Characterization of "peak E," a novel amino acid associated with eosinophilia-myalgia syndrome.

Epidemiologic studies strongly associate eosinophilia-myalgia syndrome (EMS) with ingestion of tryptophan containing a contaminant ("peak E"). Prior reports have suggested that peak E is the di-tryptophan N alpha-animal of acetaldehyde. Spectral and chemical studies now demonstrate that peak E is 1,1'-ethylidenebis[tryptophan]. This novel amino acid may be the etiological agent responsible for EMS, or it may be a marker of a still unidentified causal agent.

Role of N-methylolpentamethylmelamine in the metabolic activation of hexamethylmelamine.

Hexamethylmelamine (HMM) is metabolized by rat hepatic microsomal preparations to reactive species which covalently bind to microsomal protein and to calf thymus DNA added to microsomal incubation mixtures. Covalent binding to macromolecules is dependent on the presence of molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate and is catalyzed by cytochrome P-450 monooxygenases. Reduced nicotinamide adenine dinucleotide-dependent covalent binding of [methyl-14C]HMM to microsomal protein is greater than that of [ring-14C]HMM. Reduced nicotinamide adenine dinucleotide phosphate-dependent covalent binding of [ring-14C]HMM and [methyl-14C]HMM to calf thymus DNA added to microsomal incubation mixtures are approximately equal. The [ring-14C]-labeled carbinolamine intermediate in HMM demethylation, N-methylolpentamethylmelamine, covalently binds to microsomal protein and, to a much greater extent, to calf thymus DNA.

Pharmacological characterization of teroxirone, a triepoxide antitumor agent, in rats, rabbits, and humans.

Teroxirone is an experimental triepoxide antitumor agent currently undergoing evaluation in clinical trials. We have developed an assay based on derivatization with diethyldithiocarbamate followed by normal-phase high-performance liquid chromatographic analysis. When 14C-labeled teroxirone is administered to rabbits by rapid i.v. infusion, plasma disappearance of parent drug is very rapid (t1/2 less than 5 min), while plasma 14C-labeled drug equivalents are eliminated at a much slower rate (t1/2 greater than 60 min). Twenty-four-hr urinary recovery of parent drug is less than 1%, while recovery of 14C total radioactivity is 60 to 70%. Rapid plasma elimination (t1/2 less than 5 min) and total body clearance (greater than 5 liters/min) are observed following rapid i.v. administration of teroxirone to humans. When teroxirone is administered to humans at constant rates of infusion, plateau concentrations are rapidly achieved and maintained during infusion. Plasma concentrations rapidly decrease upon cessation of infusion. Less than 1% parent drug is recovered in 24-hr urine. Teroxirone is relatively stable in fresh human plasma and whole blood. Teroxirone is metabolized by rat liver, but not lung, microsomal preparations by an NADPH-independent pathway. Epoxide hydrolysis metabolites are detected in microsomal incubations, and cyclohexene oxide inhibits teroxirone metabolism, suggesting that epoxide hydrase may be responsible for teroxirone biotransformation. Cytotoxicity of teroxirone against continuous human tumor cell lines is abolished in the presence of 9000 X g rat liver supernatant preparations but partially restored when cyclohexene oxide is added to incubation mixtures.

Metabolic conversion of indicine N-oxide to indicine in rabbits and humans.

Indicine N-oxide, a pyrrolizidine alkaloid N-oxide that exhibits antitumor activity without some of the toxic effects associated with other pyrrolizidine alkaloids, is metabolized to indicine in rabbits and humans. Indicine can be detected in the plasma and is excreted in the urine in a dose-dependent manner following the i.v. administration of indicine N-oxide. The p.o. administration of indicine N-oxide leads to an increased plasma concentration and an increased urinary excretion of indicine. The hepatic microsomal fraction and the gut flora both catalyze the anaerobic reduction of indicine N-oxide to indicine in vitro. Whole-animal studies suggest that the gut flora play a major role in the metabolic reduction of indicine N-oxide by the rabbit. Indicine N-oxide is not actively excreted in the bile, and it is probable that indicine N-oxide finds its way into the gut by passive diffusion following i.v. administration. Neomycin and erythromycin, which reduce the number of anaerobic bacteria in the gut, lead to decreased plasma levels and a decreased urinary excretion of indicine.

