Farnesoid X Receptor Agonists Obeticholic Acid and Chenodeoxycholic Acid Increase Bile Acid Efflux in Sandwich-Cultured Human Hepatocytes: Functional Evidence and Mechanisms.

The farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in bile acid homeostasis. FXR agonists, obeticholic acid (OCA) and chenodeoxycholic acid (CDCA), increase mRNA expression of efflux transporters in sandwich-cultured human hepatocytes (SCHH). This study evaluated the effects of OCA and CDCA treatment on the uptake, basolateral efflux, and biliary excretion of a model bile acid, taurocholate (TCA), in SCHH. In addition, changes in the protein expression of TCA uptake and efflux transporters were investigated. SCHH were treated with 1 µM OCA, 100 µM CDCA, or vehicle control for 72 hours followed by quantification of deuterated TCA uptake and efflux over time in Ca2+-containing and Ca2+-free conditions (n = 3 donors). A mechanistic pharmacokinetic model was fit to the TCA mass-time data to obtain estimates for total uptake clearance (CLUptake), total intrinsic basolateral efflux clearance (CLint,BL), and total intrinsic biliary clearance (CLint,Bile). Modeling results revealed that FXR agonists significantly increased CLint,BL by >6-fold and significantly increased CLint,Bile by 2-fold, with minimal effect on CLUptake Immunoblotting showed that protein levels of the basolateral transporter subunits organic solute transporter α and ß (OSTα and OSTß) in FXR agonist-treated SCHH were significantly induced by >2.5- and 10-fold, respectively. FXR agonist-mediated changes in the expression of other TCA transporters in SCHH were modest. In conclusion, this is the first report demonstrating that OCA and CDCA increased TCA efflux in SCHH, which contributed to reduced intracellular TCA concentrations. Increased basolateral efflux of TCA was consistent with increased OSTα/ß protein expression in OCA- and CDCA-treated SCHH.

Muscle cramp susceptibility increases following a volitionally induced muscle cramp.

INTRODUCTION: Muscle cramping may increase peripheral nervous system excitability. It is unknown if, and how long, cramp susceptibility is affected by previous cramping. We tested whether volitionally induced muscle cramps (VIMCs) lowered cramp threshold frequency (TFc ) and how long TFc was affected post-VIMC. METHODS: Fifteen cramp-prone participants volitionally induced a flexor hallucis brevis (FHB) cramp on 4 separate days. FHB TFc was measured before VIMC (i.e., baseline) and 5, 30, and 60 min post-VIMC. VIMC electromyography (EMG) amplitude, VIMC duration, and perceived VIMC intensity were measured to ensure consistency of VIMC between days. RESULTS: VIMC EMG amplitude, duration, and perceived intensity were similar between days (P > 0.05). VIMC lowered TFc ; baseline TFc (18 ± 6 Hz) was higher than 5-min (14 ± 6 Hz), 30-min (14 ± 5 Hz), and 60-min TFc (14 ± 5 Hz; P < 0.05). DISCUSSION: Acute VIMCs increase cramp susceptibility. Clinicians should apply treatments for at least 60 min postcramp to decrease the probability of cramp recurrence. Muscle Nerve 56: E95-E99, 2017.

Does a Reduction in Serum Sodium Concentration or Serum Potassium Concentration Increase the Prevalence of Exercise-Associated Muscle Cramps?

CLINICAL SCENARIO: Although exercise-associated muscle cramps (EAMC) are common in ultradistance runners and athletes in general, their etiology remains unclear. EAMC are painful, sudden, involuntary contractions of skeletal muscle occurring during or after exercise and are recognized by visible bulging or knotting of the whole, or part of, a muscle. Many clinicians believe EAMC occur after an imbalance in electrolyte concentrations, specifically serum sodium concentration ([Na+]s) and serum potassium concentration ([K+]s). Studies that have established a link between EAMC occurrence and serum electrolyte concentrations after an athletic event are unhelpful. Focused Clinical Question: Are [Na+]s and [K+]s different in athletes who experience EAMC than noncrampers?

Identification of biaryl sulfone derivatives as antagonists of the histamine H3 receptor: discovery of (R)-1-(2-(4'-(3-methoxypropylsulfonyl)biphenyl-4-yl)ethyl)-2-methylpyrrolidine (APD916).

