The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

ABSTRACT:The metabolism and disposition of [ 14 C]apixaban, an orally bioavailable, highly selective, and direct acting/reversible factor Xa inhibitor, was investigated in 10 healthy male subjects without (group 1, n ‫؍‬ 6) and with bile collection (group 2, n ‫؍‬ 4) after a single 20-mg oral dose. Urine, blood, and feces samples were collected from all subjects. Bile samples were also collected for 3 to 8 h after dosing from group 2 subjects. There were no serious adverse events or discontinuations due to adverse effects. In plasma, apixaban was the major circulating component and O-demethyl apixaban sulfate, a stable and water-soluble metabolite, was the significant metabolite. The exposure of apixaban (C max and area under the plasma concentration versus time curve) in subjects with bile collection was generally similar to that in subjects without bile collection. The administered dose was recovered in feces (group 1, 56.0%; group 2, 46.7%) and urine (group 1, 24.5%; group 2, 28.8%), with the parent drug representing approximately half of the recovered dose. Biliary excretion represented a minor elimination pathway (2.44% of the administered dose) from group 2 subjects within the limited collection period. Metabolic pathways identified for apixaban included O-demethylation, hydroxylation, and sulfation of hydroxylated O-demethyl apixaban. Thus, apixaban is an orally bioavailable inhibitor of factor Xa with elimination pathways that include metabolism and renal excretion.Thromboembolic events, including acute myocardial infarction, unstable angina, deep vein thrombosis, pulmonary embolism, and ischemic stroke continue to be the leading cause of morbidity and mortality in the United States and other Western countries (Heit et al., 2005;Rosamond et al., 2007). Current therapies for the treatment and prevention of thromboembolic events, such as vitamin K antagonists (e.g., warfarin), heparin, and low-molecular-weight heparin (e.g., enoxaparin), are suboptimal (O'Donnell and Weitz, 2004;Wittkowsky, 2004;Campbell, 2006). However, the requirement for intravenous or subcutaneous injection and/or the need for careful monitoring because of the risk of excessive bleeding or unpredictable/inconsistent pharmacokinetics (PK) can complicate administration and present barriers to the use of these agents (O'Brien and Caro, 2002;Wittkowsky, 2004;Campbell, 2006). Therefore, new, orally active anticoagulants with predictable pharmacokinetic profiles that can be administered with a reduced need for monitoring are needed.Factor Xa is a key serine protease in the coagulation cascade and is a promising target enzyme for new therapeutic agents for the treatment and prevention of arterial and venous thrombosis (Kaiser, 2002;Samama, 2002;Walenga et al., 2003). In particular, factor Xa plays a critical role in blood coagulation, serving as the juncture between the extrinsic (tissue factor initiated) and intrinsic (surface activation and amplification) systems (Mann et al., 2003). Factor Xa forms the prothrombinase complex with phospholi...

Multiple oral doses of apixaban were safe and well tolerated over a 10-fold dose range, with pharmacokinetics with low variability and concentration-related increases in clotting time measures.

We created transgenic mice that overexpress WT androgen receptor (AR) exclusively in their skeletal muscle fibers. Unexpectedly, these mice display androgen-dependent muscle weakness and early death, show changes in muscle morphology and gene expression consistent with neurogenic atrophy, and exhibit a loss of motor axons. These features reproduce those seen in models of Kennedy disease, a polyglutamine expansion disorder caused by a CAG repeat expansion in the AR gene. Kennedy disease ͉ neuromuscular ͉ skeletal muscle ͉ spinal and bulbar muscular atrophy ͉ axonopathy A polymorphism in exon 1 of the androgen receptor (AR) gene, consisting of a variable number of glutamine (Q) repeats, affects AR function. Very long polyglutamine repeat (polyQ) tracts are associated with a progressive neuromuscular disease known as Kennedy disease (KD, or spinal bulbar muscular atrophy) (1). The etiological mechanism mediating polyQ toxicity is uncertain, but is generally thought to confer novel toxic functions to the protein, because expansion of polyQ tracts beyond 40 repeats in other proteins also cause neurodegenerative disease, including Huntington's disease (HD), and several autosomal dominant forms of spinocerebellar ataxia (SCA) (2). Histopathological studies of KD patients suggest ''neurogenic'' responses to denervation, and the etiology of this disease is therefore generally thought to begin with motoneuron pathology (3).Explicit mouse models of KD, in which polyQ AR alleles containing 60 CAG repeats or more are expressed, develop a disease phenotype that includes a marked reduction in body weight, kyphosis, and striking deficits in muscle strength and motor coordination (4-8). Androgen dependence, motoneuron and muscle pathology, and/or inclusions containing AR are also observed in these models (4,5,7,8). Our studies of AR in skeletal muscle (9, 10) led us to generate transgenic (Tg) mice in which AR is overexpressed solely in skeletal muscle fibers using an expression cassette containing the human skeletal ␣-actin (HSA) promoter. We discovered a striking phenotypic resemblance between these HSA-AR mice and mouse models of KD. This similarity is surprising given that the Q repeat in this AR transgene comprises only 22 glutamines and is expressed exclusively in skeletal muscle fibers and not in motoneurons. Results HSA Promoter Drives Transgene Expression Exclusively in SkeletalMuscle Fibers. We first validated our HSA expression cassette by generating HSA-LacZ (LacZ ϭ ␤-galactosidase gene) reporter mice [supporting information (SI) Fig. 5A]. Consistent with other expression cassettes containing the HSA promoter (11, 12) these reporter mice express ␤-gal specifically in skeletal muscle fibers, starting at embryonic day 9.5-10.5, with no detectable expression in other tissues, including the heart, viscera, fat or spinal cord ( SI Fig 5 B and C). We also created Tg mice in which a rat WT AR cDNA is driven by this same HSA expression cassette (SI Fig 6A), resulting in selective overexpression of AR in skeletal muscle fiber...

