Charles K. Grieshaber

Echinomycin: The first bifunctional intercalating agent in clinical trials

SummaryEchinomycin is a quinoxaline antibiotic that was originally isolated from Streptomyces echinatus. Based on its antitumor activity against two i.p. implanted murine tumors, the B16 melanoma, and the P388 leukemia, it was brought into clinical trials by the National Cancer Institute. Recent studies on its cytotoxic action have related its antitumor activity with its ability to bifunctionally intercalate with double stranded DNA.Toxicologic studies were carried out in CDF1 mice and beagle dogs using intravenous injections. For the mice studies the dose ranges were 288–692 mcg/kg (864–2076 mcg/m2) by single bolus, and 112–254 mcg/kg/day (336–762 mcg/m2/day) for five consecutive days. In the dog, dose ranges studied were 8.9–89.4 mcg/kg (178–1788 mcg/m2) by single bolus, and 3.4–33.5 mcg/kg/day (68–670 mcg/m2/day) for five consecutive days. The major toxic effects were found in the gastrointestinal, hepatic, and lymphoreticular systems. These were reversible at all but the highest dose, in dogs that had been treated for five consecutive days.Phase I clinical trials using various intravenous schedules were sponsored by the National Cancer Institute. Nausea, vomiting, reversible liver enzyme abnormalities, and allergic reactions were the most common toxicities encountered. Based on results from these studies, the National Cancer Institute has recently begun phase II trials in a broad range of diseases. These trials will further characterize echinomycins toxic effects and its antitumor activity.

Flavone acetic acid (LM 975, NSC 347512) A novel antitumor agent

SummaryFlavone acetic acid (FAA) is a synthetic flavonoid compound which has recently begun clinical trials as an antitumor agent based on its striking activity in solid tumor model systems. The pharmacologic behavior of FAA in animals appears to be predictive of both its cytotoxic efficacy and its toxicity to normal tissues (principally the central nervous system and gastrointestinal tract). The design and conduct of phase I studies in man are based upon these principles, with the goal of maximizing their safety and efficacy.

Merbarone: an antitumor agent entering clinical trials.

SummaryMerbarone was developed to clinical trial stage on the basis of its ‘curative’ activity against P388 and L1210 leukemias and moderate activity against B16 melanoma and M5076 sarcoma. Its activity appears to be schedule-dependent favoring a longer duration of administration. The mechanism of action of merbarone is not yet established but it does induce single strand breaks in DNA apparently without binding to DNA.The pharmacokinetic data in the dog indicate that clearance mechanisms may be saturable. Merbarone is hydroxylated at the 4′ position in the rat, mouse and dog, and glucuronidated in the dog. Parent drug and the hydroxy metabolite are excreted in the urine. If saturable clearance mechanisms also pertain to man, this will mean that infusion rate (and therefore steady state concentrations reached) may be a significant factor in determining acute toxicity.Preclinical toxicology studies revealed that major target tissues are in the lymphoid organs, bone marrow, gastrointestinal tract and kidney. Some behavioral signs of reversible central nervous system toxicity were observed.Phase I trials have commenced using only a 5-day continuous intravenous infusion schedule based on the preclinical data. The pharmacokinetic information from these trials will be crucial for further clinical development of the compound, including selection of the optimal schedule(s) for phase II/III evaluation.

