Mouse pharmacokinetics and metabolism of the phenylurea thiocarbamate NSC 161128.

PURPOSE: NSC 161128, a phenylurea thiocarbamate, displays activity against the NCI60 anti-cancer cell line panel and xenograft models. The metabolite N-methyl-N'-phenylurea (M10) has been detected in animal plasma; however, detection and quantification of other putative NSC 161128 metabolites have not been undertaken. The purpose of this study was to characterize the pharmacokinetics and metabolism of NSC 161128 in mice and under in vitro conditions. METHODS: An LC-MS/MS assay was developed to evaluate stability and in vitro metabolism of NSC 161128 in liver microsomes and S9 fractions. Single-dose pharmacokinetic profiles for NSC 161128 and its metabolite M10 were obtained following intraperitoneal (I.P.) administration. RESULTS: A sensitive and specific positive ionization LC-MS/MS method for measuring NSC 161128 and its metabolites was developed. HPLC separation was achieved under gradient elution using an aqueous methanol mobile phase containing 0.05% formic acid and 0.05% ammonium hydroxide. The assay was linear over the range 1.0-1000 ng/mL. NSC 161128 was stable in aqueous solution and tissue culture media, but not in plasma, where rapid degradation of NSC 161128 to the metabolite M10 was observed. Following I.P. administration of a 200 mg/kg dose to male CD1 mice, the peak plasma concentration of NSC 161128 was 255 ng/mL after 5 min with a plasma half-life of 138 min. Potential bioactivation of NSC 161128 was explored using mouse S9. CONCLUSIONS: An analytical LC-MS/MS method was successfully developed for the detection and quantification of NSC 161128 and its metabolites. These results increase the understanding of NSC 161128 pharmacokinetic and metabolic properties.

Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs.

Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.

Population Pharmacokinetics of Z-Endoxifen in Patients With Advanced Solid Tumors.

The purpose of this study was to develop and validate a population pharmacokinetic model for Z-endoxifen in patients with advanced solid tumors and to identify clinical variables that influence pharmacokinetic parameters. Z-endoxifen-HCl was administered orally once a day on a 28-day cycle (±3 days) over 11 dose levels ranging from 20 to 360 mg. A total of 1256 Z-endoxifen plasma concentration samples from 80 patients were analyzed using nonlinear mixed-effects modeling to develop a population pharmacokinetic model for Z-endoxifen. A 2-compartment model with oral depot and linear elimination adequately described the data. The estimated apparent total clearance, apparent central volume of distribution, and apparent peripheral volume of distribution were 4.89 L/h, 323 L, and 39.7 L, respectively, with weight-effect exponents of 0.75, 1, and 1, respectively. This model was used to explore the effects of clinical and demographic variables on Z-endoxifen pharmacokinetics. Weight, race on clearance, and aspartate aminotransferase on the absorption rate constant were identified as significant covariates in the final model. This novel population pharmacokinetic model provides insight regarding factors that may affect the pharmacokinetics of Z-endoxifen and may assist in the design of future clinical trials.

Development and validation of an LC-MS/MS generic assay platform for small molecule drug bioanalysis.

AIM: We developed a generic high-performance liquid chromatography mass spectrometry approach for quantitation of small molecule compounds without availability of isotopically labelled standard. METHODS: The assay utilized 50 µL of plasma and offers 8 potential internal standards (IS): acetaminophen, veliparib, busulfan, neratinib, erlotinib, abiraterone, bicalutamide, and paclitaxel. Preparation consisted of acetonitrile protein precipitation and aqueous dilution in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 µm, 2 mm x 50 mm) column and a gradient of 0.1 % formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. Performance of the generic approach was evaluated with seven drugs (LMP744, olaparib, cabozantinib, triapine, ixabepilone, berzosertib, eribulin) for which validated assays were available. RESULTS: The 8 IS covered a range of polarity, size, and ionization; eluted over the range of chromatographic retention times; were quantitatively extracted; and suffered limited matrix effects. The generic approach proved to be linear for test drugs evaluated over at least 3 orders of magnitude starting at 1-10 ng/mL, with extension of assay ranges with analyte isotopologue MRM channels. At a bias of less than 16 % and precision within 15 %, the assay performance was acceptable. CONCLUSION: The generic approach has become a useful tool to further define the pharmacology of drugs studied in our laboratory and may be utilized as described, or as starting point to develop drug-specific assays with more extensive performance characterization.

