Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein.

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supraproportional at the lower doses (50-200 mg) and approximately dose-proportional at the higher doses (200-600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model on the basis of the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics on the basis of the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-glycoprotein inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute an element in the PBPK models critical to understanding the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates, such as bosutinib, that exhibit nonlinear pharmacokinetics owing largely to a saturation of intestinal P-glycoprotein-mediated efflux.

Application of Physiologically Based Pharmacokinetic Modeling to the Understanding of Bosutinib Pharmacokinetics: Prediction of Drug-Drug and Drug-Disease Interactions.

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Philadelphia chromosome-positive chronic myelogenous leukemia. Bosutinib is predominantly metabolized by CYP3A4 as the primary clearance mechanism. The main objectives of this study were to 1) develop physiologically based pharmacokinetic (PBPK) models of bosutinib; 2) verify and refine the PBPK models based on clinical study results of bosutinib single-dose drug-drug interaction (DDI) with ketoconazole and rifampin, as well as single-dose drug-disease interaction (DDZI) in patients with renal and hepatic impairment; 3) apply the PBPK models to predict DDI outcomes in patients with weak and moderate CYP3A inhibitors; and 4) apply the PBPK models to predict DDZI outcomes in renally and hepatically impaired patients after multiple-dose administration. Results showed that the PBPK models adequately predicted bosutinib oral exposures in patients after single- and multiple-dose administrations. The PBPK models also reasonably predicted changes in bosutinib exposures in the single-dose DDI and DDZI results, suggesting that the PBPK models were sufficiently developed and verified based on the currently available data. Finally, the PBPK models predicted 2- to 4-fold increases in bosutinib exposures by moderate CYP3A inhibitors, as well as comparable increases in bosutinib exposures in renally and hepatically impaired patients between single- and multiple-dose administrations. Given the challenges in conducting numerous DDI and DDZI studies of anticancer drugs in patients, we believe that the PBPK models verified in our study would be valuable to reasonably predict bosutinib exposures under various scenarios that have not been tested clinically.

Physiologically Based Pharmacokinetic Modeling of Palbociclib.

Palbociclib is an orally available CDK4/6 inhibitor. In humans, palbociclib undergoes metabolism mediated primarily by CYP3A and SULT2A1, and it is also a weak time-dependent CYP3A inhibitor. The objectives of the current study are to (1) develop a physiologically based pharmacokinetic (PBPK) model of palbociclib based on the in silico, in vitro, and in vivo pharmacokinetic data of palbociclib, (2) verify the PBPK model with clinical drug-drug interaction (DDI) results of palbociclib with strong CYP3A inhibitor (itraconazole), inducer (rifampin), and a sensitive CYP3A substrate (midazolam), and (3) predict the DDI risk of palbociclib with moderate/weak CYP3A inhibitors. The developed PBPK model adequately described the observed pharmacokinetics of palbociclib after administration of a single oral or intravenous dose of palbociclib. The model-predicted DDIs of palbociclib with itraconazole, rifampin, and midazolam were consistent with the observed DDIs, with the discrepancies of the predicted vs observed AUCR and Cmax R within 20%, except for the AUC ratio of palbociclib with coadministration of rifampin. Using this final PBPK model, it was predicted that weak CYP3A inhibitors (fluoxetine and fluvoxamine) are anticipated to have negligible DDI risk with palbociclib, whereas moderate CYP3A inhibitors (diltiazem and verapamil) may increase plasma palbociclib AUC by ∼40%. A moderate CYP3A inducer (efavirenz) may decrease plasma palbociclib AUC by ∼40%. The established model is considered sufficiently robust for other applications in support of the continued development for palbociclib.

Assessment of drug metabolism enzyme and transporter pharmacogenetics in drug discovery and early development: perspectives of the I-PWG.

Genetic variants of drug metabolism enzymes and transporters can result in high pharmacokinetic and pharmacodynamic variability, unwanted characteristics of efficacious and safe drugs. Ideally, the contributions of these enzymes and transporters to drug disposition can be predicted from in vitro experiments and in silico modeling in discovery or early development, and then be utilized during clinical development. Recently, regulatory agencies have provided guidance on the preclinical investigation of pharmacogenetics, for application to clinical drug development. This white paper summarizes the results of an industry survey conducted by the Industry Pharmacogenomics Working Group on current practice and challenges with using in vitro systems and in silico models to understand pharmacogenetic causes of variability in drug disposition.

Cardiovascular differentiation of imatinib and bosutinib in the rat.

