(CANCER RESEARCH 49. 3696-3701. July I. 1989]

Biochemically Directed Therapy of Leukemia with Tiazofurin, a Selective Blockerof 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 HoffmanDivision 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.]: andDepartment 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

Tiazofurin (2-/3-i>-ribofuranosylthiazole-4-carboxamide, NSC 286193),a selective inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205),the rate-limiting enzyme of de novo GTP biosynthesis, provided in endstage leukemic patients a rapid decrease of IMP dehydrogenase activityand GTP concentration in the blast cells and a subsequent decline inblast cell count. Sixteen consecutive patients with end stage acute non-lymphocytic leukemia or myeloid blast crisis of chronic granulocyticleukemia were treated with tiazofurin. Allopurinol was also given toinhibit xanthine oxidase activity to decrease uric acid excretion and toelevate the serum concentration of hypoxanthine, which should competitively inhibit the activity of hypoxanthine-guanine phosphoribosyltrans-ferase (EC 2.4.2.8), the salvage enzyme of guanylate synthesis. Assaysof IMP dehydrogenase activity and GTP concentration in leukemic cellsprovided a method to monitor the impact of tiazofurin and allopurinoland to adjust the drug doses. In this group of patients with poor prognosis,five attained a complete hematological remission and one showed ahematological improvement. A marked antileukemic effect was seen intwo other patients. All five évaluablepatients with myeloid blast crisisof chronic granulocytic leukemia reentered the chronic phase of theirdisease. Five patients with acute nonlymphocytic leukemia were refractory to tiazofurin and three were unevaluable for hematological effectbecause of early severe complications. Responses with intermittent 5- to15-day courses of tiazofurin lasted 3-10 months. Tiazofurin had a clearantiproliferative effect, but the pattern of hematological response indicated that it appeared to induce differentiation of leukemic cells. In spiteof tnvidt) with severe or life-threatening complications in 11 of 16patients, tiazofurin was better tolerated in most patients than otherantileukemic treatment modalities and provided a rational, biochemicallytargeted, and biochemically monitored chemotherapy which should be ofinterest in the treatment of leukemias and as a paradigm in enzymepattern-targeted chemotherapy.

INTRODUCTION

Conventional antileukemic chemotherapy in relapsed or refractory ANLL' patients is seldom curative and remissions, if

attained, are almost always of short duration. Myeloid blastcrisis of CGL is even more resistant to chemotherapy, withcomplete hematological remission rates varying from 4 to 30%and median survivals being 6-29 weeks (1-4). A primary goalof antileukemic therapy in these patients with poor prognosisshould be the identification of agents that are more selectiveand better targeted in their actions. Such therapy should bebased upon our increasing understanding of the biology, enzy-

Received 10/17/88: revised 3/30/89; accepted 4/6/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by USPHS, National Cancer Institute. Outstanding InvestigatorGrant CA-42510 and Grant SO7 RR-5371 to G.W.

1To whom requests for reprints should be addressed, at the Division ofHemalology/Oncology, 541 Clinical Drive. Room 379, Indiana University Schoolof Medicine. Indianapolis. IN 46223.

'The abbreviations used are: ANLL, acute nonlymphocytic leukemia;IMPDH, inosine 5'-phosphate dehydrogenase; CGL, chronic granulocytic leukemia: CGL-BC, blast crisis of chronic granulocytic leukemia; HGPRT, hypoxanthine-guanine phosphoribosyltransferase: MDS, myelodysplastic syndrome.

mology, and biochemistry of cancer cells (5, 6).In various cancer cells there is a marked enzymic imbalance

