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Supplementary Figure 1. Sample simulated time-course of paclitaxel and MLN8237 PK after and the pharmacokinetic parameters used for MLN8237 and paclitaxel required to generate the exposure - efficacy model. Supplementary Figure 2. Predicted versus observed % TGI resulting from the best fit of the dose response surface to the xenograft tumor growth data. Supplementary Figure 3. Cleaved caspase 3 staining of tumor xenografts following single agent or combination treatment with MLN8237 and docetaxel in the PHTX-14B and MDA-MB-231 xenografts. Supplementary Figure 4. MLN8237 administered on an intermittent 3 days on / 4 days off schedule combined with paclitaxel results in additive antitumor activity in the MDA-MB-231 model. Supplementary Figure 5. MLN8237 and paclitaxel exposures are similar when dosed alone or in combination.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 μCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

BackgroundThe effectiveness of many targeted therapies is limited by toxicity and the rise of drug resistance. A growing appreciation of the inherent redundancies of cancer signaling has led to a rise in the number of combination therapies under development, but a better understanding of the overall cancer network topology would provide a conceptual framework for choosing effective combination partners. In this work, we explore the scale-free nature of cancer protein-protein interaction networks in 14 indications. Scale-free networks, characterized by a power-law degree distribution, are known to be resilient to random attack on their nodes, yet vulnerable to directed attacks on their hubs (their most highly connected nodes).ResultsConsistent with the properties of scale-free networks, we find that lethal genes are associated with ∼5-fold higher protein connectivity partners than non-lethal genes. This provides a biological rationale for a hub-centered combination attack. Our simulations show that combinations targeting hubs can efficiently disrupt 50% of network integrity by inhibiting less than 1% of the connected proteins, whereas a random attack can require inhibition of more than 30% of the connected proteins.ConclusionsWe find that the scale-free nature of cancer networks makes them vulnerable to focused attack on their highly connected protein hubs. Thus, we propose a new strategy for designing combination therapies by targeting hubs in cancer networks that are not associated with relevant toxicity networks.

Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. MMAE was incubated at 1–1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-β-tubulin antibody and imaging. MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of β-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [

Orteronel is a nonsteroidal, selective inhibitor of 17,20-lyase that was recently in phase 3 clinical development as a treatment for castration-resistant prostate cancer. In humans, the primary clearance route for orteronel is renal excretion. Human liver microsomal studies indicated that orteronel weakly inhibits CYP1A2, 2C8, 2C9 and 2C19, with IC50 values of 17.8, 27.7, 30.8 and 38.8 µm, respectively, whereas orteronel does not inhibit CYP2B6, 2D6 or 3A4/5 (IC50 > 100 µm). Orteronel also does not exhibit time-dependent inhibition of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 or 3A4/5. The results of a static model indicated an [I]/Ki ratio >0.1 for CYP1A2, 2C8, 2C9 and 2C19. Therefore, a physiologically based pharmacokinetic (PBPK) model was developed to assess the potential for drug–drug interactions (DDIs) between orteronel and theophylline, repaglinide, (S)-warfarin and omeprazole, which are sensitive substrates of CYP1A2, 2C8, 2C9 and 2C19, respectively. Simulation of the area under the plasma concentration–time curve (AUC) of these four CYP substrates in the presence and absence of orteronel revealed geometric mean AUC ratios

Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure–efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m2 of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m2 of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents. Mol Cancer Ther; 13(9); 2170–83. ©2014 AACR . This article is featured in Highlights of This Issue, [p. 2139][1] [1]: /lookup/volpage/13/2139?iss=9

6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 μg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 μg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors.

I do hope the DML Reader will find all twelve papers included in this issue, to be quite interesting: Urs A. Boelsterli et al.. studied the bioactivation of leflunomide and the metabolic degradation to its major metabolite, A771726. Leflunomide was rapidly metabolized in human hepatocytes to A771726, but its toxicity was dependent on other, CYP-dependent intermediates. James F. Rusling and colleagues used rat liver microsomes attached to nanoparticles for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC50 values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were also estimated. Lee Jia et al. investigated the effects of CsA and ITZ on 1) intestinal permeability of amlodipine (a calcium channel blocker used as a cardiovascular agent) in isolated rat everted gut sac model, and 2) biliary excretion and pharmacokinetics of amlodipine in rats. Pretreatment of rats with ITZ increased plasma levels and biliary excretion of amlodipine in a dose-dependent manner. In contrast, pretreatment with CsA slightly decreased biliary excretion of amlodipine and made no changes in its plasma levels. Michael W. Sinz et al. demonstrated that the humanized SXR mouse can be used as a model to predict human CYP3A4 induction and the resulting pharmacokinetic changes of CYP3A4 substrates in humans. Jasminder Sahi et al. compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. They also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. Their data showed major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. David Rodrigues et al. worked on troglitazone (TGZ) induced hepatotoxicity, which has been linked to cytochrome P450 (CYP)- catalyzed reactive metabolite formation. They concluded that both CYP3A4/5 and CYP2C8 play a major role in the formation of TGZ adduct in HLM. However, the contribution of these CYPs varies depending on their relative expression and the concentration of TGZ.

The pH of ex vivo plasma, bile and urine was monitored at different times and temperatures of storage, and following different sample processing methods such as ultrafiltration, centrifugation, precipitation and evaporation. The results showed that the pH of ex vivo plasma, bile and urine increased upon storage, and following sample processing and could lead to significant degradation of pH-labile compounds. Several compounds were used to illustrate the impact of pH shifts on drug stability and interpretation of results obtained from in vivo studies. For example, after 1 h of incubation (37 degrees C) in rat plasma (pH 8.3), about 60%, of I was lost. However, in phosphate buffer, losses were about 12% at pH 7.4 and 40% at pH 8.0. Plasma pH also increased during ultrafiltration, centrifugation and extraction. After methanol precipitation of plasma proteins, and evaporation of the supernatant and redissolution of the residue, the resulting solution had a pH of 9.5. Under these conditions, II was degraded by 60% but was stable when phosphate buffer was used to maintain the pH at 7.4. The shift in plasma pH can yield misleading results from in vivo studies if the pH is not controlled. For example, the major circulating metabolite of II was also formed in plasma ex-vivo. This ex vivo degradation was prevented when blood samples were collected into tubes containing 0.1 volume of phosphate buffer (0.3 M, pH 5). The pH of ex vivo plasma can best be stabilized at physiological conditions using 10% CO2 atmosphere in a CO2 incubator. Changes in pH of ex vivo urine and bile samples can have similar adverse effect on pH-labile samples. Thus, processing of plasma samples under a 10% CO2 atmosphere is a method of choice for stability or protein binding studies in plasma, whereas citrate or phosphate buffers are suitable for stabilizing pH in bile and urine and for plasma samples requiring extensive preparations or long term storage.

Sumatriptan and butorphanol nasal sprays are commonly used agents for the management of migraine headaches. Under certain circumstances, these two agents may be administered closely in time. However, the possibility of a pharmacokinetic interaction and the safety of this regime have not been examined. In this crossover design study, 24 healthy subjects received the following four treatments, each separated by at least 7 days: 1 mg butorphanol (Stadol NS7®); 20 mg sumatriptan (Imitrex® Nasal Spray); or both formulations together with butorphanol administered either 1 or 30 min after sumatriptan. Serial plasma samples were collected for 24 h post-dose and analysed for butorphanol and/or sumatriptan by HPLC-MS/MS. Butorphanol plasma concentrations were reduced when it was administered 1 min (mean 28.6% decrease in AUC0-∞) , but not 30 min, after sumatriptan. The pharmacokinetics of sumatriptan were not substantially altered by butorphanol. The combination of nasally administered sumatriptan and butorphanol appeared safe. However, if butorphanol nasal spray is administered < 30 min after sumatriptan nasal spray, the analgesic effect of butorphanol may be diminished due to reduced nasal absorption resulting from probable transient vasoconstriction of nasal blood vessels by sumatriptan.