Disposition and metabolism of pentamethylmelamine and hexamethylmelamine in rabbits and humans.

The disposition and metabolism of pentamethylmelamine (PMM) and hexamethylmelamine (HMM) were studied in the rabbit, and the disposition of PMM was studied in humans. Parent compound and metabolites were identified by thin-layer chromatography, gas chromatography, and gas chromatography/mass spectrometry analyses. Plasma elimination in both species following i.v. administration of each drug was best described by a two-compartment open model. Both compounds were extensively demethylated with less than 1% of the total dose administered recovered in the urine over 24 hr. The areas under the plasma time-concentration curves of PMM and HMM following p.o. administration to rabbits were 5 and 25% of the areas following i.v. administration. Gastrointestinal absorption was rapid and efficient with 75 to 89% of drug equivalents recoverable in the urine after p.o. administration of [ring-14C]PMM or [ring-14C]HMM to rabbits. Reduced bioavailability of PMM and HMM p.o. appears to be a consequence of rapid metabolism presumably in the liver.

Phase I and clinical pharmacological evaluation of a parenteral hexamethylmelamine formulation.

Hexamethylmelamine has been evaluated in single agent and combination regimen studies for many years, but only following p.o. administration. Pharmacological studies in animals and humans have shown that systematic availability of parent drug following p.o. administration is relatively low and variable due to extensive first-pass metabolism rather than due to poor absorption. Two Phase I clinical trials, with accompanying pharmacokinetic studies, have been conducted by using a parenteral formulation in which hexamethylmelamine was prepared by Intralipid 10%. The parenteral formulation was well tolerated by all patients receiving hexamethylmelamine by 1-day and by daily for 5-days schedules. Nausea and vomiting were the dose-limiting toxicities. Maximally tolerated doses on the 1-day and daily for 5-days schedules were approximately 850 mg/m2 and 630 mg/m2/day, respectively. No responses were observed in either study. Following i.v. administration of 540 mg/m2 hexamethylmelamine, plasma elimination was best described by a three-compartment open model with terminal half-life, total body clearance, and steady-state volume of distribution values of 10.4 h, 0.75 liter/min/m2 and 460 liters/m2, respectively. Twenty-four h urinary recoveries of parent drug were less than 1% for all patients. Accumulation of hexamethylmelamine during the 5-day treatment at 945 mg/m2 suggested possible saturation of parent drug elimination at that dose. Phase II studies are currently under way with the parenteral formulation of hexamethylmelamine.

Phase 1 trial and assay of rubidazone (NSC 164011) in patients with advanced solid tumors.

A new high-pressure liquid chromatographic method was developed for the simultaneous determination of rubidazone and daunorubicin in human plasma at concentrations as low as 60 ng/ml. Clinical toxicity and the stability of rubidazone were studied in nine patients with advanced solid tumors. Rubidazone was administered by i.v. infusion over 1 hr on a single day every 4 weeks. Moderate leukopenia was the dose-limiting toxicity in four of six patients treated at 150 mg sq/m. Assay of rubidazone in plasma samples obtained after administration of rubidazone showed that the drug was stable for at least 7 hr.

Plasma pharmacokinetics and cerebrospinal fluid concentrations of idarubicin and idarubicinol in pediatric leukemia patients: a Childrens Cancer Study Group report.