in purine metabolism, with strong evidence indicating up-reg-ulation of the capacity for guanylate biosynthesis (5,6). Becausethe activity of IMPDH, the rate-limiting enzyme of GTP biosynthesis, was increased in all examined cancer cells, it wassuggested that IMPDH should be a sensitive target of chemotherapy (5-7). On the basis of this argument, a number ofcompounds were prepared to block IMPDH activity, amongthem tiazofurin (8), a C-nucleoside that is metabolized in twoenzymic steps to thiazole-4-carboxamide adenine dinucleotide,an NAD analogue that selectively blocks IMPDH activity (9,10). Tiazofurin in tissue culture and in the murine systempotently inhibited IMPDH activity, depressed GTP and dGTPconcentrations, and yielded cytotoxicity and inhibition of tumorcell proliferation (10, 11). Recent evidence indicated that, inaddition to an increase in IMPDH activity, the activity of theguanine salvage enzyme HGPRT was also elevated in humanleukemic cells (12-14). When human myeloid leukemic cellswere incubated with radiolabeled tiazofurin, over 20-fold higherconcentrations of thiazole-4-carboxamide adenine dinucleotidewere produced than in bone marrow cells from healthy volunteers (15, 16). These results provided support for the consideration of tiazofurin in the treatment of myelocytic leukemia.Inhibition of IMPDH activity by tiazofurin in blast cells shoulddecrease GTP and dGTP concentrations, which should restrainthe biosynthesis of DNA. Moreover, the blocking of guanylatebiosynthesis might critically limit the availability of GTP forexpression of the ras oncogene, G protein function, and thebiosynthesis of specific proteins (Fig. 1). This approach wasstrengthened by the observation that the guanine-salvagingactivity of HGPRT can be competitively inhibited by hypoxanthine and that the concentration of hypoxanthine can be markedly increased in the plasma by allopurinol, originally given toblock xanthine oxidase activity to decrease uric acid excretion(14). Therefore, to attain an optimal impact leading to depletionof GTP in the blast cells, simultaneous administration of tiazofurin and allopurinol is essential. Since the action of tiazofurin and allopurinol is well characterized, the biochemicalimpact of these drugs can be monitored during treatment byfrequently sampling leukemic cells for measurement of IMPDHactivity and GTP concentrations.

This paper reports our biochemically directed trial in patientswith ANLL and myeloid CGL-BC, where tiazofurin rapidlybrought down IMPDH activity and then GTP concentrations,followed by a decline in the blast cell count.

MATERIALS AND METHODS

Patients. Sixteen consecutive patients were treated with tiazofurin atIndiana University Medical Center from March 1987 to April 1988.The characteristics of the patients are listed in Table 1. This Phase I/II trial was monitored by the Cancer Therapy Evaluation Program,Division of Cancer Treatment, National Cancer Institute (Bethesda,

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TIAZOFURIN TREATMENT OF LEUKEMIA

Fig. I. Pathways of IMP and GTP metabolism and the biochemical basis forthe chemotherapeutic impact of tiazofurin and allopurinol. Gua, guanine; GPRT,guanine phosphoribosyltransferase.

MD). Patients reported on here suffered from refractory or relapsedANLL, treatment-related ANLL, ANLL after a myelodysplastic syndrome, or nonlymphoid blast crisis of CGL, based on morphology andimmunological markers (negative for Tdt and the common acute Kmphocytic leukemia antigen and positive for at least one of the myeloidmarkers, VIM-2, My 9, and My 7). Eligibility criteria required a firstremission duration of <12 months; however, the initial remissionduration in patients I and 8 was 18 and 13 months, respectively. Patient11 on admission had serum bilirubin >1.S mg/dl. The initial clinicalcourse of the first patient studied (patient 9) was published (16).

Evaluation of Response. A complete remission was defined by standard criteria (17). Hematological improvement was defined as a bonemarrow blast count of <5% within the peripheral blood, absolutegranulocyte count of > 1.0 x 10'/liter, and a platelet count of >50 xlO'/Hter (18). Antileukemic effect was defined as a complete disap

pearance of all blasts from the peripheral blood with a bone marrowblast count of <5%. Patients who did not meet these criteria wereconsidered nonresponders.

Evaluation of Toxicity. Toxicity was evaluated according to thecriteria of the Eastern Cooperative Oncology Group (19).

Informed Consent. Informed consent was obtained from all patients

according to the principles of the Declaration of Helsinki. The protocolhad been reviewed and approved by the Investigational Review Boardof the Indiana University School of Medicine.

