Joseph M. Covey

Plasma Pharmacokinetics, Oral Bioavailability, and Interspecies Scaling of the DNA Methyltransferase Inhibitor, Zebularine

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 (t1/2) 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 μg/mL min, respectively, corresponding to a total body clearance (CLtb) of 13.65 mL/min/kg, apparent total body clearance (CLapp) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t1/2 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 μg/mL min, respectively, corresponding to a CLtb of 3.99 mL/min/kg for 50 mg/kg i.v. and CLapp 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 t1/2 was 70 and 150 minutes, CLtb was 3.55 and 10.85 mL/min/kg after i.v. administration, and CLapp 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: CLtb = 6.46(weight0.9). Conclusions: Zebularine has limited oral bioavailability. Interspecies scaling projects a CLtb of 296 mL/min in humans.

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

Purpose: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Experimental design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). Results: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. Clin Cancer Res; 19(4); 938–48. ©2012 AACR.

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

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 ∼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.

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells.

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.

Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.

Modulation of Gemcitabine (2′,2′-Difluoro-2′-Deoxycytidine) Pharmacokinetics, Metabolism, and Bioavailability in Mice by 3,4,5,6-Tetrahydrouridine

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 μg/mL for 3 and 2 h, respectively, whereas concentrations of >1 μg/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.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

Electrospray LC-MS/MS quantitation, stability, and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201 (NSC 710295) in the mouse

B201 (NSC 710295), [SUIM-(Darg-Arg-Pro-Hyp-Gly-Igl-Ser-Digl-Oic-Arg)(2)], a third generation of bradykinin (BK) antagonist, has been found to possess high potency. We report the development of a highly sensitive electrospray LC-MS/MS assay method for the analysis of B201 in plasma for the first time, using an ion-trap mass spectrometer. Human or mouse plasma (0.2 ml) was spiked with B201 and the internal standard, substance P. The compounds were extracted with a preconditioned C-18 reversed-phase column and analyzed by LC-MS/MS. The analytes were separated on a 50 x 2 mm (i.d.) BetaBasic C8 column, using a gradient elution. The positive ion selected reaction monitor mode was used monitoring the transitions of ions at m/z 938.9(3+)-->816.0(2+) for B201 and 674.3(2+)-->665.7(2+) for substance P. Assay validation was performed, and the limit of quantitation (LOQ) for B201 was found to be 1 ng/ml for human plasma and 2.5 ng/ml for mouse plasma. The recovery was 78% for B201 and 88% for substance P. The assay was linear from 2.5 to 1500 ng/ml for mouse plasma monitored. Using a 0.2 ml plasma, the within-day CVs were 9.3% at 2.5 ng/ml, 6.5% at 100 ng/ml, and 3.8% at 1000 ng/ml for human plasma (n=6). For mouse plasma, the respective within-day CVs were 17.6, 9.6, and 6.2% (n=6). The between-day CVs for human plasma were 8.2, 10.9, and 2.4%, respectively, (n=3) and the respective values for mouse plasma were 11.9, 8.6 and 6.5% (n=6). Pharmacokinetics of B201 in the mouse was studied following i.v. administration at 5 mg/kg and found to conform to a two-compartment model with an initial half-life of 14 min and a terminal half-life of 44 h. Plasma B201 peak level was detected at microg/ml range and the levels were detectable for a least 24 h. Preliminary oral bioavailability was found to be about 1%. This method demonstrates that an ion trap mass spectrometer can be a powerful tool to quantify large peptides at low nanogram per milliliter with a non-isotopically labeled internal standard.

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.

CHAPTER 17 – RELEVANCE OF PRECLINICAL PHARMACOLOGY AND TOXICOLOGY TO PHASE I TRIAL EXTRAPOLATION TECHNIQUES: RELEVANCE OF ANIMAL TOXICOLOGY

The chapter reviews the studies of a wide variety of anticancer drugs investigated by the U.S. National Cancer Institute (NCI) to prove the relevance of preclinical pharmacology and toxicology to Phase I trial extrapolation techniques. The development of drugs for the treatment of patients with cancer is perhaps more difficult than that of drugs for other non-life-threatening indications as cancer drugs tend to be the most toxic agents intentionally administered to man. This is further complicated by the fact that the typical patient in a Phase I trial is not a healthy human volunteer but a very ill, terminal cancer patient for whom all other therapeutic interventions have failed. Thus, the starting dose (SD) that is selected for the first human trial of cancer drugs must not only be safe but also offer some hope of benefit to the patient. However, it can also be extremely difficult to select a high enough dose so that the number of dose escalations is kept to a minimum to achieve either a maximum tolerated dose (MTD) or a biologically effective dose in humans. The results of the NCI studies that used agent directed techniques for the pharmacologic and toxicologic evaluation of antitumor agents in preclinical animal models are impressive in their prediction of human MTDs.