Idarubicin (4-demethoxydaunomycin) is an anthracycline analogue with striking in vitro and in vivo activity against murine leukemias. Based on activity in adults with acute lymphoblastic leukemia, the Childrens Cancer Study Group initiated studies to evaluate idarubicin in children with leukemia in second or subsequent relapses. As part of those studies, we have characterized the plasma pharmacokinetics of idarubicin and the major circulating metabolite idarubicinol in 21 patients. Idarubicin plasma elimination was described by a three-compartment open model following i.v. infusion (10-15 mg/m2) on a schedule of weekly for 3 weeks and on a schedule of daily for 3 days every 3 weeks (total dose, 30-45 mg/m2). There was substantial variability in idarubicin elimination among patients, but no indication of dose-dependent or of schedule-dependent changes in pharmacokinetic parameters. The mean terminal half-life, total body clearance, and steady state volume of distribution were 17.6 h, 679 ml/min/m2, and 562 l/m2, respectively. Idarubicinol elimination was prolonged compared to that of the parent drug with a terminal half-life of 56.8 h. This metabolite clearly accumulated in plasma during the 3 days of treatment on the schedule of daily for 3 days. Urinary recoveries (48 h) of idarubicin and idarubicinol after a single dose of idarubicin were 2.4 and 10.1%, respectively. Idarubicin was detected in 2 of 21 cerebrospinal fluid samples obtained 18-30 h after administration. In marked contrast, idarubicinol was detected in 20 of those 21 samples. Concentrations in the 20 samples varied from 0.22-1.05 ng/ml with a mean value of 0.51 ng/ml.

Murine pharmacokinetics and metabolism of penclomedine [3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine, NSC 338720].

Penclomedine, a highly substituted pyridine derivative, has been selected by the National Cancer Institute for evaluation as a potential anticancer agent based on antitumor activity observed in murine tumor models following i.v., p.o., and i.p. administration. We have developed a reverse-phase high performance liquid chromatography assay for PEN, and subsequently investigated murine pharmacokinetics and metabolism. Following rapid i.v. injection of PEN (300 mg/m2) to mice, plasma elimination was best described by a 2-compartment open model with an elimination phase half-life, total body clearance, and steady-state distribution volume of 69 min, 114 ml/min/m2, and 4800 ml/m2, respectively. While PEN displayed good p.o. absorption, bioavailability of PEN after p.o. administration was approximately 2% of that observed following i.v. administration. Metabolism contributed substantially to drug clearance, and total metabolites were slowly eliminated from plasma. After i.v. and p.o. administration of radiolabeled PEN, less than 0.2% of the parent drug was excreted in the 48-h urine, and 25-30% of the total radioactivity was recovered in urine. NADPH-dependent oxidative and reductive metabolism was observed when penclomedine was incubated with mouse microsomal preparations. Microsomal reductive metabolism of PEN led to formation of a metabolite tentatively identified as a molecule formed by dimerization of the radical species produced by cleavage of chlorine from the trichloromethyl moiety of penclomedine.

Phase I and clinical pharmacological evaluation of pirozantrone hydrochloride (oxantrazole).

Pirozantrone hydrochloride, an anthrapyrazole analogue, was selected for clinical evaluation based on broad antitumor activity against murine tumor systems and on potentially less cardiotoxicity when compared to anthracyclines. This anthrapyrazole analogue is currently under clinical evaluation, and we now report results on a Phase I clinical trial incorporating a pharmacologically guided dose-escalation scheme. Dose escalation was designed to proceed by factors of 2 until the patient drug exposure (concentration x time) was 40% of the murine exposure at the LD10 dose (90 mg/m2). Thereafter, more moderate dose escalations were employed. The target concentration x time value (59 micrograms-min/ml) derived from preclinical pharmacology data was exceeded in all three patients at a dose of 90 mg/m2. A dose of 160 mg/m2 was found to reproducibly result in appropriate myelosuppression. This dose is recommended for further testing in Phase II studies. Nonhematological toxicities encountered in this trial were mild, the most notable being phlebitis at the infusion site. Objective responses were observed in two patients, one with metastatic breast cancer and another with metastatic melanoma. Following a 60-min infusion, pirozantrone hydrochloride plasma elimination was monoexponential, with a half-life of approximately 30 min, mean total body clearance of 1.29 liters/min/m2, and mean steady state volume of distribution of 29 liters/m2.

Pediatric phase I trial and pharmacokinetic study of topotecan administered as a 24-hour continuous infusion.

