ml; 5-fluorouracil, 1.5 micrograms/ml; 6-mercaptopurine, 4.7 micrograms/ml; actinomycin D, 0.00044 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 4.5 micrograms/ml; and 5-azacytidine, 0.49 micrograms/ml. Dipyridamole up to 10 micrograms/ml was inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, 0.40; 6-diazo-5-oxo-L-norleucine plus methionine sulfoximine, 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings suggest that an antimetabolite regimen exploiting glutamine sensitivity might improve the chemotherapy of some human gliomas and medulloblastomas.

Influence of glutamine on the growth of human glioma and medulloblastoma in culture.

Cellular supply of glutamine, an essential substrate for growth, is derived from extracellular fluid and de novo synthesis. We investigated the relative importance of these sources to the growth of six human anaplastic glioma- and one human medulloblastoma-derived permanent cell lines. Exogenous glutamine was limiting for the proliferation of glioma-derived lines D-54 MG, U-118 MG, and U-251 MG. In contrast, medulloblastoma-derived line TE-671 and glioma-derived lines U-373 MG, D-245 MG, and D-259 MG grew in the absence of supplemental glutamine. Two cell lines with contrasting glutamine requirements, D-54 MG and TE-671, were used to explore the pharmacological interference with glutamine metabolism. DL-alpha-Aminoadipic acid, a reported glutamic acid analogue with gliotoxic properties, significantly inhibited the growth of both lines. These effects were reversed by increasing glutamine, suggesting that the major action of DL-alpha-aminoadipic acid is as a glutamine antagonist. In contrast, the glutamine synthetase inhibitor delta-hydroxylysine demonstrated activity only against TE-671. Acivicin and 6-diazo-5-oxo-L-norleucine, glutamine analogues available for clinical use, reduced the proliferation of both cell lines at pharmacological concentrations. Methionine sulfoximine, a glutamine synthetase inhibitor previously used clinically, produced marked growth inhibition only against TE-671. These findings indicate that the synthesis and utilization of glutamine are potentially exploitable targets for the chemotherapy of some human gliomas and medulloblastomas.

Therapeutic analysis of melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR.

Investigations with the melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR were carried out to identify patterns of cross-resistance and collateral sensitivity and to define the mechanism(s) mediating melphalan resistance. TE-671 MR was cross-resistant to thio-TEPA, mitomycin, vincristine, and cisplatin, and partially resistant to chlorambucil and cyclophosphamide. TE-671 MR and the parent line TE-671 were both resistant to 1,3-bis(2-chloroethyl)-nitrosourea and expressed similar levels of O6-alkylguanine-DNA alkyltransferase. TE-671 MR retained full sensitivity to actinomycin D and demonstrated enhanced sensitivity to VP-16 compared to TE-671. Treatment of TE-671 MR with melphalan plus VP-16 resulted in greater than additive growth delays. The frequency of hypoxic regions was similar in TE-671 MR and TE-671, respectively. Measurement of tumor-to-plasma levels at 180 min following i.p. administration of melphalan at 0.5 of the 10% lethal dosage showed mean tumor-to-plasma ratios of 3.81 in TE-671 MR and 7.38 in TE-671, respectively. The lower drug levels in TE-671 MR may be contributing to the resistance to melphalan and thus indicate the need for further studies to define the reasons for these differences in tumor drug level.

Establishment of a melphalan-resistant rhabdomyosarcoma xenograft with cross-resistance to vincristine and enhanced sensitivity following buthionine sulfoximine-mediated glutathione depletion.

A melphalan-resistant human rhabdomyosarcoma xenograft, TE-671 MR, was established in athymic mice by serial melphalan treatment of the parent xenograft, TE-671, at the 10% lethal dosage (LD10); significant resistance was evident after ten passages of the tumor. TE-671 MR demonstrated a doubling time of 3.5 days and a latency period to 1000-mm3 tumors of 27.5 days. The glutathione level of TE-671 MR was 2.36 mumol/g tumor, wet weight, 2-fold higher than the parent line. The glutathione S-transferase activity of TE-671 MR was 117.8 mumol/min/mg protein, essentially unchanged from the parent line. Although TE-671 MR demonstrated cross-resistance to vincristine, dot blot analysis did not reveal an elevated expression of mdr1 mRNA in the resistant line. TE-671 MR demonstrated a 9.7-day growth delay following treatment with melphalan at the LD10 (compared to 20.9 days for the parent line). Treatment with L-buthionine-SR-sulfoximine (BSO) resulted in increased sensitivity to melphalan subsequently administered at 50% of the LD10 (melphalan alone, growth delays of 3.7 and 4.6 days in duplicate trials; melphalan plus BSO, growth delays of 7.2 and 9.8 days). Sensitivity to melphalan equal to that of the parent line TE-671 was not achieved, however. Treatment with BSO did not result in significantly enhanced sensitivity to subsequently administered vincristine (50% of the LD10) (vincristine alone, growth delays of 6.8 and 6.9 days in duplicate trials; vincristine plus BSO, growth delays of 10.9 and 7.5 days). These results suggest that generation of melphalan resistance may be associated with development of cross-resistance to vincristine; this resistance may be associated with (although not necessarily mediated by) glutathione elevation; this resistance may be partially overcome by BSO-mediated depletion of glutathione.

Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in a human medulloblastoma xenograft in athymic mice.

The effect and therapeutic consequences of buthionine-(SR)-sulfoximine (BSO)-mediated depletion of glutathione in the human medulloblastoma-derived cell line, TE-671, growing as s.c. xenografts in athymic nude mice were examined. The glutathione content of the s.c. xenografts was 1.11 +/- 0.15 mumol/g (7.79 +/- 1.61 nmol/mg of protein). Administration i.p. to tumor-bearing mice of D,L-BSO (two doses at 12-h intervals; 5 mmol/kg) depleted the glutathione content of the xenografts to 25.7% of control. Administration of a 30 mM solution of L-BSO in drinking water for 96 h depleted the glutathione content to 17.4% of control. Depletion of glutathione with these regimens resulted in a significant increase in the s.c. tumor growth delay over that produced by melphalan alone: 17.2 days versus 12.6 days for D,L-BSO (i.p.) plus melphalan versus melphalan and 22.9 days versus 16.6 days for L-BSO (p.o.) plus melphalan versus melphalan. These studies demonstrate the increased cytotoxicity of melphalan resulting from BSO-mediated depletion of glutathione in human medulloblastoma and support further efforts to modulate the chemosensitivity and radiosensitivity of this tumor by modulation of glutathione.

Experimental chemotherapy of human medulloblastoma cell lines and transplantable xenografts with bifunctional alkylating agents.

A series of bifunctional alkylators were tested against the genotypically and phenotypically heterogeneous continuous human medulloblastoma cell lines, TE-671, Daoy, and D283 Med in vitro and against TE-671 and Daoy growing as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (in vivo), and busulfan (in vivo). Melphalan and phenylketocyclophosphamide were the most active agents in vitro with drug doses at which there is a 90% reduction in the number of colonies in comparison to controls of 2.13, 5.29, and 4.72 microM for melphalan and 4.60, 5.01, and 4.34 microM for phenylketocyclophosphamide against TE-671, D283 Med, and Daoy, respectively. Melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, and thiotepa produced significant growth delays against s.c. TE-671 and Daoy xenografts, while no activity could be demonstrated for 1,3-bis(2-chloroethyl)-1-nitrosourea or busulfan. Melphalan, cyclophosphamide, iphosphamide, and thiotepa also produced significant increases in median survival in mice bearing intracranial TE-671 and Daoy xenografts. These results extend our previous studies demonstrating the antitumor activity of nitrogen and phosphoramide mustard-based bifunctional alkylating agents in the treatment of human medulloblastoma continuous cell lines and transplantable xenografts, and support the continued use of these agents in clinical trials.

Synergistic interactions between cyclophosphamide or melphalan and VP-16 in a human rhabdomyosarcoma xenograft.

Based on previous work demonstrating the activity of cyclophosphamide and melphalan in a series of human medulloblastoma and rhabdomyosarcoma cell lines and transplantable xenografts, investigations were conducted to define the effects of combining cyclophosphamide or melphalan with VP-16. These studies demonstrated a synergistic interaction between cyclophosphamide and VP-16 and melphalan and VP-16 in the treatment of the human rhabdomyosarcoma cell line TE-671 growing in athymic mice. The combination of cyclophosphamide or melphalan with VP-16 may warrant consideration as a therapeutic strategy for solid tumors sensitive to bifunctional alkylating agents.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Schedule-dependent activity of temozolomide plus CPT-11 against a human central nervous system tumor-derived xenograft.

Temozolomide, an imidazole tetrazinone, and CPT-11, a camptothecin derivative, have previously been shown to have anti-central nervous system tumor activity in laboratory and clinical studies. The current experiments were designed to evaluate the activity of temozolomide plus CPT-11 against a malignant glioma-derived xenograft, D-54 MG, growing s.c. in athymic nude mice. The initial schedule of i.p. drug administration was temozolomide at 0.1 LD10 on day 1 and CPT-11 at 0.1 LD10 on days 1-5 and 8-14. The combination of these two agents produced greater than additive activity against D-54 MG. This enhanced activity was maintained when the initial administration of CPT-11 was delayed to day 3 or day 5. However, when CPT-11 was administered first on day 1 using 0.5 LD10 (for the single dose schedule) followed by temozolomide (0.1 LD10) 5 h, 3 days, or 5 days later, the enhancement of activity was substantially reduced. These results demonstrate that the combination of temozolomide plus CPT-11 displays a schedule-dependent enhancement of antitumor activity, suggest a mechanistic explanation for the enhanced activity, and provide the rationale for a Phase I trial of this regimen.

