Abemaciclib does not increase the corrected QT interval in healthy participants.

Abemaciclib is an orally administered, potent, and selective small molecule inhibitor of cyclin-dependent kinases 4 and 6, approved for advanced or metastatic breast cancer. This study aimed to use an exposure-response approach to investigate the effect of abemaciclib and its active metabolites (M2 and M20) on QTc interval and delay in cardiac repolarization at clinically relevant exposures. This was a single-blind, randomized, and placebo-controlled study of ascending doses of abemaciclib. Thirty-five healthy participants were administered a single dose of 200-600 mg abemaciclib. Twelve-lead electrocardiogram tracings and pharmacokinetic samples were collected serially pre- and post-dose. The primary objective was to study the relationship between abemaciclib and its active metabolites (M2 and M20) and QTc interval following ascending oral doses of abemaciclib. The secondary objective included evaluating the safety and tolerability of single ascending doses of abemaciclib in healthy participants. Exposure-response analysis demonstrated that there was no significant relationship between placebo-corrected change from baseline QTcF (ΔΔQTcF), abemaciclib, and metabolite plasma concentrations. Additionally, the ΔΔQTcF slopes of abemaciclib, its metabolites, and total analyte concentrations were not statistically different from zero. Single doses of abemaciclib, up to 400 mg, were well-tolerated by healthy participants; however, at the 600 mg dose (three times the highest registered dose), the frequency and severity of treatment-related gastrointestinal events (primarily diarrhea, nausea, and vomiting) increased. In conclusion, single doses of abemaciclib, up to 400 mg, had no statistically or clinically relevant effects on QTc, and abemaciclib was well tolerated up to a dose of 400 mg in this study.

Abemaciclib Does Not Have a Clinically Meaningful Effect on Pharmacokinetics of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 Substrates in Patients with Cancer.

Abemaciclib is an orally administered, potent inhibitor of cyclin-dependent kinases 4 and 6 and is metabolized extensively by CYP3A4. The effects of abemaciclib on several CYPs were qualified in vitro and subsequently evaluated in a clinical study. In vitro, human hepatocytes were treated with vehicle, abemaciclib, or abemaciclib metabolites [N-desethylabemaciclib (M2) or hydroxyabemaciclib (M20)]. mRNA levels for eight CYPs were measured using reverse-transcription quantitative polymerase chain reaction, and, additionally, catalytic activities for three CYPs were determined. In the clinical study, adult patients with cancer received a drug cocktail containing CYP substrates [midazolam (3A), warfarin (2C9), dextromethorphan (2D6), and caffeine (1A2)] either alone or in combination with abemaciclib. Plasma pharmacokinetics (PK) samples were analyzed for all substrates, caffeine metabolite paraxanthine, and abemaciclib; polymorphisms of CYP2C9, CYP2D6, CYP3A4, and CYP3A5 were evaluated. In vitro, downregulation of CYP mRNA, including 1A2, 2B6, 2C8, 2C9, 2D6, and 3A, by abemaciclib and/or M2 and M20 was observed at clinically relevant concentrations. In humans, abemaciclib did not affect the PK of CYP2D6 or CYP2C9 substrates. Minor statistically significant but clinically irrelevant changes were observed for midazolam [area under the concentration versus time curve from zero to infinity (AUC0-inf) (13% lower), Cmax (15% lower)], caffeine [AUC0-inf (56% higher)], and paraxanthine: caffeine [area under the concentration versus time curve from 0 to 24 hours ratio (was approximately 30% lower)]. However, given the magnitude of the effect, these changes are not considered clinically relevant. In conclusion, the downregulation of CYP mRNA mediated by abemaciclib in vitro did not translate into clinically meaningful drug-drug interactions in patients with cancer. SIGNIFICANCE STATEMENT: Despite observations that abemaciclib alters the mRNA of various CYP isoforms in vitro, a clinical study using a drug cocktail approach found no clinically meaningful drug-drug interactions between abemaciclib and a range of CYP substrates [midazolam (CYP3A4), S-warfarin (CYP2C9), dextromethorphan (CYP2D6), and caffeine (CYP1A2)]. This lack of translation suggests greater understanding of mechanisms of CYP downregulation is needed to accurately predict clinical drug-drug interaction risk from in vitro data.

Predicting Clinical Effects of CYP3A4 Modulators on Abemaciclib and Active Metabolites Exposure Using Physiologically Based Pharmacokinetic Modeling.