Toxicity often limits the utility of oncology drugs, and optimization of dose schedule represents one option for mitigation of this toxicity. Here we explore the schedule-dependency of neutropenia, a common dose-limiting toxicity. To this end, we analyze previously published mathematical models of neutropenia to identify a pharmacokinetic (PK) predictor of the neutrophil nadir, and confirm this PK predictor in an in vivo experimental system. Specifically, we find total AUC and Cmax are poor predictors of the neutrophil nadir, while a PK measure based on the moving average of the drug concentration correlates highly with neutropenia. Further, we confirm this PK parameter for its ability to predict neutropenia in vivo following treatment with different doses and schedules. This work represents an attempt at mechanistically deriving a fundamental understanding of the underlying pharmacokinetic drivers of neutropenia, and provides insights that can be leveraged in a translational setting during schedule selection.

Orteronel is a nonsteroidal, selective inhibitor of 17,20-lyase that was recently in phase 3 clinical development as a treatment for castration-resistant prostate cancer. In humans, the primary clearance route for orteronel is renal excretion. Human liver microsomal studies indicated that orteronel weakly inhibits CYP1A2, 2C8, 2C9 and 2C19, with IC50 values of 17.8, 27.7, 30.8 and 38.8 µm, respectively, whereas orteronel does not inhibit CYP2B6, 2D6 or 3A4/5 (IC50 100 µm). Orteronel also does not exhibit time-dependent inhibition of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 or 3A4/5. The results of a static model indicated an [I]/Ki ratio0.1 for CYP1A2, 2C8, 2C9 and 2C19. Therefore, a physiologically based pharmacokinetic (PBPK) model was developed to assess the potential for drug-drug interactions (DDIs) between orteronel and theophylline, repaglinide, (S)-warfarin and omeprazole, which are sensitive substrates of CYP1A2, 2C8, 2C9 and 2C19, respectively. Simulation of the area under the plasma concentration-time curve (AUC) of these four CYP substrates in the presence and absence of orteronel revealed geometric mean AUC ratios1.25. Therefore, in accordance with the 2012 US FDA Draft Guidance on DDIs, orteronel can be labeled a 'non-inhibitor' and further clinical DDI evaluation is not required. In PBPK models of moderate and severe renal impairment, the AUC of orteronel was predicted to increase by 52% and 83%, respectively. These results are in agreement with those of a clinical trial in which AUC increases of 38% and 87% were observed in patients with moderate and severe renal impairment, respectively.

A sensitive, selective, accurate, precise and reproducible triple-quadrupole liquid chromatographic-mass spectrometric assay was developed and validated for BMS-181885 (I), a 5HT1 agonist, in human plasma using BMS-181101 as the internal standard (IS). The method involved solid phase extraction of plasma containing I and the IS using Isoelute CN cartridges. The supernatant was then evaporated to dryness at 40 degrees C. The residue was dissolved in 100 microL of the injecting solvent. The HPLC column was ODS-3, 2 x 100 mm. The mobile phase comprised 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, used in an isocratic condition. The mass spectrometer was programmed to admit the protonated molecules at m/z 461 (I) and m/z 370 (IS) via the first quadrupole filter and to select reaction monitoring of ions at m/z 152 for I and IS for the quantification. Standard curves were fitted to a weighted quadratic function over the concentration range 0.2-200 ng/mL. The lowest standard concentration (0.2 ng/mL) was experimentally established as the lower limit of quantitation of the assay. The mean predicted quality control concentrations deviated within +/- 11% of the corresponding nominal values; the intra-assay and inter-assay precisions were within 7.0% relative standard deviation. I was stable in the injection solvent at 4 degrees C for at least 24 h and for at least three freeze-thaw cycles. Freezer stability of I in plasma was demonstrated for at least 3 months. The extraction recovery of I was established as 97%. The validated assay was applied to a pharmacokinetic study of I in humans.

A sensitive, selective, accurate, precise and reproducible high-performance liquid chromatographic-mass spectrometric (LC-MS) assay was developed and validated for the simultaneous determination of nefazodone (NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (I.S.). The method involved simultaneous protein precipitation with acetonitrile and liquid-liquid extraction with methylene chloride, after which the organic layer was evaporated to dryness. The residue was reconstituted in 25% acetonitrile in 10 mM ammonium formate (pH 4.0), and an aliquot was injected onto a BDS Hypersil C18 column at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile in 55:45 (v/v) was used in an isocratic condition. The mass spectrometer was programmed to admit the protonated molecules at m/z 197.0 (mCPP), 372.0 (I.S.) 470.4 (NEF), 458.1 (Dione), and 486.2 (OH-NEF). Standard curves were linear (r2or = 0.995) over the concentration range of 4-1000 ng/ml for Dione and 2-500 ng/ml for other analytes. The lowest standard concentrations were the lower limit of quantitation for each analyte. The mean predicted quality control (QC) concentrations for all analytes deviated less than -12.1% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay for all analytes were within 7.0% relative standard deviation. All analytes including I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 67.3 to 86.5%. The validated assay was applied to a pharmacokinetic study of nefazodone.

This study was conducted to evaluate the effects of a high-fat meal on the oral bioavailability of an 300-mg irbesartan tablet in healthy male volunteers. Sixteen healthy young male volunteers participated in this single-center, open-label, single-dose, crossover study. Each volunteer received a single 300-mg irbesartan tablet under fasted conditions and 5 minutes after a high-fat breakfast, with administrations separated by a 7-day washout period. Serial blood samples were collected over a 72-hour period, and plasma samples were analyzed for irbesartan using a validated high-performance liquid chromatography/fluorescence procedure. Food had no statistically significant effects on the peak concentration (Cmax) and area under the concentration-time curve (AUC) of irbesartan. The presence of food was associated with a slightly prolonged time to maximum concentration (tmax) and half-life (t1/2), but the differences were not statistically significant. The results of this study indicate that food does not affect the bioavailability of irbesartan. Thus, irbesartan can be administered without regard to meals.

The relative contribution of the gut and the liver to the first-pass metabolism of BMS-181101 (3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-5-fluoro-1H-ind ole dihydrochloride), a potential antidepressant agent, has been evaluated in rats. Nine male Sprague-Dawley rats were divided into three groups of three and each rat received a single 20 mg kg(-1) dose of [14C]BMS-181101 via a 30 min constant-rate intravenous infusion, a 30-min constant-rate intraportal infusion or oral gavage. Serial blood samples were collected for 8 h after dosing and plasma was analysed for unchanged BMS-181101 and total radioactivity. Extraction ratios for BMS-181101 by the gut and liver were calculated on the basis of ratios of the area under the plasma BMS-181101 concentration-time curve. The gut had a high intrinsic capacity for metabolizing BMS-181101-extraction ratios were 93% and 10% for the gut and liver, respectively. After oral administration BMS-181101 is sequentially exposed to the gut then the liver. As a result, the contribution of the gut to the overall first-pass effect (ca. 93%) was significantly greater than that of the liver (ca. 0.7%). The estimated total first-pass effect of 94% for BMS-181101 in rats is in excellent agreement with the observed absolute oral bioavailability of 6%. These results clearly illustrate the importance of metabolic activity in the gut for orally administered BMS-181101.