Arabinosyl-5-azacytosine: A novel nucleoside entering clinical trials

Arabinosyl-5-azacytosine is a new compound which has been selected by the Division of Cancer Treatment, National Cancer Institute for clinical development as an antineoplastic agent based on its high degree of activity against a broad range of tumor types in preclinical studies. Therapeutic activity has been observed against murine and human leukemias, transplantable murine solid tumors, and human tumor xenografts. Arabinosyl-5-azacytosine exhibited a broader spectrum of activity against human solid tumors than cytosine arabinoside. Arabinosyl-5-azacytosine is phosphorylated to the nucleotide level by deoxycytidine kinase. Upon further anabolism to the triphosphate level, it can be incorporated into DNA. The mechanism of cytotoxicity is thought to be related to inhibition of DNA synthesis. Leukemic and solid tumor cell lines that are resistant to cytosine arabinoside due to deletion of deoxycytidine kinase activity are cross-resistant to arabinosyl-5-azacytosine. Unlike cytosine arabinoside, arabinosyl-5-azacytosine does not readily undergo deamination. Schedule dependence has been demonstrated in mice bearing L1210 leukemia, with superior activity seen with multiple doses administered on each treatment day compared to administration of larger but less frequently administered doses. From preliminary data in solid tumor models, however, antitumor activity did not appear to be superior with continuous infusion compared to that observed on a bolus schedule. Preclinial toxicology studies indicated that the bone marrow and gastrointestinal tract were the main target organs. A single large dose of arabinosyl-5-azacytosine could be tolerated by both mice and dogs. When administered as a continuous infusion, the toxicity was related to both the dose and duration of exposure, suggesting that toxicity resulted from a critical time above a threshold concentration as opposed to the total area under the concentration-time curve. Phase I clinical trials have been initiated to determine the maximum tolerated dose on a low dose continuous infusion schedule for 72 hours and also on a high dose short infusion daily times five schedule.

In vitro toxicity of 3′-azido-3′-deoxythymidine, carbovir and 2′, 3′-didehydro-2′, 3′-dideoxythymidine to human and murine haematopoietic progenitor cells

Summary The myelotoxicities of three antiretroviral agents, 3′‐azido‐3′‐deoxythymidine (AZT), carbovir (CBV) and 2′,3′‐didehydro‐2′,3′‐dideoxythymidine (d4T), were evaluated in vitro with normal human and murine haematopoietic progenitor cells. These studies demonstrated that continuous AZT exposure was more inhibitory to human and murine colony formation than 1 h exposure, with murine and human progenitors similarly inhibited by continuous AZT exposure. These in vitro results on AZTs myelotoxicity correlate with both human and murine in vivo studies. CBV was only moderately toxic to human and murine cells following either 1 h or continuous exposure, with human and murine progenitors similarly suppressed by continuous CBV exposure. 1 h d4T exposure was less toxic to both human and murine marrow cells than continuous exposure and both species were equivalently inhibited when continuously exposed to d4T. In general, CBV was the least toxic agent to human and murine haematopoietic cells and AZT the most toxic. The study establishes CBV and d4T as less myelotoxic agents to human and murine haematopoietic progenitor cells in vitro than AZT which therefore could be considered as alternatives to AZT for the treatment of HIV infection.

Role of preclinical pharmacology in phase I clinical trials: Considerations of schedule-dependence

Antitumor activity or host toxicity can be increased or decreased by changing the rate of drug delivery. This phenomenon is known as schedule-dependence, and the most familiar examples occur with the use of methotrexate (MTX), fluorodeoxyuridine (FdUrd), and cytosine arabinoside (ara-C). The general motivation for the study of schedule-dependence is to improve the therapeutic index of a drug, i.e., to maximize the ratio of therapeutic effects to toxic effects.

In vitro characterization of the myelotoxicity of cyclopentenyl cytosine

We studied the toxicity of a new experimental anticancer drug, cyclopentenyl cytosine (CPE-C), to human and murine hematopoietic progenitor cells in vitro. Due to CPE-Cs in vivo myelotoxicity, it was important to characterize its potential adverse effects on human marrow cells during preclinical development of the drug. Marrow cells were exposed to CPE-C for either 1 h prior to addition in clonal assays or continuously during their culture period. The inhibitory effects of CPE-C on myeloid (CFU-gm) and erythroid (CFU-e, BFU-e) colony formation were concentration- and time-dependent, with continuous CPE-C exposure being significantly more inhibitory than 1-h exposure. The results of both exposure experiments were combined to investigate colony inhibition as a function of overall drug exposure (concentration x time, AUC) and data analyzed by the nonlinear Emax equation. Human and murine CFU-gm had similar AUC-response curves and IAUC70 values (i.e., AUC at 70% colony inhibition) of 40.8 and 41.9 microM h, respectively. In contrast, murine CFU-e and BFU-e were more sensitive to CPE-C, having lower IAUC70 values (both, 21.1 microM h) than human CFU-e and BFU-e (107.8 and 33.0 microM h, respectively). This difference was most prominent with the late erythroid progenitor, CFU-e, in that the human cells were 5 times more resistant to inhibition by CPE-C. CPE-C was myelotoxic in vitro to human and murine marrow cells and toxicity correlated with overall drug exposure.

