Potentiation of the Cytotoxic Action of Mafosfamide by Jv-Isopropyl-/;- Formylbenzamide, a Metabolite of Procarbazine1

[CANCER RESEARCH 51, 4170-4175, Augusl 15. 1991] Potentiation of the Cytotoxic Action of Mafosfamide by jV-Isopropyl-/;- formylbenzamide, a Metabolite of Procarbazine1

Patricia A. Maki2 and Norman E. Sladek3

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT (reviewed in Ref. 8). Cyclophosphamide is a prodrug. It is converted to the primary circulating metabolite, 4-hydroxycy- Several mouse aldehyde dehydrogenases catalyze the detoxification of clophosphamide, which exists in equilibrium with its ring- aldophosphamide, the pivotal metabolite of the prodrugs cyclophosphamide, mafosfamide, and other oxazaphosphorines. /V-Isopropyl-/>-for- opened tautomer aldophosphamide; neither of these, per se, is mylbenzamide, a major metabolite of procarbazine, was found to be an toxic to cells. Aldophosphamide undergoes one of two fates: ÃŸ excellent substrate ( A',,,= 0.84 ^M) for at least one of these enzymes, elimination to phosphoramide mustard, the ultimate toxic spe namely, mouse aldehyde dehydrogenase-2. The A',,,for mouse aldehyde cies, or oxidation to carboxyphosphamide, a nontoxic product. dehydrogenase-2-catalyzed detoxification of aldophosphamide is 16 n\\. The latter process is catalyzed by NAD(P)-linked aldehyde Thus, competition between /V-isopropyl-p-formylbenzamide and aldo dehydrogenases. phosphamide for the catalytic site on the enzyme should strongly favor Greater expression of the relevant aldehyde dehydrogenase(s) the former, and the rate at which aldophosphamide is detoxified should in critical normal tissues versus that in tumor tissue has been be markedly retarded. Mouse L1210/OAPand P388/CLA leukemia cells suggested as the basis for the favorable margin of safety exhib are relatively insensitive to the oxazaphosphorines because they contain large amounts of mouse aldehyde dehydrogenase-2. As predicted, .V- ited by cyclophosphamide and other oxazaphosphorine nitro isopropyl-p-formylbenzamide markedly potentiated the cytotoxic action gen mustards, e.g., mafosfamide (reviewed in Ref. 8). Expres of mafosfamide against these cells. Mouse LI 210/0 and P388/0 lack the sion of the relevant aldehyde dehydrogenase activity has been enzyme. Again as expected, /Y-isopropyl-p-formylbenzamide essentially demonstrated in murine and human multipotent and committed did not potentiate the cytotoxic action of mafosfamide against these cells. hematopoietic progenitor cells (9-13), and in murine intestinal Certain mouse and human hematopoietic progenitor cells also contain an crypt and villus cells (14), but not in a number of murine and aldehyde dehydrogenase that catalyzes the detoxification of aldophos human tumor cells lines (10, 15). These differences in expres phamide, but the specific identity of this enzyme remains to be estab sion of the relevant aldehyde dehydrogenase activity can, at lished. /V-Isopropyl-p-formylbenzamide potentiated the cytotoxic action least partially, explain the relative differences in sensitivity of of mafosfamide against these cells as well. Clinically, procarbazine and these cells to the oxazaphosphorines. the oxazaphosphorines are used to treat certain neoplastic diseases. Aldehyde dehydrogenases, each exhibiting unique character Frequently, they are used in combination. Our findings demonstrate the istics, e.g., substrate preferences/specificity, tissue distribution, potential for both desirable and undesirable drug interactions when these agents are used concurrently. Similar drug interactions can be expected and sensitivity to inhibitors, have been identified in a variety of when