G. Wynne Aherne

CYTOTOXIC DRUGS AND THE AQUATIC ENVIRONMENT : ESTIMATION OF BLEOMYCIN IN RIVER AND WATER SAMPLES

Abstract— A radioimmunoassay has been used to determine levels of the anticancer drug bleomycin in sewage treatment works effluent, river and potable water samples. Samples were concentrated 100‐fold by lyophilisation and a final limit of detection of 5 ng L−1 was achieved. Concentrations of immunoreactive bleomycin of between 11 and 19 ng L−1 were found in the effluents but a lower concentration range < 5–17 ng L−1 was found in river and potable water samples. The risk to human health of ingesting water (in SE England) with such low levels of this cytotoxic drug appears to be minimal in relation to the normal chemotherapeutic doses administered (20–30 mg m−2).

The effect of food on the oral administration of 6-mercaptopurine

SummaryThe effect of food on the bioavailability of 6-mercaptopurine (6-MP) has been investigated. Seven patients were studied on two separate occasions. On the first occasion 6-MP was administered p. o. after an overnight fast and on the second, 15 min after a standard breakfast. 6-MP concentrations were determined by high-performance liquid chromatography. Variable plasma drug levels were observed between individual subjects in the fasting state. The peak levels of 6-MP were lower and took longer to be achieved following administration after a standard breakfast than after an overnight fast. In two subjects levels were undetectable (<20 ng/ml). In view of these observations it is suggested that 6-MP should be administered before food if maximum blood levels are to be achieved.

The effect of timing of a single injection on the toxicity of methotrexate in the rat

SummaryThe possibility of a circadian rhythm in the toxicity of methotrexate was investigated in rats after a single intravenous bolus. Indices of haematological, renal and hepatic toxicity were studied, as were the pharmacokinetics of the drug. All the parameters showed a circadian variation, with maximum toxicity occurring after dosage at 06.00 h and minimum toxicity after dosage at midnight. Administration at the other two time points, 12.00 h and 18.00 h, gave intermediate results.

The quantitative determination of 2'-deoxycytidine-5'-triphosphate in cell extracts by radioimmunoassay.

A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.

The quantitation by radioimmunoassay of 2'-deoxyuridine 5'-triphosphate in extracts of thymidylate synthase-inhibited cells

A radioimmunoassay (RIA) for dUTP, with a sensitivity of 3.78 fmol, has been developed. The antibody cross-reacted with dTTP so that affinity purification of the immunoglobulin G fraction was required before its use in the RIA. Cross-reactivity with UTP and with mono- and diphosphodeoxyuridylates has necessitated respectively sodium periodate oxidation and anion exchange chromatography of cell extracts, prior to RIA quantitation of dUTP directly in fractions from the chromatography column. Mean recovery rate of a range of concentrations of extracted dUTP standard taken through the entire procedure is 63.7% (7.8-31.3 pmol dUTP) although at a lower concentration (3.11 pmol) the recovery was only 36.2%. Results are reproducible with CV values of between 3.1 and 9.5%. The assay has been used to assess the presence of dUTP in A549 human lung carcinoma cells exposed to the thymidylate synthase inhibitor CB3717. The high sensitivity of the quantitation step has made it possible to measure dUTP in relatively small numbers (10(6)) of cells.

The effect of abolition of the endogenous corticosteroid rhythm on the circadian variation in methotrexate toxicity in the rat

SummaryMonitoring of indices of haematological, renal and hepatic toxicity in rats after a single i.v. bolus of methotrexate has shown that they vary with the time of day at which the drug is administered. Maximum toxicity occurs after administration at 0600 h. Further experimentation has shown that the amount of corticosteroid present in the blood has a profound effect on the toxicity of methotrexate in the rat. If the endogenous production of corticosterone is suppressed by treatment with dexamethasone the toxicity of methotrexate is markedly increased at whatever clock time it is administered. However, if constantly high plasma levels are achieved by giving supplementary corticosterone methotrexate toxicity is diminished regardless of what time it is given.Since the timing of maximum methotrexate toxicity corresponds to the circadian nadir of endogenous plasma corticosterone concentration in the rat the possibility that it might be related to corticosterone production must be considered. Whether this phenomenon occurs in man and has any clinical relevance has yet to be investigated.

A radioimmunoassay for VP16-213 in plasma

SummaryA radioimmunoassay for the semi-synthetic podophyllotoxin VP16-213 has been developed which is suitable for pharmacokinetic studies of the drug. A high titre antiserum was produced in a sheep in response to a VP16-213-BSA conjugate prepared using sodium periodate. Podophyllotoxin does not cross-react with the antiserum and VM26 cross-reacts to only a small extent (<0.6%). In the absence of a high specific activity tritium label, a radioiodinated histamine ligand was produced which was only partially displaced from antibody by native drug. VP16-213 can be measured in plasma without prior drug extraction with a theoretical limit of detection of 5–10 μg/l. VP16-213 cis (picro) hydroxy acid is recognised by the antiserum to a greater extent than the drug itself. Thus, in order to eliminate any interference from the trans hydroxy acid metabolite chloroform extraction of plasma samples was carried out.

Enhanced chemiluminescent enzyme immunoassay for cannabinoids in urine

An enhanced chemiluminescent enzyme immunoassay was developed for the detection of cannabinoids in urine. It utilises an antiserum specific for tetrahydrocannabinol and its major metabolite, a donkey anti-sheep antiserum and a horseradish peroxidase labelled antigen conjugate. The bound enzyme is detected via its catalytic activity on the chemiluminescent luminol-H2O2 reaction in the presence of an enhancer. This immunoassay employs mild experimental conditions and is extremely sensitive (0.13 microgram l(-1), making it suitable for the detection of cannabinoids in samples obtained several days following drug use. None of several medicinal and other drugs of abuse tested interfered in the assay. Greater sensitivity and simplicity make it a feasible non-isotopic alternative to radioimmunoassay and it is amenable to automation and routine screening to large sample batches.

Serum kinetics of the anti-cancer agent 4-hydroxyandrostenedione in the rat

SummaryA previously described radioimmunoassay (RIA) method for the measurement of 4-hydroxyandrostenedione (4-OHA) was used to investigate the serum drug levels attained after a single oral dose in male and female rats. Marked variability of serum drug concentrations and their time course were evident in male animals at all dose levels. In the female rat, in contrast, serum 4-OHA showed fewer individual differences, rose more rapidly and was sustained at substantially higher concentrations. In all animals, 4-OHA appeared in the serum within 0.5 h following the oral dose and persisted for at least 48 h. Doubling the dose from 8 mg/kg produced a disproportionately large elevation in serum drug levels, but a further increase to 32 mg/kg did not further increase serum levels.

The effect of cotrimoxazole on the absorption of orally administered 6-mercaptopurine in the rat

SummaryThe effect of cotrimoxazole (CTX) on plasma levels of 6-mercaptopurine (6-MP) was studied in the rat. Animals receiving CTX in conjunction with 6-MP were found to have a marked but non-significant decrease in the area under the plasma time curve as compared with animals receiving 6-MP alone. It is suggested that the bioavailability and thereby, the antileukaemic effect) during maintenance therapy of ALL of 6-MP may be decreased by the co-administration of CTX.