Phase 1 study of Z-endoxifen in patients with advanced gynecologic, desmoid, and hormone receptor-positive solid tumors.

BACKGROUND: Differential responses to tamoxifen may be due to inter-patient variability in tamoxifen metabolism into pharmacologically active Z-endoxifen. Z-endoxifen administration was anticipated to bypass these variations, increasing active drug levels, and potentially benefitting patients responding sub-optimally to tamoxifen. MATERIALS AND METHODS: Patients with treatment-refractory gynecologic malignancies, desmoid tumors, or hormone receptor-positive solid tumors took oral Z-endoxifen daily with a 3+3 phase 1 dose escalation format over 8 dose levels (DLs). Safety, pharmacokinetics/pharmacodynamics, and clinical outcomes were evaluated. RESULTS: Thirty-four of 40 patients were evaluable. No maximum tolerated dose was established. DL8, 360 mg/day, was used for the expansion phase and is higher than doses administered in any previous study; it also yielded higher plasma Z-endoxifen concentrations. Three patients had partial responses and 8 had prolonged stable disease (≥ 6 cycles); 44.4% (8/18) of patients at dose levels 6-8 achieved one of these outcomes. Six patients who progressed after tamoxifen therapy experienced partial response or stable disease for ≥ 6 cycles with Z-endoxifen; one with desmoid tumor remains on study after 62 cycles (nearly 5 years). CONCLUSIONS: Evidence of antitumor activity and prolonged stable disease are achieved with Z-endoxifen despite prior tamoxifen therapy, supporting further study of Z-endoxifen, particularly in patients with desmoid tumors.

Intravenous 5-fluoro-2'-deoxycytidine administered with tetrahydrouridine increases the proportion of p16-expressing circulating tumor cells in patients with advanced solid tumors.

PURPOSE: Following promising responses to the DNA methyltransferase (DNMT) inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) combined with tetrahydrouridine (THU) in phase 1 testing, we initiated a non-randomized phase 2 study to assess response to this combination in patients with advanced solid tumor types for which tumor suppressor gene methylation is potentially prognostic. To obtain pharmacodynamic evidence for DNMT inhibition by FdCyd, we developed a novel method for detecting expression of tumor suppressor protein p16/INK4A in circulating tumor cells (CTCs). METHODS: Patients in histology-specific strata (breast, head and neck [H&N], or non-small cell lung cancers [NSCLC] or urothelial transitional cell carcinoma) were administered FdCyd (100 mg/m2) and THU (350 mg/m2) intravenously 5 days/week for 2 weeks, in 28-day cycles, and progression-free survival (PFS) rate and objective response rate (ORR) were evaluated. Blood specimens were collected for CTC analysis. RESULTS: Ninety-three eligible patients were enrolled (29 breast, 21 H&N, 25 NSCLC, and 18 urothelial). There were three partial responses. All strata were terminated early due to insufficient responses (H&N, NSCLC) or slow accrual (breast, urothelial). However, the preliminary 4-month PFS rate (42%) in the urothelial stratum exceeded the predefined goal-though the ORR (5.6%) did not. An increase in the proportion of p16-expressing cytokeratin-positive CTCs was detected in 69% of patients evaluable for clinical and CTC response, but was not significantly associated with clinical response. CONCLUSION: Further study of FdCyd + THU is potentially warranted in urothelial carcinoma but not NSCLC or breast or H&N cancer. Increase in the proportion of p16-expressing cytokeratin-positive CTCs is a pharmacodynamic marker of FdCyd target engagement.

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma.

PURPOSE: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. EXPERIMENTAL DESIGN: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. RESULTS: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. CONCLUSIONS: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors.