Imatinib and bosutinib were administered to rats for up to 6 months at clinically relevant exposures to investigate the effects on the cardiovascular system. Imatinib treatment resulted in increased volume, wall thickness and mass suggesting a hypertrophic heart in male and female rats at one and fivefold clinical exposures, respectively. Bosutinib treatment resulted in milder cardiac hypertrophy in female rats only at fivefold clinical exposures. Analysis of excised hearts and cultured myocytes demonstrated increased expression of hypertrophic genes with imatinib or analogs, but not bosutinib or c-Abl RNAi treatment. The current dataset suggests that cardiovascular liability of imatinib and bosutinib are differentiated preclinically and c-Abl independent.

A phase I, open-label, mass balance study of [(14)C] dacomitinib (PF-00299804) in healthy male volunteers.

PURPOSE: This study aimed to characterize the primary routes of elimination of the pan-HER tyrosine kinase inhibitor, dacomitinib (PF-00299804), to evaluate the pharmacokinetics of total radioactivity and of dacomitinib and to identify the metabolites of dacomitinib in plasma, urine, and feces in the healthy volunteers. METHODS: Six male healthy volunteers (mean age 31.5 years) received a single 45-mg oral dose containing ~100 µCi [(14)C] dacomitinib. Whole blood, urine, and fecal samples were collected throughout the study and analyzed for total radioactivity by liquid scintillation counting. Safety evaluations included vital signs, 12-lead ECGs, safety laboratory tests, and monitoring of adverse events. RESULTS: 78.8 % of the radiolabeled material was excreted in feces, and 3.2 % was recovered in urine. Peak concentrations of dacomitinib in plasma occurred 12 h (median) after oral dosing. Mean terminal plasma half-life was 55 and 182 h for dacomitinib and total plasma radioactivity, respectively. Geometric mean C max was approximately 2-fold higher, and total exposure (AUCinf) was almost 6-fold higher for total radioactivity than for dacomitinib in plasma. O-desmethyl dacomitinib (PF-05199265) was the major circulating metabolite. T max of this metabolite occurred 6 h after oral dosing with dacomitinib. Plasma exposure for the metabolite was one-third that of the parent compound. There were no serious/severe adverse events or deaths during the study. Dacomitinib was well tolerated. CONCLUSIONS: In humans, [(14)C] dacomitinib underwent oxidative and conjugative metabolism. Most of the administered dose was eliminated via the fecal route, and the major circulating metabolite was PF-05199265.

Which metabolites circulate?

Characterization of the circulating metabolites for a new chemical entity in humans is essential for safety assessment, an understanding of their contributions to pharmacologic activities, and their potential involvement in drug-drug interactions. This review examines the abundance of metabolites relative to the total parent drug [metabolite-to-parent (M/P) ratio] from 125 drugs in relation to their structural and physicochemical characteristics, lipoidal permeability, protein binding, and fractional formation from parent (fm). Our analysis suggests that fm is the major determinant of total drug M/P ratio for amine, alcohol, N- and S-oxide, and carboxylic acid metabolites. Passage from the hepatocyte to systemic circulation does not appear to be limiting owing to the vast majority of metabolites formed being relatively lipid permeable. In some cases, active transport plays an important role in this process (e.g., carboxylic acid metabolites). Differences in total parent drug clearance and metabolite clearance are attenuated by the reduction in lipophilicity introduced by the metabolic step and resultant compensatory changes in unbound clearance and protein binding. A small subclass of these drugs (e.g., terfenadine) is unintentional prodrugs with very high parent drug clearance, resulting in very high M/P ratios. In contrast, arenol metabolites show a more complex relationship with fm due largely to the new metabolic routes (conjugation) available to the metabolite compared with the parent drug molecule. For these metabolites, a more thorough understanding of the elimination clearance of the metabolite is critical to discern the likelihood of whether the phenol will constitute a major circulating metabolite.

How current understanding of clearance mechanisms and pharmacodynamics of therapeutic proteins can be applied for evaluation of their drug-drug interaction potential.

Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth.

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Cross-species comparison of the metabolism and excretion of zoniporide: contribution of aldehyde oxidase to interspecies differences.

