Ghanem Atassi

A new algorithm for computing the parameters of linear compartment models in pharmacokinetics

SummaryA new algorithm (FADHA) for computing pharmacokinetic parameter estimates has been developped.This technique is based on the simplex method which is used to minimize a nonlinear cost function. An important property of this program is that the convergence is ensured contrary to the well-known linear or nonlinear least-squares regression analysis which lead to a lack of convergence or to a false one. Two investigations of the comparative performances of FADHA program and other algorithms were undertaken (hexamethylmelamine and Piracetam ® pharmacokinetics). Least square analysis of data yielded biased estimates whereas FADHA estimates were unbiased and more precise. This new technique, takes into account all the possible observation errors and uses the concept of a weighting function rather than weights as such.

Simultaneous determination of cytotoxic (adriamycin, vincristine) and modulator of resistance (verapamil, S 9788) drugs in human cells by high-performance liquid chromatography and ultraviolet detection

Multidrug resistance (MDR), which was described for structurally and mechanistically unrelated anticancer agents, was modulated in vitro by a series of compounds which were of different chemical origin. In this situation, the selection of a correct assay dosage to study the MDR modulation mechanism was a problem. We developed a high-performance liquid chomatography (HPLC) method which enabled the simultaneous determination of three major cytotoxins (adriamycin, daunorubicin, vincristine) and two well-known modulators (S 9788, verapamil). This assay was fully validated and was used to follow, for the first time, the uptake and accumulation behaviour of adriamycin and S 9788 co-incubated with resistant and sensitive cell lines (KB-3-1; KB-A1).

Chlordiazepoxide photoisomerization kinetics into oxaziridine. A HPLC study.

It was proved that the N(4)-oxide group included in chlordiazepoxide (CDZ) is involved in its phototoxicity. At a wavelength of 350 nm, CDZ photoisomerizes only into oxaziridine (OXA) which is not available as standard. In the course of cytotoxicity investigations, the optimal CDZ irradiation conditions were established as acetonitrile as solvent, 10 degrees C as temperature of the irradiated solutions and 70-90 min as irradiation time for solutions in the range of 12.2-152.0 microg/ml. The kinetic parameters of the CDZ photodegradation reaction order have been calculated using an appropriate algorithm. In all cases, the first order reversible or irreversible was selected by Akaïkes criteria. The percentage of undecomposed CDZ and OXA generated after irradiation were determined by a reversed HPLC method. The latter also permitted the separation of CDZ major impurities in aqueous solutions (demoxepam and 2-amino-5-chlorobenzophenone) as well as the oxaziridine of demoxepam. In this study, the experimental irradiation conditions allowed us to produce 98% pure OXA from CDZ. This HPLC method could be easily extended to the analysis of the molecules in pharmaceutical studies.

Determination of Doxorubicin, Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.

Pharmacokinetics and metabolism of hexamethylmelamine in mice after IP administration.

SummaryThe pharmacokinetics of hexamethylmelamine (HMM) and its first metabolite (hydroxymethylpentamethylmelamine HMPMM) following IP bolus dose of 200 mg/kg were studied in mice. The drug concentrations were determined by a sensitive reversed-phase HPLC assay. Thus, for the first time, HMM major hydroxylated and demethylated metabolite plasma levels canbedetermined at the same time. Pharmacokinetic data were analyzed by an original method using a nonlinear cost function minimized by a simplex algorithm. An important property of this computer program is that convergence is ensured in contrast to linear or nonlinear least-square regression analysis, which leads to lack of convergence or to false convergence. Both HMM and HMPMM data fit a one-compartment open model. The parameters obtained indicate that the parent drug would probably be rapidly and completely transformed by the human body into HMPMM.

HPLC determination of a new multidrug resistance modulator (S9788) extracted from cancer cells in vitro.

S9788 is a novel triazinodiaminopiperidine derivative which reverses the multidrug resistance of tumour cells to anticancer drugs. In this study, a new HPLC method was developed to determine this compound in P388 leukaemia cells. The influence of various parameters (composition and pH of the mobile phase, nature of the column) on the separation of S9788 and derivatives was investigated. Using a microsphere C18 column and the optimal mobile phase (acetonitrile-0.4 M phosphate buffer containing 0.2% triethylamine, 40:60 v/v, pH 6.5) it was possible to separate S9788 and seven hypothetical metabolites and derivatives in 15 min. The limits of detection and quantification of S9788 are 75 and 250 pg, respectively. This MDR modulator was extracted from biological media by a rapid two-step procedure which removed proteins before direct injection of the sample. Absolute recoveries ranged from 90 to 100% with a mean RSD (%) lower than 5.

Pharmacokinetics and metabolism of hexamethylmelamine in mice bearing renal cell tumors.

SummaryThe pharmacokinetics of hexamethylmelamine (HMM) and its main metabolites hydroxymethylpentamethylmelamine (HMPMM), pentamethylmelamine (PMM), and 2,2,4,6 tetramethylmelamine (2,2,4,6 TetrMM) were studied in renal cell (RC) tumor tissues and plasma of CDF1 mice that had received IP bolus injections of the maximally tolerated dose (200 mg/kg) of HMM. HMM, PMM, and 2,2,4,6 Tetr MM concentrations determined in RC tissues were much higher than the plasma values, as indicated by the pharmacokinetic parameters (Cmax and AUC). On the other hand, very low levels of HMPMM, generally considered to be a potentially active antitumor compound, were detected in the target tissues, whereas this hydroxylated metabolite was stable and easily determined in plasma. High HMM concentrations in RC tissues could correlate with the high sensitivity of the tumor to this drug. However, the behavior of HMPMM remains unclear; related hypotheses are presented in this paper.

Pharmacokinetics of daunorubicin and daunorubicinol in plasma, P388 and B16 tumours. Comparison with in vitro cytotoxicity data

SummaryThe comparison of pharmacokinetics of DNR in mouse plasma, in theDNR naturally resistant B16 melanoma and in theDNR naturally sensitive P388 leukemia showed that there is no direct correlation between total concentrations of this drug in tumours and the sensitivity resistance of these tissues.A finding which demonstrates the inadequacy of distribution models to select new potential anticancer drugs. Cytotoxicity of DNR and its metabolites to B16 melanoma and P388 leukemia cell lines were determined in vitro. Calculated inhibitory concentrations SO (IC50) were compared to maximal concentrations determined by pharmacokinetic studies.In all cases in vitro IC50 were lower than Cmax values. Moreover, resistant cells in vivo were found to be sensitive to DNR and metabolites when they are propagated in vitro.Tissue concentrations, as well as in vitro data, were fitted to appropriate models by an original program (FADHA) which uses the simplex method to minimize a non-linear cost function. Best fit models were chosen by statistical criteria.