Topotecan, a water-soluble semisynthetic analogue of camptothecin, is the first topoisomerase I inhibitor to undergo evaluation in pediatric patients with refractory malignancies. A phase I and pharmacokinetic study was performed to determine the maximum tolerated dose (MTD) and dose-limiting toxicities, the incidence and severity of other toxicities, and the pharmacokinetics of topotecan in children. Twenty-nine patients received 42 courses of i.v. topotecan administered as a 24-h continuous infusion every 21 days at doses ranging from 2.0 to 7.5 mg/m2. Dose-related hematological toxicity was the dose-limiting toxicity. Leukopenia, neutropenia, and thrombocytopenia occurred sporadically at the 3.0- to 5.5-mg/m2 dose levels, but at 7.5 mg/m2 4 of 5 patients experienced dose-limiting thrombocytopenia (grade 4) and 2 of 5 had dose-limiting neutropenia (grade 4). No other dose-limiting toxicities were observed. Nausea and vomiting were mild and occurred in < 20 and 10% of patients, respectively. Grade 2 hematuria occurred in one patient. No objective responses were observed. Pharmacokinetic studies revealed a linear relationship between the steady-state topotecan concentration and dose. The mean steady-state concentration at the MTD was 18.2 +/- 3.7 nmol/liter and the total body clearance was 28.3 +/- 6.5 liters/h/m2. Elimination was biexponential with a t1/2 alpha of 14.4 +/- 1.8 min and a t1/2 beta of 2.9 +/- 1.1 h. The recommended starting dose for phase II pediatric trials is 5.5 mg/m2. Although this dose exceeds the MTD identified in heavily pretreated adult patients receiving topotecan on the same schedule, it is less than the MTD for minimally pretreated adult patients. Therefore, dose escalation to 7.5 mg/m2 in phase II pediatric trials should be considered for patients who tolerate treatment well at the 5.5-mg/m2 dose.

Phase I study of combined 2-chlorodeoxyadenosine and chlorambucil in chronic lymphoid leukemia and low-grade lymphoma.

PURPOSE: We conducted a dose-escalation study of 2-chlorodeoxyadenosine (2-CdA) added to a standard fixed dose of chlorambucil to evaluate the toxicity of the combined regimen in patients with chronic lymphocytic leukemia (CLL) and low-grade lymphoma. PATIENTS AND METHODS: A total of 15 adults with relapsed or refractory CLL or low-grade lymphoma were to receive chlorambucil orally (30 mg/m2) every 2 weeks. Also, groups of three patients were to receive escalating doses of continuous intravenously administered 2-CdA at 1, 2, or 4 mg/m2/d for 7 days repeated at monthly intervals. All patients had pharmacokinetic studies during the first cycle of 2-CdA therapy; the results were compared with other institutional phase I studies in which comparable amounts of 2-CdA were administered by bolus infusion for 5 days. RESULTS: The maximal-tolerated dose of 2-CdA that could be combined with chlorambucil was 2 mg/m2/d for 7 days for a maximum of two courses. At a higher dose level of 2-CdA or at the maximal-tolerated dose level given for more than two courses, severe and protracted pancytopenia, sometimes associated with life-threatening infections, was noted. Nonhematologic toxicities were limited to moderate degrees of nausea, vomiting, anorexia, and fatigue. Results of the pharmacologic studies suggested that 2-CdA clearance was independent of administration schedule and was unaffected by concomitant administration of chlorambucil. CONCLUSION: These observations suggest the possibility of combining two cycles of continuous intravenously administered 2-CdA (2 mg/m2/d for 7 days) with standard doses of oral chlorambucil to test the efficacy of the combination regimen in phase II trials.

Phase I study of temozolomide in children and adolescents with recurrent solid tumors: a report from the Children's Cancer Group.

PURPOSE: The Children's Cancer Group conducted a phase I trial of temozolomide stratified by prior craniospinal irradiation (CSI). PATIENTS AND METHODS: Children and adolescents with recurrent or progressive cancer were enrolled. Temozolomide was administered orally daily for 5 days, with subsequent courses administered every 21 to 28 days after full hematologic recovery. Dose levels tested included 100, 150, 180, 215, 245, and 260 mg/m2 daily. RESULTS: Twenty-seven patients on the non-CSI stratum were assessable for hematologic toxicity. During the first three dose levels (100, 150, and 180 mg/m2 daily), only grades 1 and 2 hematologic toxicity occurred. One patient at 215 mg/m2 daily had grade 3 hematologic toxicity. Three of eight patients (38%) treated at 245 to 260 mg/m2 daily had dose-limiting toxicity (DLT), which included both neutropenia and thrombocytopenia. Twenty-two patients on the CSI stratum were assessable for hematologic toxicity. Hematologic DLT occurred in one of six patients (17%) at 100 mg/m2 daily and in two of four patients (50%) at 215 mg/m2 daily. No nonhematologic DLT occurred; nausea and vomiting occurred in more than half of the patients. After two courses of temozolomide, 10 patients had stable disease (SD), and three patients had a partial response (PR), one of whom subsequently had a complete response (CR) that persists through 24 months of follow-up. CONCLUSION: The maximum-tolerated dose (MTD) of temozolomide for children and adolescents without prior CSI is 215 mg/m2 daily and for those with prior CSI is 180 mg/m2 daily for 5 days, with subsequent courses that begin on day 28. Temozolomide is well tolerated and should undergo phase II testing in children and adolescents.

