John H. Peters

The polymorphic acetylation of dapsone in man

The characteristics of the acetylation of dapsone (avlosulfon) were found to parallel those of isoniazid and sulfamethazine in 19 subjects, thereby establishing the genetic polymorphism for the acetylation of dapsone. This polymorphism was revealed by the distribution of the ratios of the plasma concentration of acetylated to parent drug. The acetylation capacity for dapsone was shown to be a reproducible, individual characteristic. Acetylation of dapsone and deacetylation of monoacetyl dapsone occurred concurrently. Constant plasma ratios of acetylated to parent drug characteristic for the individual were attained immediately after administration of dapsone but only after several hours following administration of monoacetyl dapsone. The available data suggest that acetylation rather than deacetylation is the primary determinant of these ratios. Rates of disappearance of dapsone and monoacetyl dapsone from the plasma were the same regardless of which of the two was administered or of the acetylator phenotype of the subject. After dapsone, no differences between rapid and slow acetylators were found in the 24 hour urinary excretion of dapsone and its conjugates hydrolyzed by mild or strong acid treatment. Rapid acetylators excreted significantly more monoacetyl dapsone and its acidlabile conjugates than slow acetylators. Because these compounds accounted for only a very small fraction of the dose, it was not possible to phenotype individuals by these measurements. More dapsone and acid‐hydrolyzable conjugates of dapsone were found in 120 hour urine collections after monoacetyl dapsone than after dapsone in both phenotypes.

Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation

To explore the structural characteristics of various derivatives of the anticancer drugs, doxorubicin and daunorubicin, for exhibiting redox activities believed to be associated with toxic radical production, we tested over fifty derivatives in a rapid screening procedure for augmenting oxygen consumption by rat liver microsomes. Measurement of parent drug disappearance and of metabolite appearance for fourteen anthracyclines with a broad range of activities for augmenting oxygen consumption indicated that a single reaction, conversion to the 7-deoxyaglycone, occurred. Multiple tests of selected compounds showed that the liver microsome system exhibited saturation kinetics, and calculated values of Vmax/Km gave the same relative order of activities as did the screening test. The liver microsome system was not found to be stereoselective. Measurements of the abilities of a number of the anthracycline derivatives after chemical activation by reduction with sodium borohydride to convert oxygen to superoxide anion, or to the hydroxyl radical, were also made. The reactivities of the anthracyclines in these latter two assays were positively related to the activities obtained in the rat liver microsome screening test, suggesting that all three tests were measuring various steps in the sequence from anthracycline semiquinone radical formation through oxygen activation and radical formation. Superoxide anion generation from chemically reduced anthracyclines was inhibited by the addition of calf thymus DNA, and the extent of inhibition was positively correlated with the measured DNA association constants of the anthracyclines. However, the DNA association constants were unrelated to superoxide anion generation in the absence of DNA or to the augmentation of oxygen consumption in liver microsomes. Half-wave potentials were negatively correlated with both the results of the microsomal oxygen consumption test and the production of superoxide anion in the chemical test system. No relationships were discerned among the DNA association constants, half-wave potentials, or reoxidizabilities of the anthracyclines tested. Comparisons of the relatively low activities of certain of the anthracyclines in the biochemical and chemical tests for oxygen activation with their known high activities against murine tumors in vivo, but low cardiotoxicities in animal model systems, suggest that the separation of the cytotoxic antitumor and cardiotoxic actions of these derivatives may have been achieved.

Comparative cytotoxicities of various morpholinyl anthracyclines

SummaryA series of quinone- and sugar-modified analogs of adriamycin have been tested for growth inhibition of adriamycin-sensitive (P388/S) and -resistant (P388/ADR) sublines of P388 murine leukemia cells in vitro. P388/ADR is less resistant to analogs of adriamycin containing either a 3′-deamino-3′-(4″-morpholinyl) group, MRA; or a-(3″-cyano-4″-morpholinyl) group, MRA-CN, than to adriamycin. However, MRA-CN was the most potent growth inhibitor of either subline. This potency is reduced by either modification of the quinone unit with a 5-imino substituent or restriction of the cyano-morpholinyl ring by an oxygen bridge to the daunosamine sugar. The calcium antagonist verapamil substantially increases the cytotoxicity of adriamycin to P388/ADR but has no appreciable effect on the cytotoxicity of either MRA or MRA-CN. The results suggest that increased uptake and retention by both MRA and MRA-CN may contribute to their increased cytotoxicity, but that the intense potency of the cyano-morpholinyl analogs must be due to other unique properties of these compounds.

The measurement of urinary oxalic acid by derivatization coupled with liquid chromatography

Abstract A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o -phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C 8 column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.

Uptake and retention of morpholinyl anthracyclines by adriamycin-sensitive and -resistant P388 cells.