Objective: The effects of food on the pharmacokinetics of an irbesartan/ hydrochlorothiazide combination tablet were assessed in 16 healthy young male volunteers.

The effect of food on the pharmacokinetics of BMS-181101, a new anti-depressant under development, was investigated in 12 healthy male volunteers at steady state. Each subject received a 15 mg oral dose of BMS-181101 twice a day (q 12 h) for 11 days and a morning dose of BMS-181101 on day 12. Six subjects were randomly assigned to receive BMS-181101 under fasted conditions from days 1 to 6 and then crossed over to fed conditions from days 7 to 12. The other six subjects received the reverse conditions, fed for days 1-6 and fasted for days 7-12. Serial blood samples were collected up to 12 h on days 6 and 12 following the administration of the morning dose. In addition, trough blood samples were collected on days 4, 5, 10, and 11 prior to the morning dose. Plasma samples were analyzed for intact BMS-181101 using a validated high-performance liquid chromatography method with an electrochemical detector. BMS-181101 was well tolerated both with and without ingestion of food. The statistical evaluation of the C min values indicated that steady state of BMS-181101 was achieved by the fourth day of dosing regardless of whether the subject was fasted or fed. When BMS-181101 was administered with food, C max was reduced by about 25% and t max was prolonged by 1 h. However, AUC tau , t 1/2 , and time to attain steady state of BMS-181101 were not altered by ingestion of food. In summary, BMS-181101 can be given with food without adversely impacting the safety or pharmacokinetic profiles of the drug.

During drug development, trough concentrations are usually monitored following repeated dosing to ensure that steady state has been achieved. Further utility of this information is explored in the estimation of intrasubject variability. Intrasubject variability is essential to the determination of sample sizes for planning within-subject (ie, crossover) studies of the effects of meals, concomitant drugs, or formulation changes (ie, bioequivalence). The utility of steady state trough concentration was evaluated as an indicator of intrasubject variability compared to a conventional approach of using a fraction of intersubject variability and to estimates obtained directly from crossover studies. Based on available data, estimates of intrasubject variability by the trough concentration approach were comparable to those from the other two approaches.

The pharmacokinetics of butorphanol nasal spray, with and without the coadministration of metoclopramide, were studied in 24 healthy women. In this crossover study all volunteers received 3 treatments: a single, 1-mg dose of butorphanol nasal spray, a single, 10-mg oral dose of metoclopramide, and a combination of a single, 1-mg dose of butorphanol nasal spray and a single, 10-mg oral dose of metoclopramide. There was at least a one-week washout period between sessions. Serial blood samples were collected and plasma samples analyzed using a validated radioimmunoassay to determine the concentration of butorphanol, or a high-performance liquid chromatography/ultraviolet procedure was used to determine the concentration of metoclopramide. There were no statistically significant differences in the pharmacokinetic parameters, Cmax, tmax, AUC, and t1/2, for butorphanol with or without metoclopramide. Similarly, except for a delay in tmax of metoclopramide with coadministration of butorphanol, the pharmacokinetic parameters of metoclopramide were not significantly different between two treatments. Thus, the pharmacokinetics of both butorphanol and metoclopramide were not significantly altered when administered in combination. The incidence of nausea/vomiting after butorphanol administration was substantially reduced by coadministration of metoclopramide. Based on the pharmacokinetic and safety results, it can be concluded that butorphanol nasal spray and metoclopramide can be administered in combination without altering the dose regimen of either drug.

BMS-180680 is a monobactam antibiotic currently under development for the treatment of gram negative bacteria including Pseudomonas aeruginosa. Simple, rapid, sensitive, precise, and reproducible assay methods have been developed for the quantification of BMS-180680 in dog and rat plasma using aztreonam as the internal standard. The assay methods involve a single-step protein precipitation by addition of acetonitrile, followed immediately by centrifugation, after which the supernatant is evaporated to dryness (65°C) under a gentle stream of nitrogen. The residue is re-constituted in the mobile phase, transferred to a micro-WISP vial and injected on to a Zorbax It, C-18 HPLC column preceded by a guard column. Mobile phase comprised 17% acetonitrile and 83% of 40 mM dibasic ammonium phosphate and 6 mM tetrabutylammonium hydrogen sulfate. A small amount of tetrahydrofuran (20 mL/liter of mobile phase) was added and the apparent pH of the mobile phase was adjusted to 4.1 with 85% phosphoric acid. The...

The single-dose and steady state pharmacokinetics of butorphanol and its metabolites, hydroxybutorphanol (HO-B) and norbutorphanol (NOR-B), were studied in nine healthy male volunteers. Each subject received a single 1 mg dose of butorphanol on days 1 and 6, and a 1 mg dose every 6 h (q6h) on days 2-5, via nasal administration. Serial blood and urine samples were collected for 24 h after the first dose on day 1 and for 72 h at steady state on day 6. Plasma and urine samples were analyzed for free and conjugated butorphanol, HO-B, and NOR-B. The plasma samples were analyzed using validated gas chromatography-electron capture negative chemical ionization-mass spectrometric methods and the urine samples were analyzed using a validated HPLC procedure. In the plasma, conjugated metabolites were not detected and only trace amounts of NOR-B were present. Therefore, pharmacokinetic parameters could not be estimated for NOR-B and conjugated metabolites. AUC0-->infinity of butorphanol after the first dose and AUC0-->tau at steady state were not statistically different, indicating that the kinetics of butorphanol were not significantly altered after repeated dosing. Steady state levels of butorphanol were attained within 3 days (d) of q6h dosing and the accumulation index was 1.2 for butorphanol. Due to a relatively long t1/2 of 15 h of HO-B compared to the dosing interval (q6h), the accumulation index was 6.0 for this metabolite. The evaluation of the molar plasma concentration ratio of HO-B to butorphanol as a function of time revealed that HO-B exhibits elimination-rate-limited kinetics. Similarly to butorphanol, steady state levels of HO-B were attained within 3 d of q6h dosing.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantitation of human CTLA4Ig in mouse serum was validated. The EIA method employed a technique in which a monoclonal anti-CTLA4 antibody was adsorbed onto 96-well polystyrene microtiter plates and used to capture the CTLA4Ig in mouse serum samples. The captured CTLA4Ig was then detected using a goat anti-human IgGFc antiserum conjugated to the enzyme horseradish peroxidase. The validation included assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLQ), inter-analyst robustness, storage stability in mouse serum and assay specificity. The results indicate that this validated assay is precise, accurate, and reproducible. This EIA has a range of reliable response in 10% mouse serum of 0.14-4.58 ng ml-1 resulting in a 100% serum equivalent curve of 1.4-45.8 ng ml-1. Assessment of individual standard curve variations indicated a reproducible response with R2 values ofor = 0.995. The LLQ was established at 1.4 ng ml-1. The accuracy and precision estimates, based on the quality control values, were within 3.8% and 5.2% respectively, for CTLA4Ig. Stability of CTLA4Ig was established in mouse serum for 5 days at both 4 degrees C and room temperature, for 2 months at -70 degrees C and through five freeze-thaw cycles. This validated assay was successfully employed in the assessment of pharmacokinetic characteristics of CTLA4Ig in mice and to aid in the selection of an optimal CTLA4Ig-producing cell line.