Selected pharmacologic characteristics of idarubicin and idarubicinol.

The pharmacology of ID and IDOL are of interest in light of the potential utility of ID in the treatment of adult and pediatric leukemia patients. Preclinical activity and cellular pharmacology of ID were suggestive of greater clinical activity when compared with several standard anthracyclines. Most intriguing were data comparing in vitro and in vivo activity data and cellular pharmacology of IDOL to other anthracycline alcohol metabolites. Given the pharmacokinetics of IDOL, there is continued interest in the unique aspects of IDOL pharmacology as an important element of ID pharmacology.

The aromatic-L-amino acid decarboxylase inhibitor carbidopa is selectively cytotoxic to human pulmonary carcinoid and small cell lung carcinoma cells.

The carcinoid tumor is an uncommon neuroendocrine neoplasm the hallmark of which is excessive serotonin production. In studying kinetics of tryptophan hydroxylase and aromatic-L-amino acid decarboxylase (AAAD) in human carcinoid hepatic metastases and adjacent normal liver (J. A. Gilbert et al, Biochem. Pharmacol., 50: 845-850, 1995), we identified one significant difference: the Vmax of carcinoid AAAD was 50-fold higher than that in normal liver. Here, we report Western and Northern analyses detecting large quantities of AAAD polypeptide and mRNA in human carcinoid primary as well as metastatic tumors compared with normal surrounding tissues. To assess the feasibility of targeting these high AAAD levels for chemotherapy, AAAD inhibitors carbidopa (alpha-methyl-dopahydrazine), alpha-monofluoromethyldopa (MFMD), and 3-hydroxybenzylhydrazine (NSD-1015) were incubated (72 h) with NCI-H727 human lung carcinoid cells. Carbidopa and MFMD were lethal (IC50 = 29 +/- 2 microM and 56 +/- 6 microM, respectively); NSD-1015 had no effect on proliferation. On exposure to other human tumor lines, carbidopa was lethal only to NCI-H146 and NCI-H209 small cell lung carcinoma (SCLC) lines (IC50 = 12 +/- 1 microM and 22 +/- 5 microM, respectively). Carbidopa (100 microM) decreased growth of (but did not kill) SK-N-SH neuroblastoma and A204 rhabdomyosarcoma cells and did not affect proliferation of DU 145 prostate, MCF7 breast, or NCI-H460 large cell lung carcinoma lines. The rank order of lines by AAAD activity was NCI-H146 > NCI-H209 > SK-N-SH > NCI-H727, whereas A204, DU 145, MCF7, and NCI-H460 had no measurable activity. For lung tumor lines (carcinoid, two SCLC, and one large cell lung carcinoma), AAAD activity was correlated with the potency of carbidopa-induced cytotoxicity. However, carcinoid cell death was not solely attributable to complete inhibition of either AAAD activity or the serotonin synthetic pathway. In further evaluating potential applications of these findings with carbidopa, we determined that sublethal doses of carbidopa produced additive cytotoxic effects in carcinoid cells in combination with etoposide and cytotoxic synergy in SCLC cells when coincubated with topotecan.

Pharmacokinetics of 3-methyl-(triazen-1-yl)imidazole-4-carboximide following administration of temozolomide to patients with advanced cancer.