Summary3′-Deamino-3′-(4-morpholinyl)adriamycin (MRA) and 3′-deamino-3′(3-cyano-4-morpholinyl)adriamycin (MRA-CN) were compared with adriamycin (ADR) in ADR-sensitive (P388/S) and-resistant (P388/ADR) murine leukemia cell lines with respect to cytotoxicity and cellular accumulation. MRA is only two- to threefold more cytotoxic to P388/S in culture than ADR, whereas MRA-CN is 500-fold more cytotoxic than ADR to this cell line. Yet both MRA and MRA-CN retain their potency against P388/ADR in spite of a 150-fold decrease in potency for ADR. The observed noncross-resistance of both MRA and MRA-CN in P388/ADR correlates with their increased cellular uptake and retention relative to ADR and the inability of P388/ADR to exclude these analogs as readily as it does ADR. The decreased uptake of MRA and MRA-CN in P388/ADR relative to P388/S (1.5 to 2.0-fold), the increased efflux, and the ability of verapamil to enhance cellular uptake of these analogs in P388/ADR, as it does with ADR, all indicate that the mechanism of ADR-resistance effects ADR and the morpholino analogs in a similar manner but to far different extents. The potent cytotoxicity of MRA-CN appears to be related to strong cellular interactions of the drug with macromolecules that are characterized by its nonextraction from cells by chloroform: methanol or 10 M urea and may therefore represent covalent binding.

Tissue distribution of doxorubicin and doxorubicinol in rats receiving multiple doses of doxorubicin

SummaryPlasma and tissue levels of doxorubicin (DXR) and doxorubicinol (DXR-OL) were measured fluorometrically after high-pressure liquid chromatography at 1, 3, and 24 h following one, nine, and 24 doses of 1.0 mg DXR/kg or one and eight doses of 4.0 mg DXR/kg, IP, to rats. Comparison of plasma levels of DXR found following single and multiple doses suggests significant build-up of DXR at 1 h with successive doses, but not at 3 h. Liver exhibited substantially higher levels of DXR (on a per gram of protein basis) than did plasma, and multiple doses did not produce higher levels than did a single dose. In contrast, the heart accumulated DXR slowly, attaining levels after multiple dosing in excess of those found in the liver. Skeletal muscle exhibited dose-related levels similar to those for heart but the absolute levels of DXR in muscle were only about one-tenth of those observed in heart. DXR-OL was at very low levels of ≤4% of the DXR levels in the tissues; it was, however, a major circulatory metabolite, attaining levels in the plasma as high as 85% of the concentration of DXR.

Amino acids, including asparagine and glutamine, in plasma and urine of normal human subjects.

Summary The use of lithium citrate buffer systems for the analyses of acidic and neutral amino acids in plasma and urine was tested and found superior to the usually employed sodium citrate buffer systems. The lithium systems have the advantage of resolving asparagine and glutamine without loss of resolution of other acidic and neutral amino acids. Normal levels of these amides in plasma and urine were established, and the relative contributions of the aspartic and glutamic acids, their amides, and unknown conjugates (peptides?) to the total measurable acids in protein-free filtrates after hydrolysis were determined. The general pattern of excretion of amino acids in urine collected for 4 hr from fasting individuals was shown to be quite similar to the pattern found in 24-hr urines collected from nonfasting subjects.

Plasma Histaminase Activity in Various Mammalian Species; A Rapid Method of Assay.

Summary A rapid microcolorimetric meth-od for measurement of plasma histaminase has been described. Its lower limit of sensitivity is 0.1 mμM histamine oxidized/ml of plasma/minute and its reproducibility, ± 0.1 mμM. In a series of mammalian species surveyed, no activity was detected in the plasma of mice and rats; plasma from guinea pigs, dogs, squirrel monkeys, and man exhibited relatively low activities; plasma activity of rabbits and cats was somewhat higher; plasma from rhesus monkeys was more active than that of any other species. Studies of pH optimum, aminoguanidine and substrate inhibition, and substrate specificity using human plasma indicate that the primary activity measured was histaminase activity.

Mutagenic activities of fecapentaene derivatives in the Ames/Salmonella test system

The direct mutagenic activities of a pair of naturally-occurring and several synthetic fecapentaenes were measured in the Ames/Salmonella test system. We found that strain TA100 with preincubation was the most sensitive procedure for the naturally-occurring fecapentaene-12 (FP-12). Its natural analog, FP-14, and the synthetic isomer, cis-FP-12, yielded similar mutagenic activities to FP-12 in the range of 1000-2000 TA100 revertants per microgram of compound. The synthetic analogs of FP-12 and FP-14, MFP-12 and MFP-14, wherein the glycerol moiety was replaced by methoxy, exhibited consistently higher mutagenic activities than their parent fecapentaenes (MFP-12 was about 20 times more potent than FP-12; MFP-14 was about twice the potency of FP-14). The standard rat liver metabolizing system (S9) reduced the activities of all the fecapentaenes in a dose-related manner.

Effects of 5-iminodaunorubicin on nucleoli of rats

SummaryWe have confirmed that doxorubicin induces irreversible changes in the nucleolar ultrastructure of myocardial cells of rats. Similar changes were not caused by an equal dose of the synthetic analogue, 5-iminodaunomycin. These results combined with previous and current comparative tests with this analogue, doxorubicin, and daunomycin suggest that 5-iminodaunomycin may serve as a less cardiotoxic anthracycline derivative.