Publications by authors named "Joseph M Covey"

41 Publications

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

Oncotarget 2021 Feb 16;12(4):268-277. Epub 2021 Feb 16.

Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.18632/oncotarget.27887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899551PMC
February 2021

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

Cancer Chemother Pharmacol 2020 05 20;85(5):979-993. Epub 2020 Apr 20.

Division of Cancer Treatment and Diagnosis, National Cancer Institute, 31 Center Drive, Bldg. 31 Room 3A-44, Bethesda, MD, 20892, USA.

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/m) and THU (350 mg/m) 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.
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http://dx.doi.org/10.1007/s00280-020-04073-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188725PMC
May 2020

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

Clin Cancer Res 2018 12 30;24(23):5830-5840. Epub 2018 Jul 30.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda.

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/m for LMP 776 and 100 mg/m for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312717PMC
December 2018

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

Mol Cancer Ther 2018 03 14;17(3):698-709. Epub 2018 Feb 14.

Clinical Pharmacodynamics Biomarker Program, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.

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 pYMET/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 -amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pYMET/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. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935559PMC
March 2018

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

J Drug Metab Toxicol 2016 Jun 8;7(2). Epub 2016 May 8.

Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.

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.
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http://dx.doi.org/10.4172/2157-7609.1000203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948755PMC
June 2016

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

Cancer Chemother Pharmacol 2016 Jul 11;78(1):73-81. Epub 2016 May 11.

Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, 31 Center Drive, Room 3A44, Bethesda, MD, 20814, USA.

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.
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http://dx.doi.org/10.1007/s00280-016-2998-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199138PMC
July 2016

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

AAPS J 2016 05 4;18(3):737-45. Epub 2016 Mar 4.

College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio, 43210, USA.

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.
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http://dx.doi.org/10.1208/s12248-016-9876-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256597PMC
May 2016

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

Cancer Chemother Pharmacol 2015 Oct 1;76(4):803-11. Epub 2015 Sep 1.

Cancer Therapeutics Program, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA, 15213, USA.

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.
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http://dx.doi.org/10.1007/s00280-015-2857-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573928PMC
October 2015

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

Cancer Chemother Pharmacol 2015 May 17;75(5):1015-23. Epub 2015 Mar 17.

Life Sciences Group, IIT Research Institute, 10 West 35th Street, Chicago, IL, 60616, USA,

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.
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http://dx.doi.org/10.1007/s00280-015-2722-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420695PMC
May 2015

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

Int J Toxicol 2014 Mar-Apr;33(2):75-85. Epub 2014 Mar 17.

Division of Pre-Clinical Innovations, National Center for Advancing Translational Sciences, Bethesda, MD, USA. Email:

Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-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.
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http://dx.doi.org/10.1177/1091581814524994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001115PMC
November 2014

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

J Chromatogr B Analyt Technol Biomed Life Sci 2013 May 4;926:1-8. Epub 2013 Mar 4.

College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

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.
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http://dx.doi.org/10.1016/j.jchromb.2013.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3955120PMC
May 2013

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

Cancer Chemother Pharmacol 2013 Jan 5;71(1):73-83. Epub 2012 Oct 5.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15213, USA.

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.
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http://dx.doi.org/10.1007/s00280-012-1980-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536923PMC
January 2013

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

AAPS J 2011 Sep 16;13(3):347-56. Epub 2011 Apr 16.

College of Pharmacy, The Ohio State University, Columbus, 43210, USA.

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-β-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.
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http://dx.doi.org/10.1208/s12248-011-9273-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160157PMC
September 2011

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

Cancer Chemother Pharmacol 2011 Feb 5;67(2):421-30. Epub 2010 May 5.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Hillman Research Pavilion, Room G27D, 5117 Centre Avenue, Pittsburgh, PA 5213-1863, USA.

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.
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http://dx.doi.org/10.1007/s00280-010-1337-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954253PMC
February 2011

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

J Pharm Biomed Anal 2010 Sep 23;52(5):714-20. Epub 2010 Feb 23.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.