Abemaciclib, a selective inhibitor of cyclin-dependent kinases 4 and 6, is metabolized mainly by cytochrome P450 (CYP)3A4. Clinical studies were performed to assess the impact of strong inhibitor (clarithromycin) and inducer (rifampin) on the exposure of abemaciclib and active metabolites. A physiologically based pharmacokinetic (PBPK) model incorporating the metabolites was developed to predict the effect of other strong and moderate CYP3A4 inhibitors and inducers. Clarithromycin increased the area under the plasma concentration-time curve (AUC) of abemaciclib and potency-adjusted unbound active species 3.4-fold and 2.5-fold, respectively. Rifampin decreased corresponding exposures 95% and 77%, respectively. These changes influenced the fraction metabolized via CYP3A4 in the model. An absolute bioavailability study informed the hepatic and gastric availability. In vitro data and a human radiolabel study determined the fraction and rate of formation of the active metabolites as well as absorption-related parameters. The predicted AUC ratios of potency-adjusted unbound active species with rifampin and clarithromycin were within 0.7- and 1.25-fold of those observed. The PBPK model predicted 3.78- and 7.15-fold increases in the AUC of the potency-adjusted unbound active species with strong CYP3A4 inhibitors itraconazole and ketoconazole, respectively; and 1.62- and 2.37-fold increases with the concomitant use of moderate CYP3A4 inhibitors verapamil and diltiazem, respectively. The model predicted modafinil, bosentan, and efavirenz would decrease the AUC of the potency-adjusted unbound active species by 29%, 42%, and 52%, respectively. The current PBPK model, which considers changes in unbound potency-adjusted active species, can be used to inform dosing recommendations when abemaciclib is coadministered with CYP3A4 perpetrators.

Abemaciclib Inhibits Renal Tubular Secretion Without Changing Glomerular Filtration Rate.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6, is indicated for metastatic breast cancer treatment. Reversible increases in serum creatinine levels of ~15-40% over baseline have been observed following abemaciclib dosing. This study assessed the in vitro and clinical inhibition of renal transporters by abemaciclib and its metabolites using metformin (a clinically relevant transporter substrate), in a clinical study that quantified glomerular filtration and iohexol clearance. In vitro, abemaciclib inhibited metformin uptake by organic cation transporter 2, multidrug and toxin extrusion (MATE)1, and MATE2-K transporters with a half-maximal inhibitory concentration of 0.4-3.8 µM. Clinically, abemaciclib significantly increased metformin exposure but did not significantly affect measured glomerular filtration rate, serum neutrophil gelatinase-associated lipocalin (NGAL), serum cystatin-C, or the urinary markers of kidney tubular injury, NGAL and kidney injury molecule-1.

Phase 1 and pharmacokinetic study of LY3007113, a p38 MAPK inhibitor, in patients with advanced cancer.

Background The signaling protein p38 mitogen-activated protein kinase (MAPK) regulates the tumor cell microenvironment, modulating cell survival, migration, and invasion. This phase 1 study evaluated the safety of p38 MAPK inhibitor LY3007113 in patients with advanced cancer to establish a recommended phase 2 dose. Methods In part A (dose escalation), LY3007113 was administered orally every 12 h (Q12H) at doses ranging from 20 mg to 200 mg daily on a 28-day cycle until the maximum tolerated dose (MTD) was reached. In part B (dose confirmation), patients received MTD. Safety, pharmacokinetics, pharmacodynamics, and tumor response data were evaluated. Results MTD was 30 mg Q12H. The most frequent treatment-related adverse events (>10%) were tremor, rash, stomatitis, increased blood creatine phosphokinase, and fatigue. Grade ≥ 3 treatment-related adverse events included upper gastrointestinal haemorrhage and increased hepatic enzyme, both occurring at 40 mg Q12H and considered dose-limiting toxicities. LY3007113 exhibited an approximately dose-proportional increase in exposure and time-independent pharmacokinetics after repeated dosing. Maximal inhibition (80%) of primary biomarker MAPK-activated protein kinase 2 in peripheral blood mononuclear cells was not reached, and sustained minimal inhibition (60%) was not maintained for 6 h after dosing to achieve a biologically effective dose (BED). The best overall response in part B was stable disease in 3 of 27 patients. Conclusions The recommended phase 2 dosage of LY3007113 was 30 mg Q12H. Three patients continued treatment after the first radiographic assessment, and the BED was not achieved. Further clinical development of this compound is not planned as toxicity precluded achieving a biologically effective dose.

