Indirect oxidation of the antitumor agent procarbazine by tyrosinase--possible application in designing anti-melanoma prodrugs.

The interaction of tyrosinase with the anticancer drug procarbazine has been investigated. In the presence of the enzyme alone no oxidation of this dialkylhydrazine above the background level was observed. However, when phenolic substrates (4-tert-butylcatechol or N-acetyl-l-tyrosine) were included in the reaction mixture, procarbazine was rapidly degraded. Oxygen consumption measurements showed that in a mixture both the phenolic substrate and the drug were oxidized. The major product of procarbazine degradation was isolated and identified as azoprocarbazine, the first active metabolite of this drug detected in previous in vivo and in vitro studies. This indirect oxidation of the hydrazine group in this anticancer agent indicates possible application of a hydrazine linker in construction of tyrosinase-activated anti-melanoma prodrugs.

Major isozymes of rat liver microsomal cytochrome P-450 involved in the N-oxidation of N-isopropyl-alpha-(2-methylazo)-p-toluamide, the azo derivative of procarbazine.

Seven isozymes of cytochrome P-450 were tested to establish whether they could N-oxidize azoprocarbazine to form the two isomeric azoxy metabolites after optimizing the reconstitution of various purified isozymes with regard to substrate concentration, exogenous lipid, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c (P-450) reductase concentration. Two isozymes, cytochromes P-450PB-C (an isozyme present in untreated rats or in rats treated with phenobarbital or beta-naphthoflavone) and P-450 beta NF-B (the major beta-naphthoflavone-induced isozyme), had appreciable turnover numbers for the N-oxidation reaction. The product ratio [N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide formation relative to N-isopropyl-alpha-(methyl-NNO-azoxy)-p-toluamide formation] obtained with cytochrome P-450PB-C was nearly identical to those values obtained with liver microsomes from untreated and phenobarbital-treated rats. In addition, cytochrome P-450 beta NF-B and liver microsomes from beta-naphthoflavone-treated rats had nearly identical product ratios. Specific inhibitory antibodies to four purified isozymes were used to titrate the N-oxidase activity of liver microsomes from untreated, phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats. Anti-cytochrome P-450PB-C globulin inhibited more than 70 to 90% of the formation of N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide in microsomes from untreated, phenobarbital-, and pregnenolone-16 alpha-carbonitrile-treated rats, respectively, but only 20 to 50% of N-isopropyl-alpha-(methyl-NNO-azoxy)-p-toluamide formation. A small amount (25 to 30%) of inhibition was observed with anti-cytochrome P-450PB/PCN-E globulin. Anti-cytochrome P-450 beta NF-B globulin inhibited more than 85% of the synthesis of either azoxy isomer catalyzed by liver microsomes from beta-naphthoflavone-treated rats. These results demonstrate that two isozymes are responsible for the oxidative metabolism of azoprocarbazine and can account for the major portion of this N-oxidase activity in liver microsomes from untreated and phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats.

Methylazoxyprocarbazine, the active metabolite responsible for the anticancer activity of procarbazine against L1210 leukemia.

Procarbazine is a 1,2-disubstituted hydrazine derivative that is used to treat human leukemias. The anticancer activity of procarbazine results from bioactivation to reactive intermediates. It is first oxidized to azoprocarbazine and further N-oxidized to a mixture of methylazoxyprocarbazine and benzylazoxyprocarbazine isomers. In this study the azoxyprocarbazine isomers were synthesized and purified. The cytotoxic effect of the metabolites on the L1210 murine leukemia cell line were then evaluated in vitro by use of a colorimetric assay using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. The results of this study showed that the methylazoxyprocarbazine isomer was the most cytotoxic metabolite (IC50, 0.2 mM). The benzylazoxy isomer had an insignificant cytotoxic effect, and a mixture of the two isomers was intermediate in effectiveness. This assay, however, could not be used to determine the cytotoxicity of procarbazine since the drug itself (not the live cells) reduced the dye. A soft-agar clonogenic assay demonstrated that procarbazine was cytotoxic only at higher concentrations (IC50, 1.5 mM) than methylazoxyprocarbazine (IC50, 0.15 mM). The effect of procarbazine and its metabolites on the survival of L1210 tumor-bearing mice was determined, and methylazoxyprocarbazine was again the most effective compound. These studies demonstrate that the methylazoxyprocarbazine metabolite is probably the major cytotoxic intermediate involved in the mechanism of anticancer action of procarbazine.