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.
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http://dx.doi.org/10.1016/j.jpba.2010.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865235PMC
September 2010

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

Rapid Commun Mass Spectrom 2008 Nov;22(21):3371-81

College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

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.
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http://dx.doi.org/10.1002/rcm.3744DOI Listing
November 2008

Modulation of gemcitabine (2',2'-difluoro-2'-deoxycytidine) pharmacokinetics, metabolism, and bioavailability in mice by 3,4,5,6-tetrahydrouridine.

Clin Cancer Res 2008 Jun;14(11):3529-35

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Room G.27d, Hillman Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA 15213-1863, USA.

Purpose: In vivo, 2',2'-difluoro-2'-deoxycytidine (dFdC) is rapidly inactivated by gut and liver cytidine deaminase (CD) to 2',2'-difluoro-2'-deoxyuridine (dFdU). Consequently, dFdC has poor oral bioavailability and is administered i.v., with associated costs and limitations in administration schedules. 3,4,5,6-Tetrahydrouridine (THU) is a potent CD inhibitor with a 20% oral bioavailability. We investigated the ability of THU to decrease elimination and first-pass effect by CD, thereby enabling oral dosing of dFdC.

Experimental Design: A liquid chromatography-tandem mass spectrometry assay was developed for plasma dFdC and dFdU. Mice were dosed with 100 mg/kg dFdC i.v. or orally with or without 100 mg/kg THU i.v. or orally. At specified times between 5 and 1,440 min, mice (n = 3) were euthanized. dFdC, dFdU, and THU concentrations were quantitated in plasma and urine.

Results: THU i.v. and orally produced concentrations >4 microg/mL for 3 and 2 h, respectively, whereas concentrations of >1 microg/mL have been associated with near-complete inhibition of CD in vitro. THU i.v. decreased plasma dFdU concentrations but had no effect on dFdC plasma area under the plasma concentration versus time curve after i.v. dFdC dosing. Both THU i.v. and orally substantially increased oral bioavailability of dFdC. Absorption of dFdC orally was 59%, but only 10% passed liver and gut CD and eventually reached the systemic circulation. Coadministration of THU orally increased dFdC oral bioavailability from 10% to 40%.

Conclusions: Coadministration of THU enables oral dosing of dFdC and warrants clinical testing. Oral dFdC treatment would be easier and cheaper, potentially prolong dFdC exposure, and enable exploration of administration schedules considered impractical by the i.v. route.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-4885DOI Listing
June 2008

Liquid chromatography-mass spectrometric assay for quantitation of the short-chain fatty acid, 2,2-dimethylbutyrate (NSC 741804), in rat plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Feb 8;862(1-2):168-74. Epub 2007 Dec 8.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.
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http://dx.doi.org/10.1016/j.jchromb.2007.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2254528PMC
February 2008

Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice.

Cancer Chemother Pharmacol 2008 Aug 15;62(3):457-64. Epub 2007 Nov 15.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU) is a potent CD inhibitor that has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU pharmacokinetics has not been fully characterized, which has impaired the optimal preclinical evaluation and clinical use of THU. Therefore, we characterized the THU pharmacokinetics and bioavailability in mice. Mice were dosed with THU iv (100 mg/kg) or po (30, 100, or 300 mg/kg). Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma pharmacokinetic parameters were calculated compartmentally and non-compartmentally. THU, at 100 mg/kg iv had a 73 min terminal half-life and produced plasma THU concentrations >1 microg/ml, the concentration shown to effectively block deamination, for 4 h. Clearance was 9.1 ml/min/kg, and the distribution volume was 0.95 l/kg. Renal excretion accounted for 36-55% of the THU dose. A three-compartment model fit the iv THU data best. THU, at 100 mg/kg po, produced a concentration versus time profile with a plateau of approximately 10 mug/ml from 0.5-3 h, followed by a decline with an 85 min half-life. The oral bioavailability of THU was approximately 20%. The 20% oral bioavailability of THU is sufficient to produce and sustain, for several hours, plasma concentrations that inhibit CD. This suggests the feasibility of using THU to decrease elimination and first-pass metabolism of cytidine analogues by CD. THU pharmacokinetics are now being evaluated in humans.
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http://dx.doi.org/10.1007/s00280-007-0625-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677692PMC
August 2008

Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

Rapid Commun Mass Spectrom 2007 ;21(13):1991-7

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

A number of anticancer drugs are cytidine analogues that undergo metabolic deactivation catalyzed by cytidine deaminase (CD). 3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD, by acting as a transition-state analogue of its natural substrate cytidine. However, to date its pharmacokinetic properties have not been fully characterized, which has impaired its optimal preclinical evaluation and clinical use. We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of THU in mouse plasma. Validation was performed according to FDA guidelines. The assay employed deuterated THU as the internal standard and an acetonitrile protein precipitation step. Separation, based on hydrophilic interaction chromatography, was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water followed by a wash. Chromatographic separation was followed by positive-mode electrospray ionization MS/MS detection in the multiple reaction monitoring (MRM) mode. The assay was accurate (92.5-109.9%) and precise (2.1-9.0%) in the concentration range of 0.2-50 microg/mL. Recovery from plasma was near-complete (92.9-119.3%) and ion suppression was negligible (-17.5 to -0.2%). Plasma freeze/thaw stability (93.1-102.1%), stability for 3 months at -80 degrees C (99.5-110.9%), and stability for 4 h at room temperature (92.1-102.4%) were all in order. This assay is currently being used to quantitate THU in ongoing pharmacokinetic studies. In addition, the assay is expected to be a useful tool in any future studies involving co-administration of THU with cytidine analogues.
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http://dx.doi.org/10.1002/rcm.3054DOI Listing
August 2007

Pharmacokinetics, metabolism, and oral bioavailability of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine in mice.

Clin Cancer Res 2006 Dec 30;12(24):7483-91. Epub 2006 Nov 30.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, PA 15213-1863, USA.

Purpose: In vivo, 5-fluoro-2'-deoxycytidine (FdCyd) is rapidly and sequentially converted to 5-fluoro-2'-deoxyuridine, 5-fluorouracil, and 5-fluorouridine. The i.v. combination of FdCyd and 3,4,5,6-tetrahydrouridine (THU), a cytidine deaminase (CD) inhibitor that blocks the first metabolic step in FdCyd catabolism, is being investigated clinically for its ability to inhibit DNA methyltransferase. However, the full effects of THU on FdCyd metabolism and pharmacokinetics are unknown. We aimed to characterize the pharmacokinetics, metabolism, and bioavailability of FdCyd with and without THU in mice.

Experimental Design: We developed a sensitive high-performance liquid chromatography tandem mass spectrometry assay to quantitate FdCyd and metabolites in mouse plasma. Mice were dosed i.v. or p.o. with 25 mg/kg FdCyd with or without coadministration of 100 mg/kg THU p.o. or i.v.

Results: The oral bioavailability of FdCyd alone was approximately 4%. Coadministration with THU increased exposure to FdCyd and decreased exposure to its metabolites; i.v. and p.o. coadministration of THU increased exposure to p.o. FdCyd by 87- and 58-fold, respectively. FdCyd exposure after p.o. FdCyd with p.o. THU was as much as 54% that of i.v. FdCyd with i.v. THU.

Conclusions: FdCyd is well absorbed but undergoes substantial first-pass catabolism by CD to potentially toxic metabolites that do not inhibit DNA methyltransferase. THU is sufficiently bioavailable to reduce the first-pass effect of CD on FdCyd. Oral coadministration of THU and FdCyd is a promising approach that warrants clinical testing because it may allow maintaining effective FdCyd concentrations on a chronic basis, which would be an advantage over other DNA methyltransferase inhibitors that are currently approved or in development.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-1250DOI Listing
December 2006

A mass balance and disposition study of the DNA methyltransferase inhibitor zebularine (NSC 309132) and three of its metabolites in mice.