A Population Pharmacokinetic and Pharmacodynamic Analysis of Abemaciclib in a Phase I Clinical Trial in Cancer Patients.

BACKGROUND AND OBJECTIVES: Abemaciclib, a dual inhibitor of cyclin-dependent kinases 4 and 6, has demonstrated clinical activity in a number of different cancer types. The objectives of this study were to characterize the pharmacokinetics of abemaciclib in cancer patients using population pharmacokinetic (popPK) modeling, and to evaluate target engagement at clinically relevant dose levels. METHODS: A phase I study was conducted in cancer patients which incorporated intensive pharmacokinetic sampling after single and multiple oral doses of abemaciclib. Data were analyzed by popPK modeling, and patient demographics contributing to pharmacokinetic variability were explored. Target engagement was evaluated by combining the clinical popPK model with a previously developed pre-clinical pharmacokinetic/pharmacodynamic model. RESULTS: The pharmacokinetic analysis incorporated 4012 plasma concentrations from 224 patients treated with abemaciclib at doses ranging from 50 to 225 mg every 24 h and 75 to 275 mg every 12 h. A linear one-compartment model with time- and dose-dependent relative bioavailability (F rel) adequately described the pharmacokinetics of abemaciclib. Serum albumin and alkaline phosphatase were the only significant covariates identified in the model, the inclusion of which reduced inter-individual variability in F rel by 10.3 percentage points. By combining the clinical popPK model with the previously developed pre-clinical pharmacokinetic/pharmacodynamic model, the extent of target engagement in skin in cancer patients was successfully predicted. CONCLUSION: The proportion of abemaciclib pharmacokinetic variability that can be attributed to patient demographics is negligible, and as such there are currently no dose adjustments recommended for adult patients of different sex, age, or body weight. TRIAL REGISTRATION: NCT01394016 (ClinicalTrials.gov).

Pharmacokinetic and screening studies of the interaction between mononuclear phagocyte system and nanoparticle formulations and colloid forming drugs.

Studies have shown that nanoparticles (NPs) are cleared through the mononuclear phagocyte system (MPS). Pharmacokinetic studies of Doxil, DaunoXome, micellar doxorubicin (SP1049C) and small molecule (SM) doxorubicin were performed in SCID mice, Sprague-Dawley rats, and beagle dogs. An ex vivo MPS profiling platform was used to evaluate the interaction between the same agents, as well as colloid-forming and non-colloid forming SM drugs. In all species, the systemic clearance was highest for SP1049C and lowest for Doxil. With the exception of dog blood, the MPS screening results of mouse and rat blood showed that the greatest reduction in phagocytosis occurred after the ex vivo addition of SM-doxorubicin>SP1049C>DaunoXome>Doxil. The MPS profiling platform in rats, but not dogs, could differentiate between colloid forming and non-colloid forming drugs. The results of the MPS profiling platform were generally consistent with in vivo clearance rates of NP and SM anticancer drugs in mice and rats. This study suggests the MPS profiling platform is an effective method to screen and differentiate the important characteristics of NPs and colloid-forming drugs that affect their in vivo clearance. Implications of these findings on preclinical prediction of human clearance are discussed.

Comparison between Radioanalysis and 19F Nuclear Magnetic Resonance Spectroscopy in the Determination of Mass Balance, Metabolism, and Distribution of Pefloxacin.

Mass balance and metabolism studies using radiolabeled substances are well recognized as an important part of the drug development process. In this study, we directly assessed the use of fluorine nuclear magnetic resonance (19F NMR) to achieve quantitative mass balance, metabolism, and distribution information for fluorinated compounds, without the need for radiolabeled synthesis or study. As a test case, the disposition of pefloxacin, a fluoroquinolone antibiotic, was evaluated in rats using quantitative 19F NMR in parallel with a radiolabeled study. Urine, bile, and feces samples were collected over specific periods after oral administration of either 25 mg/kg [14C]pefloxacin or 25 mg/kg pefloxacin and were subsequently profiled by radioactivity or 19F NMR, respectively. The percentage of dose excreted in each matrix was comparable between the two methods, with the total dose recovered by radioactivity and 19F NMR determined to be 86.8% and 81.8%, respectively. In addition, plasma samples were collected to determine the exposure of pefloxacin and its circulating metabolites. The plasma exposure of pefloxacin determined by 19F NMR was within 5% to that calculated by a validated liquid chromatography-tandem mass spectrometry bioanalytical method. By both methods, pefloxacin was identified as the major circulating entity, with pefloxacin glucuronide as the major circulating metabolite. Quantitative analysis of metabolites in excreta was generally comparable between the two methods. In selected tissues, both methods indicated that the parent drug accounted for most of the drug-related material. In summary, we have demonstrated that 19F NMR can be used as an alternative method to conventional radiolabeled studies for compounds containing fluorine without the need for radiolabeled synthesis/study.

Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors.

UNLABELLED: We evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months. SIGNIFICANCE: Abemaciclib represents the first selective inhibitor of CDK4 and CDK6 with a safety profile allowing continuous dosing to achieve sustained target inhibition. This first-in-human experience demonstrates single-agent activity for patients with advanced breast cancer, NSCLC, and other solid tumors. Cancer Discov; 6(7); 740-53. ©2016 AACR.See related commentary by Lim et al., p. 697This article is highlighted in the In This Issue feature, p. 681.

Optimising the combination dosing strategy of abemaciclib and vemurafenib in BRAF-mutated melanoma xenograft tumours.

BACKGROUND: Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation. METHODS: A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival. RESULTS: The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression. CONCLUSIONS: The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.

A First-in-Human Phase I Study of the Oral p38 MAPK Inhibitor, Ralimetinib (LY2228820 Dimesylate), in Patients with Advanced Cancer.

PURPOSE: p38 MAPK regulates the production of cytokines in the tumor microenvironment and enables cancer cells to survive despite oncogenic stress, radiotherapy, chemotherapy, and targeted therapies. Ralimetinib (LY2228820 dimesylate) is a selective small-molecule inhibitor of p38 MAPK. This phase I study aimed to evaluate the safety and tolerability of ralimetinib, as a single agent and in combination with tamoxifen, when administered orally to patients with advanced cancer. EXPERIMENTAL DESIGN: The study design consisted of a dose-escalation phase performed in a 3+3 design (Part A; n = 54), two dose-confirmation phases [Part B at 420 mg (n = 18) and Part C at 300 mg (n = 8)], and a tumor-specific expansion phase in combination with tamoxifen for women with hormone receptor-positive metastatic breast cancer refractory to aromatase inhibitors (Part D; n = 9). Ralimetinib was administered orally every 12 hours on days 1 to 14 of a 28-day cycle. RESULTS: Eighty-nine patients received ralimetinib at 11 dose levels (10, 20, 40, 65, 90, 120, 160, 200, 300, 420, and 560 mg). Plasma exposure of ralimetinib (Cmax and AUC) increased in a dose-dependent manner. After a single dose, ralimetinib inhibited p38 MAPK-induced phosphorylation of MAPKAP-K2 in peripheral blood mononuclear cells. The most common adverse events, possibly drug-related, included rash, fatigue, nausea, constipation, pruritus, and vomiting. The recommended phase II dose was 300 mg every 12 hours as monotherapy or in combination with tamoxifen. Although no patients achieved a complete response or partial response,19 patients (21.3%) achieved stable disease with a median duration of 3.7 months, with 9 of these patients on study for ≥ 6 cycles. CONCLUSIONS: Ralimetinib demonstrated acceptable safety, tolerability, and pharmacokinetics for patients with advanced cancer.

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.

Utilizing 19F NMR to investigate drug disposition early in drug discovery.

1. Nuclear magnetic resonance (NMR), a non-selective and inherently quantitative method, has not been widely used as a quantitative tool for characterizing the disposition of lead molecules prior to clinical development. As a test case, we have chosen a fluoropyrimidine compound in lead optimization phase and evaluated its disposition following oral administration to rats using 19F NMR. 2. Urine, bile and feces from individual rats were profiled and the amount of dose eliminated in each matrix was calculated. The results indicated that, in male rats, the mean dose eliminated over 0-48 h was 40%, with 28% in urine, 9% in bile and 3% in feces. In female rats, the mean dose recovered in excreta over the same period was 55%, with 40% in urine, 8% in bile and 7% in feces. 3. In addition, plasma from rats and plasma from toxicology study in dogs were also profiled and exposure of circulating entities was determined. Plasma exposure determined by 19F NMR was in good agreement with those determined by conventional LC-MS/MS method, suggesting quantitative 19F NMR can be reliably used to estimate single dose or steady-state systemic exposure of circulating entities in animals and humans.

Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure.