Purpose. The objectives of this study were: to delineate the pharmacokinetics of CTLA4Ig in rats after single and multiple intravenous (IV) and subcutaneous (SC) doses; to assess the relationship of the pharmacokinetic parameters of CTLA4Ig vs dose; to calculate the SC absolute bioavailability; and to assess the antibody response of CTLA4Ig. Methods. A total of 48 (24 male and 24 female) Sprague Dawley rats were divided into eight treatments with 3 rats per gender in each group: a single dose of 10, 80, or 200 mg/kg of CTLA4Ig given either IV or SC and a repeated dose of 10 mg/kg (once every other day for 7 doses over 13 days) given either SC or IV. Serial blood samples were collected up to 43 days after single dose administration and up to 50 days following the administration of the last multiple dose on day 13. The serum concentration of CTLA4Ig and anti-CTLA4Ig antibodies were measured using ELISA assays. Results. After single IV doses, Cmax and AUCinf increased in a dose proportional manner; CL appeared to be dose independent, while both Vss and T1/2 increased as the administered dose increased. Following single SC doses, Cmax and AUCinf increased in a linear manner but not proportionally; mean Tmax values were prolonged but similar among the three dose levels, while T1/2 increased as the administered dose increased. The absolute SC bioavailability of CTLA4Ig decreased as the dose increased from 10 (62.5%), 80 (55.7%), and 200 mg/kg (41.1%). Comparison of the AUCtau values between the first and last doses suggested an accumulation (3.1−4.7) of CTLA4Ig. However, regardless of the route of dosing, AUCtau after the last dose were comparable to AUCinf values following the single dose. Anti-CTLA4Ig antibodies were detected at the 10 mg/kg dose level after single or multiple doses for both routes of administration. However, regardless of single or multiple doses, antibody titers were relatively greater for the SC compared to the IV administration. Conclusions. The key findings of this study were: (i) the elimination characteristics of CTLA4Ig were comparable between the SC and IV routes; (ii) the repeated dosing did not alter the pharmacokinetics of CTLA4Ig; (iii) the SC absolute bioavailability tended to decrease as the administered dose increased; and (iv) a greater formation of anti-CTLA4Ig antibodies was observed after SC compared to IV at a single 10 mg/kg dose level; however, after multiple dosing, the formation of antibodies from either of the two routes was relatively slower, and (v) during the study period, no antibodies were observed at either the 80 or 200 mg/kg dose levels regardless of the route of administration.

Alisertib (MLN8237) is an investigational inhibitor of Aurora A kinase (AAK). Aurora A plays an essential role in the regulation of spindle assembly and chromosome alignment during mitosis. Inhibition of Aurora A by alisertib in tissue culture has previously been demonstrated to lead to improper chromosomal alignment and disruption of spindle organization, resulting in a transient mitotic delay. The spindle organization defects induced by alisertib have been used to develop a pharmacodynamic (PD) assay for Aurora A inhibition based on the percentage of mitotic cells with proper chromosomal alignment at the metaphase plate (% aligned spindles, abbreviated as AS). The transient mitotic delay that occurs with AAK inhibition permits the use of the mitotic index (the fraction of cells in the population currently undergoing mitosis, abbreviated as MI) as an additional PD assay. When the two PD assays were used in Phase I clinical trials, the reduction in AS was strongly correlated with dose levels and exposures in patients from single time point PD measurements; however, MI failed to show any correlation. To further understand this clinical finding, we constructed PK/PD/efficacy models for AS and MI that can precisely capture the temporal dynamics of the PD markers from in vivo xenograft studies. A PK/PD study was conducted using a single oral dose of alisertib at 3, 10, and 20 mg/kg in HCT-116 xenografts implanted subcutaneously in mice. An extravascular, two-compartmental pharmacokinetic (PK) model was used to describe the drug kinetics. Consistent with the mechanistic hypothesis for AAK inhibition, the PD biomarkers such as AS and MI were fitted to PK using a direct response inhibitory sigmoid model and an indirect response turnover model, respectively. The antitumor activity of alisertib dosed orally for 21 days with different dose levels and schedules was evaluated. The PK/PD models showed a fast, sustained response for AS after alisertib administration, whereas MI exhibited a slow, transient response. The PK/efficacy relationship for alisertib in HCT-116 xenografts closely corresponds to the PK/PD relationship for the PD markers, with all three IC50s in close agreement (303, 270, and 280 nM, respectively). The PK/PD and PK/efficacy models show that both AS and MI are equally relevant as mechanism-based PD markers to capture drug activity. However, of the two PD markers, the fast, sustained response of AS makes it the only clinically viable PD marker for defining a dose–response relationship, as its maximal effect can be captured from a wider time window with a single PD sampling; while the window to capture dose-related MI response is narrower.

Objective The objective of the study was to investigate the effects of hepatic impairment on the absolute transnasal bioavailability and pharmacokinetics of butorphanol. Study Design Twelve (eight men and four women) helathy subjects and 12 (eight men and four women) patients with hepatic impairment received a 1 mg dose of butorphanol by intravenous or transnasal administration on two separate occasions. Hepatic function was assessed by antipyrine and indocyanine green clearance tests. Serial blood and urine samples were collected after each dose. Plasma samples were analyzed for butorphanol, and urine samples were analyzed for butorphanol and its metabolites. Results No statistical difference in maximum plasma concentration (Cmax) for butorphanol was observed between the two groups of volunteers after transnasal administration. However, total plasma clearance (CL), steady-state volume of distribution, area under the concentration-time curve [AUC(0-∞)], and elimination half-life of butorphanol in patients with hepatic impairment were significantly altered (~two-fold to threefold). The absolute transnasal bioavailability of butorphanol was significantly higher (~20%) in patients with hepatic impairment. A greater fraction of the administered dose was recovered from the urine in hepatically impaired patients compared to that in healthy subjects (23% to 31% versus 10% to 11%). There was a significant reduction in CL of indocyanine green and antipyrine in hepatically impaired patients. The percentage of reduction in butorphanol CL was highly correlated to the estimated degree of portosystemic shunts in the patients with hepatic impairment. Conclusion Based on the comparable Cmax but the increased AUC in patients with liver dysfunction, the initial dose of butorphanol nasal spray may not need to be adjusted. However, the subsequent dosing intervals for butorphanol should be prolonged. Clinical Pharmacology & Therapeutics (1996) 60, 283–294; doi

Two separate analytical methods have been developed for the determination of butorphanol and its metabolites in human plasma. One method is specific for butorphanol (I) while the other determines the metabolites, hydroxybutorphanol (II) and norbutorphanol (III). Both procedures incorporate solid-phase extraction, chemical derivatization and separation, and detection using gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry (GC-ECNCI-MS). Both methods use the cyclopropyl analog of I (BC-2605, IV) as the internal standard and the procedures for extraction of the analytes from plasma are identical. However, following extraction, either the pentafluorobenzoyl ester of I or the tris and bis-trifluoroacetyl esters of II and III, respectively, were prepared. The derivatives were analyzed by GC-ECNCI-MS with selected-ion monitoring of the molecular ions. The standard curves were linear over the concentration ranges of 20-2000, 20-1000 and 50-1000 pg/ml for I, II and III, respectively. All standard curves from the assay validation had r2 values of > or = 0.994, 0.991 and 0.985 for I, II and III, respectively. For all three compounds, the intra- and inter-assay precisions (C.V.) and inter-assay accuracy (deviation from nominal) were within 12% for plasma quality control samples. All derivatives were stable in the reconstitution solvent for at least 24 h. The assays are being used for the determination of plasma concentrations of I, II and III in humans following repeated administration of nasal spray.

