Quantitation of uridine and L-dihydroorotic acid in human plasma by LC-MS/MS using a surrogate matrix approach.

Uridine and L-dihydroorotate (DHO) are important intermediates of de novo as well as salvage pathways for the biosynthesis of pyrimidines, which are the building blocks of nucleic acids - DNA and RNA. These metabolites are known to be significant biomarkers of pyrimidine synthesis during the development of DHODH inhibitor drugs for treatment of several cancers and immunological disorders. Here we are reporting a validated LC-MS/MS assay for the quantitation of uridine and DHO in K2EDTA human plasma. Due to presence of endogenous uridine and DHO in the biological matrix, a surrogate matrix approach with bovine serum albumin (BSA) solution was used. Human plasma samples were spiked with stable isotope labeled internal standards, processed by protein precipitation, and analyzed using LC-MS/MS. Parallelism was successfully demonstrated between human plasma (the authentic matrix) and BSA (the surrogate matrix). The linear analytical ranges of the assay were set at 30.0-30,000 ng/mL for uridine and 3.00-3,000 ng/mL for DHO. This validated LC-MS/MS method demonstrated excellent accuracy and precision. The overall accuracy was between 91.9 % and 106 %, and the inter-assay precision (%CV) were less than 4.2 % for uridine in human plasma. The overall accuracy was between 92.8 % and 106 %, and the inter-assay precision (%CV) were less than 7.2 % for DHO in human plasma. Uridine and DHO were found to be stable in human plasma for at least 24 h at room temperature, 579 days when stored at -20 °C, 334 days when stored at -70 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human plasma samples to support clinical studies. Novel Aspect: A surrogate matrix approach to quantify endogenous uridine and DHO concentrations in human plasma.

Quantitation of 2-hydroxyglutarate in human plasma via LC-MS/MS using a surrogate analyte approach.

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC-MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC-MS/MS method was developed and validated for measuring 2-HG in human plasma.

A phase I study of an oral selective gamma secretase (GS) inhibitor RO4929097 in combination with neoadjuvant paclitaxel and carboplatin in triple negative breast cancer.

Upregulation of Notch pathway is associated with poor prognosis in breast cancer. We present the results of a phase I study of an oral selective gamma secretase (GS) inhibitor (critical to Notch signaling), RO4929097 in combination with neoadjuvant chemotherapy for operable triple negative breast cancer. The primary objective was to determine the maximum tolerated dose (MTD) of RO4929097. Secondary objectives were to determine real-time pharmacokinetics of RO4929097 and paclitaxel, safety and pathologic (pCR) complete response to study treatment. Eligible patients, initiated carboplatin at AUC 6 administered intravenously (IV) on day 1, weekly paclitaxel at 80 mg/m2 IV and RO4929097 10 mg daily given orally (PO) on days 1-3, 8-10 and 15-17 for six 21-day cycles. RO4929097 was escalated in 10 mg increments using the 3 + 3 dose escalation design. Two DLTs were observed in 14 patients - Grade (G) 4 thrombocytopenia in dose level 1 (10 mg) and G3 hypertension in dose level 2 (20 mg). Protocol-defined MTD was not determined due to discontinuation of RO4929097 development. However, 4 of 5 patients enrolled to 20 mg dose of RO4929097 required dose reduction to 10 mg due to toxicities (including neutropenia, thrombocytopenia and hypertension) occurring during and beyond the DLT observation period. Thus, 10 mg would have been the likely dose level for further development. G3 or higher hematologic toxicities included neutropenia (N = 8, 57%) and thrombocytopenia (N = 5, 36%) patients. Six (43%) patients had G2-3 neuropathy requiring paclitaxel dose reduction. No signs of drug-drug interaction between paclitaxel and RO4929097 were evident. Five patients (36%) had pCR.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

BACKGROUND: STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. METHODS AND FINDINGS: An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. CONCLUSIONS: LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50-10,000ng/mL and 25-5000ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.

Preferential Delivery of an Opioid Antagonist to the Fetal Brain in Pregnant Mice.

