Richard Wiegand

Phase I Clinical Trial of BMS-247550, A Derivative of Epothilone B, Using Accelerated Titration 2B Design

Purpose: BMS-247550 is a semisynthetic derivative of epothilone B with mechanism of action analogous to paclitaxel. It has shown impressive antitumor activity in preclinical studies including in taxane-resistant models. We conducted a phase I trial, based on accelerated titration “2B” design, of BMS-247550 given as a 1-hour infusion every 3 weeks. Experimental Design: Seventeen patients (M:F, 10:7; median age, 54 years; performance status, 0-2) were treated on the trial. Forty-five cycles (1-9 cycles) of BMS-247550 were given at dosages ranging from 7.4 to 56 mg/m2. All patients received prophylaxis for hypersensitivity reactions, related to Cremophor-EL, with steroids and histamine antagonists. Results: First-course dose-limiting toxicity (DLT) was observed in two of three patients at 56 mg/m2 (neutropenic sepsis, prolonged grade 4 neutropenia) and in one of six patients at 40 mg/m2. Nonhematologic grade 3 to 4 toxicities observed were emesis and fatigue and they occurred only at 56 mg/m2. Grade 1 to 2 peripheral neuropathy was also observed. Other grade 1 to 2 toxicities were myalgias, arthralgias, rash, hand/foot syndrome, and mucositis. AUC and Cmax seemed proportional to the dose and the DLT. Development of neutropenia with BMS-247550 is related to the duration of drug exposure above a threshold. Conclusions: The maximum tolerated dose (MTD) of BMS-247550 is 40 mg/m2 given every 3 weeks. Neutropenia is the DLT. The accelerated titration “2B” design may help in determining MTD with fewer patients enrolled and more being treated closer to the MTD. However, the accelerated titration design did not seem to shorten the study duration.

Methylation suppresses the proteasome -inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer‐preventive activity. Although a recent study suggested that methylated polyphenols are less cancer‐protective, the molecular basis is unknown. We previously reported that ester bond‐containing GTPs, for example (−)‐epigallocatechin‐3‐gallate [(−)‐EGCG] or (−)‐epicatechin‐3‐gallate [(−)‐ECG], potently and specifically inhibit the proteasomal chymotrypsin‐like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome‐inhibitory abilities. To test this hypothesis, methylated (−)‐EGCG and (−)‐ECG analogs that can be found in vivo were synthesized and studied for their structure‐activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (−)‐EGCG or (−)‐ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate‐protected (−)‐EGCG molecule, Pro‐EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro‐EGCG (1) to (−)‐EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate‐protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate‐protected, monomethylated (−)‐EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate‐protected, trimethylated (−)‐EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome‐inhibitory activity, contributing to decreased cancer‐preventive effects of tea consumption. J. Cell. Physiol. 213: 252–260, 2007.

Complex Disease–, Gene–, and Drug–Drug Interactions: Impacts of Renal Function, CYP2D6 Phenotype, and OCT2 Activity on Veliparib Pharmacokinetics

Purpose: Veliparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, undergoes renal excretion and liver metabolism. This study quantitatively assessed the interactions of veliparib with metabolizing enzyme (CYP2D6) and transporter (OCT2) in disease settings (renal impairment). Experimental Design: Veliparib in vitro metabolism was examined in human liver microsomes and recombinant enzymes carrying wild-type CYP2D6 or functional defect variants (CYP2D6*10 and *4). Plasma pharmacokinetics were evaluated in 27 patients with cancer. A parent–metabolite joint population model was developed to characterize veliparib and metabolite (M8) pharmacokinetics and to identify patient factors influencing veliparib disposition. A physiologically based pharmacokinetic model integrated with a mechanistic kidney module was developed to quantitatively predict the individual and combined effects of renal function, CYP2D6 phenotype, and OCT2 activity on veliparib pharmacokinetics. Results: In vitro intrinsic clearance of CYP2D6.1 and CYP2D6.10 for veliparib metabolism were 0.055 and 0.017 μL/min/pmol CYP, respectively. Population mean values for veliparib oral clearance and M8 clearance were 13.3 and 8.6 L/h, respectively. Creatinine clearance was identified as the significant covariate on veliparib oral clearance. Moderate renal impairment, CYP2D6 poor metabolizer, and co-administration of OCT2 inhibitor (cimetidine) increased veliparib steady-state exposure by 80%, 20%, and 30%, respectively. These factors collectively led to >2-fold increase in veliparib exposure. Conclusions: Renal function (creatinine clearance) is a significant predictor for veliparib exposure in patients with cancer. Although a single factor (i.e., renal impairment, CYP2D6 deficiency, and reduced OCT2 activity) shows a moderate impact, they collectively could result in a significant and potentially clinically relevant increase in veliparib exposure. Clin Cancer Res; 20(15); 3931–44. ©2014 AACR.