A specific, accurate, precise, and reproducible High-performance liquid chromatographic-Ultraviolet (HPLC-UV) method was developed for the simultaneous quantitation of BMS-181101 (I), a new antidepressant, and its putative metabolites, 6'-hydroxy (II) and 7'-hydroxy (III) of BMS-181101 in rat and monkey plasma. The assay procedure involved solid-phase extraction of the three analytes and the internal standard (IS; BMY-42568) on 1 mL Bond Elut CN cartridge using an automated solid phase extraction controller (ASPEC) system. The final elution of the analytes was performed using 0.25% triethylamine in methanol. The eluate mixture was evaporated to dryness, the residue was reconstituted in the mobile phase and injected onto a Zorbax Phenyl column (4.6 x 250 mm; 5 microns) at a flow-rate of 1.2 mL/min. The mobile phase consisted of 20% acetonitrile, 10% methanol, 69% water and 1% 1.0 M ammonium phosphate and 1.0 M tetramethylammonium hydroxide mixture adjusted to pH 3 by phosphoric acid. An ultraviolet absorbance detector set at 287 nm was used to detect the analytes. The nominal retention times were 5, 8, 15, and 18 min for II, III, I, and IS, respectively. The standard curves for the three analytes were linear in the concentration range of 50-1000 ng/mL. The lower limit of quantitation was 50 ng/mL for each analyte. The analyses of quality control (QC) samples indicated that the nominal values could be predicted with an accuracy of (+/-) 10.5% for all three analytes in rat and monkey plasma. The precision values of the QC samples for all three analytes were within 12.7% RSD for rat and monkey plasma. All three analytes and the IS were stable in the autosampler for at least 38 h; freeze/thaw stability of the 3 analytes was established for three cycles. Stability of BMS-181101 was established for one month at -20 degrees C. The application of the assay to a pharmacokinetic study in monkey is described.

Nadolol, a nonspecific beta-blocker, is a racemate composed of equal amounts of four stereoisomers, namely, SQ-12148, SQ-12149, SQ-12150, and SQ-12151. In an open-label, randomized, four-period crossover study, the pharmacokinetics of nadolol and its stereoisomers and the bioequivalence of two formulations of nadolol were assessed in 20 healthy male subjects following a single dose (80 mg) and multiple doses (80 mg; once daily for 7 days). A standard granulated tablet and direct compressed tablet formulations, each containing 80 mg of nadolol, with different in vitro dissolution profiles that met current USP requirements were used. The four treatments were single and multiple doses of granulated tablet, and single and multiple doses of compressed tablet. There was a 7 day washout period between successive treatments. All doses of nadolol were administered after an overnight fast. Serial blood samples were collected up to 72 h following the single dose and during multiple dose treatments, following day 6 and 7 doses. Validated high-performance liquid chromatographic assays were applied to measure nadolol and its stereoisomers in the study samples. Plasma concentration data were subjected to noncompartmental pharmacokinetic analysis. Both C(max) and AUC values were significantly greater for SQ-12150 when compared to other nadolol stereoisomers obtained after a single dose or at steady state. However, T(max) and T1/2 values were similar among the four isomers. The observed steady state AUC tau values for nadolol (2278-2331 ng h/ML) or its stereoisomers (550-874 ng h/ML) were significantly greater than those predicted from the single dose AUCinf values (nadolol, 1840-1845 ng h/ML; isomers, 450-713 ng h/ML). The intrasubject variability, computed from multiple dose data, was generally greater for the stereoisomers (17-40%) than for nadolol (10-32%). The two formulations were bioequivalent for nadolol (C(max) = 0.98 [84%, 117%]; AUCinf = 1.03 [93%, 116%]) and SQ-12150 (C(max) = 1.12 [89%, 122%]; AUCinf = 0.98 [82%, 119%]) after a single dose, and only for nadolol (C(max) = 1.07 [84%, 118%]; AUCinf = 1.02 [91%, 113%]) at steady state.

During preclinical development of investigational compound, MLN1117, combination efficacy studies of MLN1117 and Taxotere in mice bearing lung cancer tumor xenografts indicated the presence of a scheduling effect. In many instances, pre-dosing Taxotere resulted in improved tumor growth inhibition as compared to concomitant dosing with MLN1117. Furthermore both in vitro and in vivo PD studies demonstrated that sequential administration of MLN1117 (PI3Ka inhibitor) and Taxotere resulted in increased apoptosis as compared to concomitant treatment. To further investigate and translate these observations, combination PK/E modeling was performed on preclinical PK and efficacy data. A scheduling efficacy study was executed in small cell lung cancer NCI-H1048 model. The study was designed to compare the anti-tumor activity of the combination under varying levels of PK concomitance between the two compounds. Using a non-linear mixed effects approach, dynamic PK/E modeling was performed to describe the individual mouse tumor growth curves as a function of the instantaneous plasma drug concentration of MLN1117 and Taxotere. The combination effect in the dynamic model was described using the following expression: (PK/E)MLN1117 + (PK/E)Taxotere + Tau* (PK/E)Taxotere * (PK/E)MLN1117 The combination interaction (Tau) between MLN1117 and Taxotere was estimated to be negative but associated with significant inter-tumor variability. A negative Tau implied the combination behaved sub-additively under conditions of concomitant dosing. Pre-dosing Taxotere made the combination non-concomitant which explains the improved anti-tumor activity associated with it. The variability associated with the positive effects of pre-dosing Taxotere or non-concomitant dosing was attributed to the variability in Tau. In general, the modeling results favored the use of non-concomitant dosing for the MLN1117/Taxotere combination to eliminate the dependence of the combination efficacy on the negative Tau. Although the emphasis of this study has been on SCLC cancer model NCI-H1048, early preclinical data from other cancer models of different origin indicate that this phenomenon could be ubiquitous. The combination PK/E model though empirical in nature, provided a useful and translatable tool to guide combination schedule selection. The modeling framework focused on understanding the behavior of the combination i.e. combination interaction term (Tau) and translating this understanding to optimize combination scheduling choices for the clinic. Citation Format: Ekta Kadakia, Natasha Iartchouk, Karuppiah Kannan, Keli Song, Dong Mei Zhang, Christopher Zopf, Christopher Zopf, Munjal Patel, Chirag Patel, Swapan Chowdhury, Wen Chyi Shyu, Jing-Tao Wu, Arijit Chakravarty. Application of preclinical combination pharmacokinetic(PK)/efficacy(E) modeling to investigate and translate the preclinical scheduling effect for MLN1117 and Taxotere combination. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B154.

A series of three-way crossover randomized studies were conducted to evaluate the absolute bioavailability of butorphanol, a potent agonist-antagonist analgesic, from transnasal, sublingual, and buccal disk formulations in order to identify a practical alternative to oral administration. In each study, healthy male volunteers received 2 mg doses of butorphanol tartrate intravenously and either transnasally, sublingually or buccally. Serial blood samples were collected over 12 h and butorphanol plasma concentrations were determined by radioimmunoassay. The plasma concentration data were subjected to non-compartmental pharmacokinetic analysis. The elimination half-life of butorphanol was about 3-5 h and was independent of the route of administration. Absorption of butorphanol following transnasal administration was faster than that observed following sublingual or buccal administration. Mean absolute bioavailabilities of sublingual tablet and buccal disk formulation were only 19 per cent and 29 per cent, respectively, but for transnasal administration the value rose significantly, to 70 per cent. Based on the results of these studies, transnasal dosage form of butorphanol was selected for further clinical trials of treatment of moderate to severe pain.

