Gertrude B. Elion

A comparison of the specificities of xanthine oxidase and aldehyde oxidase

Abstract This study directly compares the specificities of the structurally similar hydroxylating enzymes, aldehyde oxidase and xanthine oxidase. Michaelis-Menten constants for a variety of substrates of xanthine oxidase were in general lower than those of aldehyde oxidase. With respect to the rates of oxidation, the basic similarity was a preference for compounds having a substituted pyrimidine ring structure. Outstanding among the differences were the effects of the number and position of the ring substituents. Both enzymes readily oxidized a variety of unsubstituted and C-monosubstituted heterocycles, but only xanthine oxidase readily oxidized C-disubstituted derivatives. Certain N-substitutions, however, enhanced substrate activity with aldehyde oxidase, but markedly decreased it with xanthine oxidase. Although both enzymes preferred oxo over amino substituents, there were some specificity differences with respect to the chemical nature of substituents. Aldehyde oxidase, but not xanthine oxidase, tolerated 6-substitution of purine by alkyl, halogeno, cyano, or methylthio groups, while 6-hydroxyl or 6-methylamino substituents were tolerated only by xanthine oxidase. The position at which oxidation occurred was influenced by both the chemical nature and the positions of substituents. With some purines a different site was initially hydroxylated by each enzyme.

Potentiation by inhibition of drug degradation : 6-substituted purines and xanthine oxidase

Abstract The administration of the xanthine oxidase inhibitor. 4-hydroxypyrazolo (3, 4-d)pyrimidine, concurrently with 6-mercaptopurine, results in a marked decrease in the metabolic oxidation of the latter to 6-thiouric acid in both the mouse and man. The inhibition of metabolic degradation by this means results in several-fold potentiations of 6-mercaptopurine, 6-methylthiopurine, 6-propylthiopurine, and 6-chloropurine in trials against adenocarcinoma 755 and of 6-mercaptopurine and 6-chloropurine as inhibitors of the immune response of mice to sheep erythrocytes.

Metabolic studies of allopurinol, an inhibitor of xanthine oxidase

Abstract The metabolic disposition of allopurinol [4-hydroxypyrazolo(3,4-d)-pyrimidine) was determined in mice, dogs, and human subjects. The drug is a substrate for, as well as an inhibitor of, xanthine oxidase and is converted in all species to the corresponding xanthine analog, alloxanthine, which is its major metabolite. Neither is bound to human plasma proteins, and both are distributed more or less equally in total body water in the mouse. Both analogs are cleared rapidly by the mouse and dog kidney. In the human subject allopurinol is cleared rapidly, but alloxanthine resembles uric acid in having a slow clearance responsive to probenecid. The accumulation of alloxanthine during prolonged therapy with allopurinol may contribute significantly to the therapeutic effects of the drug in the control of hyperuricemias.

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.

Renal clearance of oxipurinol, the chief metabolite of allopurinol

Abstract Oxipurinol (4,6-dihydroxypyrazolo(3,4-d)pyrimidine; alloxanthine) is the chief metabolite of allopurinol in vivo . Like allopurinol, although to a lesser degree, oxipurinol suppresses xanthine oxidase and lowers the serum and urinary uric acid levels; indeed, for reasons indicated, much of the effect of allopurinol may well be due to oxipurinol. Unlike allopurinol, but like uric acid, oxipurinol appears to be reabsorbed by the tubules, since its renal clearance is much less than the glomerular filtration rate in man and the mongrel dog, although it is not bound to plasma proteins. Moreover, uricosuric drugs increase the clearance of oxipurinol in man, but not in the Dalmatian dog. Oxipurinol apparently is not secreted by the tubules. In the presence of marked kidney damage the renal clearance of oxipurinol is impaired and the plasma oxipurinol concentration in patients given usual allopurinol dosages is elevated. Patients with marked renal insufficiency appear to exhibit a higher incidence of side reactions than do patients with normal renal function.

Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities

Abstract The inhibitor and substrate specificities of deoxythymidine (dThd) kinase purified from herpes simplex virus (HSV Type 1) were studied. A number of nucleosides and nucleoside analogs were phosphorylated by the virus coded enzyme. These included several compounds structurally related to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), a potent inhibitor of HSV replication. Some contained guanine with 9-substituents differing from that of acyclovir by methylene additions, methylene and thioether substitutions for the ether oxygen, and branching on the distal side of the ether oxygen. Others were various 2-substituted 6-hydroxypurines with the 9-(2-hydroxyethoxymethyl) substituent. A limitation of the specificity of the enzyme with guanine derivatives was the lack of phosphorylation of any derivative with an acyclic moiety branched on the proximal side of the ether oxygen. Many of the compounds that were phosphorylated were subsequently found to inhibit HSV replication. Such compounds apparently inhibited HSV replication via the same route of activation previously described for acyclovir [G. B. Elion, P. A. Furman, J. A. Fyfe, P. de Miranda, L. Beauchamp and H. J. Schaeffer, Proc. natn. Acad. Sci. U.S.A. 74 , 5716 (1977)]. Moreover, several compounds not phosphorylated by the enzyme did not inhibit replication. However, some other acyclic nucleoside analogs that were phosphorylated were not good antivirals, indicating that phosphorylation catalyzed by the HSV dThd kinase was not sufficient for inhibition of viral replication to occur. These results emphasize the importance of the specificity of cellular kinases and the HSV DNA polymerase to the mechanism of antiviral activity. The dThd kinase from Vero cells was also purified. With this host cell enzyme, kinetic constants of known antiviral compounds were determined and compared to those of dThd (relative V ′ max ; k ′ m ): dThd (100; 1.3 μM), 5-iodo-2′-deoxyuridine (87; 1.8 μM), 5-trifluoromethyl-2′-deoxyuridine (91; 1.2 μM), 5-bromo-2′-deoxycytidine (5; 580 μM), and 9-β- d -arabinofuransoylthymine (23; 2300 μM). None of the purine acyclic nucleoside analogs tested (at 1000 μM) was detectably phosphorylated by the Vero cell enzyme, and all had apparent K i values >300 μM. The phosphorylation catalyzed by host cell dThd kinase correlated with the toxicity of some pyrimidine nucleoside analogs.

Phase I Study of 506U78 Administered on a Consecutive 5-Day Schedule in Children and Adults With Refractory Hematologic Malignancies

PURPOSE A phase I study was conducted to determine the maximum-tolerated dose (MTD), toxicity profile, and pharmacokinetics of a novel purine nucleoside, nelarabine, a soluble prodrug of 9-beta-D-arabinosylguanine (araG; Nelarabine), in pediatric and adult patients with refractory hematologic malignancies. PATIENTS AND METHODS Between April 1994 and April 1997, 93 patients with refractory hematologic malignancies were treated with one to 16 cycles of study drug. Nelarabine was administered daily, as a 1-hour intravenous infusion for 5 consecutive days, every 21 to 28 days. First-cycle pharmacokinetic data, including plasma nelarabine and araG levels, were obtained on all patients treated. Intracellular phosphorylation of araG was studied in samples of leukemic blasts from selected patients. RESULTS The MTDs were defined at 60 mg/kg/dose and 40 mg/kg/dose daily x 5 days in children and adults, respectively. Dose-limiting toxicity (DLT) was neurologic in both children and adults. Myelosuppression and other significant organ toxicities did not occur. Pharmacokinetic parameters were similar in children and adults. Accumulation of araGTP in leukemic blasts was correlated with cytotoxic activity. The overall response rate was 31%. Major responses were seen in patients with T-cell malignancies, with 54% of patients with T-lineage acute lymphoblastic leukemia achieving a complete or partial response after one to two courses of drug. CONCLUSION Nelarabine is a novel nucleoside with significant cytotoxic activity against malignant T cells. DLT is neurologic. Phase II and III trials in patients with T-cell malignancies are encouraged.

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with Herpesvirus genetic information

Abstract Thymidine kinase-deficient LM cells (LMTK−-) biochemically transformed to the TK+ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK+ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.