Clin Cancer Res 2006 Oct;12(19):5826-33

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.

Purpose: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer.

Experimental Design: Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine.

Results: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine.

Conclusions: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-1234DOI Listing
October 2006

LC-MS/MS assay and dog pharmacokinetics of the dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501).

J Chromatogr B Analyt Technol Biomed Life Sci 2006 Aug 24;840(1):56-62. Epub 2006 May 24.

Department of Oncology, Division of Developmental Oncology Research, Mayo Clinic and Foundation, 200 First Street S.W., Rochester, MN 55905, United States.

The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G-G interstrand cross-links via reactive N(10)-C(11)/N(10')-C(ll') imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557-m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8-1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22-720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 microg/kg doses for five consecutive days.
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http://dx.doi.org/10.1016/j.jchromb.2006.04.031DOI Listing
August 2006

Determination of desoxyepothilone B in nude mice plasma by liquid-liquid extraction and reversed-phase high-performance liquid chromatography.

J Pharm Biomed Anal 2006 Sep 17;42(2):272-6. Epub 2006 Apr 17.

Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, and Comprehensive Cancer Center, University of Alabama at Birmingham, 35294-0019, USA.

A novel reversed-phase high-performance liquid chromatographic (HPLC) method has been established for the determination of a newly developed anti-cancer agent desoxyepothilone B (dEpo B) in nude mice plasma. The sample preparation involved deproteination of 200 microl of plasma sample first, followed by liquid-liquid extraction of the resultant supernatant with chloroform. The compound taxol was used as the internal standard. Chromatographic separations were carried out on a 250 mm x 4.6 mm Zorbax SB-phenyl column with acetonitrile-0.25% orthophosphoric acid (50/50, v/v) as mobile phase and UV detection at 250 nm. For dEpo B and taxol at the concentration level of 10 microg/ml in nude mice plasma, the absolute extraction recoveries were 85.3 and 87.2%, respectively. The linear quantification range of the method was 0.1-100 microg/ml in nude mice plasma with linear correlation coefficients greater than 0.999. The within-day and between-day relative standard deviations (R.S.D.s) for dEpo B at 0.5, 2.5 and 10 microg/ml levels in nude mice plasma fell in the range of 2.8-4.8 and 1.5-4.6%, and the within-day and between-day recoveries were in the range of 96.5-101.7 and 97.7-101.2%, respectively.
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http://dx.doi.org/10.1016/j.jpba.2006.02.047DOI Listing
September 2006

High performance liquid chromatographic analysis and preclinical pharmacokinetics of the heteroarotinoid antitumor agent, SHetA2.

Cancer Chemother Pharmacol 2006 Nov 14;58(5):561-9. Epub 2006 Mar 14.

College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

Background: SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methane-thione], NSC 726189} is a sulfur-containing heteroarotinoid, which selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective was to develop and validate a HPLC/UV method for the determination of SHetA2, and study the pharmacokinetics of SHetA2 in the mouse.

Methods: SHetA2 and the internal standard, methylated XK469 (MeXK469) were isolated from 0.2 ml of mouse plasma by solid phase extraction. The analytes were separated on a narrow-bore C18 column, with the mobile phase consisting of 60% acetonitrile in water at a flow rate of 0.2 ml/min. UV detection was set at 341 nm. Pharmacokinetic studies of SHetA2 were carried out in mice following i.v. bolus dose at 20 mg/kg and oral administrations at 20 and 60 mg/kg.