The antitumor activity of temozolomide (TMZ) is believed to arise via formation of the reactive, alkylating metabolite 3-methyl-(triazen-1-yl)imidazole-4-carboximide (MTIC), which is produced by chemical hydrolysis of the parent drug. MTIC has not been quantitated in plasma or urine following administration of TMZ to patients. We developed a sensitive, specific method for the determination of MTIC levels in plasma, based on reverse-phase high-pressure liquid chromatography of the supernatant that is obtained by methanol precipitation of plasma proteins. Due to poor stability under physiological conditions, determination of MTIC required rapid specimen processing, precipitation of plasma proteins with methanol, and storage of the methanolic supernatant at -70 degreesC. The pharmacokinetics of MTIC were studied in 15 patients who received 125-250 mg/m2 TMZ. Peak plasma concentrations of 0.07-0.61 microgram/ml MTIC were observed approximately 1 h after a p.o. dose of TMZ. Appearance and disappearance (t1/2, 88 min) of the reactive metabolite paralleled the appearance and disappearance of TMZ in plasma. The mean values of the metabolite peak plasma concentration and AUC were 2.6% (range, 1.6-4.6%) and 2.2% (range, 0.8-3.6%), respectively, of the values for TMZ. MTIC did not accumulate in plasma following five consecutive daily doses of TMZ.

Metabolic activation of dacarbazine by human cytochromes P450: the role of CYP1A1, CYP1A2, and CYP2E1.

Dacarbazine (DTIC), a widely used anticancer agent, is inactive until metabolized in the liver by cytochromes P450 to form the reactive N-demethylated species 5-[3-hydroxymethyl-3-methyl-triazen-1-yl]-imidazole-4-carboxamide (HMMTIC) and 5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide (MTIC). The modest activity of DTIC in the treatment of cancer patients has been attributed in part to lower activity of cytochromes P450 (P450) in humans when compared with rodents. Importantly, the particular P450 isoforms involved in the activation pathway have not been reported. We now report that the DTIC N-demethylation involved in MTIC formation by human liver microsomes is catalyzed by CYP1A1, CYP1A2, and CYP2E1. The most potent inhibitors of DTIC N-demethylation were alpha-naphthoflavone (CYP1A1 and CYP1A2), quercetin (CYP1A2), chlorzoxazone (CYP1A2 and CYP2E1), and di-sulfiram (CYP2E1). Antihuman CYP1A2 antiserum also inhibited DTIC N-demethylation. DTIC N-demethylation in a panel of 10 human liver microsome preparations was correlated with the catalytic activities for CYP1A2 (ethoxyresorufin O-deethylation and caffeine N3-demethylation) in the absence of alpha-naphthoflavone and with the catalytic activities for CYP2E1 (chlorzoxazone 6-hydroxylations) in the presence of alpha-naphthoflavone. DTIC metabolism was catalyzed by recombinant human CYP1A1, CYP1A2, and CYP2E1. The Km (Vmax) values for metabolism of DTIC by recombinant human CYP1A1 and CYP1A2 were 595 microM (0.684 nmol/min/mg protein) and 659 microM (1.74 nmol/min/mg protein), respectively. The CYP2E1 Km value exceeded 2.8 mM. Thus, we conclude that (a) CYP1A2 is the predominant P450 that catalyzes DTIC hepatic metabolism; (b) CYP2E1 contributes to hepatic DTIC metabolism at higher substrate concentrations; and (c) CYP1A1 catalyzes extrahepatic metabolism of DTIC.

Phase I study of the duocarmycin semisynthetic derivative KW-2189 given daily for five days every six weeks.