The design of a new clinical candidate histamine-H(3) receptor antagonist for the potential treatment of excessive daytime sleepiness (EDS) is described. Phenethyl-R-2-methylpyrrolidine containing biphenylsulfonamide compounds were modified by replacement of the sulfonamide linkage with a sulfone. One compound from this series, 2j (APD916) increased wakefulness in rodents as measured by polysomnography with a duration of effect consistent with its pharmacokinetic properties. The identification of a suitable salt form of 2j allowed it to be selected for further development.

Threshold frequency of an electrically induced cramp increases following a repeated, localized fatiguing exercise.

Though clinical observations and laboratory data provide some support for the neuromuscular imbalance theory of the genesis of exercise-associated muscle cramps, no direct evidence has been published. The purpose of this study was to determine the effect of local muscle fatigue on the threshold frequency of an electrically induced muscle cramp. To determine baseline threshold frequency, a cramp was electrically induced in the flexor hallucis brevis of 16 apparently healthy participants (7 males, 9 females; age 25.1 +/- 4.8 years). The testing order of control and fatigue conditions was counterbalanced. In the control condition, participants rested in a supine position for 30 min followed by another cramp induction to determine post-threshold frequency. In the fatigue condition, participants performed five bouts of great toe curls at 60% one-repetition maximum to failure with 1 min rest between bouts followed immediately by a post-threshold frequency measurement. Repeated-measures analysis of variance and simple main effects testing showed post-fatigue threshold frequency (32.9 +/- 11.7 Hz) was greater (P < 0.001) than pre-fatigue threshold frequency (20.0 +/- 7.7 Hz). An increase in threshold frequency seems to demonstrate a decrease in one's propensity to cramp following the fatigue exercise regimen used. These results contradict the proposed theory that suggests cramp propensity should increase following fatigue. However, differences in laboratory versus clinical fatiguing exercise and contributions from other sources, as well as the notion of a graded response to fatiguing exercise, on exercise-associated muscle cramp and electrically induced muscle cramp should be considered.

APD791, 3-methoxy-n-(3-(1-methyl-1h-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a novel 5-hydroxytryptamine 2A receptor antagonist: pharmacological profile, pharmacokinetics, platelet activity and vascular biology.

We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.

A new family of H3 receptor antagonists based on the natural product Conessine.

A new family of Histamine H(3) receptor antagonists (5a-t) has been prepared based on the structure of the natural product Conessine, a known H(3) antagonist. Several members of the new series are highly potent and selective binders of rat and human H(3) receptors and display inverse agonism at the human H(3) receptor. Compound 5n exhibited promising rat pharmacokinetic properties and demonstrated functional antagonism of the H(3) receptor in an in-vivo pharmacological model.

Novel H3 receptor antagonists with improved pharmacokinetic profiles.

A new series of H(3) antagonists derived from the natural product Conessine are presented. Several compounds from these new series retain the potency and selectivity of earlier diamine based analogs while exhibiting improved PK characteristics. One compound (3u) demonstrated functional antagonism of the H(3) receptor in an in vivo pharmacological model.

Assessment of Pharmacokinetic Interactions Between Obeticholic Acid and Caffeine, Midazolam, Warfarin, Dextromethorphan, Omeprazole, Rosuvastatin, and Digoxin in Phase 1 Studies in Healthy Subjects.

INTRODUCTION: Obeticholic acid (OCA), a potent and selective farnesoid X receptor agonist, is indicated for the treatment of primary biliary cholangitis (PBC). We investigated the potential drug-drug interaction effect of OCA on metabolic CYP450 enzymes and drug transporters. METHODS: Five phase 1 single-center, open-label, fixed-sequence, inpatient studies were conducted in healthy adult subjects to evaluate the effect of oral daily doses of 10 or 25 mg OCA on single-dose plasma pharmacokinetics of specific probe substrates for enzymes CYP1A2 (caffeine, R-warfarin), CYP3A (midazolam, R-warfarin), CYP2C9 (S-warfarin), CYP2D6 (dextromethorphan), CYP2C19 (omeprazole), and drug transporters, BCRP/OATP1B1/OATP1B3 (rosuvastatin), and P-gp (digoxin). RESULTS: OCA showed no substantial suppression/inhibition of S-warfarin, digoxin, and dextromethorphan and weak interactions with caffeine, omeprazole, rosuvastatin, and midazolam. The maximal pharmacodynamic responses (E max) to warfarin-based INR, PT, and aPTT were reduced by 11%, 11%, and 1%, respectively, for the 10-mg dose group and by 7%, 7% and 0%, respectively, for the 25-mg dose group. Overall, drugs dosed in combination with OCA were well tolerated, and most adverse events were mild in severity. No clinically important trends were noted in laboratory evaluations, vital signs, or 12-lead ECGs. CONCLUSION: In these studies, OCA showed weak to no suppression/inhibition of metabolic enzymes and drug transporters at the highest recommended therapeutic dose in patients with PBC. On the basis on these analyses, monitoring and maintenance of target INR range are required during coadministration of OCA with drugs that are metabolized by CYP1A2 (R-warfarin). FUNDING: Intercept Pharmaceuticals, Inc.