Kennedy disease, a degenerative disorder characterized by androgen-dependent neuromuscular weakness, is caused by a CAG/glutamine tract expansion in the androgen receptor (Ar) gene. We developed a mouse model of Kennedy disease, using gene targeting to convert mouse androgen receptor (AR) to human sequence while introducing 113 glutamines. AR113Q mice developed hormone and glutamine length-dependent neuromuscular weakness characterized by the early occurrence of myopathic and neurogenic skeletal muscle pathology and by the late development of neuronal intranuclear inclusions in spinal neurons. AR113Q males unexpectedly died at 2-4 months. We show that this androgen-dependent death reflects decreased expression of skeletal muscle chloride channel 1 (CLCN1) and the skeletal muscle sodium channel α-subunit, resulting in myotonic discharges in skeletal muscle of the lower urinary tract. AR113Q limb muscles show similar myopathic features and express decreased levels of mRNAs encoding neurotrophin-4 and glial cell line-derived neurotrophic factor. These data define an important myopathic contribution to the Kennedy disease phenotype and suggest a role for muscle in non-cell autonomous toxicity of lower motor neurons.

Summary Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here we tested this hypothesis using AR113Q knock-in and human BAC transgenic mice that express the full-length polyQ AR and display androgen-dependent weakness, muscle atrophy and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery, but not CNS, following subcutaneous administration. Suppression of polyQ AR in the periphery rescued deficits in muscle weight, fiber size and grip strength, reversed changes in muscle gene expression, and extended lifespan of mutant males. We conclude that polyQ AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Aim Apixaban is an orally active inhibitor of coagulation factor Xa and is eliminated by multiple pathways, including renal and non‐renal elimination. Non‐renal elimination pathways consist of metabolism by cytochrome P450 (CYP) enzymes, primarily CYP3A4, as well as direct intestinal excretion. Two single sequence studies evaluated the effect of ketoconazole (a strong dual inhibitor of CYP3A4 and P‐glycoprotein [P‐gp]) and diltiazem (a moderate CYP3A4 inhibitor and a P‐gp inhibitor) on apixaban pharmacokinetics in healthy subjects. Method In the ketoconazole study, 18 subjects received apixaban 10 mg on days 1 and 7, and ketoconazole 400 mg once daily on days 4–9. In the diltiazem study, 18 subjects received apixaban 10 mg on days 1 and 11 and diltiazem 360 mg once daily on days 4–13. Results Apixaban maximum plasma concentration and area under the plasma concentration–time curve extrapolated to infinity increased by 62% (90% confidence interval [CI], 47, 78%) and 99% (90% CI, 81, 118%), respectively, with co‐administration of ketoconazole, and by 31% (90% CI, 16, 49%) and 40% (90% CI, 23, 59%), respectively, with diltiazem. Conclusion A 2‐fold and 1.4‐fold increase in apixaban exposure was observed with co‐administration of ketoconazole and diltiazem, respectively.

This open-label study evaluated apixaban pharmacokinetics, pharmacodynamics, and safety in subjects with mild, moderate, or severe renal impairment and in healthy subjects following a single 10-mg oral dose. The primary analysis determined the relationship between apixaban AUC∞ and 24-hour creatinine clearance (CLcr ) as a measure of renal function. The relationships between 24-hour CLcr and iohexol clearance, estimated CLcr (Cockcroft-Gault equation), and estimated glomerular filtration rate (modification of diet in renal disease [MDRD] equation) were also assessed. Secondary objectives included assessment of safety and tolerability as well as international normalized ratio (INR) and anti-factor Xa activity as pharmacodynamic endpoints. The regression analysis showed that decreasing renal function resulted in modestly increased apixaban exposure (AUC∞ increased by 44% in severe impairment with a 24-hour CLcr of 15 mL/min, compared with subjects with normal renal function), but it did not affect Cmax or the direct relationship between apixaban plasma concentration and anti-factor Xa activity or INR. The assessment of renal function measured by iohexol clearance, Cockcroft-Gault, and MDRD was consistent with that determined by 24-hour CLcr . Apixaban was well tolerated in this study. These results suggest that dose adjustment of apixaban is not required on the basis of renal function alone.