other substrates for, or inhibitors of, the relevant aldehyde dehy animal species (16, 17). Ten different aldehyde dehydrogenases drogenases, e.g., chloramphenicol, chloral hydrate, and methyltetrazole- that are capable of catalyzing the detoxification of aldophos thiol-containing cephalosporins, are co-administered with the phamide have been identified in mouse tissues (18). Nine are oxazaphosphorines. found in the liver. One, namely, AHD-2,4 accounts for the bulk of the total hepatic activity when the aldophosphamide concen INTRODUCTION tration is pharmacological (17, 18). It is found in the cytosol. This is not the hepatic aldehyde dehydrogenase that is most Cyclophosphamide and procarbazine are antineoplastic important in catalyzing the oxidation of acetaldehyde. That agents that are frequently included in combination chemother distinction goes to a mitochondrial enzyme, AHD-5 (17, 18). apy regimens, e.g., COPP (cyclophosphamide, vincristine, pred- Expression of a relevant aldehyde dehydrogenase has also nisone, procarbazine) (1); CAMP (cyclophosphamide, Adria- been implicated as a mechanism of acquired resistance to the mycin, methotrexate, procarbazine) (2); PROVECIP (procar oxazaphosphorines. Certain murine tumor cell lines, e.g., bazine, vinblastine, cyclophosphamide, prednisone) (3, 4); and LI210 and P388, that ordinarily lack the relevant aldehyde CABOPP (cyclophosphamide, doxorubicin, bleomycin, vincris dehydrogenase activity and are sensitive to the oxazaphosphor tine, procarbazine, prednisone (5, 6). The combination of cyclo ines, become resistant to them when repeatedly exposed to phosphamide and procarbazine may also have some utility in increasing amounts of these agents. In at least some cases, e.g., preventing the graft rejection that otherwise often occurs after L1210/OAP and P388/CLA, cells made resistant by this treat bone marrow transplantation (7). ment express the relevant aldehyde dehydrogenase(s) (10, 14, The utility of cyclophosphamide in the treatment of cancer 15, 19-22). The aldehyde dehydrogenase expressed in L1210/ is in large part due to the favorable margin of safety exhibited OAP has been identified as AHD-2 (10, 14, 15, 22). by this agent, a property which is a function of its metabolism Like cyclophosphamide, procarbazine is a prodrug. The par ent compound is oxidized to the primary circulating metabolite, Received 2/1/91; accepted 6/3/91. azoprocarbazine, by the hepatic microsomal P-450 system and 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 in by monoamine oxidase. Azoprocarbazine can be further metab- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'Supported by USPHS Grant CA 21737. A description of parts of this ' The abbreviations used are: AHD-2, mouse aldehyde dehydrogenase-2; LC,9, investigation has appeared in abstract form (41). drug concentration required to effect a 99% cell-kill; LC50, drug concentration 1 Present address: Department of Radiation Oncology, Division of Oncology required to effect a 50% cell-kill; CFU-GEMM, colony-forming unit, granulocy- Research, University of Pennsylvania. 530 Clinical Research Building, 422 Curie toid/erythroid/monocytoid/megakaryocytoid; CFU-Mk, colony-forming unit, Boulevard, Philadelphia. PA 19104-6142. megakaryocytoid; CFU-GM, colony-forming unit, granulocytoid/monocytoid; 3To whom requests for reprints should be addressed, at the University of BFU-E, colony-forming unit, erythroid; CFU-Mix, colony-forming unit, granu- Minnesota, Department of Pharmacology. 3-249 Millard Hall. 435 Delaware locytoid/erythroid/monocytoid, with or without megakaryocytes; CFU-S, colony- Street S.E., Minneapolis, MN 55455. forming unit, spleen. 4170