Biochemical Assays. Preparation of bone marrow and peripheralblood mononuclear cells and determination of the concentration ofGTP were carried out as reported (15). Briefly, mononuclear cells wereseparated by a Ficoll-Paque density gradient. An aliquot of cells wasthen extracted with 10% trichloracetic acid, neutralized immediatelywith tri-n-octylamine in Freon, and analyzed by high pressure liquidchromatography for GTP concentrations, using a buffer system ofammonium phosphate. Another aliquot of cells was assayed directlyfor IMPDH activity as described (20). During treatment with tiazofurin,IMPDH activity and GTP pools in leukemic cells were measured atleast 3 times/day in peripheral blood samples.

Chromosome Preparation and Analysis. Cytogenetic investigationswere performed on bone marrow or peripheral blood using directanalysis, 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 wasobtained from the National Cancer Institute (Bethesda, MD). The drugwas administered daily, over a period of 1 h, by an infusion pump (ImedCo., San Diego, CA) under sterile conditions. Uniformity in treatmentprotocol was provided by biochemical targeting to decrease GTP concentrations to <20% of control; therefore, individual schedules wererequired, involving variations in tiazofurin dose. In the first patient inthe study (patient 9), the initial dose was 1100 mg/nr, based on datafrom previous Phase I trials. This dose was too low to decrease GTPlevels. Therefore, all subsequent patients were initially given 2200 mg/m2 (an exception was patient 1, who received 5800 mg/nr the first day

because of a mistake made in the hospital pharmacy). The differenttimings of dose escalations were based on the biochemical response;the goal was to avoid unnecessary dose escalations. If an insufficientdecline occurred in GTP levels, further dose escalations were initiated.At the end of the study, we concluded that the best results are obtainedby escalating uniformly after 3 days in the case of lack of decrease inGTP concentration to <20% and that each treatment course should beno longer than 15 days.

Tiazofurin at the effective dose was then administered until theperipheral blood was cleared of blast cells. This pattern of administration was followed in all the patients except for the first one (patient 9)who was treated with tiazofurin for only 5 days at the effective dose.Patients who attained a complete remission or hematological improvement received further courses of tiazofurin as consolidation treatment(patient 1) or whenever an increase in absolute numbers of blast cells(> 5 x lO'/Hter) was noted in the peripheral blood (patients 2-6 and

Table 1 Patients' characteristics, total dose and number of days of initial tiazofurin treatment, outcome, and duration of response

PatientSex/AgeDiseaseStatusTotal

dose oftiazofurin(mg/m2)No.

ofdaysResponse

du-Outcome ration (mo)Total

survival(mo)Responders12345678F/53M/21F/20M/44M/56F/58M/70M/55ANLLCGLCGLCGLCGLCGLANLLANLLFirst

relapseMyeloidblastcrisisMyeloidblastcrisisMyeloidblastcrisisMyeloidblastcrisisMyeloidblastcrisisSecondary

leukemiaFirstrelapse19.00041.80022,00024.20046,20020.90033.00089.10071181167926Complete

remission10Completeresponse3Completeresponse6Completeresponse7Completeresponse6Hematological

improvement10Antileukemiceffect<1Antileukemiceffect<11546'131316<1<lNonresponders910111213M/48F/21F/37M/24M/41ANLLANLLANLLANLLANLLSecond

relapseSecondrelapseRefractory

firstrelapseFirstrelapseFirst

relapse23,65049.50052,50027,50020.9001118201010No

responseNoresponseNoresponseNoresponseNo

response42<126Unevaluablefor hematologicaleffect141516M/83F/50M/27ANLLANLLCGLPost

MDSPostMDSMyeloid

blast crisis14,3005,5002.200531UnevaluableUnevaluableUnevaluable<18<1" Patient returned to primary1 physician, in California, who treated her there with mitoxanthrone to prepare her for bone marrow transplantation, but patient died

during mitoxanthrone chemotherapy.

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TIAZOFURIN TREATMENT OF LEUKEMIA

9). During these intermittent courses, the dose of tiazofurin was escalated in some patients to 6600 mg/nr, if an inadequate decline in GTPlevels was seen.

On admission, a permanent central line was placed in all patients.When neutropenic, patients received antimicrobial prophylaxis withnorfloxacin and ketoconazole. While receiving tiazofurin, all patientswere placed on allopurinol, 300 mg/day or 100 mg every 4-6 h.Allopurinol was given initially to prevent hyperuricemia (300 mg/day).Subsequently, divided doses of allopurinol (100 mg, 4 to 8 times/day)were given in an attempt to maintain high and more constant hypoxan-thine levels in the serum. Depletion of GTP in blast cells depended onboth tiazofurin and allopurinol, to block the de novo and salvagepathways, respectively.