Pyrazole: preclinical reassessment

SummaryPyrazole (NSC-45410) is a low molecular weight, heterocyclic compound which has been considered for reevaluation in the clinic as a potential cytotoxic agent (Fig. 1). Discovered in 1893 [1], pyrazole is best known as an inhibitor of liver alcohol dehydrogenase (ki= 0.2 uM), and as a result, has been used extensively in studies of alcohol metabolism [2]. In 1960, pyrazole was identified as being active in preclinical antitumor models [3], which led to preliminary clinical testing [4,5]. The early Phase I studies were not followed by disease specific Phase II trials, and the clinical activity of the drug has never been evaluated. This omission was noted by the National Cancer Institutes Project for the Review of Old Drugs (PROD), at which time it was also noted that pyrazole is selectively toxic to thyroid tissue in an animal model [6]. Hence, interest in pyrazole was revived for two reasons: (a) failure to screen it for clinical activity in the 1960s, and (b) current interest in discovering drugs with selective toxicity to specific tissues for evaluation of their activity in malignancies arising in the target tissue. In this review, we summarize the evidence which has accumulated concerning pyrazoles potential role as an anticancer agent.

The effect of the monoamine oxidase inhibitor isocarboxazid on the canine metabolism of the cell-differentiating agent hexamethylene bisacetamide

SummaryThe acute toxicities of the cellular differentiating agent hexamethylene bisacetamide (HMBA) in humans and animals include CNS toxicity (agitation, somnolence, seizures, hallucinations) and an anion-gap metabolic acidosis,N-Acetyl-1,6-diaminohexane (NADAH), the first metabolite of HMBA, is as active as the parent compound in causing differentiation of leukemic cells in vitro, whereas 6-acetamidohexanoic acid (6AcHA), which is formed by the oxidation of NADAH in the presence of monoamine oxidase (MAO) and aldehyde dehydrogenase, is inactive. To test whether the inhibition of MAO blocks the production of an inactive and possibly toxic HMBA metabolite (6AcHA) or increases the amount of active compounds (HMBA+NADAH) in vivo, we investigated the effect of the MAO inhibitor isocarboxazid on the metabolism and toxicity of HMBA in beagle dogs. Two groups of dogs, composed of one male and one female dog per group, were used in the study. One group received isocarboxazid (3.3 mg/kg p.o. q8h×9) beginning at 24 h before the initiation of a 48-h i.v. infusion of HMBA (40 mg kg−1 h−1), whereas the other received placebo in an identical fashion prior to the start of an identical HMBA infusion. The mean plasma steady-state concentration (css) of HMBA was 0.91 mM in dogs given HMBA and isocarboxazid as opposed to 0.78mm in those given HMBA and placebo. As measured spectrophotometrically, plasma MAO activity was inhibited by 86%±3% in dogs receiving isocarboxazid. Gas chromatography/mass spectrometry detected 6AcHA in the plasma of animals that were given placebo but not in the plasma of dogs that received isocarboxazid. Gas chromatographic analysis of urine samples revealed that the total amount of 6AcHA and of NADAH excreted in urine was 8 times less and 3 times greater, respectively, in isocarboxazid-treated dogs than in animals that received HMBA and placebo. One dog was excitable after the initial two doses of isocarboxazid and developed seizures at the end of the HMBA infusion. Another dog was agitated during treatment with HMBA and isocarboxazid. No CNS toxicity occurred in animals that were treated with HMBA and placebo. We conclude that isocarboxazid inhibits the production of 6AcHA in vivo, thus supporting the involvement of MAO in HMBA metabolism. Because the combination of HMBA and isocarboxazid produces CNS toxicity, 6AcHA is probably not the neurotoxic agent in dogs.