A sensitive liquid chromatography-mass spectrometry bioanalytical assay for a novel anticancer candidate--ZMC1.

ZMC1 {azetidinecarbothioic acid, [1-(2-pyridinyl) ethylidene] hydrazide} is a lead compound being developed as one of the first mutant p53 targeted anti-cancer drugs. Establishing a precise quantitative method is an integral component of this development. The aim of this study was to develop a sensitive LC/MS/MS assay suitable for assessing purity, stability and preclinical pharmacokinetic studies of ZMC1. Acetonitrile protein precipitation extraction was chosen for plasma sample preparation with satisfactory recovery (84.2-92.8%) for ZMC1. Chromatographic separation was achieved on an Xterra C18 column (50 × 4.6 mm, 3.5 µm) using a gradient elution with mobile phase of 0.1% formic acid in water and acetonitrile. ZMC1 and internal standard 2-amino-6-bromobenzothiazole were identified using selected-ion monitoring mode at m/z 235.2/178.2 and m/z 231.0/150.0 at retention times of 5.2 and 6.3 min, respectively. The method was validated with a linearity range of 3.9-500.0 ng/mL in human plasma and showed acceptable reproducibility with intra- and interday precisions <5.9 and 10.5%, and accuracy within ±5.4% of nominal values. This analytical method together with basic stability data in plasma and plasma binding experiments provides a reliable protocol for the study of ZMC1 pharmacokinetics. This will greatly facilitate the pre-clinical development of this novel anti-cancer drug.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma.

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C18 reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors.

Phase I and pharmacokinetic study of imatinib mesylate (Gleevec) and gemcitabine in patients with refractory solid tumors.

PURPOSE: Preclinical data shows improvements in response for the combination of imatinib mesylate (IM, Gleevec) and gemcitabine (GEM) therapy compared with GEM alone. Our goals were to determine the maximum tolerated dose of GEM and IM in combination, the pharmacokinetics of GEM in the absence and in the presence of IM, and IM pharmacokinetics in this combination. PATIENTS AND METHODS: Patients with refractory malignancy, intact intestinal absorption, measurable/evaluable disease, adequate organ function, Eastern Cooperative Oncology Group PS 0-2, and signed informed consent were eligible. Initially, treatment consisted of 600 mg/m2 of GEM (10 mg/m2/min) on days 1, 8, and 15, and 300 mg of IM daily every 28 days. Due to excessive toxicity, the schedule was altered to IM on days 1 to 5 and 8 to 12, and GEM on days 3 and 10 every 21 days. Two final cohorts received IM on days 1 to 5, 8 to 12, and 15 to 19. RESULTS: Fifty-four patients were treated. IM and GEM given daily at 500 to 600 mg/m2 on days 1, 8, and 15 produced frequent dose-limiting toxicities. With the modified scheduling, GEM given at 1,500 mg/m2/150 min was deliverable, along with 400 mg of IM, without dose-limiting toxicities. Three partial (laryngeal, renal, and mesothelioma) and two minor (renal and pancreatic) responses were noted at GEM doses of 450 to 1,500 mg/m2. Stable disease >24 weeks was seen in 17 patients. CA19-9 in 7 of 10 patients with pancreatic cancer was reduced by approximately 90%. IM did not significantly alter GEM pharmacokinetics. CONCLUSION: The addition of intermittently dosed IM to GEM at low to full dose was associated with broad antitumor activity and little increase in toxicity.

Transcription-dependent degradation of topoisomerase I-DNA covalent complexes.

Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR.

Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin.

Breast cancer resistance protein (BCRP)/MXR/ABCG2 is a new member of the family of ATP-dependent drug efflux proteins. Whereas overexpression of another member of this family, P-glycoprotein, minimally affects the cytotoxicity of camptothecins (CPTs), overexpression of wild-type as well as certain mutant BCRPs confers resistance to CPT analogues that are used clinically, including topotecan and irinotecan. Relatively little is known regarding the effects of BCRP on other CPT analogues. We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). The results indicate that overexpression of either P-glycoprotein, multidrug resistance protein type 1, or multidrug resistance protein type 2 has little effect on the cytotoxicity of 9-NC or 9-AC. By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Furthermore, overexpression of wild-type or mutant BCRP is associated with reduced intracellular accumulation of 9-AC, but not 9-NC. In addition, immunoblotting studies indicate that whereas increased BCRP expression is evident in cells selected for resistance to irinotecan, BCRP expression is not detectable in two different cell lines selected for resistance to 9-NC. Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Furthermore, the results suggest that polar groups at the 9 or 10 position of the CPT A ring facilitate interaction with BCRP and have implications for the clinical development of new CPT analogues.