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1991 American Association for Cancer Research. POTENTIATION OF MAFOSFAMIDE BY A PROCARBAZINE METABOLITE olized along any of four pathways (23-26). Of the four, two suspensions for these assays was as previously described (II). Drug give rise to the aldehyde metabolite, /V-isopropyl-p-formylben- exposure was for 30 min beginning immediately following the addition zamide. This compound is rapidly converted to A'-isopropylter- of cytotoxic agent. Control values ranged from 6-16 (CFU-GEMM), 25-41 (CFU-Mk), and 200-235 (CFU-GM) colonies/1 x IO5nucleated ephthalamic acid by aldehyde dehydrogenase(s) (26, 27). In view of the foregoing, the potential for a significant cyclo- cells plated. The sensitivity of human hematopoietic stem cells to mafosfamide phosphamide-procarbazine drug interaction is obvious. Thus, with or without A'-isopropyl-p-formylbenzamide was determined as the purpose of the present investigation was to test the notion previously described (10). Human bone marrow cells were obtained that jV-isopropyl-p-formylbenzamide, acting as an alternative from a healthy adult volunteer and were prepared for assay as previously substrate, inhibits aldehyde dehydrogenase-catalyzed detoxifi described (10). cation of cyclophosphamide, thereby sensitizing cells wherein Aldehyde dehydrogenase-catalyzed oxidation of acetaldehyde and N- this reaction ordinarily occurs, to this agent. isopropyl-p-formylbenzamide was quantified as described by Manthey et al. ( 18, 28). Km values for the catalysis of 7V-isopropyl-/j-formylbenzamide by AHD-2 were determined by the integrated Michaelis method MATERIALS AND METHODS of analysis of single enzyme progress curves (31). Computer-assisted unweighted regression analysis was carried out Azoprocarbazine, azoxy-I-procarbazine, and /V-isopropyl-/>-formyl- by using the Statview (Brainpower, Inc., Calabasas, Ã‡A)statistical benzamide were provided by Dr. Russell A. Prough (University of program to generate all straight-line functions. The method of Bliss Louisville, Louisville, KY). Mafosfamide (2-[bis-(2-chloroethyl)-amino] -4-(2-sulfoethylthio)-tetrahydro-2//-1,3,2-oxazaphosphorine-2-oxide â€¢ (32) was used to generate LCÂ«Â«andLC50 values and 90% confidence cyclohexylamine was provided by Dr. P. Hilgard (Asta-Werke AG, intervals. Bielefeld, Federal Republic of Germany). Procarbazine was obtained from Mr. Frank F. Sorter, (Hoffmann-La Roche Inc., Nutley, NJ). RESULTS Phosphoramide mustard â€¢cyclohexylamineand conditioned medium from phytohemagglutinin-stimulated human peripheral blood leuko Preliminary experiments revealed that /V-isopropyl-p-formyl- cytes were supplied by Mr. L. H. Kedda (Drug Synthesis and Chemistry benzamide, but not procarbazine itself, inhibited the oxidation Branch, Division of Cancer Treatment, National Cancer Institute, of acetaldehyde to acetic acid catalyzed by a crude soluble Bethesda, MD), and Dr. F. Uckun (Department of Therapeutic Ra diology, University of Minnesota, Minneapolis, MN), respectively. fraction obtained from mouse liver, and that it was a substrate NAD+ was purchased from Sigma Chemical Co., St. Louis, MO. for the aldehyde dehydrogenase(s) present therein (data not Procarbazine, azoprocarbazine, azoxy-I-procarbazine, and /V-isopro- presented). pyl-p-formylbenzamide were dissolved in 95% ethanol. All other drugs jV-Isopropyl-/j-formylbenzamide was an excellent substrate were dissolved in triple-distilled water or drug-exposure media and for AHD-2; the Km value was 0.84 MM,Fig. 1. The Km value sterilized by passage through 0.22-^m Millipore filters. Drugs were defining the metabolism of an agent, e.g., jY-isopropyl-p-for- used within l h of preparation and were kept on ice until use. Drug- mylbenzamide, equals the K, value defining competitive inhi exposure medium was a phosphate-buffered, saline-based solution, pH bition by this agent of the metabolism of a second agent, e.g., 7.4, prepared as previously described (20). aldophosphamide, when the two are alternative substrates for Preparation of mouse liver soluble fractions was as described previ ously (28). Semipurified AHD-2 was provided by Dr. Carl Manthey, the same enzyme (33). Given the preceding and the fact that the Kmvalue for AHD-2-catalyzed oxidation of aldophospham University of Minnesota, Minneapolis, MN. A detailed description of its preparation is available (18). Selected physical and kinetic charac ide is 16 fiM(18), /V-isopropyl-p-formylbenzamide should mark- teristics of this enzyme have been published (15, 17. 18). Male BALB/c mice, 12-16 weeks old, were used as bone marrow donors for the murine CFU-S, CFU-GEMM, CFU-Mk, and CFU-GM experiments. They were also used as bone marrow recipients in the CFU-S experiments. These animals were obtained from Taconic, Ger- 1.0 mantown, NY. Animals were housed in plastic cages fitted with filtered lids and were given standard laboratory food and water ad libitum. A strict 12-h photoperiod was maintained. Cultured mouse LI210 and P388 cells, sensitive (LI210/0, P388/0) and resistant to oxazaphosphorines specifically (L1210/OAP) or to cross-linking agents in general (P388/CLA) (20), were obtained from 0.6 Southern Research Institute, Birmingham, AL, through the courtesy of Drs. R. F. Struck and L. J. Wilkoff. Culture and growth conditions were as previously described (20). The back-extrapolation assay of Alexander and Mikulski (29) was used as described previously (20) to determine the sensitivity of tumor cells to drugs. /V-Isopropyl-p-formylbenzamide or vehicle was added 0.2 exactly 3 min prior to the addition of cytotoxic agent or vehicle, after which the cells were incubated at 37Â°Cfor 30 min. The sensitivity of murine CFU-S to cytotoxic agents was determined by the method of Till and McCulloch (30) as modified by Kohn and 1.8 2.0 2.2 Sladek (9, 12). Preparation of bone marrow cell suspensions for this umol/min/mg assay was as previously described (9, 12). A'-Isopropyl-p-formylben- Fig. 1. jV-isopropyl-p-formylbenzamide oxidation catalyzed by AHD-2: inte zamide was added exactly 3 min prior to the addition of cytotoxic agent grated Michaelis equation kinetic analysis. A complete enzyme progress curve of or vehicle, after which the cells were incubated at 37Â°Cfor 30 min. the total consumption of initially saturating concentrations (15 /JM)of A'-isopro- Control values ranged from 16-33 colonies formed/8 x 10J nucleated pyl-p-formylbenzamide by AHD-2 was generated and analyzed as described in "Materials and Methods." Points represent a single analysis: the line was drawn cells injected. by least squares regression analysis from which the A'm(negative inverse of the The sensitivity of murine CFU-GEMM, CFU-Mk, and CFU-GM to slope) was determined. This experiment was repeated 2 more times. The mean drug treatment was determined, and preparation of bone marrow cell Km value for the 3 determinations was 0.84 Â±0.37 ^M. 4171