Potentiation of the cytotoxic action of mafosfamide by N-isopropyl-p-formylbenzamide, a metabolite of procarbazine.

Several mouse aldehyde dehydrogenases catalyze the detoxification of aldophosphamide, the pivotal metabolite of the prodrugs cyclophosphamide, mafosfamide, and other oxazaphosphorines. N-Isopropyl-p-formylbenzamide, a major metabolite of procarbazine, was found to be an excellent substrate (Km = 0.84 microM) for at least one of these enzymes, namely, mouse aldehyde dehydrogenase-2. The Km for mouse aldehyde dehydrogenase-2-catalyzed detoxification of aldophosphamide is 16 microM. Thus, competition between N-isopropyl-p-formylbenzamide and aldophosphamide for the catalytic site on the enzyme should strongly favor the former, and the rate at which aldophosphamide is detoxified should be markedly retarded. Mouse L1210/OAP and P388/CLA leukemia cells are relatively insensitive to the oxazaphosphorines because they contain large amounts of mouse aldehyde dehydrogenase-2. As predicted, N-isopropyl-p-formylbenzamide markedly potentiated the cytotoxic action of mafosfamide against these cells. Mouse L1210/0 and P388/0 lack the enzyme. Again as expected, N-isopropyl-p-formylbenzamide essentially did not potentiate the cytotoxic action of mafosfamide against these cells. Certain mouse and human hematopoietic progenitor cells also contain an aldehyde dehydrogenase that catalyzes the detoxification of aldophosphamide, but the specific identity of this enzyme remains to be established. N-Isopropyl-p-formylbenzamide potentiated the cytotoxic action of mafosfamide against these cells as well. Clinically, procarbazine and the oxazaphosphorines are used to treat certain neoplastic diseases. Frequently, they are used in combination. Our findings demonstrate the potential for both desirable and undesirable drug interactions when these agents are used concurrently. Similar drug interactions can be expected when other substrates for, or inhibitors of, the relevant aldehyde dehydrogenases, e.g., chloramphenicol, chloral hydrate, and methyltetrazolethiol-containing cephalosporins, are co-administered with the oxazaphosphorines.

A structural modification study of procarbazine.

Eight analogues of the antineoplastic compound procarbazine were prepared by varying one portion of the molecule, keeping either the methylhydrazinomethyl or the N-(1-methylethyl)benzamido portion of procarbazine intact. Preliminary screening results indicated that none of the analogues tested in leukemias L1210 and P388 were as active as the original compound.

The in vivo cytotoxic activity of procarbazine and procarbazine metabolites against L1210 ascites leukemia cells in CDF1 mice and the effects of pretreatment with procarbazine, phenobarbital, diphenylhydantoin, and methylprednisolone upon in vivo procarbazine activity.