RESULTS

Biochemical Parameters during Tiazofurin Treatment. A correlation between biochemical and hematological responses wasseen in all patients. Figs. 2 and 3 are typical of positive responses; they were absent when clinical treatment failed. Institution of tiazofurin therapy resulted in a rapid decrease inIMPDH activity. The decline in GTP pools occurred moreslowly, and dose escalations were necessary in most patients toreach a >80% decline. The decline in GTP pools was followedby a rapid decrease in the absolute numbers of peripheral WBC

TIAZOFURIN (mg/m )

2200 3300n rrr~n

UJ

Z01SH

UJ

UJU.0.LL

Oaf>

120-1

100-

80-

60-

10-

20-

<H

•WBC

O BLAST

A CTP

IMP DH

ODd-DDCr10

TREATMENT (days)

Fig. 2. Changes in hematological and biochemical parameters during tiazofurin therapy in patient 2. The values of WBC, blast cells, GTP pools, andIMPDH activity are expressed as a percentage of the pretreatment values. Thepretreatment value of WBC was 19.3 x 10'/liter; blast cells, 7.3 X lO'/liter; GTPpools, 205.8 nmol/10* leukemic cells; and IMPDH activity, 33.4 nmol/h/mg

protein.

3698

and blast cells. In patients 11 and 12, who were refractory totiazofurin treatment, IMPDH activity remained elevated andGTP pools were never depressed to <20% of pretreatmentvalues. Patients 1-6 and 9 received multiple courses of tiazofurin treatment. Clinical relapse of the disease in patients 2-6and 9 was always associated with marked increases in IMPDHactivity and GTP pools in the leukemic cells. Reinstitution oftiazofurin (Fig. 3) was accompanied by a biochemical andhematological response similar to that observed during theinitial treatment. This pattern was seen with all retreatmentcourses except during the fifth course of treatment in patient 9;he became refractory to tiazofurin by both biochemical andhematological parameters.

Clinical Response to Tiazofurin. Outcome of treatment andduration of response are provided in Table 1. Peripheral bloodcell counts obtained from responders prior to, at the end of,and 7-10 days after treatment are given in Table 2. Bonemarrow cellularity, bone marrow cell differential counts, andcytogenetic analyses of these responders are shown in Table 3.

Five patients, one with ANLL and four with CGL-BC, attained a complete hematological remission. The patient withANLL also entered a cytogenetic remission, while chromosomeabnormalities persisted in the patients with CGL-BC. Onepatient with CGL-BC showed a hematological improvement.In two patients with ANLL a marked antileukemic effect wasseen; both patients died, however, from invasive fungal infections. Five patients were classified as therapy failures, althoughsome antileukemic effect was seen in three of these (patients 9-11), with a >75% decrease in the absolute number of peripheralblasts in all three and a >75% decrease in bone marrow blastsin patient 9 (16).

The hematological effect of tiazofurin was not évaluableinthree patients because of early severe or life-threatening toxic-ity, consisting of seizures (patient 14), pleuropericarditis (patient IS), and sudden coma (patient 16).

Most of the responders maintained a granulocyte count of>0.5 x 109/liter during and after treatment. Patients 1, 3, and

4 showed an increase in platelet counts after tiazofurin treatment.

Bone marrow specimens obtained during and shortly aftertreatment remained normo- or hypercellular in all respondersexcept patients 4 and 8 (Table 3). Posttreatment bone marrowaspirates in responders showed a shift from blast cells to moremature cells, except in patient 8, who received 25 days oftiazofurin and became severely hypoplastic after treatment.

The complete remission in patient 1 lasted for 10 months.The duration of response in the CGL-BC patients was 3, 6, 6,7, and 10 months. Patient 2 became clinically refractory totreatment after 3 months; at that time, he had developed anadditional chromosome abnormality. After four cycles of goodresponse, patient 3 discontinued treatment to undergo an allo-geneic bone marrow transplantation with an unrelated donor.Patients 4-6 remained clinically and biochemically sensitive totiazofurin but preferred to be switched to oral antileukemictherapy. Two of these patients were refractory to hydroxyureaand 6-thioguanine before they began to be treated with tiazofurin; retreatment with these agents resulted in an adequatecontrol of peripheral WBC and blast count.