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1991 American Association for Cancer Research. POTENTIATION OF MAFOSFAMIDE BY A PROCARBAZINE METABOLITE phorine nitrogen mustard which spontaneously gives rise to 4- hydroxycyclophosphamide in physiological solutions, against cultured oxazaphosphorine-resistant L1210/OAP cells (data not presented). However, a marked, dose-dependent potentiation of the cytotoxic activity of mafosfamide against L1210/ OAP, as well as against oxazaphosphorine-resistant P388/ CLA, cells, was observed when /V-isopropyl-p-formylbenzamide was included in the incubation media; Fig. 2. Like L1210/OAP cells, P388/CLA cells contain an aldehyde dehydrogenase that catalyzes the detoxification of aldophosphamide (14, 20, 21). In contrast, potentiation by A'-isopropyl-p-formylbenzamide of the cytotoxic activity of mafosfamide against the oxazaphosphorine-sensitive LI210/0 and P388/0 cells was marginal at best. These cells lack aldehyde dehydrogenases capable of ca talyzing the oxidation of aldophosphamide (10, 19-21). A 40 SO 40 80 50 150 50 150 relatively small amount of potentiation of the cytotoxic action N-ISOPROPYL-p-FORMYLBENZAMIDE, nM of phosphoramide mustard, a cyclophosphamide metabolite not Fig. 2. Sensitivity of cultured murine tumor cells to mafosfamide and phos- susceptible to oxidation catalyzed by aldehyde dehydrogenases, phoramide mustard in the presence of iV-isopropyl-p-formylbenzamide. Vehicle or AMsopropyl-yj-formylbenzamide was added to tumor cell suspensions. Exactly was also observed. Collectively, these observations are consist 3 min later, vehicle (O). mafosfamide (â€¢),or phosphoramide mustard (D) was ent with the finding that /V-isopropyl-p-formylbenzamide is an added, and the cells were incubated for 30 min at 37Â°C.Concentrations of excellent substrate for AHD-2, and the notion that it competi cytotoxic agents were 21 (LI210/0). 155 (L1210/OAP), 5 (P388/0), and 35 tively inhibits AHD-2-catalyzed detoxification of aldo (P388/CLA) MMmafosfamide,and 114(L1210/0), 110(L1210/OAP), 50(P388/ 0), and 100 (P388/CLA) UMphosphoramide mustard. Cells were harvested at the phosphamide. end of the incubation period and were subsequently resuspended and grown in In order to quantify the effect of /V-isopropyl-/>-formylben- drug-free medium. Surviving fractions were determined by the back-extrapolation method as described in "Materials and Methods." Points represent the mean of zamide on the cytotoxic action of mafosfamide and phosphor- measurements on duplicate cultures. amide mustard, L1210/OAP and P388/CLA cells were exposed to increasing concentrations of mafosfamide or phosphoramide mustard in the presence of 200 uM /V-isopropyl-/7-formylbenzamide; Figs. 3 and 4. LC99 values for mafosfamide and phos phoramide mustard in the presence and absence of /V-isopropyl- /7-formylbenzamide were calculated from these data. The pres ence of jV-isopropyl-p-formylbenzamide reduced the LC99 val ues for mafosfamide from 176 to 17 MM(LI210/OAP), and from 44 to 6 MM(P388/CLA). These observations reflect essen tially full restoration of sensitivity to mafosfamide by yV-isopropyl-/7-formylbenzamide, since LC99 values for mafosfamide against wild type LI210/0 and P388/0 cells are 14 and 7 MM, respectively. The presence of /V-isopropyl-p-formylbenzamide also reduced