Excretion and metabolism of zoniporide were investigated in humans after intravenous infusion of [(14)C]zoniporide at an 80-mg dose. Bile was the primary route of excretion because 57% of dose was recovered in the feces after intravenous infusion. Zoniporide was primarily cleared via metabolism in humans. 2-Oxozoniporide (M1) was the major excretory and circulating metabolite in humans and was catalyzed by aldehyde oxidase (K(m) of 3.4 microM and V(max) of 74 pmol/min/mg protein). Metabolites M2 (17% of the dose) and M3 (6.4% of circulating radioactivity), in which the guanidine moiety was hydrolyzed to a carboxylic acid, were also detected in human feces and plasma, respectively, suggesting that hydrolysis was another route of metabolism of zoniporide in humans. The metabolism and excretion of [(14)C]zoniporide in rats and dogs were also evaluated. As in humans, bile was the primary route of excretion of the radiolabeled material in both species, and metabolism was the primary route of clearance. A comparison of plasma metabolites showed that for M3, rats had a higher concentration than human or dog. M1 was absent in dog and present in human and rat plasma at comparable levels, whereas comparison of excreta showed that the total body burden of M1 was greater in rat than that in human. No further evaluation of M2 was considered because it was detected only in the human fecal extracts. Hence, no further toxicological evaluation of the three human metabolites was undertaken.

Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites.

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.

Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members. Oral administration of PF-00299804 causes significant antitumor activity, including marked tumor regressions in a variety of human tumor xenograft models that express and/or overexpress erbB family members or contain the double mutation (L858R/T790M) in erbB1 (EGFR) associated with resistance to gefitinib and erlotinib. Furthermore, PF-00299804 shows exceptional distribution to human tumor xenografts and excellent pharmacokinetic properties across species.

Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailability of rats and humans.

The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

N-(6,7-dichloro-2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-N-alkylsulfonamides as peripherally restricted N-methyl-D-aspartate receptor antagonists for the treatment of pain.

It has been hypothesized that peripherally restricted NMDA receptor antagonists may be effective analgesics for osteoarthritis pain. A class of novel quinoxalinedione atropisomers, first discovered for an NMDA receptor antagonist program for the treatment of stroke, was evaluated and further optimized with the goal of finding peripherally restricted NMDA receptor antagonists.

Atorvastatin glucuronidation is minimally and nonselectively inhibited by the fibrates gemfibrozil, fenofibrate, and fenofibric acid.

Gemfibrozil coadministration generally results in plasma statin area under the curve (AUC) increases, ranging from moderate (2- to 3-fold) with simvastatin, lovastatin, and pravastatin to most significant with cerivastatin (5.6-fold). Inhibition of statin glucuronidation has been postulated as a potential mechanism of interaction (Drug Metab Dispos 30:1280-1287, 2002). This study was conducted to determine the in vitro inhibitory potential of fibrates toward atorvastatin glucuronidation. [(3)H]Atorvastatin, atorvastatin, and atorvastatin lactone were incubated with human liver microsomes or human recombinant UDP-glucuronosyltransferases (UGTs) and characterized using liquid chromatography (LC)/tandem mass spectrometry and LC/UV/beta-radioactivity monitor/mass spectrometry. [(3)H]Atorvastatin yields a minor ether glucuronide (G1) and a major acyl glucuronide (G2) with subsequent pH-dependent lactonization of G2 to yield atorvastatin lactone. Atorvastatin lactonization best fit substrate inhibition kinetics (K(m) = 12 microM, V(max) = 74 pmol/min/mg, K(i) = 75 microM). Atorvastatin lactone yields a single ether glucuronide (G3). G3 formation best fit Michaelis-Menten kinetics (K(m) = 2.6 microM, V(max) = 10.6 pmol/min/mg). Six UGT enzymes contribute to atorvastatin glucuronidation with G2 and G3 formation catalyzed by UGTs 1A1, 1A3, 1A4, 1A8, and 2B7, whereas G1 formation was catalyzed by UGTs 1A3, 1A4, and 1A9. Gemfibrozil, fenofibrate, and fenofibric acid inhibited atorvastatin lactonization with IC(50) values of 346, 320, and 291 microM, respectively. Based on unbound fibrate concentrations at the inlet to the liver, these data predict a small increase in atorvastatin AUC (approximately 1.2-fold) after gemfibrozil coadministration and no interaction with fenofibrate. This result is consistent with recent clinical reports indicating minimal atorvastatin AUC increases ( approximately 1.2- to 1.4-fold) with gemfibrozil.

Pharmacodynamic and toxicokinetic evaluation of the novel MEK inhibitor, PD0325901, in the rat following oral and intravenous administration.

