Daniel S. Zaharko

Studies of the metabolism of methotrexate by intestinal flora—I: Identification and study of biological properties of the metabolite 4-amino-4-deoxy-N10-methylpteroic acid

Abstract Incubation of tritium-labeled methotrexate (MTX) with caecal contents obtained from CDF 1 mice results in the production of at least three labeled metabolites separable from MTX by chromatography on DEAE-cellulose. The principal metabolite has been isolated and identified by means of its Chromatographic properties and its ultraviolet and infra-red absorption spectra as 4-amino-4-deoxy- N 10 -methylpteroic acid (APA). In studies in vivo , APA has been recovered from the urine and feces of CDF 1 mice in the 6 and 12-hr period after the intraperitoneal administration of tritiumlabeled MTX (3 mg/kg). While APA is the major metabolite present in the feces, only small amounts are detectable in the urine of CDF 1 mice previously injected with labeled MTX. The major portion of the radioactivity associated with MTX metabolites in urine migrated prior to APA on DEAE-cellulose. Some of the biological properties of APA were determined. The compound was found to be a moderately effective inhibitor of dihydrofolate reductase (apparent K i = 1.3 × 10 −9 M) and a substrate for aldehyde oxidase (apparent K m = 2.2 × 10 −4 M); the product of oxidation of APA by the latter enzyme was 4-amino-4-deoxy-7-hydroxy- N 10 -methylpteroic acid. In studies in vivo , APA was found to be considerably less toxic than MTX in CDF 1 mice. These studies provide direct experimental support for the hypothesis of Zaharko et al. ∗ that in the mouse MTX is subject to extensive metabolism by intestinal bacteria.

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.

The kinetics of methotrexate distribution in spontaneous canine lymphosarcoma

A mathematical model is presented to simulate the time-dependent uptake of methotrexate in spontaneous canine lymphosarcomas in vivo.Blood flow rates in these tumors are high so that transport to the tumor is limited by cell membrane resistance. A significant amount of rapidly exchangeable methotrexate appears to exist in extracellular space loosely bound to proteins or cell membranes. Transmembrane drug transport follows Michaelis-Menten kinetics, with the maximum facilitated transport ranging from 0.002 to 0.007 μg/min/ml for the separate tumors studied and a Michaelis constant for transport equal to 0.2 μg/ml. This is in the range of Michaelis constants reported for normal tissues in rats in vivoand in several cell lines in vitro.

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.

Could interspecies differences in the protein binding of flavone acetic acid contribute to the failure to predict lack of efficacy in patients

SummaryWe investigated the differences in plasma protein binding of flavone acetic acid (FAA) in mice and men in an attempt to explain the inter-species differences in response. In vitro data indicate both qualitative and quantitative differences in FAA protein binding: approximately 80% is bound in humans, with two different types of binding site identified; in mice, 70% is bound and only one binding site could be described. Protein binding is dose-dependent in both species. Plasma samples from 20 patients receiving FAA showed that most achieved levels that would be active in mice. We conclude that these differences in protein binding are insufficient to explain totally the observed differences in response.

Chloroquinoxaline sulfonamide: A sulfanilamide antitumor agent entering clinical trials

SummaryChloroquinoxaline sulfonamide (CQS) has been developed to the clinical trial stage based on its activity in the Human Tumor Colony Forming Assay (HTCFA). In the HTCFA, CQS demonstrated inhibition of colony formation against breast, lung, melanoma and ovarian carcinomas. The mechanism of action of CQS is unknown. It does not appear to inhibit folate metabolism as does the structurally similar sulfaquinoxaline. Preclinical toxicology studies in dogs and rats have shown that CQS is toxic to lymphoid organs, bone marrow, gastrointestinal tract, pancreas, CNS, adrenal glands and testes. Toxicity was generally reversible with the exception of testicular atrophy in dogs and rats which occurred late and was not reversible within the study time frame.The pharmacokinetic data indicate that CQS binds to serum proteins in a dose and species specific manner. Terminal half-lives appear to vary between species from 60 hours in mice, 15 hours in rats, and 45–132 hours in dogs. Preliminary data indicate a longer terminal half-life in humans.Two phase I trials are ongoing using a 60 min infusion schedule once every 28 days. The starting dose for each trial was 18 mg/m2.