The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify "biologically effective doses" (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pY1234/1235MET/total MET ratio) in a phase 0-like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a MET-amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pY1234/1235MET/total MET of 95%-99% were achievable with tolerable doses of EMD1214063/MSC2156119J (tepotinib), XL184 (cabozantinib), and XL880/GSK1363089 (foretinib), but not ARQ197 (tivantinib), which did not alter the pharmacodynamic biomarker. Duration of kinase suppression and rate of kinase recovery were specific to each agent, emphasizing the importance of developing customized dosage regimens to achieve continuous suppression of the pharmacodynamic biomarker at the required level (here, ≥90% MET kinase suppression). The customized dosage regimen of each inhibitor yielded substantial and sustained tumor regression; the equivalent effectiveness of customized dosage regimens that achieve the same level of continuous molecular target control represents preclinical proof-of-concept and illustrates the importance of proper scheduling of targeted agent BEDs. Pharmacodynamics-guided biologically effective dosage regimens (PD-BEDR) potentially offer a superior alternative to pharmacokinetic guidance (e.g., drug concentrations in surrogate tissues) for developing and making head-to-head comparisons of targeted agents. Mol Cancer Ther; 17(3); 698-709. ©2018 AACR.

Comparative Metabolism of Batracylin (NSC 320846) and N-acetylbatracylin (NSC 611001) Using Human, Dog, and Rat Preparations In Vitro.

BACKGROUND: Batracylin is a heterocyclic arylamine topoisomerase inhibitor with preclinical anticancer activity. Marked species differences in sensitivity to the toxicity of batracylin were observed and attributed to differential formation of N-acetylbatracylin by N-acetyltransferase. A Phase I trial of batracylin in cancer patients with slow acetylator genotypes identified a dose-limiting toxicity of hemorrhagic cystitis. To further explore the metabolism of batracylin and N-acetylbatracylin across species, detailed studies using human, rat, and dog liver microsomal and hepatocyte preparations were conducted. METHODS: Batracylin or N-acetylbatracylin was incubated with microsomes and hepatocytes from human, rat, and dog liver and with CYP-expressing human and rat microsomes. Substrates and metabolites were analyzed by HPLC with diode array, fluorescence, radiochemical, or mass spectrometric detection. Covalent binding of radiolabeled batracylin and N-acetylbatracylin to protein and DNA was measured in 3-methylcholanthrene-induced rat, human, and dog liver microsomes, and with recombinant human cytochromes P450. RESULTS: In microsomal preparations, loss of batracylin was accompanied by formation of one hydroxylated metabolite in human liver microsomes and five hydroxylated metabolites in rat liver microsomes. Six mono- or di-hydroxy-N-acetylbatracylin metabolites were found in incubations of this compound with 3MC rat liver microsomes. Hydroxylation sites were identified for some of the metabolites using deuterated substrates. Incubation with recombinant cytochromes P450 identified rCYP1A1, rCYP1A2, hCYP1A1 and hCYP1B1 as the major CYP isoforms that metabolize batracylin and N-acetylbatracylin. Glucuronide conjugates of batracylin were also identified in hepatocyte incubations. NADPH-dependent covalent binding to protein and DNA was detected in all batracylin and most N-acetylbatracylin preparations evaluated. CONCLUSIONS: Microsomal metabolism of batracylin and N-acetylbatracylin results in multiple hydroxylated products (including possible hydroxylamines) and glutathione conjugates. Incubation of batracylin with hepatocytes resulted in production primarily of glucuronides and other conjugates. There was no clear distinction in the metabolism of batracylin and N-acetylbatracylin across species that would explain the differential toxicity.

Clinical and pharmacologic evaluation of two dosing schedules of indotecan (LMP400), a novel indenoisoquinoline, in patients with advanced solid tumors.

PURPOSE: Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. METHODS: The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. RESULTS: An MTD of 60 mg/m(2)/day was established for the daily regimen, compared to 90 mg/m(2) for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4-6 h) was observed following treatment. CONCLUSIONS: We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.

Preclinical Pharmacokinetics Study of R- and S-Enantiomers of the Histone Deacetylase Inhibitor, AR-42 (NSC 731438), in Rodents.

AR-42, a new orally bioavailable, potent, hydroxamate-tethered phenylbutyrate class I/IIB histone deacetylase inhibitor currently is under evaluation in phase 1 and 2 clinical trials and has demonstrated activity in both hematologic and solid tumor malignancies. This report focuses on the preclinical characterization of the pharmacokinetics of AR-42 in mice and rats. A high-performance liquid chromatography-tandem mass spectrometry assay has been developed and applied to the pharmacokinetic study of the more active stereoisomer, S-AR-42, when administered via intravenous and oral routes in rodents, including plasma, bone marrow, and spleen pharmacokinetics (PK) in CD2F1 mice and plasma PK in F344 rats. Oral bioavailability was estimated to be 26 and 100% in mice and rats, respectively. R-AR-42 was also evaluated intravenously in rats and was shown to display different pharmacokinetics with a much shorter terminal half-life compared to that of S-AR-42. Renal clearance was a minor elimination pathway for parental S-AR-42. Oral administration of S-AR-42 to tumor-bearing mice demonstrated high uptake and exposure of the parent drug in the lymphoid tissues, spleen, and bone marrow. This is the first report of the pharmacokinetics of this novel agent, which is now in early phase clinical trials.