The MEK-mitogen-activated protein kinase (MAPK) signal transduction pathway is involved with numerous cellular processes including cell growth and differentiation. Phosphorylation of MAPK (pMAPK) by MEK results in activation of this pathway. In various solid tumors, the MEK-MAPK pathway is constitutively active; therefore inhibition of this pathway may provide a therapeutic strategy for treating cancer. The objective of this study was to determine the extent and duration of inhibition of pMAPK in selected normal tissues in rats following single oral or intravenous (IV) doses of the novel MEK inhibitor, PD0325901. Male Sprague-Dawley rats (9/group) received either single oral (PO) or IV doses of PD0325901 at 10, 30, or 100 mg/kg (60, 180, and 600 mg/m(2), respectively). Controls received vehicle alone which was aqueous 0.5% hydroxypropylmethyl-cellulose/0.2% Tween 80 for PO dosing and 20% beta-cyclodextran sulfobutyl ether in water (w:v) for IV dosing. Animals (3/group/day) were euthanized on Days 2, 3, and 4, at approximately 24, 48, and 72 h after dosing, respectively. The effects on pMAPK in liver and lung were determined by Western blot analysis and compared with plasma PD0325901 levels. Satellite animals (6/dose/route) received single PO or IV doses and serial blood samples were collected for determination of toxicokinetic parameters of PD0325901 and its major metabolite. In general, systemic exposure to PD0325901 was comparable between routes of administration due to high PO bioavailability (56-109%). Plasma area under the concentration-time curve values of the pharmacologically inactive carboxylic acid metabolite ranged from 18 to 40% of PD0325901. Clinical signs of toxicity occurred at 100 mg/kg PO or IV, indicating the maximum-tolerated dose had been achieved. On Day 2, pMAPK was inhibited 57-95% in liver and 86-99% in lung at all doses, irrespective of route of administration. On Day 3, lung pMAPK remained inhibited 75-91% at all IV doses and by 88% after the 100-mg/kg PO dose. Liver pMAPK remained inhibited 79 and 91% on Day 3 after 100 mg/kg by IV and PO doses, respectively. On Day 4, liver pMAPK was still inhibited 66% after the 100-mg/kg PO dose. The EC(50) and EC(90) plasma drug levels for inhibition of lung pMAPK were calculated to be 20 and 99 ng/ml, respectively. Liver pMAPK levels were inhibited at least 50% at plasma PD0325901 concentrations > or =50 ng/ml. In conclusion, single PO or IV doses of PD0325901 resulted in dose-dependent inhibition of pMAPK in liver and lung. Inhibition of pMAPK in liver was comparable between routes of administration at < or =30 mg/kg, whereas inhibition of pMAPK in lung occurred for a longer duration following IV administration. Measurement of pMAPK in normal tissues served as a means for assessing the pharmacologic activity of PD0325901 and should be included in toxicity studies to evaluate toxicity-pharmacology relationships.

Application of in-line liquid chromatography-accurate radioisotope counting-mass spectrometry (LC-ARC-MS) to evaluate metabolic profile of [3H]-mefenamic acid in rat plasma.

Profiling of rat plasma using a highly sensitive LC-ARC-MS technique showed that [(3)H] mefenamic acid was metabolized to several products, including a sulfate conjugate and a hydroxylated analogue as major metabolites. This technique of detecting low levels of radioactivity in plasma was superior to previously used methods, such as beta-RAM detectors.

Roles of exercise and pharmacokinetics in cerivastatin-induced skeletal muscle toxicity.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are associated with adverse skeletal muscle effects, but the underlying mechanisms remain unclear. To determine whether toxicity involves the level of drug exposure in muscle tissue and to test the effect of exercise on cerivastatin (CVA)-induced skeletal muscle damage, female rats were administered vehicle or CVA at 0.1, 0.5, and 1.0 mg/kg/day by gavage for two weeks and exercised or not on treadmills for 20 min/day. Clinical chemistry and plasma and tissue pharmacokinetics were evaluated; light and transmission electron microscopy (TEM) of Type I and Type II fiber-predominant skeletal muscles were performed. Serum levels of AST, ALT, CK, and plasma lactic acid were significantly elevated dose-dependently. CVA treatment decreased psoas and quadriceps weights. At 1 mg/kg all muscles except soleus demonstrated degeneration. Exercise-exacerbated severity of CVA-induced degeneration was evident in all muscles sampled except soleus and quadriceps. Early mitochondrial involvement in toxicity is suggested by the numerous membranous whorls and degenerate mitochondria observed in muscles at 0.5 mg/kg. No significant differences in CVA concentrations between either EDL and soleus or plasma and muscle were found. We found that CVA had no effect on cleaved caspase 3. In summary, we found that treadmill exercise exacerbated the incidence and severity of CVA-induced damage in Type II fiber-predominant muscles. Tissue exposure is likely not the key factor mediating CVA-induced skeletal muscle toxicity.