Penetration of cefprozil into tonsillar and/or adenoidal tissues was investigated for patients undergoing tonsillectomy and/or adenoidectomy. A total of 29 patients ranging in age from 2 to 14 years participated in the study. The tonsils and/or the adenoids were removed at times ranging from 0.33 to 3.17 h after oral administration of a dose of either 7.5 or 20 mg/kg of body weight. A blood sample was also collected as soon as the tissue sample was removed. Plasma, tonsil, and adenoid samples were analyzed for cis and trans isomers of cefprozil by high-performance liquid chromatographic assays. The concentrations of the cis isomer of cefprozil in plasma ranged from 0.60 to 9.87 micrograms/ml at the 7.5-mg/kg dose level and from 1.04 to 20.40 micrograms/ml at the 20-mg/kg dose level. The corresponding concentrations of the cis isomer in tonsil tissue ranged from 0.48 to 2.42 micrograms/g and from 1.00 to 4.29 micrograms/g, respectively. The corresponding concentrations of the cis isomer in adenoid tissue ranged from 0.40 to 4.20 micrograms/g and from 1.74 to 4.94 micrograms/g, respectively. The concentrations of the trans isomer were about 1/10 of those observed for the cis isomer. The median ratios of the cefprozil concentration in tonsillar tissue to that in plasma were 0.37 and 0.47 for patients receiving a 7.5- or a 20-mg/kg oral dose of cefprozil, respectively. The corresponding median ratios for the adenoidal tissue were 0.46 and 0.82, respectively. The cefprozil concentrations in either the tonsillar or the adenoidal tissue at both dose levels over 3.17 h after dosing are much higher than the MICs for common pathogens which cause pharyngitis or tonsillitis.

Didanosine is a purine nucleoside analogue approved for the treatment of human immunodeficiency virus infection. It is extremely unstable at pH values less than 3 and requires protection against gastric acid-induced hydrolysis. Beagle dogs pretreated with pentagastrin, an analogue of gastrin that reproducibly stimulates gastric acid secretion, have been used to screen different didanosine formulations. The absolute bioavailability of didanosine from a saline solution decreased from approximately 43% in untreated dogs to 8% after pretreatment with pentagastrin. Administration of buffered solution of didanosine to untreated and pretreated dogs yielded bio-availability estimates of 37 and 30%, respectively. In humans, the bioavailability from a similar buffered solution was approximately 40%. Pentagastrin-pretreated dogs were used to evaluate four new products relative to a citrate-phosphate buffer sachet, the formulation selected for large-scale clinical trials in humans. Two of these new formulations, a chewable tablet and an antacid suspension, were more bioavailable then the reference sachet. This also proved to be true in man, necessitating an adjustment in the dose of didanosine when administered as the chewable tablet. Dogs pretreated with pentagastrin accurately predicted the improved bioavailability of new didanosine formulations prior to clinical use. This animal model may be helpful in evaluating the biopharmaceutics of other acid-labile drugs.

The effect of antacid on the bioavailability of cefprozil was investigated in a two-way crossover study. Eight healthy male subjects received a single 500-mg oral dose of cefprozil with and without coadministration of 30 ml of an antacid suspension containing magnesium hydroxide and aluminum hydroxide (Maalox). Cefprozil consists of cis and trans isomers in an approximate 90:10 ratio. When cefprozil was administered alone (treatment A), the mean maximum concentrations (Cmax) of the cis and trans isomers were 9.2 and 1.2 micrograms/ml, respectively. When cefprozil was coadministered with Maalox (treatment B), the Cmax values of the cis and trans isomers were 8.7 and 1.3 micrograms/ml, respectively. The mean values of the area under the curve from time zero to infinity (AUC0-infinity) were 27.7 and 3.5 micrograms.h/ml for treatment A and 27.5 and 3.5 micrograms.h/ml for treatment B for the cis and trans isomers, respectively. The other pharmacokinetic parameters, time to Cmax, elimination half-life, mean residence time, renal clearance, and percent urinary excretion, were essentially the same for the two isomers. The respective values of the elimination half-life for the cis and trans isomers were 1.36 and 1.32 h for treatment A and 1.36 and 1.42 h for treatment B. Mean urinary excretion was 63 and 60% for treatment A and 58 and 56% for treatment B for the cis and trans isomers, respectively. No significant differences between the two treatments were found for any of the pharmacokinetic parameters for either isomer. For the cis isomer, bioavailability point estimates (90% confidence intervals) of the mean Cmax and AUG0-infinity values for the Maalox treatment relative to those for the reference treatment were 95% (87%, 103%) and 99% (95%, 104%), respectively. For the trans isomer, the value were 109% (92%, 126%) for Cmax and 97% (88%, 106%) for AUC0-infinity. On the basis of the results of this study, it is concluded that the bioavailability of cefprozil is not affected by the coadministration of Maalox.

Monitoring the exposure of a drug and its metabolites in humans and preclinical species during drug development is required to ensure that the safety of drug-related components in humans are adequately assessed in the standard toxicology studies. Recently published FDA guidance on metabolites in safety testing (MIST) has generated broad discussion from various perspectives. Most of the opinions and experiences shared among the scientific community are scientifically sound and practical. There are various approaches to assess the metabolite exposure margin between toxicology species and humans: either by direct or indirect comparison or by qualitative or quantitative comparison. The choice of when and how to pursuit metabolite assessment is based on the overall development strategy of the compound. Therefore, it is important to understand the utility and limitations of analytical instruments in order to apply an appropriate analytical tool to address specific questions posed at different stages of drug development. The urgency of metabolite monitoring depends on the intrinsic nature of the compound, therapeutic intent and objective of the clinical development. The strategy for assessing metabolite exposure in humans should be a holistic approach considering clinical situations and cumulative knowledge of the metabolism of the drug in order to appropriately address metabolite safety in humans. A one-size-fits-all approach is rarely the best use of resources.

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

Cefprozil, a new broad-spectrum oral cephalosporin, is composed of cis and trans isomers in an approximate 90:10 ratio. The pharmacokinetics of a single oral 1000-mg dose of cefprozil were evaluated in 6 healthy subjects and 24 patients with various degrees of renal impairment. Six of these subjects were studied both while receiving hemodialysis and during an interdialytic period. Plasma, urine, and hemodialysate that were collected at predetermined times were analyzed for concentrations of the cis and trans isomers of cefprozil using reverse-phase HPLC assay with UV detection. The maximum plasma concentration of the cis isomer ranged from 12.3 micrograms/mL in subjects with normal renal function to 36.7 micrograms/mL in hemodialysis patients. Similarly the area under the plasma concentration-time curve and the elimination half-life increased from 46 micrograms.h/mL to 373 micrograms.h/mL and from 1.72 hours to 5.94 hours, respectively. Renal clearance of the cis isomer decreased from 198 mL/min in normal subjects to 19 mL/min in volunteers with creatinine clearances of less than or equal to 30 mL/min; there was a strong correlation (r2 greater than or equal to .93) between the renal clearance of the cis isomer and creatinine clearance. Urinary recovery of the cis isomer decreased from 57% in those with normal renal function to 24% in the group with a creatinine clearance of less than or equal to 30 mL/min. Hemodialysis decreased the half-life of the cis isomer to 2 hours and removed approximately 55% of it from the body during a 3-hour dialysis period (hemodialysis clearance equaled approximately 87 mL/min). The pharmacokinetics of the trans isomer were similar to those observed for the cis isomer and were affected similarly by declining renal function. A reduction in dosage is recommended in patients with a creatinine clearance of 30 mL/min or less. It may be necessary to administer a dose after hemodialysis to maintain therapeutic plasma concentrations.

