Concise Review International Journal of Cell Cloning 8:161-170 (1990)
Tiazofurin: Biological Effects and Clinical Uses Guih Tricot”, Hiremagalur N J a y a d , George Wberb,Ronald Hoa Division of HematologyIOncology, Department of Medicine, bLaboratory for Experimental Oncology, ‘Indiana Elks Cancer Research Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
Key M& Leukemia Tmfurin Inosine 5’-phosphatedehydrogenase Guanosine triphosphate Antiproliferative effect Chronic myeloid leukemia Toxicity
Abstract. Inosine 5’-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferativeeffect on tumor cells in vitro and in the murine system.Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but wefe discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.
Introduction In various cancer cells a marked enzymic imbalance exists. There is strong evidence to indicate an up-regulation of the capacity for guanylate biosynthesis in cancer cells [1, 21. The activity of inosine 5’-monophosphate dehydrogenase (IMPDH) is increased in many cancer cells [2]. IMPDH is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis (Fig. 1). Weber and colleagues have therefore suggested that IMPDH might be a sensitive target for antineoplastic chemotherapy [l-31. Based on this hypothesis, a series of compounds were synthesized; tiazofurin, which might alter IMPDH function [4], is among these. Tiazofurin, a c-nucleoside, is metabolized in two enzymic steps to thiazole-4Correspondence: Guido J. Tricot, M.D., Ph.D., Indiana University Hospital, 926 W. Indianapolis, IN 46202-5250, USA. Michigan Street, Room W-608, Received March 28, 1990; accepted for publication March 28, 1990. 0737-1454/90/$2.OO/O @AlphaMedPress
Tiazofurin: Biological Effects and Clinical Uses
162
Fig. 1. Combination chemotherapy with tiazofurin and allopurinol resulting in a decrease in Cirp concentrationin mononuclearcells. IMPDH = IMP dehydrogenase; G P E = guanine phosphoribosyltransferase;Gua = guanine; HX = hypoxanthine; X = xanthine; XO = xanthine oxidase. (Reprinted with permission from Cancer Research 1989;49: 3696-3701.)
carboxamide adenine dinucleotide (TAD), a nicotinamide adenine dinucleotide (NAD) analogue that selectively blocks IMPDH activity [5, 61. TAD can be degraded in turn into inactive metabolites by a phosphodiesterase [7]. Tiazofurin in vitro and in vivo potently inhibits IMPDH activity, depresses GTP and dGTP concentrations, and results in inhibition of tumor cell proliferation [6, 71. The sensitivity of a particular tumor to tiazofurin depends primarily on the cellular concentration of TAD achieved [5, 7, 91. In addition to an increase in IMPDH activity [10-12], the activity of the guanine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase(HGPRT) is also elevated in leukemic cells [El. When leukemic cells are incubated with radiolabeled tiazofurin, more than a 20-fold elevation of TAD concentration is produced as compared to that achieved in bone marrow cells from healthy volunteers [D]. In vitro studies have demonstrated that low concentrations of tiazofurin are able to induce differentiation of the human leukemic cell lines HL-60 and K-562
TricotlJayaramNeberlHoffman
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[14-171. Induction of differentiation in K-562cells is preceded by reduction of IMPDH activity and GTP concentrations, and the possible down-regulation of c-rus proto-oncogene expression [17]. All these findings provide support for the use of tiazofurin for the treatment of leukemia. Inhibition of IMPDH activity by tiazofurin would hopefully decrease GTP and dGTP concentrations in leukemic blast cells and thereby restrain DNA synthesis. Moreover, the blockage of guanylate synthesis might result in induction of differentiation of leukemic blast cells. To successfully deplete the leukemic blast cells of GTP and dGTP, it is mandatory to block the guanine-salvage activity of HGPm. HGPRT activity can be competitively inhibited by hypoxanthine; serum concentrations of hypoxanthine can be markedly increased by the administrationof allopurinol [l2]. Therefore, simultaneous administration of tiazofurin and allopurinol is likely essential for a therapeutically significant antileukemic effect to be achieved. Since the action of tiazofurin and allopurinol is well characterized, the biochemical impact of these drugs can be monitored during treatment by frequent determination of leukemic cell IMPDH activity and GTP concentration; hypoxanthine concentrations in the plasma can also be determined.