50 100 150 PHOSPHORAMIDE MUSTARD, uM Fig. 3. Sensitivity of murine L1210/OAP cells to mafosfamide or phosphoramide mustard in the presence of /V-isopropyl-p-formylbenzamide. Vehicle (O) or 200 MMA/-isopropyl-/>-formylbenzamide (â€¢)wasadded to tumor cell suspensions. Exactly 3 min later, vehicle or cytotoxic agent was added and cells were incubated at 37Â°Cfor 30 min. Cells were harvested at the end of the incubation period and were subsequently resuspended and grown in drug-free medium. Surviving frac tions were determined by the back-extrapolation assay as described in "Materials and Methods." Points represent the mean of measurements on duplicate cultures. The responses of cultured L1210/0 cells to mafosfamide are included for com parative purposes (D).

edly reduce the rate at which AHD-2 catalyzes the oxidation of aldophosphamide when both agents are present. 100 200 300 Effect of /V-Isopropyl-p-formylbenzamide on the Cytotoxic MAFOSFAMIDE, PHOSPHORAMIDE MUSTARD, M Action of Mafosfamide against Murine Tumor Cell Lines. Pre Fig. 4. Sensitivity of murine P388/CLA cells to mafosfamide or phosphor- liminary experiments established that 30-min exposure to pro- amide mustard in the presence of iV-isopropyl-p-formylbenzamide. Vehicle (O) or carbazine (1 rriM), or any of three of its metabolites, namely, 200 uM A'-isopropyl-p-formylbenzamide (â€¢)wasadded to tumor cell suspensions. Exactly 3 min later, vehicle or cytotoxic agent was added and cells were incubated azoprocarbazine (0.3 ITIM),azoxy-I-procarbazine (0.3 HIM),and at 37Â°Cfor 30 min. Cells were harvested at the end of the incubation period and yV-isopropyl-p-formylbenzamide (1 ITIM),was not toxic to cul were subsequently resuspended and grown in drug-free medium. Surviving frac tions Â»eredetermined by the back-extrapolation assay as described in "Materials tured LI210 cells (data not presented). Moreover, azoprocar and Methods." Points represent the mean of measurements on duplicate cultures. bazine (0.3 HIM) and azoxy-I-procarbazine (0.3 ITIM)did not The response of cultured P388/0 cells to mafosfamide are included for compar potentiate the cytotoxic action of mafosfamide, an oxazaphos- ative purposes (G). 4172

