PK/TD modeling for prediction of the effects of 8C2, an anti-topotecan mAb, on topotecan-induced toxicity in mice.

To facilitate the development of an inverse targeting strategy, where anti-topotecan antibodies are administered to prevent systemic toxicity following intraperitoneal topotecan, a pharmacokinetic/toxicodynamic (PK/TD) model was developed and evaluated. The pharmacokinetics of 8C2, a monoclonal anti-topotecan antibody, were assessed following IV and SC administration, and the data were characterized using a two compartmental model with nonlinear absorption and elimination. A hybrid PK model was constructed by combining a PBPK model for topotecan with the two-compartment model for 8C2, and the model was employed to predict the disposition of topotecan, 8C2, and the topotecan-8C2 complex. The model was linked to a toxicodynamic model for topotecan-induced weight-loss, and simulations were conducted to predict the effects of 8C2 on the toxicity of topotecan in mice. Increasing the molar dose ratio of 8C2 to topotecan resulted in a dose-dependent decrease in the unbound (i.e., not bound to 8C2) topotecan exposure in plasma (AUCf) and a decrease in the extent of topotecan-induced weight-loss. Consistent with model predictions, toxicodynamic experiments showed substantial reduction in the percent nadir weight loss observed with 30 mg/kg IP topotecan after co-administration of 8C2 (20 ± 8% vs. 10 ± 8%). The investigation supports the use of anti-topotecan mAb to reduce the systemic toxicity of IP topotecan chemotherapy.

Topotecan enhances immune clearance of gliomas.

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3(+) T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3(+) T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-gamma, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling.

High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma.

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm x 150 mm, 4 microm) protected by a Nova-Pak C18 guard column (3.9 mm x 20 mm, 4 microm), where 10 mM KH(2)PO(4)-methanol-triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 microl mouse plasma. The assay was shown to be linear over a concentration range of 1-500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT.

Determination of topotecan by ELISA.

A highly sensitive ELISA for the determination of (s)-9-dimethylaminomethyl-10-hydroxy-camptothecin (topotecan) capable of measuring as low as 80 pg/ml was developed. Anti-topotecan antibody was obtained by immunizing rabbits with topotecan conjugated with bovine serum albumin using diazotized m-aminobenzoic acid as a cross-linker. Enzyme labeling of topotecan with beta-D-galactosidase was performed by utilizing another cross-linker, N-(4-diazophenyl)maleimide. The specificity of this ELISA appears to be primarily toward the lactone moiety of topotecan, showing a very slight cross-reactivity with the lactone opened-ring of topotecan. The values for the topotecan concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. These findings suggest that this ELISA can detect the natural amounts of the lactone form. Using this assay, drug levels were easily determined in the blood and urine of rats for 5 h after i.v. administration of topotecan at a single dose of 1 mg/kg. The sensitivity and specificity of the ELISA should provide a useful tool for developing pharmacokinetic and pharmacodynamic studies of topotecan.