(CANCER RESEARCH 49. 3696-3701. July I. 1989] Biochemically Directed Therapy of Leukemia with Tiazofurin, a Selective Blocker of Inosine 5'-Phosphate Dehydrogenase Activity1 Guido J. Tricot,2 Hiremagalur N. Jayaram, Elizabeth Lapis, Yutaka Natsumeda, Craig R. Nichols, Pat Kneebone, Nyla Heerema, George Weber, and Ronald Hoffman Division of Hematology/Oncology, Department of Medicine [G. J. T., C. R. N., P. K., R. H.]; Laboratory for Experimental Oncology [H. N. J., E. L., Y. N., G. W.]: and Department of Medical Genetics /A". //./ and the Indiana Elks Cancer Research Center f(i. J. T., R. H.¡,Indiana University School of Medicine, Indianapolis, Indiana 46223 ABSTRACT mology, and biochemistry of cancer cells (5, 6). In various cancer cells there is a marked enzymic imbalance Tiazofurin (2-/3-i>-ribofuranosylthiazole-4-carboxamide, NSC 286193), in purine metabolism, with strong evidence indicating up-reg- a selective inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), ulation of the capacity for guanylate biosynthesis (5,6). Because the rate-limiting enzyme of de novo GTP biosynthesis, provided in end the activity of IMPDH, the rate-limiting enzyme of GTP bio stage leukemic patients a rapid decrease of IMP dehydrogenase activity synthesis, was increased in all examined cancer cells, it was and GTP concentration in the blast cells and a subsequent decline in blast cell count. Sixteen consecutive patients with end stage acute non- suggested that IMPDH should be a sensitive target of chemo lymphocytic leukemia or myeloid blast crisis of chronic granulocytic therapy (5-7). On the basis of this argument, a number of leukemia were treated with tiazofurin. Allopurinol was also given to compounds were prepared to block IMPDH activity, among inhibit xanthine oxidase activity to decrease uric acid excretion and to them tiazofurin (8), a C-nucleoside that is metabolized in two elevate the serum concentration of hypoxanthine, which should compet enzymic steps to thiazole-4-carboxamide adenine dinucleotide, itively inhibit the activity of hypoxanthine-guanine phosphoribosyltrans- an NAD analogue that selectively blocks IMPDH activity (9, ferase (EC 2.4.2.8), the salvage enzyme of guanylate synthesis. Assays 10). Tiazofurin in tissue culture and in the murine system of IMP dehydrogenase activity and GTP concentration in leukemic cells potently inhibited IMPDH activity, depressed GTP and dGTP provided a method to monitor the impact of tiazofurin and allopurinol concentrations, and yielded cytotoxicity and inhibition of tumor and to adjust the drug doses. In this group of patients with poor prognosis, cell proliferation (10, 11). Recent evidence indicated that, in five attained a complete hematological remission and one showed a hematological improvement. A marked antileukemic effect was seen in addition to an increase in IMPDH activity, the activity of the two other patients. All five évaluablepatients with myeloid blast crisis guanine salvage enzyme HGPRT was also elevated in human of chronic granulocytic leukemia reentered the chronic phase of their leukemic cells (12-14). When human myeloid leukemic cells disease. Five patients with acute nonlymphocytic leukemia were refrac were incubated with radiolabeled tiazofurin, over 20-fold higher tory to tiazofurin and three were unevaluable for hematological effect concentrations of thiazole-4-carboxamide adenine dinucleotide because of early severe complications. Responses with intermittent 5- to were produced than in bone marrow cells from healthy volun 15-day courses of tiazofurin lasted 3-10 months. Tiazofurin had a clear teers (15, 16). These results provided support for the consider antiproliferative effect, but the pattern of hematological response indi ation of tiazofurin in the treatment of myelocytic leukemia. cated that it appeared to induce differentiation of leukemic cells. In spite of tnvidt) with severe or life-threatening complications in 11 of 16 Inhibition of IMPDH activity by tiazofurin in blast cells should decrease GTP and dGTP concentrations, which should restrain patients, tiazofurin was better tolerated in most patients than other antileukemic treatment modalities and provided a rational, biochemically the biosynthesis of DNA. Moreover, the blocking of guanylate targeted, and biochemically monitored chemotherapy which should be of biosynthesis might critically limit the availability of GTP for interest in the treatment of leukemias and as a paradigm in enzyme expression of the ras oncogene, G protein function, and the pattern-targeted chemotherapy. biosynthesis of specific proteins (Fig. 1). This approach was strengthened by the observation that the guanine-salvaging