Acyclovir kinetics after intravenous infusion.

The disposition and safety of the antiviral drug acyclovir were studied in 14 subjects with advanced malignancies. Acyclovir was administered by a 1-hr intravenous infusion at doses of 0.5, 1.0, 2.5, and 5.0 mg/kg. At the end of infusion, mean peak plasma levels (+/- SEM), determined by radioimmunoassay, were 6.4 +/- 0.7, 12.1 +/- 2.3, 14.9 +/- 2.7, and 33.7 +/- 7.1 microM. The plasma concentration-time profiles could be described by a biexponential equation. The half-life of acyclovir in the slow disposition phase ranged from 2.2 to 5 hr and the drug was detected in the plasma for at least 18 hr after infusion. The total body clearance ranged from 117 to 396 ml/min/1.73 m2. A proportionality between area under the curve and dose suggests that acyclovir exhibits dose-independent kinetics in the dose range studied. There was wide variation in cumulative urinary excretion of unchanged drug, ranging from 30 to 69% of the dose. From renal clearances of acyclovir, which were higher than creatinine clearances, it appears that both glomerular filtration and tubular secretion contribute to its renal excretion. Analysis of the urine by reverse-phase high-performance liquid chromatography revealed the presence of the metabolite 9-carboxymethoxymethylguanine. There was no indication of toxicity either clinically or from laboratory findings in any of the study subjects. This study demonstrates that in addition to selectivity and low toxicity, the kinetic profile and metabolic disposition of acyclovir make it an attractive candidate for therapy in a variety of herpes infections.

Mechanism of action and selectivity of acyclovir.

Acyclovir, an acrylic purine nucleoside analog, is a highly potent inhibitor of herpes simplex virus (HSV), types 1 and 2, and varicella zoster virus, and has extremely low toxicity for the normal host cells. This selectivity is due to the ability of these viruses to code for a viral thymidine kinase capable of phosphorylating acyclovir to a monophosphate; this capability is essentially absent in uninfected cells. The acyclovir monophosphate (acyclo-GMP) is subsequently converted to acyclovir triphosphate (acyclo-GTP) by cellular enzymes. Acyclo-GTP persists in HSV-infected cells for many hours after acyclovir is removed from the medium. The amounts of acyclo-GTP formed in HSV-infected cells are 40 to 100 times greater than in uninfected Vero cells. Acyclo-GTP acts as a more potent inhibitor of the viral DNA polymerases than of the cellular polymerases. The DNA polymerases of HSV-1 and HSV-2 also use acyclo-GTP as a substrate and incorporate acyclo-GMP into the DNA primer-template to a much greater extent than do the cellular enzymes. The viral DNA polymerase binds strongly to the acyclo-GMP-terminated template, and in thereby inactivated.

Effect of acyclovir combined with other antiherpetic agents on varicella zoster virus in vitro.

The sensitivity of varicella zoster virus (VZV) strain Ellen to acyclovir in combination with other antiherpetic agents in vitro has been examined by the plaque-reduction and infectious center assay methods. (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), trifluorothymidine (TFT), or 5-iodo-2'-deoxyuridine (IdUrd) produced, in general, additive antiviral activity when examined by either assay method. Acyclovir in combination with phosphonoacetate (PAA) resulted in additive effects as measured by the plaque-reduction assay. The combination of acyclovir with 9-beta-D-arabinofuranosyladenine (ara-A) gave generally additive or occasionally synergistic antiviral activity when tested in MRC-5 or WI-38 cells respectively, regardless of assay method.

Effect of acyclovir on various murine in vivo and in vitro immunologic assay systems.

In two in vitro tests, lymphocyte-mediated cytotoxicity and neutrophil chemotaxis, acyclovir showed no inhibitory effects at concentrations as high as 600 microM. The compound inhibited rosette formation with nonimmune mouse lymphocytes in vitro by approximately 50 percent at 15.8 microM. The significance of this inhibition is unclear. In four in vivo tests in mice which measured humoral and cell-mediated immunity (complement-dependent cellular cytotoxicity, complement-independent cellular cytotoxicity, delayed hypersensitivity and graft versus host reaction) acyclovir showed no inhibitory effects at single doses up to 200 mg/kg given on day 2 after antigenic stimulation. Four daily doses of acyclovir at 50 mg/kg per day had no effect on the numbers of hemolytic IgM antibody-forming cells in the spleen when assayed on day 4. At the higher dosage of 100 mg/kg per day for four days, there was a slight reduction in the numbers of these cells. There was no significant decrease in hemagglutinin or hemolysin antibody titers after four daily doses of acyclovir up to 200 mg/kg.

