Methods for the analysis of the new vinca alkaloid derivative, S 12363, in plasma by high-performance liquid chromatography with fluorescence detection.

Two sensitive analytical methods for the analysis of S 12363 in plasma are described. A highly sensitive procedure for human and dog plasma using cyanopropyl solid-phase extraction with ion pairing chromatography and fluorescence detection, has a limit of quantification of 0.1 ng ml-1. The technique has an overall precision and accuracy of 4.8 and 5.4% respectively over the concentration range 0.1-20 ng ml-1. A second, less sensitive, assay specifically adapted for rodent plasma, uses benzene sulphonyl cation-exchange solid-phase extraction followed by reversed-phase chromatography, with post-column fluorescence enhancement. This method has a limit of quantitation of 1.0 ng ml-1, with overall accuracy and precision of 7.2 and 11.6% respectively, over the concentration range 1.0-20.0 ng ml-1. Both assays have been successfully applied to dog and mouse toxicokinetic studies.

Method for the analysis of S9788, a drug to reverse resistance to anticancer agents, in animal plasma and human plasma and serum by high-performance liquid chromatography with ultraviolet detection.

An extraction method has been developed using benzene sulphonyl cation-exchange sample preparation cartridges and reversed-phase high-performance liquid chromatography with ultraviolet detection for the measurement of S9788, a drug to reverse resistance to anticancer agents, in plasma and serum. This includes a toxicokinetic assay which has a mean precision and accuracy of 11.7% and 7.9%, respectively, over the range 10-1000 ng ml-1 and a quantification limit of 10 ng ml-1 and a more sensitive pharmacokinetic procedure with a mean precision and accuracy of 5.0% and 7.9%, respectively, over the range 1-500 ng ml-1 and a quantification limit of 1 ng ml-1. The specificity of the procedure has been demonstrated by mass and ultraviolet spectrometry, and linearity, precision, accuracy, recovery and sensitivity have been established. The assays have been successfully applied to toxicokinetic and pharmacokinetic studies.

Monoclonal antibodies to human eosinophil plasma membrane antigens enhance the secretion of eosinophil cationic protein.

This study was done to examine the nature of the membrane constituents involved in the secretion of eosinophil cationic protein (ECP) from human blood eosinophils. Three mouse monoclonal antibodies were used, which showed greater binding to membrane antigens on activated, and light density eosinophils from patients with an eosinophilia, than on nonactivated or normal density eosinophils. All three antibodies (EoN4, EoN5 & EoN6) stimulated normal density human eosinophils to secrete ECP, either alone or in association with sepharose-C3b. The antibodies bound to at least two separate sites on the membrane, which were distinct from the receptors for immunoglobulins, C3b, and eosinophil activating factor. One combination of antibodies increased the amount of ECP which was secreted. The membrane antigen recognized by antibody EoN4 was a glycoprotein, molecular weight 75 kD. These findings showed that ECP secretion may be induced by a wider range of stimuli than has been previously recognized, and that the antigens recognized by these monoclonal antibodies may play an important role in the induction of eosinophil degranulation.

Plasma membrane antigens on light density and activated human blood eosinophils.

In order to study the membrane events which lead to human blood eosinophil activation and degranulation, six clone of mouse monoclonal antibodies were made to isolated blood eosinophils from a patient with the hypereosinophilic syndrome (HES). The antibodies were specific for the plasma membranes of blood eosinophils and neutrophils, eosinophil myelocytes and haemopoietic cell lines. A higher proportion of blood eosinophils from eight patients with the HES bound these antibodies compared to normal individuals. Five antibodies stained intermediate density eosinophils preferentially, and one stained the light density eosinophils most strongly. Normal blood eosinophils were induced to express more of these antigens either by culture alone (two antibodies) or culture with monocyte culture supernatant which activates eosinophils for increased cytotoxic capacity (four antibodies). It was concluded that several of the principal antigens on the eosinophil plasma membrane are also present on neutrophils and immature haemopoietic cells, and that their expression in mature blood eosinophils is related to the extent of eosinophil activation and degranulation. It is suggested that these antigens may be useful in studying the ways in which eosinophils alter their plasma membrane during activation and degranulation in vitro, and in the blood of patients with an eosinophilia.

Eosinophil membrane antigens: phenotypic frequencies in normal individuals and patients with the hypereosinophilic syndrome.

The membrane antigen phenotype of eosinophils from six normal individuals and eight patients with the hypereosinophilic syndrome (HES) were examined to see whether subpopulations of eosinophils exist. Experiments were done with a panel of 6 monoclonal antibodies, using the fluorescent activated cell sorter, and immunocytochemistry. All six antibodies bound eosinophils and neutrophils, but not lymphocytes, monocytes, platelets or erythrocytes. The phenotypic frequencies of five of the six antibodies were increased in patients' eosinophils (p less than 0.005). This increase was associated with the intermediate-density eosinophils, while the antigen detected by antibody Eon 7 was associated with the light-density eosinophils. Normal eosinophils could be induced to increase their expression of these membrane antigens by incubation with mononuclear cell supernatants which are known to increase the cytotoxic capacities of eosinophil, 'activation'. It was concluded that there is a single eosinophil which undergoes post-mitotic differentiation in the blood, leading to activation and degranulation. The heterogeneity seen in patients' eosinophils reflect different stages of cell maturation and activation.