activity of HGPRT can be competitively inhibited by hypoxan INTRODUCTION thine and that the concentration of hypoxanthine can be mark edly increased in the plasma by allopurinol, originally given to Conventional antileukemic chemotherapy in relapsed or re fractory ANLL' patients is seldom curative and remissions, if block xanthine oxidase activity to decrease uric acid excretion (14). Therefore, to attain an optimal impact leading to depletion attained, are almost always of short duration. Myeloid blast of GTP in the blast cells, simultaneous administration of tia crisis of CGL is even more resistant to chemotherapy, with zofurin and allopurinol is essential. Since the action of tiazo complete hematological remission rates varying from 4 to 30% and median survivals being 6-29 weeks (1-4). A primary goal furin and allopurinol is well characterized, the biochemical impact of these drugs can be monitored during treatment by of antileukemic therapy in these patients with poor prognosis frequently sampling leukemic cells for measurement of IMPDH should be the identification of agents that are more selective activity and GTP concentrations. and better targeted in their actions. Such therapy should be based upon our increasing understanding of the biology, enzy- This paper reports our biochemically directed trial in patients with ANLL and myeloid CGL-BC, where tiazofurin rapidly Received 10/17/88: revised 3/30/89; accepted 4/6/89. brought down IMPDH activity and then GTP concentrations, The costs of publication of this article were defrayed in part by the payment followed by a decline in the blast cell count. of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS, National Cancer Institute. Outstanding Investigator Grant CA-42510 and Grant SO7 RR-5371 to G.W. MATERIALS AND METHODS 1To whom requests for reprints should be addressed, at the Division of Hemalology/Oncology, 541 Clinical Drive. Room 379, Indiana University School Patients. Sixteen consecutive patients were treated with tiazofurin at of Medicine. Indianapolis. IN 46223. 'The abbreviations used are: ANLL, acute nonlymphocytic leukemia; Indiana University Medical Center from March 1987 to April 1988. IMPDH, inosine 5'-phosphate dehydrogenase; CGL, chronic granulocytic leu The characteristics of the patients are listed in Table 1. This Phase I/ kemia: CGL-BC, blast crisis of chronic granulocytic leukemia; HGPRT, hypo II trial was monitored by the Cancer Therapy Evaluation Program, xanthine-guanine phosphoribosyltransferase: MDS, myelodysplastic syndrome. Division of Cancer Treatment, National Cancer Institute (Bethesda, 3696 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. TIAZOFURIN TREATMENT OF LEUKEMIA according to the principles of the Declaration of Helsinki. The protocol had been reviewed and approved by the Investigational Review Board of the Indiana University School of Medicine. Biochemical Assays. Preparation of bone marrow and peripheral blood mononuclear cells and determination of the concentration of GTP were carried out as reported (15). Briefly, mononuclear cells were separated by a Ficoll-Paque density gradient. An aliquot of cells was then extracted with 10% trichloracetic acid, neutralized immediately with tri-n-octylamine in Freon, and analyzed by high pressure liquid chromatography for GTP concentrations, using a buffer system of ammonium phosphate. Another aliquot of cells was assayed directly for IMPDH activity as described (20). During treatment with tiazofurin, IMPDH activity and GTP pools in leukemic cells were measured at least 3 times/day in peripheral blood samples. Chromosome Preparation and Analysis. Cytogenetic investigations were performed on bone marrow or peripheral blood using direct analysis, short term incubation for 24 li. or culturing without phyto- hemagglutinin for 48 h. Chromosome preparations were studied at >400-band level (21). Drug and Administration. Tiazofurin in sterile injectable form was obtained from the National Cancer Institute (Bethesda, MD). The drug was administered daily, over a period of 1 h, by an infusion pump (Imed Fig. I. Pathways of IMP and GTP metabolism and the biochemical basis for the chemotherapeutic impact of tiazofurin and allopurinol. Gua, guanine; GPRT, Co., San Diego, CA) under sterile conditions. Uniformity in treatment guanine phosphoribosyltransferase. protocol was provided by biochemical targeting to decrease GTP con centrations to <20% of control; therefore, individual schedules were MD). Patients reported on here suffered from refractory or relapsed required, involving variations in tiazofurin dose. In the first patient in ANLL, treatment-related ANLL, ANLL after a myelodysplastic syn