An in vivo assay of the activity of procarbazine, N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride, and several metabolic intermediates against IP-implanted L1210 leukemia cells in CDF1 male mice is described. Treatment of tumor-bearing mice with procarbazine at doses of 300-500 mg/kg IP increased the mean lifespan of treated mice by 29%-32% relative to that of untreated animals. Procarbazine treatment with doses of 200-400 mg/kg/day given IP for 3 consecutive days increased mean lifespan by 39%-46%. The major circulating metabolite, azoprocarbazine (N-isopropyl-alpha-(2-methylazo)-p-toluamide), was as active as procarbazine when administered at equivalent doses for 3 consecutive days. A 2:1 mixture of azoxyprocarbazines (N-isopropyl-alpha-(2-methyl-ONN-azoxy)-: and N-isopropyl-alpha-(2-methyl-NNO-azoxy)-p-toluamide) was more active than procarbazine, increasing mean lifespan by 76% using the 3-consecutive-day dose schedule. The effects of pretreatment with procarbazine and drugs that are often co-administered with procarbazine, i.e., phenobarbital, diphenylhydantoin, and methylprednisolone, upon procarbazine anticancer activity against L1210 ascites leukemia cells was also determined. Pretreatment of CDF1 male mice with phenobarbital and diphenylhydantoin for 7 days was found to increase the antineoplastic activity of procarbazine by 13%-24%. Pretreatment with methylprednisolone did not significantly alter procarbazine activity. The effects of pretreatment with procarbazine, which is often administered daily for a period of 2-4 weeks, on procarbazine antineoplastic activity were varied. The results of these preliminary pretreatment studies combined with the finding that procarbazine metabolites have antitumor activity that is equal to or greater than that of the parent drug suggest that current clinical protocols that use procarbazine along with agents capable of altering procarbazine metabolism may involve drug interactions that alter the efficacy of procarbazine as an anticancer agent.

Non-enzymatic activation of procarbazine to active cytotoxic species.

The cellular cytotoxicity of procarbazine is thought to result from bioactivation of the parent compound through reactive intermediates to an ultimate alkylating species. Procarbazine is converted initially to azoprocarbazine, which is then N-oxidized through a cytochrome P-450-mediated process to a mixture of the positional isomers, benzylazoxyprocarbazine and methylazoxyprocarbazine. In order to define the bioactivation events that lead to the cytotoxic species, the in vitro cytotoxicities of the purified azoxy isomers as well as of the parent compound, procarbazine, were evaluated with the human leukemia cell line, CCRF-CEM. The methylazoxy isomer was found to be the most active species. Procarbazine inhibited the growth of CCRF-CEM cells but at a concentration much higher than that required for the methylazoxy isomer. Since procarbazine must be metabolized to form the cytotoxic species, we sought to determine if the active metabolite, methylazoxyprocarbazine, was being formed in the incubations. Solutions of procarbazine incubated with and without cells at 37 degrees C were analyzed by combined liquid chromatography-mass spectrometry with a thermospray interface. The azoxy metabolites of procarbazine appeared rapidly in cellular incubations and in the aqueous solutions without cells. More of the methylazoxy isomer was formed initially, but by 72 hr the benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was non-enzymatically oxidized to the two azoxyprocarbazine isomers and that the methylazoxy compound was the most cytotoxic to CCRF-CEM cells.

Potential antitumor agents: procarbazine analogs and other methylhydrazine derivatives.

With the objective of developing new antitumor agents, two groups of hydrazine compounds, having structural features in common with the antitumor agents procarbazine and 1-acetyl-2-picolinoylhydrazine, were synthesized. The L-1210 leukemia system was used to evaluate compounds of both groups. The aliphatic procarbazines also were screened for antitumor activity as bis(benzyloxycarbonyl) derivatives and as derivatives having a phthalazine nucleus. No L-1210 antitumor activity was exhibited by these compounds.

Effects in-vitro of procarbazine metabolites on some amine oxidase activities in the rat.

The effects were examined of four metabolites of the anticancer agent, procarbazine (N-isopropyl-alpha-(2-methyl hydrazino)-p-toluamide hydrochloride) on semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase-A and -B (MAO-A and -B) activities in rat brown adipose tissue and liver homogenates, respectively. Azoprocarbazine (AZO) and monomethylhydrazine (MMH) inhibited selectively the deamination of benzylamine by SSAO, when compared with their effects on MAO activities. The IC50 values against SSAO, of 32.7 nM (AZO) and 7.0 nM (MMH), were more than three orders of magnitude lower than those exhibited against MAO. Neither isomer of azoxyprocarbazine was an effective inhibitor of rat amine oxidase activities. The inhibition of SSAO by AZO was reversed very slowly by dialysis, in contrast to results seen for MMH. The non-competitive kinetics of MMH and the ability of B24, a rapidly reversible SSAO inhibitor, to protect SSAO against inhibition by MMH are consistent with the view that this compound binds to the enzyme cofactor at, or near, the active site.