Cefprozil, a new oral cephalosporin, consists of a 90:10 cis:trans isomer mixture. Sensitive, specific and reproducible high performance liquid chromatographic methods have been developed for the simultaneous quantification of the two stereoisomers of cefprozil in plasma and urine samples from human and rats. Cephalexin acted as the internal standard. Plasma protein was precipitated with acetonitrile and trichloracetic acid with subsequent extraction of acetonitrile. After vortexing and centrifuging, the aqueous phase was injected onto a reverse phase C8 column. Urine samples were acidified with sodium acetate buffer (pH 3.8) and then directly injected onto a reverse phase C18 column. The detector was set at 280 nm. These methods were applied to determine protein binding of both isomers in human and rat sera, and to perform a pharmacokinetic study in human. Results showed that both isomers bound moderately to serum proteins with no interference by the other isomer. The pharmacokinetic study in human indicated that cefprozil was well absorbed and the cis and trans isomers have similar pharmacokinetics.

The objectives of this study were to assess the safety and tolerance of cefprozil, to characterize the pharmacokinetics of cefprozil after administration of multiple doses of the drug, and to compare these pharmacokinetic parameters with those obtained with cefaclor. The volunteers received 28 doses of 250, 500, or 1,000 mg of cefprozil or 500 mg of cefaclor every 8 h for 10 days. Serial blood samples and the total volume of urine voided by each individual were collected for pharmacokinetic evaluation on days 1, 5, and 10. Both cephalosporins were well tolerated after multiple oral dosing. The peak levels in plasma (Cmax) of cefprozil ranged from 5.7 to 18.3 micrograms/ml after oral administration of 250- to 1,000-mg doses. The regression analysis of Cmax on cefprozil dose showed a dose-linear response. The mean Cmax of cefaclor ranged from 15.2 to 16.7 micrograms/ml and did not change significantly on multiple dosing. The overall mean terminal half-life of cefprozil was 1.2 h and was invariant with respect to dose or duration of dosing. The area under the plasma-concentration-versus-time curve from 0 h to infinity (AUC0-infinity) of cefprozil increased in a dose-proportional manner with an increase in dose. The overall urinary recovery (61% of dose) and renal clearance values of cefprozil were generally invariant with respect to dose and duration of dosing. While cefprozil was apparently absorbed less rapidly and achieved lower Cmax values than cefaclor, the AUC0-infinity of cefprozil was nearly twofold greater than that of cefaclor. The half-life of cefprozil was also twofold longer than that observed for cefaclor. Although the urinary recovery of cefaclor (75% of dose) was significantly higher than that of cefprozil (61% of dose), the concentrations of cefprozil in urine remained significantly higher than those of cefaclor from 2 to 8 h postdosing. If the therapeutic concept is maintained that levels of beta-lactam antibiotics in plasma should exceed the MIC for the offending organisms over a period that approximates the dosing interval, then cefprozil would appear to be suitable for twice-daily administration, whereas cefaclor should probably be administered three or even four times a day.

The objective of this study was to assess the effects of food on the pharmacokinetics of cefprozil and cefaclor. A group of 12 healthy male volunteers received a single 250-mg dose of cefprozil or cefaclor under fasting conditions as well as after the intake of food. There was a 1-week washout period between each treatment. Serial blood samples were collected and assayed for cefprozil or cefaclor by specific high-pressure liquid chromatographic methods. The mean +/- standard deviation peak concentration (Cmax) of cefprozil in plasma was 6.13 +/- 1.22 micrograms/ml under the fasting condition and 5.27 +/- 1.06 micrograms/ml after breakfast, and these values were not significantly different from each other. The corresponding median time to reach Cmax was prolonged after food intake, but this difference was not significant. The mean Cmax values of cefaclor decreased significantly from 8.70 +/- 2.72 micrograms/ml under the fasting condition to 4.29 +/- 1.52 micrograms/ml after breakfast, and the corresponding median times to reach Cmax were significantly prolonged. The mean half-lives of cefprozil and cefaclor were nearly identical for the two treatments, suggesting that the elimination kinetics of these cephalosporins remained unaltered when the drugs were administered with food. The area under the plasma-concentration-versus-time curves for fasted and fed conditions were not significantly different for both drugs. The results of this study indicate that the extent of absorption and rate of elimination of both cephalosporins remain unaltered in the presence of food. However, the absorption rate of cefaclor is significantly reduced in the presence of food, while that of cefprozil remains unaltered. As a result, the Cmax of cefaclor is significantly reduced in the presence of food, whereas that of cefprozil is not significantly affected. Cefprozil can be administered with a meal without markedly affecting levels in blood.

Aims Single dose pharmacokinetics and safety of irbesartan, an angiotensin II receptor antagonist, were evaluated in healthy young and elderly male and female subjects. Methods Irbesartan was administered as two 25 mg capsules after a 10 h fast to 12 young men, 12 young women, 12 elderly men and 12 elderly women. Serial blood and urine sample were collected up to 96 h after the dose. Plasma and urine samples were analysed for irbesartan by h.p.l.c./fluorescence methods. Results No statistically significant gender effects were observed in peak plasma concentration (Cmax ), area under the curve (AUC), and terminal elimination half-life (t1/2 ) of irbesartan. The geometric mean AUC and Cmax increased by about 43% and 49%, respectively, in the elderly subjects. Also the time to peak was significantly shorter in the elderly subjects compared with that observed in the young subjects. Renal clearance of irbesartan was significantly reduced in the elderly females but this reduction is not likely to be of any clinical relevance since less than 3% of the administered dose of irbesartan is excreted unchanged in the urine. Conclusions Although there was an effect of age on the pharmacokinetics of irbesartan, based on the safety and efficacy profile, no adjustment in irbesartan dosage is necessary with respect to age or gender.

2-{Butyryl-[2'-(4,5-dimethyl-isoxazol-3-ylsulfamoyl)-biphenyl-4-ylmethyl]-amino}-N-isopropyl-3-methyl-butyramide (BMS-1) is a potent dual acting angiotensin-1 and endothelin-A receptor antagonist. The compound was subject to rapid metabolic clearance in monkey and human liver microsomes and exhibited low systemic exposure and marked interanimal variability in cynomolgus monkeys after p.o. administration. The variability pattern was identical to that of midazolam given p.o. in the same monkeys, as measured by area under the curve and Cmax values, suggesting that CYP3A-mediated metabolism might play a role in the rapid clearance and observed interanimal variability. Subsequent in vitro metabolism studies using human liver microsomes and cDNA-expressed human cytochrome P450 (P450) enzymes revealed that BMS-1 was a CYP3A4 substrate and was not metabolized by other human P450 enzymes. Mass spectral and NMR analyses of key metabolites led to the identification of the dimethyl isoxazole group as a major metabolic soft spot for BMS-1. Replacement of the 4-methyl group on the isoxazole ring with halogens not only improved overall metabolic stability but also decreased CYP3A-mediated hydroxylation of the isoxazole 5-methyl group. As exemplified by 2-{butyryl-[2'-(4-fluoro-5-methyl-isoxazol-3-ylsulfamoyl)-biphenyl-4-ylmethyl]-amino}-N-isopropyl-3-methyl-butyramide (BMS-3), a fluorinated analog of BMS-1, the structural modification resulted in an increase in the systemic exposure relative to previous analogs and a dramatic reduction in interanimal variability in the monkeys after p.o. administration. In addition, BMS-3 could be metabolized by both CYP2C9 and CYP3A4, thus avoiding the reliance on a single P450 enzyme for metabolic clearance. Integration of results obtained from in vitro metabolism studies and in vivo pharmacokinetic evaluations enabled the modulation of site-specific CYP3A-mediated metabolism, yielding analogs with improved overall metabolic profiles.