LY2090314 (3-[9-fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenous glycogen synthase kinase-3 inhibitor in oncology trials. Drug disposition was characterized after intravenous infusion of [(14)C]LY2090314 to rats and dogs, and was related to available clinical data. LY2090314 exhibited high clearance (approximating hepatic blood flow) and a moderate volume of distribution (∼1-2 l/kg) resulting in rapid elimination (half-life ∼0.4, 0.7, and 1.8-3.4 hours in rats, dogs, and humans, respectively). Scaled clearance from liver microsomes accurately predicted perfusion-limited clearance across species. LY2090314 was cleared by extensive metabolism, and the numerous metabolites were rapidly excreted into feces via bile (69-97% of dose; 62-93% within 0-24 hours); urinary recovery of drug-related material was low (≤3% of dose). Despite extensive metabolism, in rats and humans the parent compound was the sole identifiable drug-related moiety in plasma. Even in Mdr1a-, Bcrp-, and Mrp2-knockout rats, LY2090314 metabolites did not appear in circulation, and their urinary excretion was not enhanced, because the hypothesized impaired biliary excretion of metabolites in the absence of these canalicular transporters was not observed. Canine metabolite disposition was generally similar, with the notable exception of dog-unique LY2090314 glucuronide. This conjugate was formed in the dog liver and was preferentially excreted into the blood, where it accounted for the majority of circulating radioactivity at later times, and was predominantly recovered in urine (16% of dose). In conclusion, LY2090314 was rapidly cleared by extensive metabolism with negligible circulating metabolite exposures due to biliary excretion of metabolites into feces with no apparent intestinal reabsorption.

Metabolism of LY654322, a growth hormone secretagogue, to an unusual diimidazopyridine metabolite.

2-Methylalanyl-N-{1-[(1R)-1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidin-1-ylethyl]-1H-imidazol-4-yl}-5-phenyl-D-norvalinamide (LY654322) was rapidly cleared in rats and dogs by renal excretion of parent and metabolism (oxidative and hydrolytic). Among the metabolites identified in the urine of rats and dogs was M25, which was structurally unusual. Indeed, the characterization of M25 and investigation into its disposition relied on the convergence of diverse analytical methodologies. M25 eluted after the parent on reverse-phase chromatography with an MH(+) at m/z 598 (parent + 35 Da). Given its increased lipophilicity and its mass difference compared with the parent, it was evident that M25 was not a phase 2 conjugate. Subsequent liquid chromatography with multiple-stage tandem mass spectrometry and accurate mass experiments identified the structure of M25 as having two replicates of the 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl substructure flanking a central aromatic core of composition C(7)H(3)N(5) that was refractory to fragmentation. Compared with the UV spectrum of the parent (λ(max) = 213 nm), M25 displayed a bathochromic shift (λ(max) = 311 nm), which substantiated extensive conjugation within the central core. Subsequent NMR analysis of M25 isolated from dog urine coupled with molecular modeling revealed the structure to be consistent with a diimidazopyridine core with two symmetrically substituted 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl moieties. Using a structural analog with a chromophore similar to M25, LC-UV was used to quantitate M25 and determine its urinary disposition. The formation of M25 appears consistent with hydrolysis of LY654322 to an aminoimidazole, dimerization of the latter with the loss of NH(3), C-formylation, and subsequent ring closure and aromatization with loss of H(2)O.

Disposition and metabolism of semagacestat, a {gamma}-secretase inhibitor, in humans.

Semagacestat is a functional gamma-secretase inhibitor that has been shown to reduce the rate of formation of amyloid-beta in vitro and in vivo. This study was conducted to characterize the disposition of semagacestat in humans. After a single 140-mg dose of [(14)C]semagacestat administered as an oral solution to six healthy male subjects, semagacestat was rapidly absorbed (T(max) approximately 0.5 h) and eliminated from the systemic circulation (terminal t(1/2) approximately 2.4 h). The major circulating metabolites of semagacestat, M2 (hydrolysis of the amide bond proximal to the benzazepine ring) and M3 (benzylic hydroxylation of the benzazepine ring), accounted for approximately 27 and 10% of total radioactivity exposure, respectively, as calculated from relative area under the plasma concentration versus time curve from 0 to 24 h derived from the plasma radiochromatograms. The radioactive dose was almost completely recovered after 7 days postdose, with 87% of the dose in urine and 8% in feces. Unchanged [(14)C]semagacestat in urine accounted for approximately 44% of the dose, which indicates that renal excretion played an important role in elimination. Metabolites M2 and M3, with their related secondary metabolites, each accounted for approximately 20% of the dose in excreta. In vitro data indicate the formation of M3 is primarily mediated by CYP3A, with cDNA-expressed CYP3A5 approximately 2 times more efficient than CYP3A4 in forming M3. Thus, the relative content of CYP3A4 and CYP3A5 in humans will likely determine the formation clearance of M3 after exposure to semagacestat.