Prolonged fetal exposure to opioids results in neonatal abstinence syndrome (NAS), a major medical problem requiring intensive care and increased hospitalization times for newborns with NAS. Multiple strategies are currently available to alleviate withdrawal in infants with NAS. To prevent NAS caused by opioid maintenance programs in pregnant women, blocking fetal dependence without compromising the mother's opiate therapy is desirable. Here we tested in pregnant mice whether a peripherally selective opioid antagonist can preferentially enter the fetal brain and, thereby, in principle, selectively protect the fetus. We show using mass spectrometry that 6ß-naltrexol, a neutral opioid antagonist with very limited ability to cross the blood-brain barrier (BBB), readily crosses the placental barrier and enters the fetal brain at high levels, although it is relatively excluded from the maternal brain. Furthermore, owing to the late development of the BBB in postnatal mice, we show that 6ß-naltrexol can readily enter the juvenile mouse brain until at least postnatal day 14. Taking advantage of this observation, we show that long-term exposure to morphine starting in the second postnatal week causes robust and quantifiable dependence behaviors that are suppressed by concomitant administration of 6ß-naltrexol with much greater potency (ID50 0.022-0.044 mg/kg, or 1/500 the applied dose of morphine) than previously demonstrated for either the suppression of central nervous system opioid effects or the induction of withdrawal in adults. These results indicate that peripherally selective opioid antagonists capable of penetrating the placenta may be beneficial for preventing or reducing neonatal dependence and NAS in a dose range that should not interfere with maternal opioid maintenance.

Phase I dose escalation trial of the novel proteasome inhibitor carfilzomib in patients with relapsed chronic lymphocytic leukemia and small lymphocytic lymphoma.

The proteasome complex degrades proteins involved in a variety of cellular processes and is a powerful therapeutic target in several malignancies. Carfilzomib is a potent proteasome inhibitor which induces rapid chronic lymphocytic leukemia (CLL) cell apoptosis in vitro. We conducted a phase I dose-escalation trial to determine the safety and tolerability of carfilzomib in relapsed/refractory CLL or small lymphocytic lymphoma (SLL). Nineteen patients were treated with carfilzomib initially at 20 mg/m(2), then escalated in four cohorts (27, 36, 45 and 56 mg/m(2)) on days 1, 2, 8, 9, 15 and 16 of 28-day cycles. Therapy was generally well tolerated, and no dose limiting toxicities were observed. The most common hematologic toxicities were thrombocytopenia and neutropenia. All patients evaluable for response had stable disease, including patients with del17p13 and fludarabine-resistant disease. This trial shows acceptable tolerability and limited preliminary efficacy of carfilzomib in CLL and SLL.

OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells.

Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

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.

Cyclophosphamide, alvocidib (flavopiridol), and rituximab, a novel feasible chemoimmunotherapy regimen for patients with high-risk chronic lymphocytic leukemia.

Alvocidib has demonstrated efficacy in high-risk chronic lymphocytic leukemia (CLL) patients. In this phase I study, we combined cyclophosphamide, alvocidib and rituximab (CAR) in a schema designed to mitigate tumor lysis syndrome (TLS) seen previously with alvocidib. Nine nucleoside analog-naïve, high-risk patients received escalating doses of CAR therapy. Dose limiting toxicity was not experienced. No instances of TLS were observed. Patient responses included three complete remissions and four partial remissions. CAR was tolerable and active in high-risk CLL patients without TLS toxicity. With continued monitoring of toxicities, a phase Ib/II study of this combination as frontline therapy is warranted.

Pharmacokinetics of phase I nevirapine metabolites following a single dose and at steady state.

Nevirapine is one of the most extensively prescribed antiretrovirals worldwide. The present analyses used data and specimens from two prior studies to characterize and compare plasma nevirapine phase I metabolite profiles following a single 200-mg oral dose of nevirapine in 10 HIV-negative African Americans and a steady-state 200-mg twice-daily dose in 10 HIV-infected Cambodians. Nevirapine was assayed by high-performance liquid chromatography (HPLC). The 2-, 3-, 8- and 12-hydroxy and 4-carboxy metabolites of nevirapine were assayed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Pharmacokinetic parameters were calculated by noncompartmental analysis. The metabolic index for each metabolite was defined as the ratio of the metabolite area under the concentration-time curve (AUC) to the nevirapine AUC. Every metabolite concentration was much less than the corresponding nevirapine concentration. The predominant metabolite after single dose and at steady state was 12-hydroxynevirapine. From single dose to steady state, the metabolic index increased for 3-hydroxynevirapine (P < 0.01) but decreased for 2-hydroxynevirapine (P < 0.001). The 3-hydroxynevirapine metabolic index was correlated with nevirapine apparent clearance (P < 0.001). These findings are consistent with induction of CYP2B6 (3-hydroxy metabolite) and a possible inhibition of CYP3A (2-hydroxy metabolite), although these are preliminary data. There were no such changes in metabolic indexes for 12-hydroxynevirapine or 4-carboxynevirapine. Two subjects with the CYP2B6 *6*6 genetic polymorphism had metabolic indexes in the same range as other subjects. These results suggest that nevirapine metabolite profiles change over time under the influence of enzyme induction, enzyme inhibition, and host genetics. Further work is warranted to elucidate nevirapine biotransformation pathways and implications for drug efficacy and toxicity.