Simultaneous determination of ABT-888, a poly (ADP-ribose) polymerase inhibitor, and its metabolite in human plasma by liquid chromatography/tandem mass spectrometry

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 ml of plasma with 10 microl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C(18) column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1-2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration-time profile of ABT-888 after its oral administration in cancer patients.

A stable isotope-labeled internal standard is essential for correcting for the interindividual variability in the recovery of lapatinib from cancer patient plasma in quantitative LC-MS/MS analysis

The development and validation of a LC-MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC-MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29-70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16-56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5-5000ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100±10%), and precision (<11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC-MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation.

Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

A Phase I clinical trial of spicamycin derivative KRN5500 (NSC 650426) using a Phase I accelerated titration 2B design

The spicamycin derivative KRN5500 was considered as a potential anti-cancer agent based on in vitro and preclinical studies. A Phase I study involving 24 cancer patients in whom tumors were refractory to all other conventional therapies was conducted to determine the dose limiting toxicity, maximum tolerated dose, effectiveness, and pharmacokinetic parameters of this drug administered by 1-h IV infusion daily for five consecutive days every 3 weeks. Using an accelerated dose titration strategy, 8.4 mg/m2/d × 5 days was the maximum administered dose. Severe gastrointestinal and hepatic toxicities were observed at doses at or above 4.3 mg/m2/d × 5. The recommended Phase II dose is 4.3 mg/m2/d × 5. The distribution of KRN5500 followed a two-compartment model, and clearance did not decrease significantly over the dose range 0.8–8.4 mg/m2/d × 5. No significant correlation was observed between plasma levels and toxicity. No tumor responses were observed among the 14 patients evaluable for response.

Preclinical pharmacokinetic, antitumor and toxicity studies with CL-994 (N-acetyldinaline)

CI-994, a substituted benzamide derivative, is a compound that showed solid tumor selectivity for a variety of solid tumor models compared to L1210 leukemia. Due to its lack of aqueous solubility, it requires oral administration. Female B6D2F1 mice were treated with CI-994 once daily by oral administration of 50 mg/kg for 14 days. Following treatment mice were evaluated for pharmacodynamic effects as well as the pharmacokinetic behavior of CI-994 and the de-acetylated derivative dinaline. Mice samples (plasma, urine, feces) were analyzed using solid phase extraction, reverse phase HPLC and ultraviolet detection. The plasma distribution and elimination half-lives for CI-994 were 51 minutes and 9.4 hours, respectively, on D-1; 31 minutes and 3.4 hours, respectively on D-14. The apparent plasma distribution and elimination half-lives for dinaline were 27 minutes and 2.4 hours, respectively, on D-1; 40 minutes and 7.3 hours, respectively on D-14. The CI-994 AUC on D-1 and D-14 were 2879 and 2407 µ × minutes, respectively; while the dinaline AUC on D-1 and D-14 were 87 and 92 µg/ml × minutes, respectively. Urinary excretion for CI-994 and dinaline was higher on D-14, while the fecal excretion was the same on both days. The Colon #38 tumor growth in treated mice was reduced to 22% of that observed in the controls by D-19. The levels of all blood cells were reduced in the treated mice when compared to controls and the total WBC was the most affected (median 38%). Recovery to pretreatment levels occurred quickly following treatment cessation. Phase I evaluation of chronic oral administration of CI-994 is currently ongoing.

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra MS C(18) column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5-2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.

Identification and antitumor activity of a reduction product in the murine metabolism of pyrazoloacridine (NSC-366140)

Purpose. Pyrazoloacridine (PZA) is a newly developed anticancer agent currently undergoing clinical trials. Its mode of action has not been elucidated but the presence in its chemical structure of a 5-nitro functional group and its activity against oxygen-deficient cancerous cells argue in favor of enzymatic nitro reduction as a possible pathway for its antitumor activity. In order to assess the involvement of the nitro functiona lity in PZA activity, as well as to determine other metabolic products, a pharmacological and chemical study of PZA was designed. Methods. Urine and stool samples were collected from mice before and after treatment with PZA. Samples were fractionated using chromatographic methods and then evaluated using mass spectrometry (MS). One of the characterized metabolites was synthesized and tested in vitro and in vivo for anticancer activity. Results. One major fraction from mouse stool was initially characterized by MS as the 5-aminopyrazoloacridine (5-APZ). This compound was chemically synthesized by catalytic hydrogenation of PZA and stabilized as the hydrochloride salt. 5-APZ was marginally cytotoxic in vitro and was inactive in vivo against a tumor cured by PZA (Panc 03). Conclusions. Bioreduction of the nitro group to an amine compound from PZA represents a pathway in the metabolic sequence of PZA. The inactivity of the chemically generated amine product does not provide conclusive evidence that this pathway is not involved in the cytotoxicity of PZA because other intermediates in the nitro reduction pathway may have a role in the activity of PZA. In particular, the hydroxylamine derivative of PZA could give answers to the involvement of this pathway in PZA cytotoxicity.