would potentiate the toxic action of the oxazaphosphorines against critical normal tissues known to express an aldehyde dehydrogenase that catalyzes the detoxification of aldophosphamide, e.g., mouse CFU-S, CFU-GEMM, and CFU-Mk, but not mouse CFU-GM (9, 11, 12). yV-isopropyl-/7-formylbenzamide itself was not toxic to mouse CFU-S, CFU-GEMM, or CFU-Mk, and only marginally toxic to mouse CFU-GM; Fig. 5. It potentiated the cytotoxic action of mafosfamide against CFU-S, CFU-GEMM, and CFU-Mk; it did not potentiate the cytotoxic action of mafosfamide against CFU-GM, nor did it potentiate the cytotoxic action of phos phoramide mustard against CFU-S. These observations are in keeping with the notions that (a) like mouse L1210/OAP and P388/CLA leukemia cells, mouse CFU-S, CFU-GEMM, and CFU-Mk are capable of detoxifying cyclophosphamide because 40 80 40 80 50 150 50 150 N-ISOPROPYL-p-FORMYLBENZAMIDE, MM they contain an enzyme, namely, aldehyde dehydrogenase, that Fig. 5. Sensitivity of murine CFU-S, CFU-GEMM, CFU-Mk, and CFU-GM catalyzes such a detoxification; (b) yV-isopropyl-/>-formyl- to a fixed concentration of mafosfamide or phosphoramide mustard in the presence of increasing concentrations of A'-isopropyl-p-formylbenzamide. Vehicle benzamide, because it too is a substrate for the enzyme, slows or /V-isopropyl-p-formylbenzamide was added to BALB/c bone marrow cell the rate at which detoxification occurs; and (c) CFU-GM lacks suspensions. Exactly 3 min later, vehicle (O), mafosfamide (â€¢),orphosphoramide mustard (D) was added, and cells were incubated for 30 min at 37Â°C.Cells were the relevant aldehyde dehydrogenase. harvested at the end of the incubation period, resuspended in drug-free medium, To quantify the potentiation observed in the preceding ex and assayed for CFU-S, CFU-GEMM, CFU-Mk, and CFU-GM as described in periments, CFU-GEMM's present in mouse bone marrow were "Materials and Methods." Concentrations of mafosfamide were 40 (CFU-S), 50 (CFU-GEMM), 35 (CFU-Mk), and 20 (CFU-GM) MM.The concentration of exposed to increasing concentrations of mafosfamide and a phosphoramide mustard was 64 UM(CFU-S). Points are mean Â±SE of observa fixed concentration (10 MM)of /V-isopropyl-p-formylbenzamide, tions made in 3-4 mice (day 12, CFU-S), or mean Â±SEof triplicate plates (CFU- GEMM, CFU-Mk, CFU-GM). Results are expressed as percentage of control and LC5ovalues were determined; Fig. 6. This value was 56 and (exposed to vehicle only). *, significantly different from control, P < 0.05; **. 10 MMwhen /V-isopropyl-p-formylbenzamide was omitted from significantly different from mafosfamide, P s 0.05. and included in, respectively, the exposure media. Human hematopoietic progenitor cells, e.g., CPU-Mix, CFU- Mk, CFU-GM, and BFU-E, also express aldehyde dehydroge-

O nase(s) capable of catalyzing the detoxification of aldophos-

m phamide (10). N-isopropyl-p-formylbenzamide potentiated T 75 the cytotoxic action of mafosfamide against these cells as well; Fig. 7. O

E O

o Ãœ o â€ž 10 100 MAFOSFAMIDE, uM Fig. 6. Sensitivity of murine CFU-GEMM to mafosfamide in the absence and presence of a fixed concentration of /V-isopropyl-p-formylbenzamide. Vehicle (O) or 10 Â¡Â¿MW-isopropyl-/>-formylbenzamide(â€¢)wasadded to BALB/c bone marrow cell suspensions. Exactly 3 min later, vehicle or mafosfamide was added, and cells were incubated for 30 min at 37'C. Following incubation, cells were harvested, resuspended in drug-free medium, and assayed for CFU-GEMM as described in "Materials and Methods." yV-Isopyropyl-p-formylbenzamide alone was without cytotoxic activity. Results are expressed as percentage of inhibition of the relevant control. Points are the mean of three experiments; bars, SE.

20 60 100 20 60 100 20 60 100 20 60 100 the LC99 values for phosphoramide mustard but to a much N-ISOPROPYL-p-FORMYLBENZAMIDE. MM smaller extent (less than 2-fold); reduction was from 127 to 68 Fig. 7. Sensitivity of human progenitor cells to a fixed concentration of MMin the case of L1210/OAP cells, and 184 to 104 MMin the mafosfamide in the presence of increasing concentrations of /V-isopropyl-p-for- case of P388/CLA cells. mylbenzamide. Vehicle or A'-isopropyl-p-formylbenzamide was added to mono- nuclear bone marrow cell suspensions. Exactly 3 min later, vehicle (O) or 20 nM Effect of jY-Isopropyl-p-formylbenzamide on the Cytotoxic mafosfamide (â€¢)was added and cells were incubated for 30 min at 37Â°C.Cells Action of Mafosfamide against Hematopoietic Progenitor Cells. were harvested at the end of the incubation period, resuspended in drug-free The effect of /V-isopropyl-p-formylbenzamide on the toxic ac medium, and assayed for CPU-Mix, CFU-Mk, CFU-GM, and BFU-E. Control colony numbers were 24 (CPU-Mix), 40 (CFU-Mk), 200 (CFU-GM), and 175 tion of mafosfamide against hematopoietic progenitor cells was (BFU-E). Points, mean of measurements made on duplicate plates; bars, range. examined to determine if jV-isopropyl-p-formylbenzamide Results are expressed as percentage of control (exposed to vehicle only). 4173