Toxicity to Tiazofurin. Adverse events observed in the 16patients treated with tiazofurin are listed in Table 4. All adverseevents that occurred during treatment or within 4 weeks aftercessation of the therapy with tiazofurin are listed, irrespectiveof the fact that some of these toxicities might have been causedor aggravated by concomitant therapy or by the patient's un-

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2200

TIAZOFURIN (mg/m¿ )

3300 3300 3300

rm rm rm rmFig. 3. Changes in hematological and bio

chemical parameters during two consecutivecourses of tiazofurin treatment in patient 4.The values of WBC, blast cells, GTP pools,and IMPDH activity are expressed as a percentage of pretreatment values. The pretreatment value of WBC was 6.6 x lO'/Hter; blastcells, 4.0 x lO'/liter; GTP pools, 344.3 nmol/10' leukemic cells; and IMPDH activity, 12.2

nmol/h/mg protein.

250-,

200-150-120-

i 5 ; 9 24 26 28 30

TREATMENT (days)

Table 2 Effect of tiazofurin therapy on peripheral blood counts of patients who responded to tiazofurin therapyValues represent absolute numbers of specific cellular elements.

WBC(xlO'/liter) Blasts (x lO'/Hter) Granulocytes (x lO'/liter) Platelets (x lO'/liter)

Patient Pre° End* Post' Pre End Post Pre End Post Pre End Post

11.32148.036.6412.6594.0619.07111.38

17.41.522.12.41.59.85.47.50.44.04.06.03.21.64.12.60.70.440.04.01.019.77.446.512.70.00.00.10.00.00.30.00.010.00.00.00.20.30.10.00.00.422.20.24.833.06.530.63.80.315.00.61.08.93.86.00.03.12.41.61.00.83.22.10.039.0124.019.0''524.0203.065.041.0''94.062.0136.046.0134.038.050.034.0"9.0a143.0149.0125.0210.0194.050.023.0''IS.O*°Prior to the institution of therapy.* On the day therapy was discontinued.'7-10 days after discontinuation of therapy.'' Patient receiving platelet transfusions.

Table 3 Bone marrow findings before and after tiazofurin treatment in the eight responders

Cellularity"Patient1

234S678BeforeHypo

HyperHyperHyperHyperHyperHyperNormalAfterNormal

HyperHyperHypoNormalHyperHyperHypoBlasts

(%)Before36C

NE553348256082After04

22510Pro'-t-

{*Before029

288

1412

1Myelo

)After1515

2014060MM

+ Gran(%)Before22028

4145510After7875

54727493

0CytogeneticsBeforeInv(16),+22

t(9;22)t(9;22),+21t(8;22),+8,+ 12,der(22)t(9;22)t(9;22)i(17q),del(6q)-7,del(17q)NormalAfterNormal

t(9;22)t(9;22),+21t(8;22),+8,+ 12,+dt(9;22)t(9;22)i(l7q)delNDNo mitoses

" Cellularity was estimated in bone marrow biopsies or aspirates.* Pro, promyelocytes; Myelo, myelocytes; MM, metamyelocytes; Gran, granulocytes; NE, not évaluablebecause of dry tap; ND, not done.' Percentage of cells on a 200 cell marrow differential of a Wright-Giemsa-stained bone marrow aspirate specimen.

derlying hematological disorder. Most of the toxic events (66%)were mild or moderate. The most common side effect wasdrowsiness. Patients with drowsiness were easily arousable andwere fully awake by the time the next infusion was given. Othermild and moderate toxicities noted were nausea, vomiting,headaches, myalgias, conjunctivitis, skin rash, and atrial fibrillation.

Five patients developed clinical signs of pleuropericarditis.In all five patients, pain due to the serositis improved within48 h after discontinuation of tiazofurin and institution of cor-ticosteroids. Chest X-rays and echocardiograms remained normal. One patient had electrocardiogram changes suggestive ofpericarditis; in another patient the pericarditis was associatedwith atrial fibrillation requiring digitalization. Tiazofurin ther

apy could be resumed without reappearance of clinical symptoms of pleuropericarditis in three patients.