Oral and intravenous pharmacokinetics of 5-fluoro-2'-deoxycytidine and THU in cynomolgus monkeys and humans.

INTRODUCTION: 5-Fluoro-2'-deoxycytidine (FdCyd; NSC48006), a fluoropyrimidine nucleoside inhibitor of DNA methylation, is degraded by cytidine deaminase (CD). Pharmacokinetic evaluation was carried out in cynomolgus monkeys in support of an ongoing phase I study of the PO combination of FdCyd and the CD inhibitor tetrahydrouridine (THU; NSC112907). METHODS: Animals were dosed intravenously (IV) or per os (PO). Plasma samples were analyzed by LC-MS/MS for FdCyd, metabolites, and THU. Clinical chemistry and hematology were performed at various times after dosing. A pilot pharmacokinetic study was performed in humans to assess FdCyd bioavailability. RESULTS: After IV FdCyd and THU administration, FdCyd C(max) and AUC increased with dose. FdCyd half-life ranged between 22 and 56 min, and clearance was approximately 15 mL/min/kg. FdCyd PO bioavailability after THU ranged between 9 and 25 % and increased with increasing THU dose. PO bioavailability of THU was less than 5 %, but did result in plasma concentrations associated with inhibition of its target CD. Human pilot studies showed comparable bioavailability for FdCyd (10 %) and THU (4.1 %). CONCLUSION: Administration of THU with FdCyd increased the exposure to FdCyd and improved PO FdCyd bioavailability from <1 to 24 %. Concentrations of THU and FdCyd achieved after PO administration are associated with CD inhibition and hypomethylation, respectively. The schedule currently studied in phase I studies of PO FdCyd and THU is daily times three at the beginning of the first and second weeks of a 28-day cycle.

Plasma pharmacokinetics of the indenoisoquinoline topoisomerase I inhibitor, NSC 743400, in rats and dogs.

PURPOSE: NSC 743400 is a novel synthetic indenoisoquinoline analog under development as an anticancer agent. It is a potent topoisomerase I inhibitor with potential therapeutic advantages over FDA-approved camptothecin derivatives. In preparation for clinical development of NSC 743400, we determined the pharmacokinetics after administration to rats and dogs. METHODS: NSC 743400 was administered intravenously at a dose of 12 or 24 mg/m(2) to rats (single bolus) or 10, 50, 100, 215, 430, or 646 mg/m(2) (intravenous infusion) or 860 or 1720 mg/m(2) (orally) to dogs. RESULTS: Intravenously administered NSC 743400 was eliminated from both species with an estimated t 1/2 of 2-5 h in rat and 6-14 h in dog. Elimination t 1/2 increased with dose in dog. Area under the plasma concentration-versus-time curve (AUC) was comparable in both species, at about 300-400 h ng/mL for the approximately 10 mg/m(2) dose groups. Overall, AUC values increased proportionally with dose for both species but had evidence of more than proportional exposure at the highest doses. Oral dosing resulted in variable drug absorption. CONCLUSIONS: The pharmacokinetic data were used to plan first-in-human clinical trials.

Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice.

Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and ß-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts.

LC-MS/MS quantification of a neuropeptide fragment kisspeptin-10 (NSC 741805) and characterization of its decomposition product and pharmacokinetics in rats.