Species differences in the disposition of acyclovir.

The disposition of acyclovir was investigated in several species. The drug was well distributed into all the tissues, including the brain, in mice and rats. Binding of acyclovir to plasma proteins was 36 percent or lower. After intravenous bolus dosing (20 mg/kg) in dogs, the plasma acyclovir concentration-time profile, determined by radioimmunoassay, showed a biphasic decline with a half-life in the elimination phase of 2.3 +/- 0.1 hours. The volume of distribution (Vd beta) of 1.2 +/- 0.2 liters/kg indicated distribution of drug into the tissues. Marked species differences were observed in the gastrointestinal absorption and biotransformation of acyclovir. Oral dosing produced better absorption in dogs and mice than in rats and rhesus monkeys. More than 95 percent of the radioactivity in the urine derived from a 14C-acyclovir parenteral dose was recovered as the unchanged drug in mice, rats, and dogs. Minor urinary metabolites were characterized by high-performance liquid chromatography as 9-carboxymethoxymethylguanine (CMMG) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine. In other species--guinea pig, rabbit, and rhesus monkey--up to 40 percent of the radioactivity in the urine consisted of these metabolites.

Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus.

A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 microM acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay. The capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovir-sensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumable due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.

Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus.

Acyclovir inhibited the replication of a varicella-like simian virus (DHV-1) in cell culture (Vero cells) with an ED50 of 38 +/- 2 microM. The activation of acyclovir in this cell culture system was compared with that in the cell system with human varicella zoster virus (VZV). Extracts of cells infected with DHV-1 catalyzed the phosphorylation of acyclovir. The phosphorylation was inhibited by dThd, suggesting the catalyst was a dThd kinase. Electrophoresis of cytosol fractions on polyacrylamide gels corroborated the existence of a virus-associated dThd kinase. This enzyme copurified with an acyclovir-phosphorylating activity. The enzyme catalyzed the phosphorylation of acyclovir at a greater relative rate than that with the VZV enzyme, but with a higher apparent Km value for acyclovir. The relative efficiencies for the two enzymes with acyclovir were similar. Anabolic studies with cells infected with DHV-1 and incubated with [14C]acyclovir indicated that triphosphate of acyclovir did accumulate. The results indicate that acyclovir is activated in cells infected with DHV-1 in a manner similar to that in cells infected with VZV.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 2. Synthesis and activity of some nucleosides of 4-(alkylamino)-1H-pyrazolo[3, 4-d]pyrimidines.

A series of 4-(alkylamino)-1-beta-D-ribofuranosyl-1H-pyrazolo[3, 4-d]pyrimidines was synthesized by enzymatic and chemical methods. On the basis of the previous finding that 4-(alkylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidines were effective anticoccidial agents, this series was examined for efficacy against Eimera tenella in chicks. The most active anticoccidial agent in the present study was the 4-cyclopentylamino derivative (8), which cleared chicks of the parasite at 200 ppm in the diet. Some members of this series were toxic to embryonic chick liver cells, mouse cells, and human cells in vitro. The 4-diethylamino derivative (16), which was not toxic in vitro, appeared to be toxic in chicks.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 1. Synthesis and activity of some nucleosides of purines and 4-(alkylthio)pyrazolo[3,4-d]pyrimidines.

The finding that 6-(methylthio)-9-beta-D-ribofuranosyl-9H-purine (6) was more toxic to the avian coccidium, Eimeria tenella, than to embryonic chick liver host cells in vitro prompted the synthesis and testing of analogues of this compound. It was revealed that the beta-D-ribofuranosyl moiety was an important structural feature and that several types of 2-substituents in the purine ring decreased efficacy, as did 3-deaza and 8-aza ring modifications of 6. In contrast, the pyrazolo[3,4-d]pyrimidine analogue of 6 (24) was an order of magnitude more active. Moreover, this analogue was 24-fold less toxic to the host cells than was 6. A series of 4-(alkylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidines was prepared from 4-mercapto-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (23) and various alkyl halides. The most effective compound in this series in vivo, 4-(ethylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (25), cleared chicks of the parasite at 50 ppm in the diet and was much less toxic than was 24.