DISCUSSION alyzed hepatic-soluble fraction was reduced to 37% of control values when livers were obtained from female DBA/2 mice that The results of the present investigation demonstrate that a had repeatedly been given a relatively high dose of procarbazine, metabolite of procarbazine, namely, jV-isopropyl-/?-formylben- namely, 100 mg/kg, i.p., 96, 72, 48, 24, and l h before sacrifice zamide, is an excellent substrate for mouse AHD-2 (Km = 0.84 (data not presented). Vasiliou et al. (36) have made similar Ã•Â¿M).Theexpectation is that, as an alternative substrate, N- observations in rats. isopropyl-p-formylbenzamide would be a good competitive in We know of no clear clinical example of increased toxicity hibitor of aldophosphamide oxidation catalyzed by this enzyme due to the anticipated drug interaction but, perhaps relevant, (Km = 16 Â¿/M).Manthey et al., (18) demonstrated that this the in vivo toxic action of cyclophosphamide on mouse intes enzyme accounts for the bulk of total hepatic aldehyde dehy- tinal crypt cells was markedly increased when an inhibitor of drogenase-catalyzed oxidation of aldophosphamide. Taken to aldehyde dehydrogenase, namely, 4-(diethylamino)-benzalde- gether these observations suggest that A'-isopropyl-p-formyl- hyde, was coadministered with cyclophosphamide; 4-(diethyl- benzamide might reduce hepatic clearance of aldophosphamide amino)-benzaldehyde and A-isopropyl-/Â»-formylbenzamide are when procarbazine and cyclophosphamide are administered structurally quite similar (14, 37). It should also be noted that concurrently. However, because the liver contains such a large the postulated drug interaction may be advantageous when the excess of AHD-2, hepatic clearance of aldophosphamide is therapeutic objective is to prevent the graft rejection that oth thought to be a flow-limited process (28), and it is therefore erwise often occurs after bone marrow transplantation; indeed, unlikely that inhibition of hepatic AHD-2 by /V-isopropyl-p- there is some evidence to support this notion (7). formylbenzamide would potentiate the systemic action of cyclo A-isopropyl-p-formylbenzamide also potentiated the cyto- phosphamide. Further complicating this prognostication is that toxic action of mafosfamide against oxazaphosphorine-resist- several other aldehyde dehydrogenases capable of catalyzing ant mouse L1210/OAP and P388/CLA tumor cells. These cell the oxidation of aldophosphamide have been identified in lines are resistant to oxazaphosphorines because they contain mouse liver (18). It is not known whether A'-isopropyl-/Â»-for- relatively high levels of an aldehyde dehydrogenase that cata mylbenzamide is a substrate for these enzymes, although that lyzes the detoxification of aldophosphamide. High levels of a would seem likely. relevant aldehyde dehydrogenase are likely to be found in The foregoing does not, however, preclude the possibility that human tumor populations that become relatively insensitive to local inhibition of a relevant aldehyde dehydrogenase by N- the oxazaphosphorines (but not to other antitumor agents) after isopropyl-p-formylbenzamide in critical normal tissues which repeated exposure to them, although there is currently no may express smaller amounts of this activity, e.g., bone marrow evidence that this occurs clinically. However, it has been dem hematopoietic progenitor cells, might enhance the toxicity of onstrated that human mammary and melanoma cell lines cul mafosfamide against such tissues/organs. The results of the tured in media containing progressively increasing concentra present investigation suggest that this is, in fact, a possible tions of an oxazaphosphorine become resistant only to the outcome of combined administration of procarbazine and cy oxazaphosphorines (38, 39), suggesting that the underlying clophosphamide. We observed a sizeable potentiation of the biological basis for the oxazaphosphorine-specific resistance is toxicity of mafosfamide against both murine and human he an increase in a relevant aldehyde dehydrogenase. Measurable matopoietic progenitor cells at a very low concentration (10 amounts of relevant aldehyde dehydrogenase activity were pres MM)of A-isopropyl-p-formylbenzamide. ent in 4 Wilms tumors and a testicular tumor.5 Colvin and Potentiation of