The kisspeptins are critical regulators of mammalian reproduction. Kisspeptin-10 ((45)YNWNSFGLRF-NH2(54), kisspeptin-112-121 or metastin 45-54, NSC 741805), an active fragment of kisspeptin, has been shown to be a potent stimulator of gonadotropin-releasing hormone and secretion of luteinizing hormone in both rodents and primates. This shorter peptide fragment may have clinical utility potential and it is important to characterize its pharmacokinetic property. Recently, the pharmacokinetics of both kisspeptin-54 and kisspeptin-10 were characterized in humans using a radioimmunoassay (RIA), which measures only the immunoreactive kisspeptin (kisspeptin-IR). In this study, a highly sensitive and specific LC-MS/MS assay was developed to quantify kisspeptin-10 levels in rat plasma. The lower limit of quantitation (LLOQ) was 0.5 ng/mL, the within-day and between-day coefficient of variations (CVs) ranged from 5.2 to 15.4% and 1.3 to 14.2%, and the accuracy values ranged from 98 to 114% and 99 to 105%, respectively. With this method, stability studies demonstrated that kisspeptin-10 degraded rapidly with decomposition half-lives of 6.8 min, 2.9 min and 1.7 min at 4 °C, 25 °C, and 37 °C, respectively. The principal decomposition product was characterized as the N-terminal tyrosine deleted kisspeptin-10 (46)NWDSFGLRF-NH2(54). Pharmacokinetic study in rats showed that low ng/mL kisspeptin-10 was detected in the first few minutes, and eliminated rapidly and became undetectable 30 min after intravenous (i.v.) bolus administration of 1.0 mg/kg kisspeptin-10.

Formation of active products of benzaldehyde dimethane sulfonate (NSC 281612, DMS612) in human blood and plasma and their activity against renal cell carcinoma lines.

Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is an alkylating agent with activity against renal cell carcinoma and is being evaluated clinically. To support clinical trials, we developed an LC-MS/MS assay to detect and quantitate BEN and its metabolites/decomposition products. We tested the stability and products of BEN and benzoic acid dimethane sulfonate (BA) in plasma, blood and five renal carcinoma cell lines in vitro. Further, we determined the IC(50) of BEN, BA and four of their products in these cell lines. Low temperature and pH stabilized the analytes, and utilizing this resulted in an accurate, precise and reproducible assay. The half-lives of BEN and BA added to plasma in vitro were 220 and 5 min, while the half-life of BEN in whole blood was 18 min. The generation and degradation of up to 12 analytes were monitored, and structures confirmed with available authentic standards. The IC(50) for BEN was 5- to 500-fold lower than that of any of its products, while the cellular metabolic activity toward BEN correlated with ALDH activity and IC(50) values. We detected six of the in vitro products and their respective glucuronides in murine plasma after dosing BEN. The information gained from these experiments will be instrumental in the evaluation of the pharmacology of BEN in ongoing human trials.

Characterization of silvestrol pharmacokinetics in mice using liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-ß-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.

Plasma pharmacokinetics and oral bioavailability of the 3,4,5,6-tetrahydrouridine (THU) prodrug, triacetyl-THU (taTHU), in mice.

PURPOSE: Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU). METHODS: Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally. RESULTS: taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU. CONCLUSIONS: The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.

Liquid chromatography-tandem mass spectrometric assay for the quantitation in human plasma of the novel indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776.

Topoisomerase I (Topo I) is a recognized target for ovarian, lung, and colorectal cancer therapy. The FDA-approved camptothecin (CPT) Topo I inhibitors, topotecan and irinotecan are labile and their effects are rapidly reversible. The indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776, have been developed as a new generation of Topo I inhibitors and are being advanced to clinical evaluation. To support the clinical development of NSC 743400 and NSC 725776, we developed and validated, according to FDA guidelines, LC-MS/MS assays for the sensitive, accurate and precise quantitation of NSC 743400 and NSC 725776 in 0.2 mL human plasma. After ethyl acetate extraction, separation was achieved with a Synergi Polar RP column and a gradient of 0.1% formic acid in acetonitrile:water. NSC 743400 and NSC 725776 eluted at approximately 3 min, and the total run time was 14 min. Detection consisted of electrospray, positive-mode ionization mass spectrometry. Between 3 and 1000 ng/mL, accuracy was 96.9-108.2% for NSC 743400 and 95.1-106.7% for NSC 725776, and precision was <11.4% for NSC 743400 and <5.9% for NSC 725776. Extraction recovery was >80% for both analytes, and ion suppression ranged from -46.7 to 5.7%. The use of isotopically labeled internal standards and a wash phase at the end of the run were necessary to achieve adequate assay performance. Protein binding in human plasma as assessed by equilibrium dialysis showed both indenoisoquinolines to be more than 98% protein bound.

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry.

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.