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

The comparison of pharmacokinetics of DNR in mouse plasma, in the DNR naturally resistant B16 melanoma and in the DNR 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 50 (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.

Compared cytotoxicity effects of five anticancer drugs on human (HBL) and mouse (B16) melanoma cells in vitro.

In order to assess the potential interest of replacing the murine cell lines by human cell lines for in vitro cytotoxic assays, the sensitivity and the selectivity of the murine B16 and the human HBL melanomas to five chemotherapeutic drugs were investigated in vitro. The cytotoxicities of Melphalan, Daunorubicin (DNR), Hexamethylmelamine (HMM), Hydroxymethylpentamethylmelamine (HMPMM), and Dihydroxymethyltetramethylmelamine (DHTMM), 2 HMM derivatives, were measured in the two cell lines using two different techniques: reduction of a tetrazolium derivative (MTT) and tritiated thymidine uptake into DNA. The cytotoxicity at inhibitory concentration 50 (IC50) was determined after one hour as well as after 2 days exposure of cell after one hour as well as after 2 days exposure of cells to each drug. The results indicate that the HBL human melanoma was generally more sensitive to Melphalan and DHTMM than the B16 murine melanoma cells as far as the IC50 was concerned. In contrast, no difference of sensitivity was found to DNR and DHTMM. HMM was found to be inactive in both cell lines. The analysis of variance on IC50 values showed that the sensitivity of murine and human melanoma cell lines to drugs was statistically different. Despite the identical selectivity of the two cell lines, two promising observations can be made as far as the comparison of the two cell lines is concerned: 1) the higher sensitivity of HBL human cell line to Melphalan in the in vitro assays and 2) the slightly lower sensitivity of HBL to DNR, a drug without clinical activity against human melanoma.

In vitro cytotoxicity of hexamethylmelamine (HMM) and its derivatives.

The cytotoxicity of hexamethylmelamine (HMM) and its metabolites was investigated in three murine cell lines: one in vitro naturally sensitive to HMM (RC) and two in vivo naturally resistant (P388 and P388D1). The percentage of viable cells was determined both by the in situ reduction of a tetrazolium salt (MTT assay) and by the uptake of labelled thymidine into DNA (3HTdR assay). Short (1h) and long (48h) exposures of cells to drugs were considered. In all experimental conditions used, HMM was found to be inactive, whereas its hydroxylated metabolite hydroxymethylpentamethylmelamine (HMPMM) and one analog N, N 'dihydroxymethyltetramethylmelamine (DHTMM) were found to be cytotoxic. The results further indicated that HMM must be metabolized before it can exert its cytotoxic effect. The activity of HMPMM and DHTMM was found unlikely to be related to extracellular or intracellular release of formaldehyde.

Comparison of two cytotoxicity assays--tetrazolium derivative reduction (MTT) and tritiated thymidine uptake--on three malignant mouse cell lines using chemotherapeutic agents and investigational drugs.

Two different techniques [reduction of a tetrazolium derivative (MTT) and 3HTdR uptake assays] were compared in order to evaluate the cytotoxic effects of different chemotherapeutic agents in vitro. The cytotoxicities of Melphalan, hexamethylmelamine and seven derivatives, and daunorubicin were measured on P388D1 mouse macrophage-like cell line, RC mouse renal carcinoma cell line, and B16 mouse melanoma cell lines. Growth inhibition was determined after one hour as well as after continuous (48 hours) exposure to drugs. The IC50 was calculated using an appropriate algorithm (FADHA) which allowed within and between run variabilities to be taken into account. Optimal conditions had to be elucidated for culture conditions before assay: number of cells/well and assay duration for each line. The MTT and 3HTdR uptake assays were found to be similar in terms of sensitivity and reproducibility, both for adherent and floating cell lines. However, the MTT assay has the advantages of low cost and time saving. It also avoids problems related to radioactivity manipulation and counting. Both techniques rank the same chemicals as active or inactive. The algorithm Fadha was found to be a very powerful mathematical tool for comparing the IC50 values obtained by both assays.

Tumor pharmacokinetics of hexamethylmelamine in CDF1 mice bearing P388 leukemia.

The pharmacokinetics and metabolism of hexamethylmelamine (HMM) have been studied in CDF1, mice bearing P388 leukemia. The results obtained for this tumor, which is naturally resistant to HMM, were compared with data on HMM sensitive RC tumor and plasma. The pharmacokinetic parameters, estimated by an original algorithm (FADHA), indicated that HMM Cmax and AUC were very high in RC and P388 tumors as compared to plasma values, but could not be directly correlated with HMM activity. Hydroxymethylpentamethylmelamine (HMPMM), a potentially anticancer metabolite of HMM, was easily detected in P388 leukemia while very poorly detected in RC tissue. This finding led us to make the hypothesis that HMM activity could correlate with HMPMM ability to interact irreversibly with DNA and proteins in tumors.

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

The 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 TetrMM 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.

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

A new algorithm (FADHA) for computing pharmacokinetic parameter estimates has been developed. 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.

Pharmacokinetics and metabolism of hexamethylmelamine in mice after IP administration.

The 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.