Phase I Trials with Tiazofurin in Cancer Patients The National Cancer Institute initiated a number of Phase I trials to determine the toxicity and possible efficacy of tiazofurin in patients with solid tumors [18-231. Review of these Phase I trials revealed few objective tumor responses, but considerable toxicity [24]. Considerable neurotoxicity, including lethargy, headaches, seizures, agitation, confusion, hemiparesis and cortical blindness was reported. Other common toxicities included pleuropericarditis with chest pain, myelosuppression, exfoliativedermatitis, nausea and vomiting, hypertension, diarrhea, fever, liver function abnormalitiesand elevation of creatine phosphokinase (CPK; Table I). Tiazofurin was administered as a continuous infusion or as a 10 '-30' infusion for five consecutive days. The maximum tolerated dose varied between 80%decline in GTP concentrations. It is conceivable that massive destruction of DNA from the leukemic cells makes it impossible to completely block the availability of GTP during the first few days of treatment. Moreover, it usually took several days to attain adequate levels of hypoxanthine to prevent GTP synthesis through the salvage pahway. The decline in GTP pools in the leukemic cells was followed by a rapid decrease in absolute numbers of peripheral white blood cells and blast cells. In patients who were refractory to tiazofurin treatment, IMPDH activity remained high and GTP pools were never depressed to 50 x 109/l. In four patients, two with AML, one with CGL-BC and one with advanced stages of a myelodysplastic syndrome, a marked antileukemic effect was seen, defined as a complete disappearance of all blast cells from the peripheral blood and a bone marrow blast cell count of 75% decrease in blast cells in the peripheral blood and bone marrow. The hematologic effect was unevaluable in five patients because of toxicity (4 patients) or withdrawal from the study (1 patient). Most responders maintained a granulocyte count of >0.5 X 109/1during and after treatment. Bone marrow specimens remained normo- or hypercellular with a shift from blast cells to more mature cells in most patients, although one patient’s bone marrow became completely aplastic. The patient with AML maintained a complete remission for 10months. The CGL-BC patients relapsed three to five weeks after therapy, but showed further response to tiazofurin with reinstitution of the treatment. Toxicity to Eazofurin Adverse events observed in the 24 patients treated with tiazofurin are listed in Table III. The table delineates all the adverse events that occurred during or within four weeks after cessation of tiazofurin therapy, irrespectiveof the EdCt that some of these events might have been caused or aggravated by concomitant treatment or the patient’s underlying disease. Most of the severe and life-threatening toxicities were seen in the five patients who were treated for > 15 days or who entered the study in a severely compromised state. Drowsiness, nausea, vomiting, pleuropericarditis, headaches, exfoliative dermatitis, myalgias, hypertension and seizures were the primary toxicities attributed to tiazofurin. The pleuropericarditis, dermatitis and myalgias frequently responded very quickly to corticosteroid treatment. Tiazofurin treatment could frequently be resumed after one to three days in these patients. Seizures preceded by hypertension were seen in two of the first five patients treated. With aggressive treatment of the hypertension with calcium channel blockers, no seizures have been seen in the subsequent 19 patients.
Fl58
MI44 MI56
F120
MI21
Fl53
SexlAge
AML CGL CGL CGL CGL CGL CGL CGL AML AML
Disease
~
~
23,650 49,500 52,500 21,500 20.900 29.100 45,100 14,300 5,500 2.200 16.500 20;900
Second relapse Second relapse Refrac. first relapse First relapse First relapse Post MDS Refractory disease Post MDS Post MDS Myeloid blast crisis Second relame Refrac. first relapse
Total dose of tiamfurin (mp/m') 19,000 41,800 22,000 24,200 46,200 20,900 46,200 39,600 33,000 89.100 104,500 39.600
~
First relapse Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Secondary leukemia First relapse RAEB transformation Myeloid blast crisis
status
~
5 3
10 10 9 14
11 18 20
15 13 9 26 14 13
6 7
11
8
11
7
days
No.