Patient 12 developed a pericardia! friction rub during hissecond course of tiazofurin; he quickly improved on cortico-steroids and tiazofurin therapy was restarted. However, 9 daysafter the completion of treatment, he was readmitted to thehospital with fever, skin lesions, swelling of both knees, severeneutropenia, and progressive leukemia. A pericardia! frictionrub was noted. Echocardiogram performed 5 days later showeda large pericardia! effusion and the patient died from theconsequences of cardiac tamponade. Patient 15 developed acutechest pain and shortness of breath after 3 days of tiazofurin.She recovered completely but received no further treatment.Three patients developed life-threatening infections. Patients 7

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TIAZOFIJRIN TREATMENT OF LEUKEMIA

Table 4 Konhematological toxicily observed with tia:ofurin treatmentGroup A, 11 patients treated for less than 15 days and without major complicating medical problems at entrance in the protocol; group B. 5 patients treated for

longer than 15 days or entering trial with major complicating medical problems.

MildModerateSevereLife-threaten-ingSymptoms

andsignsDrowsiness,

confusionInfectionNausea/vomitingPleuropcricarditisLiver

functionabnormalitiesHeadachesCardiac

toxicitySkinrashMyalgiasHypertensionSeizuresC'omaA1030010(1(10(10B200010000000A622011111200B2i1001011000A100400000000B000020000000A010100000020B020000200002

and 8 developed invasive fungal infections and patient 14 anaspiration pneumonia. Patients 6 and 14 developed seizureswhich were difficult to control and they needed multiple anti-convulsant drugs. Patient 6 recovered completely and tiazofurintherapy could be restarted; she experienced a second episode ofseizures at a higher dose of tiazofurin. Patient 14 remainedsomnolent and confused and subsequently died of an aspirationpneumonia.

Two patients showed a marked increase in liver functionabnormalities. Patient 8 was being given broad spectrum antibiotics, acyclovir, and amphotericin B for septicemia and esoph-agitis with Candida albicans and herpes simplex; patient 11already had impaired liver function before the start of tiazofurin.

Two patients developed sudden coma. Patient 11 had aninfection, was on broad spectrum antibiotics, and was completely refractory to platelet transfusions. Patient 16 was admitted with a WBC count of 282 x IfT/liter and 79% blasts.He presented with clinical findings consistent with a hyperleu-kocytic syndrome. He was subjected to leukapheresis twice andtiazofurin therapy was then started. He abruptly developedcoma with signs of decerebration, intractable hypotension, andrespiratory failure, 6 h after the completion of the first dose oftiazofurin. He died the next day; autopsy was refused. Twopatients experienced ventricular fibrillation and cardiac arrest(patients 8 and 11).

DISCUSSION

The results of this Phase I/II trial with tiazofurin in leukemiaare very encouraging because five patients (31%) attained acomplete remission, one (6.5%) had a hematological improvement, and two showed a marked antileukemic effect (12.5%).Objective responses (complete and partial) in Phase I trialsbetween 1974 and 1982 were reported in 5.8% of leukemicpatients, while the best reported complete and partial responserates in Phase II trials with the four drugs considered to beactive in leukemia, aclarubicin, amsacrine, azacytidine, andzorubicin, were 21, 20, 14, and 44%, respectively (22). Especially the results with tiazofurin in nonlymphoid blast crisis ofCGL appear very promising. All five hematologically évaluablepatients with CGL-BC returned to the chronic phase of theirdisease. The duration of complete remission in one ANLLpatient was 10 months. The response in CGL-BC was shortlived (2-6 weeks), but patients returned to the chronic phasewith reinstitution of tiazofurin treatment. The total duration ofresponse in these patients was 3, 6, 6, 7, and 10 months. It islikely that the initial response in CGL-BC could be considerably

prolonged if an oral formulation of tiazofurin were available.An alternative approach may be to maintain the initial responsewith oral antileukemic agents such as hydroxyurea and 6-thioguanine. These agents appeared to be active in three of ourpatients after tiazofurin therapy.