cyclophosphamide-induced bone marrow tox Hilton (40) detected aldehyde dehydrogenase activity in several icity by procarbazine in vivo would be expected only if suffi human tumor cell lines, but it is not clear whether the activity ciently high concentrations of A-isopropyl-p-formylbenzamide was relevant, since the substrate used for these studies was not were generated. This would seem likely given that (a) l h after identified. Potentially, procarbazine (A-isopropyl-p-formyl- the p.o. administration of procarbazine to rats, 72% of total benzamide) could sensitize neoplastic cells that contain the drug found in plasma was /V-isopropyl-p-formylbenzamide (25); relevant aldehyde dehydrogenase activity to cyclophosphamide (b) the usual human dose of procarbazine is 100 to 200 mg/ and other oxazaphosphorines, thus providing an acceptable nr/day (34); (c) up to 70% of the administered human dose is rationale for combining these two agents when treating such recovered in the urine as A'-isopropylterephthalamic acid within neoplasias. The downside is that, as discussed above, certain 24 h of p.o. or parenteral procarbazine administration (34); (d) critical normal cells, e.g., bone marrow hematopoietic cells, A'-isopropylterephthalamic acid is the metabolite generated would also become more sensitive to the oxazaphosphorine, so when A-isopropyl-/?-formylbenzamide is oxidized (26); and (e) that there may be no net improvement in the therapeutic index in our models, substantial potentiation was observed at concen (margin of safety) of the oxazaphosphorine, and when the tumor trations of A-isopropyl-p-formylbenzamide as low as 10 n\\. cells lacked the relevant aldehyde dehydrogenase activity, the Moreover, procarbazine is known to produce a "disulfiram- margin of safety would actually decrease. The relevant aldehyde like" effect, namely, nausea, vasodilation. headache, etc., when dehydrogenase activity was not present in 87 human primary leukemias and lymphomas obtained from oxazaphosphorine- alcohol is ingested following its administration (35). This re naive patients.6 action is attributed to the accumulation of acetaldehyde result ing from the inhibition of aldehyde dehydrogenase(s). In the The significance of the present investigations extends beyond case of procarbazine, inhibition could be by A'-isopropyl-p- potential drug interactions between procarbazine and cyclo formylbenzamide, meaning that the concentration of this me phosphamide. Review of the literature reveals >30 drugs which are reported to produce a disulfiram-like effect. Many of these, tabolite is sufficiently high to do so. Alternatively, decreased e.g., chloramphenicol, chloral hydrate, and methyltetrazole- enzyme activity could be due to irreversible inhibition of the thiol-containing cephalosporins, are used as adjunctive therapy enzyme or a reduction in the amount of enzyme present effected by procarbazine or one of its metabolites. Thus, aldehyde de- *C. L. Manthey and N. E. Sladck. unpublished observations. hydrogenase-catalyzed oxidation of acetaldehyde by a nondi- 6 N. E. Sladck and F. M. Uckun, unpublished observations. 4174

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1991 American Association for Cancer Research. POTENTIATION OF MAFOSFAM1DE BY A PROCARBAZ1NE METABOLITE in the treatment of cancer patients receiving cyclophosphamide. human hematopoietic progenitor cells. Blood, 75: 1947-1950, 1990. 14. Russo. J. E., Hilton. J.. and Colvin, O. M. The role of aldehyde dehydroge Obvious are the clinical consequences if, like /V-isopropyl-p- nase isozymes in cellular resistance to the alkylating agent cyclophosphamide. formylbenzamide, these agents inhibit the human aldehyde Prog. Clin. Biol. Res.. 290: 65-79. 1989. dehydrogenase(s) that contribute to the detoxification of aldo- 15. Manthey, C. L. Resolution and characterization of the aldehyde dehydrogenases important in cyclophosphamide metabolism. Ph.D. Dissertation, Uni phosphamide. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1991 American Association for Cancer Research. Potentiation of the Cytotoxic Action of Mafosfamide by N -Isopropyl-p-formylbenzamide, a Metabolite of Procarbazine

Patricia A. Maki and Norman E. Sladek

Cancer Res 1991;51:4170-4175.

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