Therapeutic efficacy of the topoisomerase I inhibitor 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin against pediatric and adult central nervous system tumor xenografts

Abstract Therapy of patients with malignant central nervous system tumors is frequently unsuccessful, reflecting limitations of current surgical, radiotherapeutic, and pharmacotherapeutic treatments. The camptothecin derivative irinotecan (CPT-11) has been shown to possess antitumor activity in phase II trials for patients with carcinoma of the lung, cervix, ovary, colon, or rectum and for patients with non-Hodgkin’s lymphoma. The current study was designed to test the efficacy of the drug against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies. Tumors included childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D341 Med, D487 Med), ependymomas (D528 EP, D612 EP), and a rhabdomyosarcoma (TE-671), as well as sublines with demonstrated resistance to busulfan (D-456 MG (BR)), cyclophosphamide (TE-671 CR), procarbazine (D-245 MG (PR)) or melphalan (TE-671 MR), growing subcutaneously and intracranially in athymic nude mice. In replicate experiments, CPT-11 was given at a dosage of 40 mg/kg per dose via intraperitoneal injection in 10% dimethylsulfoxide on days 1–5 and 8–12, which is the dosage lethal to 10% of treated animals. CPT-11 produced statistically significant (P<0.001) growth delays in all subcutaneous xenografts tested, including those resistant to busulfan, cyclophosphamide, procarbazine, and melphalan, with growth delays ranging from 21.3 days in D487 Med to 90+ days in several tumor lines. Further, tumor regression was evident in every treated animal bearing a subcutaneous tumor, with some xenografts yielding complete tumor regression. Statistically significant (P<0.001) increases in survival were demonstrated in the two intracranial xenografts – D341 EP (73.0% increase) and D-456 MG (114.2% increase) – treated with CPT-11. These studies demonstrate that, of over 40 drugs evaluated in this laboratory, CPT-11 is the most active against central nervous system xenografts and should be advanced to clinical trial as soon as possible.

Studies on analogs of purines and pyrimidines.

A study was begun in these laboratories, in 1942, of the relationship between chemical structure and the ability of certain pyrimidine* derivatives to serve as precursors for or to modify nucleic acid synthesis. Since only brief accounts of small portions of this work have been published to the present paper is to be regarded as a preliminary report of the work as n whole. It was felt that such studies might lead to fundamental knowledge of the roles of pyrimidine and purine bases in growth, and of the part played by folk acid in the synthesis of these bases. It was felt also that new chemotherapeutic agents might be discovered by this means since, it was argued, parasitic tissues in general depend for survival on a more rapid growth, hence a more rapid synthesis of nucleic acid, than that of the host tissues. This argument applies equally well to bacterial, viral, rickettsial, and neoplastic diseases, so that, in a sense, one might say we have been searching for the philosopher’s stone, the universal panacea, of the ancients. A distinct advantage of antipurines and antipyrimidines as chemotherapeutic agents seemed to lie in the fact that the requirements of bacteria, at least, appeared to be qualitatively different from those of mammalian tissues. A considerable number of microorganisms6 were known to require preformed pyrimidines and/or purines for growth, whereas the evidence available a t that time indicated that purine and pyrimidine bases played no role in mammalian nucleic acid synthesis.6 In the interim, of course, it has become known that two purines, adenine7rR and 2,6-diaminop~rine,~ do contribute to the nucleic acid purine of mammalian tissues, and the hypothesis becomes subject to modification on this account. The effect of thymine in nutritional macrocytic anemia, sprue, and pernicious anemialo indicates that some metabolic role may have to be postulated for this pyrimidine base in the face of the studies with isotopically tagged thymine, which indicated only a catabolic elimination and no retention of exogenous thymine.6 It is conceivable that guanine, uracil, and cytosine may have metabolic roles which remain undetected in similar studies because of low turnover rates or for other reasons. The choice of Lactobacillus casei as a model biological system for the study of pyrimidine analogs was based on the known requirement of this microorganism for folk acid and the role of thymine and guanine in the satisfaction of this growth requirement:‘ This allows a study of the activity of each substance in a number of different ways in the same microorganism. TABLE 1 shows the six media used for study and the effects of various substances in each of the media. The media contain thymine or