An ELISA for the measurement of VP16 (etoposide) in unextracted plasma.

An ELISA for the anti-cancer drug VP16 in unextracted plasma has been developed using VP16-thyroglobulin-coated microtitre plates, a sheep anti-VP16 serum, and a donkey anti-sheep HRPO-labelled second antibody. The sensitivity of the assay was 0.5 ng/ml, providing a detection limit of 0.1 microgram VP16/ml plasma. Plasma interference effects were negligible and therefore the standard curve could be set up in assay buffer. A good correlation was obtained between the ELISA and established HPLC and RIA methods. No evidence was found of significant levels of cross-reacting metabolites in plasma samples obtained from patients receiving VP16 therapy. The antiserum did not cross-react with the epiaglycone of VP16.

Arginine vasopressin response to hypertonicity in hypertension studied by arginine vasopressin assay in unextracted plasma.

OBJECTIVE: To investigate whether plasma osmolality (Posm)-plasma arginine vasopressin (PAVP) response relationships with particular characteristics of sensitivity, gain or response pattern (linear/non-linear) occur more frequently in hypertensive than normotensive subjects. DESIGN: Analysis of Posm-PAVP curves observed in individual normotensive and hypertensive subjects rather than whole groups was considered appropriate for the described objective. METHODS: A sensitive and precise radioimmunoassay of PAVP in unextracted plasma was developed. Posm was raised in 11 normotensive and 19 hypertensive subjects by infusion of NaCl solution (0.86 mol/l, i.v.), and PAVP assayed at intervals. Best-fitting linear and non-linear equations for the Posm-PAVP relationship were established by computer. RESULTS: The mean rise in osmolality, blood pressure and blood volume was similar in the two groups. Hypertensive subjects showed a tendency towards higher rates of response (pmol AVP/l per mosm per kg) at Posm greater than 290 mosm/kg, leading to a non-linear response pattern compared with the normotensive subjects. CONCLUSIONS: The enhanced PAVP responses found in the hypertensive subjects at Posm greater than 290 mosm/kg may reinforce vascular and central nervous pathogenetic mechanisms.

Immunocytochemical localisation of VP16-213 in normal and malignant tissues.

Immunocytochemistry has been used to visualise the distribution of the anti-cancer drug VP16-213 in tissues from normal and leukaemic mice, and in EMT6 mammary tumours. Uptake of the drug into EMT6 tumour tissue was poor compared to uptake into normal tissues (liver, kidney, heart, small intestine). Uptake of the drug into tissues from L1210 mice was greater than that into normal tissues. Immunocytochemistry promises to be a very useful additional technique for studying the distribution of drugs given singly or in combination, specific organ toxicities, and mechanisms of resistance.

Visualisation of doxorubicin in human and animal tissues and in cell cultures by immunogold-silver staining.

In previous pharmacologic studies, the native fluorescent properties of doxorubicin (DOX) have been utilised to visualise tissue and cellular drug distribution. Such distribution studies provide valuable additional information to that obtained by measuring tissue drug concentration alone. An alternative immunocytochemical method of drug localisation using a rabbit immunoadsorbed antiserum to DOX and silver-enhanced gold-labelled second antibodies has been used to achieve visualisation of DOX in normal and malignant tissues from drug-treated animals and patients, and in human tumour cell lines treated in vitro. Non-specific staining in untreated tissues or in controls stained without primary antibody was minimal. Widespread dark brown to black specific immunostaining was observed in the normal tissues of drug-treated animals and in rat sarcoma and in the mouse EMT6 mammary tumour. In human breast tumour biopsy samples obtained at surgery 1 h following a 25 mg intravenous dose of DOX, considerable variation in drug distribution was observed which appeared to be related to drug concentration. Both nuclear and membrane staining was apparent; the latter was especially noticeable in human tumour cells grown in the presence of DOX at concentrations greater than 0.92 microM. Immunolocalisation using silver enhanced gold-labelled reagents provides an additional technique to study cell and organ specific differences in drug uptake and distribution.