Acute Passive Static Stretching and Cramp Threshold Frequency.

CONTEXT: Exercise-associated muscle cramps are a common clinical problem for athletes. OBJECTIVE: To determine whether acute passive static stretching altered cramp threshold frequency (CTF) of electrically induced muscle cramps. DESIGN: Crossover study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Seventeen healthy college-aged individuals. INTERVENTION(S): Stretching or no stretching. MAIN OUTCOME MEASURE(S): The independent variable was the static stretch versus the no-stretch condition, and the dependent variable was the CTF. RESULTS: The CTF increased in both the control (pretest: 18.12 ± 6.46 Hz, posttest: 19.65 ± 7.25 Hz; P = .033) and stretching (pretest: 18.94 ± 5.96 Hz, posttest: 20.47 ± 7.12 Hz; P = .049) groups. No difference between the groups was found (t15 = 0.035, P = .97). CONCLUSIONS: Acute passive static stretching did not seem to increase the CTF.

Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 µmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OSTα ) and OSTß increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OSTα and OSTß, by OCA reduced the intracellular concentrations of d8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases.

Comparative potency of obeticholic acid and natural bile acids on FXR in hepatic and intestinal in vitro cell models.

Obeticholic acid (OCA) is a semisynthetic farnesoid X receptor (FXR) agonist, an analogue of chenodeoxycholic acid (CDCA) which is indicated for the treatment of primary biliary cholangitis (PBC) in combination with ursodeoxycholic acid (UDCA). OCA efficiently inhibits bile acid synthesis and promotes bile acid efflux via activating FXR-mediated mechanisms in a physiologically relevant in vitro cell system, Sandwich-cultured Transporter Certified ™ human primary hepatocytes (SCHH). The study herein evaluated the effects of UDCA alone or in combination with OCA in SCHH. UDCA (≤100 µmol/L) alone did not inhibit CYP7A1 mRNA, and thus, no reduction in the endogenous bile acid pool observed. UDCA ≤100 µmol/L concomitantly administered with 0.1 µmol/L OCA had no effect on bile acid synthesis beyond what was observed with OCA alone. Furthermore, this study evaluated human Caco-2 cells (clone C2BBe1) as in vitro intestinal models. Glycine conjugate of OCA increased mRNA levels of FXR target genes in Caco-2 cells, FGF-19, SHP, OSTα/ß, and IBABP, but not ASBT, in a concentration-dependent manner, while glycine conjugate of UDCA had no effect on the expression of these genes. The results suggested that UDCA ≤100 µmol/L did not activate FXR in human primary hepatocytes or intestinal cell line Caco-2. Thus, co-administration of UDCA with OCA did not affect OCA-dependent pharmacological effects.

Peripheral ankle cooling and core body temperature.

CONTEXT: Exposure of the human body to cold is perceived as a stressor and results in a sympathetic response geared at maintaining core temperature. Application of ice to the periphery may lead to a decrease in core temperature, which may counteract the therapeutic effects of cryotherapy. OBJECTIVE: To determine if core temperature is lowered by the application of an ice bag to the ankle joint complex. DESIGN: A within-subjects, repeated-measures design. SETTING: The University of Virginia General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS: Twenty-three healthy adults aged 19 to 39 years. INTERVENTION(S): Subjects were admitted to the hospital on 2 separate occasions. During one admission, subjects had a 20-minute ice treatment applied to their ankles; in the other admission, a bag of marbles was applied. Temperature measurements were recorded at 6 time intervals: baseline (before ice application), immediately after ice application, 10 and 20 minutes after ice application, and 10 and 20 minutes after ice removal. MAIN OUTCOME MEASURE(S): We measured core temperature and ankle and soleus muscle surface temperatures. A mixed-effects model analysis of variance with repeated measures was used to determine if differences existed in core temperature and ankle and soleus surface temperatures between conditions (cryotherapy and control) and over time. RESULTS: Core temperature did not change after ice application or ice removal (P > 0.05). The average core temperatures during the cryotherapy and control conditions were 36.72 degrees C +/- 0.42 degrees C and 36.45 degrees C +/- 1.23 degrees C, respectively. CONCLUSIONS: A 20-minute cryotherapy treatment applied to the ankle did not alter core temperature.