A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds.

Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant.

Simultaneous characterization of a mixture of fluorochemicals using three-dimensional 19F-1H heteronuclear TOCSY filtered/edited NMR experiments.

Heteronuclear 19F-1H cross-polarization can be used effectively as a tool for both spectral filtering and editing in the NMR analysis of the increasing number of fluorine-containing compounds encountered in drug discovery. Combined with LC-MS, three-dimensional 19F-1H heteronuclear TOCSY filtered experiments based on this approach have enabled the simultaneous identification of a mixture of closely related dexamethasone derivatives without the need for isolation.

Dehydrogenation of indoline by cytochrome P450 enzymes: a novel "aromatase" process.

Indoline derivatives possess therapeutic potential within a variety of drug candidates. In this study, we found that indoline is aromatized by cytochrome P450 (P450) enzymes to produce indole through a novel dehydrogenation pathway. The indole products can potentially be bioactivated to toxic intermediates through an additional dehydrogenation step. For example, 3-substituted indoles like 3-methylindole and zafirlukast [4-(5-cyclopentyloxy-carbonylamino-1-methyl-indol-3-ylmethyl)-3-methoxy-N-o-tolylsulfonylbenzamide] are dehydrogenated to form 3-methyleneindolenine electrophiles, which react with protein and/or DNA nucleophilic residues to cause toxicities. Another potentially significant therapeutic consequence of indoline aromatization is that the product indoles might have dramatically different therapeutic potency than the parent indolines. In this study, indoline was indeed efficiently aromatized by human liver microsomes and by several P450s, but not by flavin-containing monooxygenase (FMO) 3. CYP3A4 had the highest aromatase activity. Four additional indoline metabolites [2,3,4,7-tetrahydro-4,5-epoxy-1H-indole (M1); N-hydroxyindole (M2), N-hydroxyindoline (M3), and M4 ([1,4,2,5]dioxadiazino[2,3-a:5,6-a']diindole)] were characterized; none was a metabolite of indole. M1 was an arene oxide from P450 oxidation, and M2, M3, and M4 were produced by FMO3. Our data indicated that indoline was oxidized to M3 and then to an intermediate indoline nitrone, which tautomerized to form M2, and subsequently dimerized to a di-indoline. This dimer was immediately oxidized by FMO3 or atmospheric oxygen to the final product, M4. No evidence was found for the P450-mediated production of an aliphatic alcohol from indoline that might dehydrate to produce indole. Therefore, P450 enzymes catalyze the novel "aromatase" metabolism of indoline to produce indole. The aromatase mechanism does not seem to occur through N-oxidation or dehydration of an alcohol but rather through a formal dehydrogenation pathway.

Selective metabolism of vincristine in vitro by CYP3A5.

Clinical outcomes of vincristine therapy, both neurotoxicity and efficacy, are unpredictable, and the reported pharmacokinetics of vincristine have considerable interindividual variability. In vitro and in vivo data support a dominant role for CYP3A enzymes in the elimination of vincristine. Consequently, genetic polymorphisms in cytochrome P450 (P450) expression may contribute to the interindividual variability in clinical response, but the contributions of individual P450s and the primary pathways of vincristine metabolism have not been defined. In the present study, vincristine was incubated with a library of cDNA-expressed P450s, and the major oxidative metabolites were identified. CYP3A4 and CYP3A5 were the only P450s to support substantial loss of parent drug and formation of the previously unidentified, major metabolite (M1). The structure of M1, arising as a result of an oxidative cleavage of the piperidine ring of the dihydro-hydroxycatharanthine unit of vincristine, was conclusively established after conversion to suitable derivatives followed by spectroscopic analysis, and a new pathway for vincristine metabolism is proposed. CYP3A5 was more efficient in catalyzing the formation of M1 compared with CYP3A4 (9- to 14-fold higher intrinsic clearance for CYP3A5). The formation of M1 was stimulated (3-fold) by the presence of coexpressed cytochrome b5, but the relative efficiencies of M1 formation by CYP3A4 and CYP3A5 were unaffected. Our findings demonstrate that in contrast to most CYP3A biotransformations, the oxidation of vincristine is considerably more efficient with CYP3A5 than with CYP3A4. We conclude that common genetic polymorphisms in CYP3A5 expression may contribute to the interindividual variability in the systemic elimination of vincristine.