Cyclopentenyl cytosine : interspecies predictions based on rodent plasma and urine kinetics

SummaryA hybrid compartmental-physiological model for cyclopentenyl cytosine (CPE-C) is designed on the basis of early limited rodent pharmacokinetic data. Application of model independent pharmacokinetics and biochemical knowledge was first used to conceptualize such a model. The approach was to scale the physiological parameters of the model (compartmental clearances) and keep constant the anatomic parameters of the model (compartment volumes). Scaling of physiological mechanisms was based on body weight/surface area ratios. Using these principles, simulations with the model can reasonably anticipate the in vivo behavior of (CPE-C) in several species (mouse, rat, dog). The model is useful in understanding species differences in pharmacokinetic behavior of CPE-C.

Tolerance of long-term methotrexate infusions by mice

Abstract Mice were chronically exposed to between 1 × 10 −8 and 5 × 10 −9 M plasma methotrexate (MTX) for 2 months. No signs of toxicity were manifested except minor facial hair loss. At the end of this time they were sacrificed, and the activity of dihydrofolate reductase in crude Tris extracts of small intestine, femur, liver and kidney was determined at pH 7.0 and 8.6. At pH 7.0 the enzyme activity was maintained at control levels in small intestine and femur but was decreased to half of controls in liver and kidney. The enzyme activity at pH 8.6 was increased significantly above controls in small intestine and femur but remained at control levels in liver and kidney. These results suggest that some organs adapt to the chronic presence of MTX by increasing their enzyme synthesis rate while other organs are able to function normally for prolonged periods of time with less than normal amounts of active enzyme.

Arabinosyl-5-azacytosine: plasma kinetics and therapeutic response (L1210) in vitro and in vivo in mice

SummaryArabinosyl-5-azacytosine (Ara-AC) was studied in vitro and in vivo in kinetic and therapeutic experiments. This compound is degraded fairly rapidly in mouse plasma in vitro at 37°C (t 1/2 = 130 min) and even more rapidly in vivo (terminal t 1/2 = 76 mins, with a three phase plasma clearance curve, single dose iv 200 mg/kg). In vitro clonogenic assays with L1210 exposed to Ara-AC indicated that cytotoxic concentrations of 1 to 10 μg/ml were optimal at exposure times of 72 hours or longer (3 to 4 logs of L1210 cell kill). Extrapolating this information to in vivo infusion therapeutic studies in mice illustrated that optimal therapy (estimated 8 logs of L1210 cell kill) was also achieved at plasma concentrations of between 1 to 10 μg/ml for 72 hours of infusion. Infusions of 96 hours resulted in some lethal toxicity and 144 hour infusions were 100% lethal.

Observations on the effects of cyclophosphamide, phosphoramide mustard and some activated oxazaphosphorines on murine L1210 leukemia

SummaryThe L1210 tumor system was used in vitro and in vivo in comparative studies with activated cyclophosphamide analogs, cyclophosphamide and phosphoramide mustard. All the above compounds gave substantial cell kills (5 logs) of L1210 in vivo at doses that were non-toxic, but slight differences were noted. ASTA Z 7557 had a slight advantage in cure rate over cyclophosphamide when these drugs were given i.v. or i.p. to early tumor (i.p.). However, cyclophosphamide had the advantage in cure rate when drug administration was i.v. to advanced tumor. At equimolar concentrations in vitro ASTA Z 7557 was more cytotoxic than either phosphoramide mustard or acrolein. In vivo, the activated cyclophosphamide derivatives caused some unusual toxicities at therapeutic doses that were not seen with cyclophosphamide. The toxicities manifested as spastic responses and acute deaths on rapid i.v. or i.p. injections and as chronic liver atrophies and fibrosis with i.p. treatment.