Results: The standard curves were linear between 25 and 2,500 nM and the lower limit of quantification (LLOQ) was 25 nM. The within-run coefficients of variation (CVs) were 11.1% at 10, 9.4% at 100, and 5.2% at 2,500 nM and the respective between-run CVs were 10.9, 3.1, and 1.5% (all n=5). The recovery was 85.8% for SHetA2 and 80.6% for MeXK469. Following i.v. bolus dose, plasma concentrations of approximately 10 microM were achieved at 5 min in mice and declined biexponentially with detectable levels at 60 h. The data were fitted with a two-compartment model, which gave a mean initial t1/2 of 40 min and terminal t1/2 of 11.4 h (n=6). The total body clearance was approximately 1.81 l/h/kg. The volume of distribution at steady state (Vdss) was 20.8 l/kg. Plasma protein binding was found to be 99.3-99.5% at low micromolar concentrations. Plasma concentration data for the i.v. and p.o. doses were also fitted interactively to a two-compartment deconvolution model. From this result, oral bioavailability values of 15% at 20 mg/kg and 19% at 60 mg/kg were obtained.

Conclusions: A highly sensitive HPLC/UV method for the quantification of SHetA2 in plasma has been developed to support pharmacokinetics of SHetA2 in the mouse. Pharmacokinetic behaviors of this drug appear to be favorable for future development.
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http://dx.doi.org/10.1007/s00280-006-0211-zDOI Listing
November 2006

Quantitative determination of zebularine (NSC 309132), a DNA methyltransferase inhibitor, and three metabolites in murine plasma by high-performance liquid chromatography coupled with on-line radioactivity detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2006 Feb 20;831(1-2):147-55. Epub 2005 Dec 20.

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.
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http://dx.doi.org/10.1016/j.jchromb.2005.11.048DOI Listing
February 2006

Preclinical pharmacology of the novel antitumor agent adaphostin, a tyrphostin analog that inhibits bcr/abl.

Cancer Chemother Pharmacol 2006 May 6;57(5):607-14. Epub 2005 Dec 6.

Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Cancer Pharmacology Laboratory, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.

Purpose: To define several pharmacological properties for the potential anticancer agent, adaphostin, in order to determine whether the compound is appropriate for clinical evaluation as an anticancer agent.

Methods: The analytical procedure involved high-performance liquid chromatography and utilized an analytical J'Sphere ODS H-80 column.

Results: The stability of adaphostin at two different concentrations was determined at temperatures of 37 degrees C, 4 degrees C, and -80 degrees C, in the plasma of mice, rats, dogs, and humans. The compound was most stable at the lower temperatures. At all temperatures, adaphostin was generally most stable in human plasma and least stable in dog plasma. Adaphostin bound strongly (>93%) to proteins in plasma from all four species. Following intravenous (i.v.) administration to mice (50 mg/kg; 150 mg/m(2)), plasma concentrations declined rapidly from 50 microM at 2 min to 1 microM at 2 h. Elimination was triexponential, with t (1/2) values of 1.1, 9.1, and 41.2 min. The Cl(tb) was 0.411 L/(min.m(2)), the V (dss) was 24.6 L/m(2), and the AUC was 927 microM.min. In a comparison of vehicles for intraperitoneal (i.p.) dosing, PEG 300 allowed the highest plasma concentrations of adaphostin. Bioavailability following an i.p. dose was greater than that following a subcutaneous dose, or that for a dose administered by oral gavage. For rats dosed i.v. with adaphostin (50 mg/kg; 300 mg/m(2)), plasma concentrations also decreased triexponentially, with t (1/2) values of 1.8, 10.6, and 136 min. Other pharmacokinetic values were Cl(tb) = 0.466 L/(min.m(2)), AUC = 1,161 microM.min, and V (dss)=8.0 L/m(2). Analysis of samples collected from two dogs dosed i.v. with adaphostin (7.5 mg/kg; 150 mg/m(2)) showed that plasma concentrations decreased in a biphasic manner, with individual values for t (1/2alpha) of 6.0 and 9.8 min for the distribution phase and t (1/2beta) of 40.6 and 66.2 min for the elimination phase. Other pharmacokinetic values were Cl(tb) = 0.565 and 0.852 L/(min.m(2)), AUC = 673 and 446 microM min, and V (dss) = 29.6 and 56.8 L/m(2).

