David W. Yesair

Role of the entero-hepatic cycle of indomethacin on its metabolism, distribution in tissues and its excretion by rats, dogs and monkeys☆

Abstract Dogs excrete most of an i.v. dose of 14 C-indomethacin unchanged in their feces. Monkeys extensively metabolize the drug to deschlorobenzoylindomethacin and excrete it in urine, and rats, without coprophagy, excrete the major metabolite, desmethylindomethacin, equally in urine and feces. 14 C-indomethacin constitutes the major radioactive species in plasma, liver and kidney of rats and monkeys. In both species, a new lipid-soluble metabolite of indomethacin was observed. The half-life of the drug in plasma varied among the three species ranging from minutes in monkeys and dogs to hours in rats. In rats, plasma clearance of indomethacin by liver, although low, was thirty times the clearance rate by kidney, and the reabsorption of indomethacin from the intestine was extensive. Desmethylindomethacin, the major metabolite, was cleared from plasma equally by liver and kidney and was not reabsorbed from the intestine of rats. Consequently, this metabolite was distributed equally in urine and feces. Indomethacin was extensively and rapidly secreted in bile by dogs, eventually excreted as unchanged drug in their feces, and minimally metabolized to deschlorobenzoylindomethacin, which was excreted in urine. Monkeys were similar to dogs in that the liver was more than ten times as effective as the kidneys in clearing total radioactivity from plasma. However, they differed from dogs in that all drug species were maximally reabsorbed from the intestine. These differences in the plasma clearance of indomethacin and its metabolites by liver and kidney and in the entero-hepatic circulation of these drug species were sufficient to account for the species differences in the distribution and excretion of indomethacin.

Comparative effects of salicylic acid, phenylbutazone, probenecid and other anions on the metabolism, distribution and excretion of indomethacin by rats☆

Abstract This report describes the effects of salicylate, phenylbutazone, probenecid and other anions on the metabolism, distribution and excretion of indomethacin by the rat. Salicylic acid (100 mg/kg) given intravenously or orally 1 or 3 hr after indomethacin administration (10 mg/kg) significantly decreased (30–60 per cent) concentrations of indomethacin in plasma. Concomitantly, urinary excretion of 14 C-indomethacin equivalents was decreased, biliary and fecal excretions were increased, and concentrations in tissues were modified. These changes after salicylic acid treatment were reflected primarily in indomethacin and secondarily in its metabolites. Other agents, such as phenylbutazone, chlorogenic acid and acetic acid, had no effect on the radioactivity in plasma. Probenecid increased the concentrations of indomethacin in plasma by about 20 per cent. The specificity of salicylic acid in decreasing concentrations of indomethacin in plasma of rats and of probenecid in increasing indomethacin concentrations in plasma of both rat and man may arise from the similarity in structure of the benzoyl group of the three compounds. Such a specificity has been discussed with respect to the controversy about the intake of salicylates during the clinical evaluation of indomethacin.

Comparative uptake, metabolism and retention of anthracyclines by tumors growing in vitro and in vivo

Abstract Adriamycin, daunomycin and their metabolites were differentially taken up in vitro by L 1210 lymphocytic leukemia cells in culture. An intracellular drug equivalent concentration of about 0.20 μg/107 cells (20 μg/g) was required to attain equivalent cell-kill for daunomycin, daunomycinol or adriamycin but, in order to attain this intracellular concentration, the extracellular concentrations of daunomycinol and adriamycin were 3 and 2 times that of daunomycin. Intracellular concentrations of daunomycin comparable to those in vitro were attained in L 1210 lymphocytic leukemia, P388 leukemia, and B16 melanoma in vivo when both drug and tumor were present in the intraperitoneal cavity, but only a fraction of that concentration was attained if the tumor was growing as a solid mass at a site distal to the site of drug administration. Daunomycin was quantitatively metabolized by the reductase in homogenates of L 1210/NSC 38280, L 1210, P388 or B16 tumors; however, the rate of metabolism by the lymphocytic leukemias was much slower than that by B16 melanoma. Daunomycinol effluxed more slowly than daunomycin from cells of all responsive tumors. In practice, chemotherapeutic response to anthracycline drugs may depend upon several dynamic factors: uptake, metabolism and retention of the drug by tumors and the pharmacokinetics of the drug and metabolites in the mammalian host.