~~
~~~
Outcome
No response No response No response No response No response No response No response
Complete remission Complete response Complete response Complete response Complete response Hematologic improvement Hematologic improvement Hematologic improvement Antileukemic effect Antileukemic effect Antileukemic effect Antileukemic effect
~
Tiazofurin: Biological Effects and Clinical Uses Guih Tricot”, Hiremagalur N J a y a d , George Wberb,Ronald Hoa Division of HematologyIOncology, Department of Medicine, bLaboratory for Experimental Oncology, ‘Indiana Elks Cancer Research Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
Key M& Leukemia Tmfurin Inosine 5’-phosphatedehydrogenase Guanosine triphosphate Antiproliferative effect Chronic myeloid leukemia Toxicity
Abstract. Inosine 5’-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferativeeffect on tumor cells in vitro and in the murine system.Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but wefe discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.
Introduction In various cancer cells a marked enzymic imbalance exists. There is strong evidence to indicate an up-regulation of the capacity for guanylate biosynthesis in cancer cells [1, 21. The activity of inosine 5’-monophosphate dehydrogenase (IMPDH) is increased in many cancer cells [2]. IMPDH is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis (Fig. 1). Weber and colleagues have therefore suggested that IMPDH might be a sensitive target for antineoplastic chemotherapy [l-31. Based on this hypothesis, a series of compounds were synthesized; tiazofurin, which might alter IMPDH function [4], is among these. Tiazofurin, a c-nucleoside, is metabolized in two enzymic steps to thiazole-4Correspondence: Guido J. Tricot, M.D., Ph.D., Indiana University Hospital, 926 W. Indianapolis, IN 46202-5250, USA. Michigan Street, Room W-608, Received March 28, 1990; accepted for publication March 28, 1990. 0737-1454/90/$2.OO/O @AlphaMedPress
Tiazofurin: Biological Effects and Clinical Uses
162
Fig. 1. Combination chemotherapy with tiazofurin and allopurinol resulting in a decrease in Cirp concentrationin mononuclearcells. IMPDH = IMP dehydrogenase; G P E = guanine phosphoribosyltransferase;Gua = guanine; HX = hypoxanthine; X = xanthine; XO = xanthine oxidase. (Reprinted with permission from Cancer Research 1989;49: 3696-3701.)
carboxamide adenine dinucleotide (TAD), a nicotinamide adenine dinucleotide (NAD) analogue that selectively blocks IMPDH activity [5, 61. TAD can be degraded in turn into inactive metabolites by a phosphodiesterase [7]. Tiazofurin in vitro and in vivo potently inhibits IMPDH activity, depresses GTP and dGTP concentrations, and results in inhibition of tumor cell proliferation [6, 71. The sensitivity of a particular tumor to tiazofurin depends primarily on the cellular concentration of TAD achieved [5, 7, 91. In addition to an increase in IMPDH activity [10-12], the activity of the guanine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase(HGPRT) is also elevated in leukemic cells [El. When leukemic cells are incubated with radiolabeled tiazofurin, more than a 20-fold elevation of TAD concentration is produced as compared to that achieved in bone marrow cells from healthy volunteers [D]. In vitro studies have demonstrated that low concentrations of tiazofurin are able to induce differentiation of the human leukemic cell lines HL-60 and K-562
TricotlJayaramNeberlHoffman
163
[14-171. Induction of differentiation in K-562cells is preceded by reduction of IMPDH activity and GTP concentrations, and the possible down-regulation of c-rus proto-oncogene expression [17]. All these findings provide support for the use of tiazofurin for the treatment of leukemia. Inhibition of IMPDH activity by tiazofurin would hopefully decrease GTP and dGTP concentrations in leukemic blast cells and thereby restrain DNA synthesis. Moreover, the blockage of guanylate synthesis might result in induction of differentiation of leukemic blast cells. To successfully deplete the leukemic blast cells of GTP and dGTP, it is mandatory to block the guanine-salvage activity of HGPm. HGPRT activity can be competitively inhibited by hypoxanthine; serum concentrations of hypoxanthine can be markedly increased by the administrationof allopurinol [l2]. Therefore, simultaneous administration of tiazofurin and allopurinol is likely essential for a therapeutically significant antileukemic effect to be achieved. Since the action of tiazofurin and allopurinol is well characterized, the biochemical impact of these drugs can be monitored during treatment by frequent determination of leukemic cell IMPDH activity and GTP concentration; hypoxanthine concentrations in the plasma can also be determined.