Conclusions: The stability of adaphostin in plasma varies with species. In mice and dogs dosed with adaphostin, plasma concentrations of the compound decreased rapidly. The clearance of adaphostin from plasma, on an m(2) basis, was equivalent for mice and rats but more rapid in dogs. These results are relevant for assessing the pharmacologic and toxicologic profiles and the antitumor activity of adaphostin in humans.
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http://dx.doi.org/10.1007/s00280-005-0094-4DOI Listing
May 2006

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance.

Cancer Chemother Pharmacol 2005 Dec 29;56(6):637-47. Epub 2005 Jun 29.

Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Executive Plaza North, Room 8040, Rockville, MD 20852, USA.

Purpose: 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs.

Methods: Doses of 0, 2.4, 12 and 24 mg/m2 per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m2 per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m2 per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5.

Results: Daily i.v. administration of 17-DMAG at doses of 24 mg/m2 per day in rats or 16 mg/m2 per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration.

Conclusions: With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m2 per day in rats and 8 mg/m2 per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m2 per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.
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http://dx.doi.org/10.1007/s00280-005-1000-9DOI Listing
December 2005

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

Clin Cancer Res 2005 May;11(10):3862-8

Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA.

Purpose: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation.

Experimental Design: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys.

Results: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)).

Conclusions: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.
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http://dx.doi.org/10.1158/1078-0432.CCR-04-2406DOI Listing
May 2005

Preclinical pharmacology of 2-methoxyantimycin A compounds as novel antitumor agents.

Cancer Chemother Pharmacol 2005 Sep 10;56(3):291-8. Epub 2005 May 10.

Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Cancer Pharmacology Laboratory, Comprehensive Cancer Center, University of Alabama, Birmingham, AL, 35294, USA.

Purpose: The present study was designed to determine pharmacological and biochemical properties of 2-methoxyantimycin A analogs (OMe-A1, OMe-A2, OMe-A3, and OMe-A5), which are novel antitumor compounds, and provide a basis for future pharmaceutical development, preclinical evaluation, and clinical trials.

Methods: A high-performance liquid chromatography (HPLC) method was established and employed to assess the biostability of these analogs and to determine their pharmacokinetic properties in mice and rats.

Results: In vitro biostability of the 2-methoxyantimycin analogs was esterase-dependent, compound-dependent, and species-dependent. In the absence of esterase inhibitors, all of the analogs were relatively unstable. Stability was greater, however, in human and dog plasma than in rat and mouse plasma. In the presence of esterase inhibitors, OMe-A1 was stable at 37 degrees C for 60 min in mouse and rat plasma, moderately stable in human plasma, and unstable in dog plasma. OMe-A2 was generally stable in all types of plasma. OMe-A3 was stable in dog and rat plasma, but not in human or mouse plasma. OMe-A5 was stable in human and dog plasma, but not in mouse or rat plasma. Each of these analogs was highly bound to plasma proteins. Of S9 fractions from four species, human S9 was least efficient in metabolizing OMe-A3. Following an intravenous dose of OMe-A1 in mice, plasma levels decreased rapidly, with an initial half-life of 2.7 min and a terminal half life of 34 min. Following an intraperitoneal dose in mice, plasma levels decreased less rapidly with a terminal half-life of 215 min. Following an intravenous dose of OMe-A1 or OMe-A3 in rats, plasma levels decreased more rapidly with initial half-lives of about 1.0 min. At an equivalent dose, OMe-A3 had a faster clearance than OMe-A1.

Conclusions: For 2-methoxyantimycin A analogs, species differences in biostability, metabolism, and pharmacokinetics may be pertinent in assessing their pharmacological and toxicological profiles and antitumor activity in humans.
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http://dx.doi.org/10.1007/s00280-004-0978-8DOI Listing
September 2005