Introduction Xenograft response has been shown to hold predictive value for the clinical efficacy of a drug, but growth sensitivity to a drug candidate can vary widely between xenografts models. As the ability to test pre-clinically for broad biological activity across many xenografts models is often resource-limited, in this work we develop a mathematical approach based on evolutionary dynamics to derive dose-response information from a single xenograft tumor. Methods We propose to estimate a dose-response curve of xenograft growth inhibition by treating a single tumor with a series of different dose strengths. Exponential tumor growth rate (GR) may be quickly estimated to determine the inhibition effect of a particular exposure during week-long intervals of constant dose strength. We next considered the possibility of a dose strength ordering effect on tumor growth due to cellular heterogeneity in GR and drug sensitivity within a tumor. To determine to what extent dose history could influence the observed growth inhibition, we developed an evolutionary model of tumor growth for a heterogeneous population of cancer cells with joint-distributed clonal GR and drug sensitivities, and simulated tumor response to treatment for distributions having variable degrees of correlation. Finally, we tested the ability to estimate dose-response curves from single tumors in vivo compared to the typical method of assessing drug effect based on multiple dose-groups. Results For the three possible orders of dosing three different treatment strengths, we found that the simulated study design with escalating dose intervals lead to the least error compared to assessing each dose in a separate tumor. Additionally, the discrepancy between the growth inhibition effects of different dose orders peaks when the correlation between GR and drug sensitivity decreases to negative. We then show that this study design allows efficient, accurate estimation of the range of dose-responses in a broad set of simulated tumor types (e.g., fast-growing and sensitive, or slow-growing and insensitive). An in vivo dose-response study validates the estimation of dose-response curves from a single xenograft by comparing favorably with the single dose per mouse method. Conclusions The mathematical approach described here allows efficient estimation of a dose-response curve for a xenograft model using a single tumor. By reducing the resources required to determine the response of a single model, a molecule's anticancer effect can be tested against a larger sample of tumor types pre-clinically. A more accurate picture of a drug candidate's broad biological activity will allow for greater confidence in translational projections of clinical efficacy. Citation Format: Andrew Chen, Christopher J. Zopf, Jing-Tao Wu, Wen Chyi Shyu, Arijit Chakravarty. Xenograft dose-response from intra-mouse dose escalation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2015-3242

Introduction Tumors are often composed of a heterogeneous cell population growing over time subject to the forces of evolutionary dynamics. These principles offer insights not only for bulk tumor growth, but also for small cell populations such as in metastases or de-bulked tumors following treatment. In this work, we investigate whether the evolutionary effects of small population sizes are evident in in vivo pre-clinically through a meta-analysis of xenograft rebound growth kinetics following treatment. Methods We first used a HCT116 cell fitness distribution we have previously measured in soft agar to demonstrate the expected population fitness trajectory as a function of starting population size. We sampled subclone fitnesses from this distribution to initialize either large or small theoretical tumors, and simulated tumor growth as a Moran process to find the probability distribution of expected outcomes in final tumor population fitness. Since initial tumor volumes in xenografts experiments have a narrow range of volume (equivalently, population size), we next identified tumor volume measurements following suspension of treatment with anticancer agents from in-house historical xenograft growth datasets. Each of 1359 tumor volume trajectories after treatment was fit using non-linear regression to determine the exponential growth rate (population fitness), and compared to both the volume at treatment end as well as the average control group growth rate from the corresponding experiment. Results The simulated tumors demonstrate the “founder effect” in heterogeneous populations: large populations become dominated by the fastest growing, most fit subclones while small populations are sensitive to initial clonal sampling as well as random births and deaths. Rebound growth in xenografts post-treatment displayed large variability compared to the growth of the corresponding experimental control group. Post-treatment and untreated growth rates are poorly correlated (R2 = 0.1) consistent with a significant change in the tumor cell population following a period of selective pressure. Two additional observations are coherent with a founder effect following a bottleneck event (i.e., treatment). The relationship between post-treatment starting volume and growth rate has a positive slope (p = 0.01, F-test) while growth rate variance decreases with starting volume (p < 0.01 F-test). Both results concur with the Moran simulations showing smaller populations have more stochastic outcomes while larger populations deterministically drift to greater fitness. Conclusions We find that xenograft growth kinetics following cessation of treatment with anticancer agents are consistent with a selection process and the founder effect. Application of evolutionary theory to small cell populations in preclinical models may yield new strategies to combat metastases and resistance emergence. Citation Format: Andrew Chen, Christopher J. Zopf, Jing-Tao Wu, Wen Chyi Shyu, Arijit Chakravarty. Xenograft rebound kinetics are consistent with the founder effect. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3759. doi:10.1158/1538-7445.AM2015-3759

Introduction Finding the ideal dosing schedule to optimize efficacy while balancing toxicity is a challenge for single agent clinical development, and takes on added complexity for combinations. In this work, we use theoretical modeling to investigate the role of schedule in determining combination activity, and propose a novel method of using dosing schedule to identify the in vivo interaction strength pre-clinically. Methods To evaluate the effect of schedule on tumor growth inhibition, we built a dynamic pharmacokinetic (PK)/effect (E) model to simulate tumor volume as a function of plasma concentration of a drug combination. The PK of each single agent was simulated using a one compartment model, and the inhibition of tumor growth rate was modeled as the sum of single agent effects and their product scaled by an interaction coefficient. We first simulated this model for various relative dosing frequencies and offsets between the two drugs to understand the relationship between schedule and activity for a synergistic combination. We next assessed the accuracy of estimating the interaction parameter using the activity discrepancy between in- and off-phase dosing schedules versus a traditional isobologram analysis. Using Fourier analysis of single agent PK profiles, we then identified an efficient study design to identify both the interaction parameter and optimal relative dosing schedule of the two drugs. Results Simulation of the PK/E model with parameter sweeps of dosing frequency and offsets led to a phase plot for combination activity showing peaks and valleys related to the concomitant exposure of the two drugs. From this, we surmised the interaction could be estimated based on the difference in tumor growth inhibition between schedules, and a comparison of in- and off-phase schedules performed well compared to isobologram analysis. Fourier analysis of simulated PK profiles of the two drugs revealed the concomitance as a function of dosing offset, and the pattern was maintained in the simulated activity. Using a study design with only four dose groups with different dosing offsets, we show it is possible to determine both the interaction coefficient (comparing one group at the peak and one at the valley of concomitance) and any effect of dose-ordering (two groups at different offsets but with the same predicted concomitance). Additionally, we demonstrate the difference in activity between synergistic and additive combinations manifests as beat frequencies present in the frequency spectrum, another possibility to identify synergy. Conclusions While combination development offers unique challenges, building an understanding of the PK/E relationship from first principles provides a framework to investigate drug interaction effects. The insights gained from studying combinations in vivo with a pre-clinical scheduling study may provide translational guidance on clinical questions around concomitance and dose-ordering. Citation Format: Andrew Chen, Jing-Tao Wu, Wen Chyi Shyu, Arijit Chakravarty, Christopher J. Zopf. Dosing schedule effects on combination activity from first principles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4527. doi:10.1158/1538-7445.AM2015-4527

e20726 Background: Therapeutic intervention in oncology often results in thrombocytopenia. Clinical schedule modification may be used to mitigate thrombocytopenia in patients. Prior knowledge of th...

e22169 Background: Metrics such as RECIST and % Change from baseline (%CFB) are regularly used as efficacy end-points in Phase-1 to estimate response rate and establish dose-response relationships....