The metabolism of nonane, a JP-8 jet fuel component, by human liver microsomes, P450 isoforms and alcohol dehydrogenase and inhibition of human P450 isoforms by JP-8.

Nonane, a component of jet-propulsion fuel 8 (JP-8), is metabolized to 2-nonanol and 2-nonanone by pooled human liver microsomes (pHLM). Cytochrome P450 (CYP) isoforms 1A2, 2B6 and 2E1 metabolize nonane to 2-nonanol, whereas alcohol dehydrogenase, CYPs 2B6 and 2E1 metabolize 2-nonanol to 2-nonanone. Nonane and 2-nonanol showed no significant effect on the metabolism of testosterone, estradiol or N,N-diethyl-m-toluamide (DEET), but did inhibit carbaryl metabolism. JP-8 showed modest inhibition of testosterone, estradiol and carbaryl metabolism, but had a more significant effect on the metabolism of DEET. JP-8 was shown to inhibit CYPs 1A2 and 2B6 mediated metabolism of DEET, suggesting that at least some of the components of JP-8 might be metabolized by CYPs 1A2and/or 2B6.

An experimental knee joint effusion does not affect plasma catecholamine concentration in humans.

Knee joint effusion causes quadriceps inhibition and is accompanied by increased soleus muscle excitability. In order to reverse the neurological alterations that occur to the musculature following effusion, we need to understand the extent of neural involvement. Ten healthy adults were tested on two occasions; during one session, subjects had their knees injected with saline and in the other admission, they did not. Soleus Hmax, Mmax, plasma epinephrine, and norepinephrine concentrations were obtained at five intervals. Results showed that Hmax increased following the effusion, while norepinephrine and epinephrine levels were not altered. We suggest that the soleus facilitation seen following knee effusion results from stimulation of joint mechanoreceptors and removal of descending spinal and supraspinal inhibition and is not the result of a sympathetic response.

Hydromorphone transfer into breast milk after intranasal administration.

STUDY OBJECTIVES: To determine the distribution of hydromorphone into breast milk and the potential exposure of the suckling infant, and whether the distribution of hydromorphone into milk can be predicted accurately by a passive diffusion model. DESIGN: Single-dose, pharmacokinetic study. SETTING: University clinical research unit. PATIENTS: Eight lactating, nonsmoking, healthy women aged 24-32 years. INTERVENTION: Hydromorphone HCl 2 mg was given intranasally to the women to characterize its pharmacokinetics and extent of its transfer into breast milk. MEASUREMENTS AND MAIN RESULTS: Plasma and milk samples were analyzed using liquid chromatography with tandem mass spectrometry detection. The milk:plasma ratio (M:P) was calculated as the total area under the concentration-time curve (AUC) of the milk divided by the total AUC of the plasma. Predicted in vitro M:P ratios were calculated using a diffusion model. Protein binding in milk and plasma, partitioning into milk fat (whole milk:skim milk ratios), as well as pH partitioning between plasma and milk were incorporated in the model. Protein binding was determined by equilibrium dialysis. Protein binding was minimal in both milk and plasma, with unbound fractions of 1 and 0.84, respectively There was little partitioning into milk fat, as demonstrated by the whole milk:skim milk ratio of 0.98. The observed and predicted M:P ratios +/- SD for hydromorphone were 2.57 +/- 0.47 and 1.11 +/- 0.28, respectively. The 95% confidence interval for the observed M:P ratio overlapped the confidence interval of the predicted M:P ratio, a finding that supports a role for both passive diffusion and active transport as mechanisms of hydromorphone transfer into milk. CONCLUSION: Hydromorphone distributes rapidly from plasma into breast milk; however, the drug does not partition into fat. The suckling infant would receive approximately 0.67% of the maternal dose of hydromorphone (adjusted for body weight). As this is a limited exposure, further studies are needed to determine any potential impact to an infant who is fed breast milk from a mother treated with hydromorphone.