Method for extraction and separation of drugs and metabolites from biological tissue

Abstract A method has been developed for the quantitative extraction, partitioning, and separation of indomethacin, its metabolites and other anionic drugs (salicylic acid and phenylbutazone) from biological tissues. Tissues are extracted with chloroform and anhydrous, acidic methanol. When the extract is made biphasic with aqueous acid, conjugated drug species are partitioned to the aqueous, acid phase while protonated and esterified drug species are partitioned to the organic phase. The ionized drugs (indo-methacin, its nonconjugated metabolites, and salicylic acid) in this phase are partitioned to an aqueous, basic phase (pH 8.6) and separated by anion-exchange chromatography. Phenylbutazone is extracted at pH 12–13 and esterified drug species remain in the organic phase. The quantitative aspects of this method have been evaluated with biological samples from rats which had received 14 C-indomethacin or 14 C-salicylic acid in vivo .

The effect of suramin-phthalanilide complexes on the chemotherapeutic activity and toxicity of 4′,4″-bis (1,4,5,6-tetrahydro-2-pyrimidinyl)terephthalanilide (NSC 57153)

Abstract Suramin forms water-insoluble complexes with 4′,4″- bis (1,4,5,6-tetrahydro-2-pyrimidinyl)terephthalanilide (NSC 57153). The mole ratio of Suramin-drug for maximum complex formation is 0·3:1 . Excess Suramin dissolves this precipitate whereas excess drug does not. The uptake of NSC 57153 by P815 mast cell leukemic cells in vitro is affected by the presence of Suramin. Excess moles of Suramin relative to drug inhibit uptake, whereas excess moles of drug enhance the uptake. Although Suramin administration has no effect on drug uptake by tumor cells in vivo and does not affect the antileukemic activity of NSC 57153, it does delay the onset of NSC 57153 toxicity in mice. Three drug components (lipid-bound, hydrophilic-bound, and “free” drug) are extracted from tumor cells and the ratio of the three drug components is not affected by the extracellular Suramin-drug ratios after the cells have been exposed to drug either in vitro or in vivo . Suramin lowers the concentration of NSC 57153 in both kidney and liver but not in muscle. The drug is extracted primarily as a lipid complex from all tissues, whether the animals have been treated with NSC 57153 or with Sumarin-NSC 57153 complexes.

Importance of schedule in adriamycin/cyclophosphamide combination chemotherapy

Abstract Use of the chemosensitive tumor P388 lymphocytic leukemia has facilitated the demonstration of small but perhaps important schedule-dependent therapeutic effects with adriamycin (ADR) and cyclophosphamide (CY) combination therapy. Administration of ADR at least 3 days pror to the administration of CY resulted in inhibition of DNA synthesis to a greater extent in tumor than in bone marrow. This preferential inhibition of thymidine incorporation into nucleic acid of tumor cells is in accord with the enhanced chemotherapeutic response observed when the agents were given in this sequence (ADR day 5 and CY on day 8, 9 or 10 ). This effect on response was more marked with lower doses of the drugs, which could be of clinical significance since the total dose of ADR is so restrictive.

COMPARATIVE STUDIES OF DRUG UPTAKE, VIABILITY, AND BIOSYNTHETIC CAPABILITIES OF P388 CELLS TREATED WITH “ACTIVE” OR “INACTIVE” TEREPHTHALANILIDES*