Phase I Trials with Tiazofurin in Cancer Patients The National Cancer Institute initiated a number of Phase I trials to determine the toxicity and possible efficacy of tiazofurin in patients with solid tumors [18-231. Review of these Phase I trials revealed few objective tumor responses, but considerable toxicity [24]. Considerable neurotoxicity, including lethargy, headaches, seizures, agitation, confusion, hemiparesis and cortical blindness was reported. Other common toxicities included pleuropericarditis with chest pain, myelosuppression, exfoliativedermatitis, nausea and vomiting, hypertension, diarrhea, fever, liver function abnormalitiesand elevation of creatine phosphokinase (CPK; Table I). Tiazofurin was administered as a continuous infusion or as a 10 '-30' infusion for five consecutive days. The maximum tolerated dose varied between 80%decline in GTP concentrations. It is conceivable that massive destruction of DNA from the leukemic cells makes it impossible to completely block the availability of GTP during the first few days of treatment. Moreover, it usually took several days to attain adequate levels of hypoxanthine to prevent GTP synthesis through the salvage pahway. The decline in GTP pools in the leukemic cells was followed by a rapid decrease in absolute numbers of peripheral white blood cells and blast cells. In patients who were refractory to tiazofurin treatment, IMPDH activity remained high and GTP pools were never depressed to 50 x 109/l. In four patients, two with AML, one with CGL-BC and one with advanced stages of a myelodysplastic syndrome, a marked antileukemic effect was seen, defined as a complete disappearance of all blast cells from the peripheral blood and a bone marrow blast cell count of 75% decrease in blast cells in the peripheral blood and bone marrow. The hematologic effect was unevaluable in five patients because of toxicity (4 patients) or withdrawal from the study (1 patient). Most responders maintained a granulocyte count of >0.5 X 109/1during and after treatment. Bone marrow specimens remained normo- or hypercellular with a shift from blast cells to more mature cells in most patients, although one patient’s bone marrow became completely aplastic. The patient with AML maintained a complete remission for 10months. The CGL-BC patients relapsed three to five weeks after therapy, but showed further response to tiazofurin with reinstitution of the treatment. Toxicity to Eazofurin Adverse events observed in the 24 patients treated with tiazofurin are listed in Table III. The table delineates all the adverse events that occurred during or within four weeks after cessation of tiazofurin therapy, irrespectiveof the EdCt that some of these events might have been caused or aggravated by concomitant treatment or the patient’s underlying disease. Most of the severe and life-threatening toxicities were seen in the five patients who were treated for > 15 days or who entered the study in a severely compromised state. Drowsiness, nausea, vomiting, pleuropericarditis, headaches, exfoliative dermatitis, myalgias, hypertension and seizures were the primary toxicities attributed to tiazofurin. The pleuropericarditis, dermatitis and myalgias frequently responded very quickly to corticosteroid treatment. Tiazofurin treatment could frequently be resumed after one to three days in these patients. Seizures preceded by hypertension were seen in two of the first five patients treated. With aggressive treatment of the hypertension with calcium channel blockers, no seizures have been seen in the subsequent 19 patients.
Fl58
MI44 MI56
F120
MI21
Fl53
SexlAge
AML CGL CGL CGL CGL CGL CGL CGL AML AML
Disease
~
~
23,650 49,500 52,500 21,500 20.900 29.100 45,100 14,300 5,500 2.200 16.500 20;900
Second relapse Second relapse Refrac. first relapse First relapse First relapse Post MDS Refractory disease Post MDS Post MDS Myeloid blast crisis Second relame Refrac. first relapse
Total dose of tiamfurin (mp/m') 19,000 41,800 22,000 24,200 46,200 20,900 46,200 39,600 33,000 89.100 104,500 39.600
~
First relapse Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Myeloid blast crisis Secondary leukemia First relapse RAEB transformation Myeloid blast crisis
status
~
5 3
10 10 9 14
11 18 20
15 13 9 26 14 13
6 7
11
8
11
7
days
No.
~~
~~~
Outcome
No response No response No response No response No response No response No response
Complete remission Complete response Complete response Complete response Complete response Hematologic improvement Hematologic improvement Hematologic improvement Antileukemic effect Antileukemic effect Antileukemic effect Antileukemic effect
~