The duocarmycins represent a new group of antitumor antibiotics produced by Streptomyces that bind to the minor groove of DNA. KW-2189 is a water-soluble semisynthetic derivative of duocarmycin B2, with significant activity in murine and human tumor models. We conducted a Phase I trial of KW-2189 in patients who had solid tumors that were refractory to standard chemotherapy or for whom no more effective therapy existed. KW-2189 was administered as a rapid i.v. bolus daily for 5 days every 6 weeks. Twenty-two patients were enrolled and received a total of 31 cycles of KW-2189. Leukopenia, neutropenia, and thrombocytopenia were the dose-limiting toxicities, with nadirs occurring at medians of 36, 38, and 29 days, respectively, at the 0.04 mg/m2/day dose level. Nonhematological toxicities were mild, although one patient developed grade 3 fatigue. Four patients had stable disease over two to four cycles of treatment and showed no cumulative toxicity. The mean t1/2, plasma clearance, and steady-state volume of distribution were 13.5 min, 1,287 ml/min/m2, and 10,638 ml/m2, respectively. Pharmacokinetics were similar on days 1 and 5, with no drug accumulation in plasma. The active metabolite DU-86 was not consistently found in patient plasma. For Phase II trials, when the 5 days every 6 weeks schedule was used, 0.04 mg/m2/day KW-2189 appears to be the maximal tolerated dose, especially for patients who have received prior chemotherapy. At this dose level, the drug was well tolerated, and the toxicities were acceptable.

Effect of pyrazoloacridine (NSC 366140) on DNA topoisomerases I and II.

Pyrazoloacridine (PA), an acridine congener with an unknown mechanism of action, has shown selective activity against solid tumor cells, cytotoxicity in noncycling and hypoxic cells, and promising antitumor activity in Phase I clinical trials. In the present study, the effect of PA on topoisomerase (topo) activity was evaluated using yeast strains lacking functional topo I or II, mammalian cell nuclear extracts, purified samples of mammalian topo I and topo II, and intact mammalian tissue culture cells. Clonogenic assays revealed that PA cytotoxicity in yeast strains was unaffected by selective loss of topo I or topo II activity. On the other hand, enzyme assays revealed that 2-4 microM PA abolished the catalytic activity of both topo I and topo II in vitro. In contrast to topotecan and etoposide, PA did not stabilize covalent topo-DNA complexes. Instead, PA inhibited topotecan-induced stabilization of covalent topo I-DNA complexes and etoposide-induced stabilization of topo II-DNA complexes in vitro and in intact cells. Consistent with these results, colony-forming assays indicated that short-term PA exposure inhibited the cytotoxicity of topotecan and etoposide, whereas prolonged PA exposure was itself toxic to these cells. Accumulation studies revealed that PA was concentrated as much as 250-fold in drug-treated cells, resulting in intranuclear concentrations that far exceeded those required to inhibit topo I and topo II. Collectively, these results not only suggest that PA can target both topo I and topo II at clinically achievable concentrations but also indicate that its mechanism is distinct from topo I and topo II poisons presently licensed for clinical use.

Phase I and pharmacological trial of fazarabine (Ara-AC) with granulocyte colony-stimulating factor.

Fazarabine (1-beta-D-arabinofuranosyl-5-aza-cytosine, or Ara-AC) is a nucleoside analogue that consists of the arabinoside ring of 1-beta-D-arabinofuranosylcytosine and the pyrimidine base of 5-azacytidine. In Phase I and Phase II trials, neutropenia was dose limiting, with minimal nonhematological toxicity. The in vitro cytotoxic concentrations of Ara-AC could not be achieved in these studies; neutropenia precluded dose escalation. The objectives of this study were: to determine either the maximum tolerated dose of Ara-AC or to safely achieve target plasma levels of 2-5 microgram/ml when Ara-AC was administered as a 24-h infusion with granulocyte colony-stimulating factor (G-CSF) to patients with advanced refractory malignancies; to characterize the pharmacokinetic behavior of Ara-AC with G-CSF; and to define the relationship of Ara-AC pharmacokinetics to toxicity. Twenty-four patients received 67 courses of Ara-AC at doses of 54-112 mg/m2/h. Dose-limiting toxicity was approached but not reached. Grade 3 or 4 neutropenia and nausea were the principle side effects. Steady-state plasma concentrations exceeded the minimum target concentration of 2 microgram/ml in all patients who received >/=78 mg/m2/h for 24 h. The maximum target concentration was approached during administration of 112 mg/m2/h for 24 h. The mean steady-state clearance was 475 +/- 103 ml/min/m2 and did not change with dose. One partial response was seen. One patient received 16 courses and another received 7 courses of therapy before progression. Ara-AC can be safely administered in doses that result in plasma concentrations of 2-5 microgram/ml, if it is given with G-CSF. Phase II trials of Ara-AC in selected malignancies are planned.