Many substituted terephthalanilides have potent activity against a broad spectrum of transplanted murine leukemias and lymphomas *-9 and against several microorganisms.l~-~~ The interaction between DNA and the terephthalanilides in vitro does not appear to correlate with the therapeutic efficacy of these compounds against mouse leukemia,’4 although the bacteriostatic effect of the terephthalanilides apparently parallels the magnitude of their interaction with DNA and RNA.“ The terephthalanilides, which are cationic at neutral pH, form ionic complexes with many biological components in vitro; e.g. anionic lipids!5.16 protein!7 and nucleic acids.”, 13. 1 4 3 la However, evidence for these ionic complexes in situ has not been obtained. The terephthalanilides are extracted from tissues, tumor cells, nuclei, and mitochondria of tumor cells and E. coli B primarily as ionic drug-lipid complexes.15~ 19-21922 The lipids that are extracted from dog brain and from leukemic cells represent a new class of p h o s p h ~ l i p i d ! ~ ~ ~ ~ ~ ~ The concentration of the extractable drug complexes is generally highest in tissues and tumors that are adversely arfected by terephthalanilide treatment. 19-21, 23-26 Since many terephthalanilides have been synthesized, it has been possible to associate particular structures or combinations thereof with the observed chemotherapy.aq9.11.14 Considering chemotherapeutic activity against L1210 growing in mice, the terephthalanilides may be divided into two broad categories: “active” compounds, which produce an increase in life-span of 125% or more, and “inactive” compounds, which produce less. We have evaluated these structureactivity relationships in terms of drug uptake, viability, and biosynthetic capabilities of P388 leukemia cells treated with structurally related “active” and “ in active” terephthalanilides. A preliminary report of this work has been made by Yesair and colleagues?

The Importance of Pharmacokinetic Principles in Characterizing Carcinogenic Thresholds for Naturally Occurring and Synthetic Chemicals

Many chemicals, both man-made and naturally occuring, can elicit a carcinogenic response either as a mutagenic response in vitro or as a frank tumor in vivo. In both instances emphasis has been placed historically on the dose which elicits the response. When the carcinogenic response is related to the pharmacokinetics of the carcinogen taking into account the dose, the frequency of dosing, andthe relative half lives of the carcinogen, its reactive metabolites and any modified macromolecules, a narrow plateau level of modified macro-molecules is found in specific responsive tissues or species of animals. This plateau threshold concept for carcinogen as defined by the pharmacokinetics, has been evaluated for both chloroform and aflatoxin. The carcinogenic risk associated with plateaus can be evaluated experimentally, and perhaps can be evaluated today with the available data at hand.

PROPOSED MECHANISM FOR THE REDUCTIVE GLYCOSIDIC CLEAVAGE OF DAUNOMYCIN (NSC 82151) AND ADRIAMYCIN (NSC 123127)

Publisher Summary This chapter presents a mechanism for the reductive glycosidic cleavage of daunomycin (NSC 82151) and adriamycin (NSC 123127). The chapter presents an experiment in which chelates of the anthracycline drugs were obtained by mixing the anthracycline drugs in ethanol with varying concentrations of cations in ethanol or by mixing aqueous buffered solutions of both. The ultraviolet and visible absorption spectra were recorded on a Beckman DK-2 recording spectrophotometer. The chloride salts of the metal ions were used, and the metabolites and parent drugs were separated by thin-layer chromatography on silica gel, using two solvent systems. To evaluate whether the protons of the dihydroquinone system of the anthracycline drugs are exchangeable, it was shown that the absorption spectra of the anthracycline drugs varied as a function of increased pH showing two new absorption maxima at 445 and 585 nm and decreasing the absorption region at 475–500 nm. Chelates of metals with the anthracycline drugs also showed two new absorption maxima at about 530 and 570 nm, and the absorption in the 475–500 nm region was only slightly decreased by metal ions.

Characterization of the urinary metabolites of 5-azacytidine in mice

Abstract The metabolism of 5-azacytidine, radiolabeled in the 4- or 6-position with carbon-14, was studied in mice. High pressure liquid Chromatographic analysis of urine from mice given [4- 14 C]- or [6- 14 C]-5-azacytidine showed that six radioactive peaks were present and that peaks V and VI were the major peaks excreted. Similar analysis of urine from mice given the labeled compounds and tetrahydrouridine, a deaminase inhibitor, indicated that peaks I, II and V were deaminated metabolites. Peak V appeared to contain components with the 6-carbon absent, whereas peak VI contained components, in a 1:1 ratio, with the 6-carbon either present or absent. Co-chromatography of urine from treated mice with authentic standards indicated that peaks I, III, IV were 5-azauracil, 5-azacytosine and 5-azacytidine, respectively. These data, along with the characterization of peaks V and VI by gasliquid chromatography and mass spectrometry, indicated that the metabolism of 5-azacytidine in mice is similar to that proposed by other investigators.