Plasma kinetics of procarbazine and azo-procarbazine in humans.

The plasma kinetics of procarbazine (PCB) and its major metabolite azo-procarbazine (azo-PCB) were systematically investigated in humans for the first time. Eight therapy-refractory tumor patients with normal liver and renal function were given a single oral dose of 300 mg PCB hydrochloride as a drinking solution under fasting conditions. With the exception of the single i.v. administration of 10 mg ondansetron hydrochloride immediately before the administration of PCB, the patients were free of any co-medication 4 weeks before and during the study. PCB and azo-PCB were determined by a specially developed HPLC-UV method. PCB was absorbed very rapidly. Mean maximum plasma concentration was 12.5 min. A high elimination rate of PCB from plasma was found. The mean apparent oral systemic clearance and the plasma elimination half-life were estimated at 35.8 l/min and 9.2 min, respectively. Considerable amounts of azo-PCB are found in the plasma of the eight tumor patients. The mean Cmax and AUC ratios of azo-PCB/PCB were estimated at 5.5 and 45.2. Azo-PCB is formed very rapidly from PCB, but eliminated much more slowly from plasma than PCB. Considerable interindividual differences in the conversion rate of azo-PCB to its further metabolites were observed which should have consequences for the individual tumor therapeutic efficiency of PCB. No toxic side-effects or symptoms such as nausea or vomiting were observed during the entire study.

Analysis of procarbazine and metabolites by gas chromatography-mass spectrometry.

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography-mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride. A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma. Studies of the degradation of procarbazine . HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine . HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine . HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.

Isolation, purification, and characterization of two new chemical decomposition products of methylazoxyprocarbazine.

We have previously reported that the antineoplastic agent, procarbazine, in aqueous solutions was chemically oxidized to its azoxy metabolites (methylazoxy and benzylazoxy). To determine if there was additional metabolism of the most active metabolite, methylazoxyprocarbazine, it was incubated in the presence and absence of CCRF-CEM human leukemia cells. Incubations were extracted, and potential metabolites were detected by HPLC with UV detection and by combined HPLC and thermospray mass spectrometric analysis. The major metabolite identified by HPLC with UV detection of the extracts was N-isopropyl-p-formylbenzamide; this was identified by comparison of its retention time with that of a synthesized standard. This identification was further corroborated by HPLC/thermospray mass spectrometry (LC/MS). Analysis of the extracts by LC/MS also showed the presence of a closely eluting peak that had a protonated molecular ion at m/z 207. This new metabolite was identified as N-isopropyl-(benzene-1,4-bis-carboxamide) by 1H NMR and gas chromatography/ion trap mass spectrometry. This metabolite is postulated to arise from breakage of the N-N bond in the hydrazine portion of the molecule. Reconstructed ion (m/z 236) current profiles from the analysis of the cell extracts indicated that there was only a trace amount of methylazoxyprocarbazine left after a 72-hr incubation. Interestingly, a peak with the same molecular weight as the parent compound (methylazoxyprocarbazine) was observed in the cellular incubations and also in extracts of control incubations in which methylazoxyprocarbazine was incubated in medium without cells. This unknown was silylated and identified as a hydroxyazo compound by an ion trap mass spectrometer operated under both single and multiple-stage mass analysis. Formation of this decomposition product appears to involve a novel intramolecular rearrangement of methylazoxyprocarbazine in solution. This pathway may be responsible for the formation of the ultimate cytotoxic species by chemical decomposition of procarbazine.

Studies on the pathway of methane formation from procarbazine, a 2-methylbenzylhydrazine derivative, by rat liver microsomes.