Development of a bioanalytical liquid chromatography method for quantitation of 9-nitrocamptothecin in human plasma.

9-Nitrocamptothecin (9-NC) is an orally administered camptothecin (CPT) that is under evaluation in clinical trials. This compound is not fluorescent, which has hampered development of a sensitive high-performance liquid chromatographic (LC) assay for measurement of drug concentrations in clinical trials. We now report development of an assay that involves reduction of 9-NC to the fluorescent compound 9-aminocamptothecin (9-AC). The method is based on enzymatic reduction of 9-NC using bovine liver S-9 fraction. This method is validated to quantitate 9-NC and 9-AC in patient samples, and yields results comparable to those obtained with an LC/MS method.

Paclitaxel in tyrosine-derived nanospheres as a potential anti-cancer agent: in vivo evaluation of toxicity and efficacy in comparison with paclitaxel in Cremophor.

Paclitaxel (PTX) has gained widespread clinical use yet its administration is associated with significant toxicity. In the present study, the toxicity and anti-tumor efficacy of tyrosine-derived nanospheres (NSP) for the delivery of PTX was compared to a clinical formulation of PTX in PBS-diluted Cremophor® EL (PTX-CrEL-D). Maximum tolerated dose was determined using a concentration series of PTX in NSP and CrEL-D, with toxicity assessed by measuring changes in body weight. Healthy mice administered PTX-NSP continued to gain weight normally while treatment with PTX-CrEL-D resulted in significant weight loss that failed to recover following treatment. Even at the dose of 50mg/kg, PTX-NSP showed better tolerance than 25mg/kg of PTX-CrEL-D. Xenograft studies of breast cancer revealed that the anti-tumor efficacy of PTX-NSP was equal to that of PTX-CrEL-D in tumors originating from both MDA-MB-435 and ZR-75-1 cancer lines. Larger volume of distribution and longer half-life were measured for PTX-NSP administration compared to those reported in the literature for a CrEL formulation. This trend suggests the potential for improved therapeutic index of PTX when administered via NSP. The findings reported here confirm that the NSP formulation is an efficient method for PTX administration with significant increase in maximum tolerated dose, offering possible clinical implications in the treatment of breast tumors.

A validated HPLC assay for the determination of R-(-)-gossypol in human plasma and its application in clinical pharmacokinetic studies.

R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiff's base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C(18) at 30 °C. The mobile phase consists of 10 mmol/L KH(2)PO(4) (pH 3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R(2)=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.

Effects of drug efflux proteins and topoisomerase I mutations on the camptothecin analogue gimatecan.

Clinically relevant resistance to the currently approved camptothecins, irinotecan and topotecan, is poorly understood but may involve increased expression of ATP-dependent drug transporters such as ABCG2 (breast cancer resistant protein, BCRP). Gimatecan (ST1481) is a lipophilic 7-substituted camptothecin derivative that exhibits potent anti-tumor activity in a variety of preclinical cancer models and is under investigation in the clinic. Previous studies reported that gimatecan cytotoxicity was not affected by expression of ABCG2. To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Expression of wild-type or mutant ABCG2 in human cell lines conferred resistance to topotecan but not to gimatecan. Similarly, intracellular accumulation of gimatecan was unaffected by expression of wild-type ABCG2. Furthermore, expression of P-glycoprotein or MRP2 did not alter gimatecan cytotoxicity. Whereas expression of MRP1 had a minor effect on gimatecan cytotoxicity, expression of ABCC4 was found to significantly reduce the anti-proliferative effects of this drug. Cells containing resistance-conferring mutations in topoisomerase I were also resistant to gimatecan. These results suggest that gimatecan may be more effective than irinotecan or topotecan in cancers that express ABCG2, but not in cancers that express high levels of ABCC4 or contain certain topoisomerase I (TOP1) mutations.