Exercise-associated muscle cramps: causes, treatment, and prevention.

CONTEXT: Exercise-associated muscle cramps (EAMC) are a common condition experienced by recreational and competitive athletes. Despite their commonality and prevalence, their cause remains unknown. Theories for the cause of EAMC are primarily based on anecdotal and observational studies rather than sound experimental evidence. Without a clear cause, treatments and prevention strategies for EAMC are often unsuccessful. EVIDENCE ACQUISITION: A search of Medline (EBSCO), SPORTDiscus, and Silverplatter (CINHAL) was undertaken for journal articles written in English between the years 1955 and 2008. Additional references were collected by a careful analysis of the citations of others' research and textbooks. RESULTS: Dehydration/electrolyte and neuromuscular causes are the most widely discussed theories for the cause of EAMC; however, strong experimental evidence for either theory is lacking. CONCLUSIONS: EAMC are likely due to several factors coalescing to cause EAMC. The variety of treatments and prevention strategies for EAMC are evidence of the uncertainty in their cause. Acute EAMC treatment should focus on moderate static stretching of the affected muscle followed by a proper medical history to determine any predisposing conditions that may have triggered the onset of EAMC. Based on physical findings, prevention programs should be implemented to include fluid and electrolyte balance strategies and/or neuromuscular training.

Role of P-glycoprotein in distribution of nelfinavir across the blood-mammary tissue barrier and blood-brain barrier.

As a first approach in understanding the possible efficacy and toxicity of human immunodeficiency virus protease inhibitors during breast feeding, the milk-to-plasma ratio of nelfinavir was determined in lactating rats. The milk-to-plasma ratio of nelfinavir was determined to be 0.56 +/- 0.10 (means +/- standard deviations). Western blotting indicated that P-glycoprotein is expressed in rat mammary and brain tissue; however, the multidrug-resistant modulator GF120918 showed a significant effect only at the blood-brain barrier and not at the mammary-epithelial tissue barrier.

Pre-synaptic modulation of quadriceps arthrogenic muscle inhibition.

Arthrogenic muscle inhibition (AMI) impedes rehabilitation following knee joint injury by preventing activation of the quadriceps. AMI has been attributed to neuronal reflex activity in which altered afferent input originating from the injured joint results in a diminished efferent motor drive to the quadriceps muscles. Beginning to understand the mechanisms responsible for muscle inhibition following joint injury is vital to control or eliminate this phenomenon. Therefore, the purpose of this investigation is to determine if quadriceps AMI is mediated by a presynaptic regulatory mechanism. Eight adults participated in two sessions: in one session their knee was injected with saline and in the other session it was not. The maximum Hoffmann reflex (H-reflex), M-wave, reflex activation history, plasma epinephrine, and norepinephrine were recorded at: baseline, post needle stick, post lidocaine, and 25 and 45 min post effusion. Measures for the control condition were matched to the effusion condition. The percent of the unconditioned reflex amplitude for reflex activation history and the maximum H-reflex were decreased at 25 and 45 min post effusion as compared to measures taken at baseline, post needle stick, and post lidocaine (P<0.05). No differences were noted for the maximum M-wave or plasma epinephrine and norepinephrine levels in either the effusion or noneffusion admission (P>0.05). No differences were detected at any time interval for any measure during the control admission (P>0.05). Quadriceps AMI elicited via an experimental knee joint effusion is, at least in part, mediated by a presynaptic mechanism.

GF120918, a P-glycoprotein modulator, increases the concentration of unbound amprenavir in the central nervous system in rats.

The goal of this study was to determine the distribution of unbound amprenavir in the central nervous system (CNS) of rats. The concentration of unbound amprenavir in the extracellular fluid of the brain and the blood was examined in the presence and absence of the MDR modulator GF120918 by microdialysis. The brain-to-blood ratio of amprenavir in the absence and presence of GF120918 was found to be significantly different (P < 0.003; 0.076 and 0.617, respectively). The use of the MDR modulator GF120918 could potentially increase the penetration of human immunodeficiency virus protease inhibitors into the CNS.