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.

A phase I study of prolonged infusion of triapine in combination with fixed dose rate gemcitabine in patients with advanced solid tumors.

PURPOSE: Prolonged exposure of cancer cells to triapine, an inhibitor of ribonucleotide reductase, followed by gemcitabine enhances gemcitabine activity in vitro. Fixed-dose-rate gemcitabine (FDR-G) has improved efficacy compared to standard-dose. We conducted a phase I trial to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of prolonged triapine infusion followed by FDR-G. EXPERIMENTAL DESIGN: Triapine was given as a 24-hour infusion, immediately followed by FDR-G (1000 mg/m(2) over 100-minute). Initially, this combination was administered days 1 and 8 of a 21-day cycle (Arm A, triapine starting dose 120 mg); but because of myelosuppression, it was changed to days 1 and 15 of a 28-day cycle (Arm B, starting dose of triapine 75 mg). Triapine steady-state concentrations (Css) and circulating ribonucleotide reductase M2-subunit (RRM2) were measured. RESULTS: Thirty-six patients were enrolled. The MTD was determined to be triapine 90 mg (24-hour infusion) immediately followed by gemcitabine 1000 mg/m(2) (100-minute infusion), every 2 weeks of a 4-week cycle. DLTs included grade 4 thrombocytopenia, leukopenia and neutropenia. The treatment was well tolerated with fatigue, nausea/vomiting, fever, transaminitis, and cytopenias being the most common toxicities. Among 30 evaluable patients, 1 had a partial response and 15 had stable disease. Triapine PK was similar, although more variable, compared to previous studies using doses normalized to body-surface-area. Steady decline in circulating levels of RRM2 may correlate with outcome. CONCLUSIONS: This combination was well tolerated and showed evidence of preliminary activity in this heavily pretreated patient population, including prior gemcitabine failure.

Development and validation of sensitive liquid chromatography/tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue.

A liquid chromatography-tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse-phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between-batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. The validated method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.

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.

Effects of human oral mucosal tissue, saliva, and oral microflora on intraoral metabolism and bioactivation of black raspberry anthocyanins.

Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried components of black raspberries (BRB) affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRB's chemopreventive effects. Consequently, functional assays, immunoblotting, and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were conducted. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles showed the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated, whereas immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. ß-Glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC/MS-MS analyses showed retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, postrinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data show that comparable to the small intestine, the requisite hydrolytic, phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, interpatient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness.

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.

A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma.

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin

A sensitive and specific liquid chromatography/tandem mass spectrometry method for quantification of nevirapine and its five metabolites and their pharmacokinetics in baboons.

A highly sensitive and specific LC-MS/MS assay was developed and validated to quantify nevirapine (NVP) and its five metabolites [2-, 3-, 8-, 12-hydroxyl NVP (OHNVP) and 4-carboxyl NVP (CANVP)] simultaneously in baboon serum and the assay was used to characterize their pharmacokinetic studies of an oral-dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolites was 1.0 ng/mL and for 4-CANVP was 5.0 ng/mL. The between-run and within-run precisions and accuracies at four quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboon serum with less than 14% variation and 93-114% accuracies (n = 6), except for the LLOQ for 2-OHNVP, which had an accuracy of 115.8% for between-run validation. The pharmacokinetics of NVP and its five metabolites in non-pregnant baboons by a single-dose escalation study were also profiled. The major metabolites detected were 4-CANVP and 12-OHNVP. 3-OHNVP and 2-OHNVP were the minor metabolites with only a trace amount of 2-OHNVP detected in some pharmacokinetic samples. No 8-OHNVP was observed in all of the pharmacokinetic samples. In addition, the fragmentation for the four hydroxyl metabolite isomers is also discussed.