The oxidative metabolism of procarbazine, its azo, hydrazone, and two azoxy derivatives, and methylhydrazine by hepatic microsomes from phenobarbital-pretreated rats was investigated to elucidate the pathway of metabolism that resulted in methane formation from procarbazine. When incubated with microsomal reaction mixtures fortified with NADPH, all of the compounds, except the azoxy isomers, were metabolized to yield methane. A lag phase in methane formation was noted for procarbazine, but not for the other compounds. Kinetic and inhibition studies utilizing methimazole and ethylhydrazine precluded methylhydrazine as an intermediate in methane formation from procarbazine. When the azo derivative was oxidatively metabolized in the presence of liver microsomes, no hydrazone tautomer was detected. Upon monitoring the production of the azo and hydrazone metabolites formed during microsomal metabolism of procarbazine, the azo derivative was formed in sufficient quantities to account for the majority of the methane produced. In addition, small amounts of hydrazone were also detected. It was concluded that both the azo and hydrazone metabolites of procarbazine contribute to methane formation from the terminal methyl group of the hydrazine with the azo derivative being the predominant source and the hydrazone derivative being a minor source of methane. Consideration of the chemical and enzymatic pathways of procarbazine oxidation and the implication of a methyl radical intermediate in methane formation are discussed.

Metabolic activation of the terminal N-methyl group of N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine).

The NADPH-dependent microsomal metabolism of [14C]procarbazine, labeled on the terminal N-methyl group, resulted in the covalent binding of the drug to exogenously added DNA; this reaction was inhibited by metyrapone. Procarbazine metabolism was also shown to result in covalent binding of the methyl group of the drug to microsomal protein upon metabolism, but the extent of protein binding was at least an order of magnitude smaller than that seen with its primary oxidative metabolite. N-isopropyl-alpha-(2-methylazo)-p-toluamide. The characteristics of the reactions leading to the covalent binding of the N-methyl group of the azo derivative to microsomal protein and its metabolism to form the hydrocarbon, methane, possessed a number of similarities in the apparent kinetic parameters (Km and Vmax), induction, and inhibition patterns indicating a common pathway of metabolism to form a reactive intermediate and the involvement of cytochrome P-450. Reduced glutathione stimulated methane formation and inhibited covalent binding to protein. One azoxy derivative, N-isopropyl-alpha-(2-methyl-ONN-azoxy)-p-toluamide, was chemically unstable and its decomposition was shown to lead to covalent binding to microsomal protein. A diazene intermediate and a methyl radical are proposed to be intermediates in the formation of methane during the oxidative metabolism of the azo derivative of procarbazine and a common intermediate in the activation of procarbazine may result in both covalent binding to cellular macromolecules and methane production. In addition, chemical decomposition of the azoxy metabolites may also contribute to a small portion of the covalent binding, but not to methane formation.

Evaluation of the dysmorphogenic effects of procarbazine, benzylazoxyprocarbazine, and methylazoxyprocarbazine in whole embryo culture.

Serum from procarbazine (PCZ)-treated rats is dysmorphogenic to rat embryos cultured in vitro, but PCZ is not effective when added directly to culture medium, even with an exogenous metabolizing system. Methylazoxyprocarbazine (MPCZ) is a metabolite which we have identified by HPLC in the dysmorphogenic serum of PCZ-treated rats. PCZ, MPCZ, and benzylazoxyprocarbazine (BPCZ, an isomer of MPCZ) were tested in rat whole embryo culture to determine their effects on embryo development. The parent compound, PCZ, produced no effect on embryo growth or development at concentrations up to 200 micrograms/ml. MPCZ proved to be the most potent of the agents tested. There was significant embryo lethality at concentrations of > or = 10 micrograms/ml while 25 micrograms/ml had significantly reduced embryonic developmental score (DEVS), and 35 micrograms/ml reduced DEVS, head length, and somite number. There was 89% embryo lethality at the 50 micrograms/ml exposure level. At concentrations > 5 micrograms/ml, there were significant increases in anomalies, primarily, failure of neural tube closure, erratic neural seam, and microcephaly. In contrast, BPCZ produced embryo lethality and reductions in DEVS only at 100 micrograms/ml. These data suggest that MPCZ, which has been identified in PCZ-treated rat serum, may be the proximate dysmorphogenic metabolite of PCZ.

In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study.

Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.