The unique aspect of the tiazofurin-allopurinol therapy wasthe manner in which the dose of therapy was guided by cellularbiochemical parameters. Drug dosage was determined by sampling residual leukemic cells from either peripheral blood orbone marrow and determining GTP pools and IMPDH activity.A marked decline in IMPDH activity and GTP pools wasfollowed by a hematological response. The duration of treatment was determined by biochemical and hematological (WBCand blast cell count) parameters.

It is yet unknown whether tiazofurin acts solely as an anti-proliferative agent or whether it may also induce maturation ofleukemic cells. Observations from an ANLL patient whoachieved complete remission with loss of her marker chromosomes and from one patient who developed severe marrowhypoplasia after prolonged tiazofurin therapy provide us withclinical evidence that this drug clearly has an antiproliferativeeffect. In contrast, the absence of bone marrow hypoplasia, themarked shift from blast cells to mature granulocytes in the bonemarrow, the persistence after the cessation of tiazofurin administration of adequate numbers of granulocytes and platelets,and the persistence of chromosome abnormalities observed inthe other responding patients provide evidence that this drugpromoted leukemic cell differentiation in vivo (Table 3). Thissuggestion is supported by in vitro studies with tiazofurin inhuman leukemic cell lines (23-25). However, the possibilitythat tiazofurin induces maturation of leukemic cells /// vivomust be substantiated by further clinical investigations.

Toxicity has been well described in Phase I trials with tiazofurin (26-31). Toxicity was also substantial in this trial. Themost severe complications, however, such as sudden coma,ventricular fibrillation, and cardiac tamponade, were seen inpatients with either complicating medical problems such ashyperviscosity syndrome, abnormal liver function, and progressive leukemia or prolonged treatment with tiazofurin (over 15days), resulting in bone marrow hypoplasia with infectiouscomplications. The exact contribution of tiazofurin to thesesevere toxicities is therefore difficult to determine. As Table 4shows, toxicity was lower in patients who entered the studywith few complications and were treated for less than 15 daysin each cycle. More severe and life-threatening toxicity occurredin patients who entered in a severely compromised status andwere treated longer than 15 days in the cycles. In future trials,we would recommend the restriction of tiazofurin treatment to

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patients in stable clinical condition without complicating medical problems; the total duration of treatment should not exceed10 to 15 days/course. Drowsiness was quickly reversible afterdiscontinuation of therapy and never resulted in medical problems. Myalgias and pleuropericarditis rapidly responded totemporary discontinuation of the tiazofurin and/or institutionof prednisone therapy; myalgias and pleuropericarditis did notprevent further therapy with tiazofurin. Seizures were precededby hypertension; since we promptly instituted antihypertensivetherapy with calcium channel blockers in patients with hypertension, no new seizures have been observed.

Novel aspects of this study include the following, (a) Theclinical trial was rationally targeted against the enzyme patternof elevated IMPDH and HGPRT activities in the leukemicblast cells, (b) Infusion of tiazofurin rapidly decreased IMPDHactivity and curtailed GTP concentrations in the blast cells,yielding a clearing of blast cells (c) Measurement of the behaviorof these biochemical targets provided monitoring of therapeuticprogress and allowed adjustment of frequency and dose ofadministration of tiazofurin and allopurinol. (d) Allopurinolnot only was used as a blocker of uric acid formation but alsowas targeted to raising serum hypoxanthine concentrations toinhibit HGPRT activity.

Tiazofurin appears to be a novel promising agent in thetreatment of leukemia, if toxicity can be better handled. Alarger number of patients will have to be studied to confirm itsactivity. Future studies should aim at improving efficacy bycombining tiazofurin with other antileukemic agents or imincers of differentiation.

ACKNOWLEDGMENTS

We are indebted to the oncology nursing siali anil medical housestaffof the Leukemia Service of the Indiana University Hospital, for theirexcellent patient care, and to Charlotte Franklin and Julie McDaniels,for preparing the manuscript.

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1989;49:3696-3701. Cancer Res   Guido J. Tricot, Hiremagalur N. Jayaram, Elizabeth Lapis, et al.   Activity

-Phosphate Dehydrogenase′Selective Blocker of Inosine 5Biochemically Directed Therapy of Leukemia with Tiazofurin, a

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