First-in-Human Pharmacokinetics and Safety Study of GSK3008356, a Selective DGAT1 Inhibitor, in Healthy Volunteers.

Diacylglycerol acyltransferase (DGAT) enzymes are involved in triglyceride (TG) biosynthesis. GSK3008356 is a potent and selective DGAT1 inhibitor that was administered orally in a 2-part study as double-blind, randomized, placebo-controlled single doses (SDs) and repeat doses (RDs) in healthy subjects to investigate its pharmacokinetics, pharmacodynamics, and safety/tolerability. Gastrointestinal adverse events were considered drug related and increased with dose and when given as multiple doses. In the SD part (n = 80), GSK3008356 was dosed from 5 to 200 mg as single or multiple doses per day. In the RD part (n = 24), GSK3008356 was dosed twice daily at 1, 3, and 10 mg for 14 days. GSK3008356 was generally well tolerated in the SD and RD parts. With single doses, absorption was rapid (median tmax , 0.5-1.5 hours), whereas single-day divided dosing resulted in higher tmax . Following 14-day RD oral administration, GSK3008356 was also rapidly absorbed, with median tmax ranging from 0.5 to 0.75 hours on days 1 and 14. Estimated mean half-life ranged from 1.5 to 4.6 hours with SDs and 1.3 to 2.1 hours with RDs. Exposure of GSK3008356 was largely dose proportional after RDs. At higher doses, there was a trend toward lower absolute postprandial TG level in some subjects.

Identification of Novel Pathways in Idelalisib Metabolism and Bioactivation.

Idelalisib (ILB) is a selective phosphatidylinositol-3-kinase delta inhibitor approved for the treatment of hematological malignancies. However, ILB frequently causes hepatotoxicity, and the exact mechanism remains unclear. The current study profiled the metabolites of ILB in mouse liver, urine, and feces. The major metabolites found in the liver were oxidized metabolite GS-563117 (M1) and ILB-glutathione (GSH) adduct (M2). These metabolic pathways were confirmed by analysis of urine and feces from mice treated with ILB. Identification of ILB-GSH adduct (M2) suggests the formation of reactive metabolites of ILB. We also found that M1 can produce reactive metabolites and form M1-GSH adducts. The GSH-conjugates identified in mouse liver were also found in the incubations of ILB and M1 with human liver microsomes. Furthermore, we illustrated that CYP3A4 and 2C9 are the key enzymes contributing to the bioactivation pathway of ILB and M1. In summary, our work revealed that both ILB and its major metabolite M1 can undergo bioactivation to produce reactive metabolites in the liver. Further studies are required to determine whether these metabolic pathways contribute to ILB hepatotoxicity.

A reliable and stable method for the determination of foretinib in human plasma by LC-MS/MS: Application to metabolic stability investigation and excretion rate.

Foretinib (GSK1363089) is a multiple receptor tyrosine kinases inhibitor. In this study, a reliable, fast liquid chromatography-tandem mass spectrometric method was described for assaying foretinib in plasma, urine, and rat liver microsome samples. Simple extraction procedure by protein preciptation with acetonitrile was implemented for foretinib and brigatinib (internal standard) analysis. Chromatographic resolution of analytes was achieved on C18 column with the help of isocratic mobile phase. The binary mobile phase consisted of 60% ammonium formate (10 mM, pH 4.2) and 40% acetonitrile at a flow rate of 0.25 mL/min. Run time was 3 min, and both foretinib and brigatinib were eluted within 0.74 and 1.95 min; they were detected in positive ion mode utilizing multiple reactions monitoring mode. Linearity of the proposed method ranged from 5 to 500 ng/mL (r2 ≥ 0.9993) in the human plasma. Lower limit of quantification and detection were 6.0 and 1.8 ng/mL, respectively. Intraday and interday precision and accuracy were 0.16 to 1.67 % and -2.39 to -0.52 %. In vitro half-life and intrinsic clearance were 24.93 min and 6.56 mL/min/kg, respectively. Literature review showed that no previous studies have been proposed for the analytical quantification of foretinib in human plasma or its metabolic stability. The established method was also applied to estimate the rate of foretinib excretion in rat urine. The developed method can be used for foretinib pharmacokinetic applications.

Sensitive analysis and pharmacokinetic study of epalrestat in C57BL/6J mice.

Epalrestat is clinically applied for the management of diabetic peripheral neuropathy, yet its pharmacokinetic properties are not well understood. In this study, a rapid and sensitive LC-MS/MS method was established for assaying epalrestat in bio-samples of mice. The method was validated and it showed a good linearity over the range of 2-5000ng/mL, a precision of less than 12.3%, and recovery and matrix effects of 112.5-123.6% and 87.9-89.5%, respectively. After administration of a single dose of epalrestat administered, the exposure level of AUC0-∞ was positively dose-dependent and the mean Cmax, AUC0-12h, T1/2, and MRT were 36.23±7.39µg/mL, 29,086.5µg/Lh, 1.2h and 1.8h, respectively. Epalrestat was highly exposed in stomach, intestine, liver and kidney, and only a small amount was detected in brain, urine and feces. Multi-dose of epalrestat significantly increased MRT and apparent volume of distribution (Vd) relative to those of a single-dose.

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

BACKGROUND: The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. CONCLUSION: These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

Pharmacokinetics of TAK-475, a Squalene Synthase Inhibitor, in Rats and Dogs.

The pharmacokinetics of TAK-475 (lapaquistat acetate), a squalene synthase inhibitor, was investigated in rats and dogs. After oral administration of (14)C-labeled TAK-475 ([(14)C]TAK-475) to rats and dogs at a dose of 10 mg/kg, the bioavailability (BA) was relatively low at 3.5 and 8.2%, respectively. The main component of the radioactivity in the plasma was M-I, which has a comparable pharmacological activity to TAK-475 in vitro. The radioactivity in the portal plasma after intraduodenal administration of [(14)C]TAK-475 to portal vein-cannulated rat was also mainly M-I, suggesting that most of the TAK-475 was hydrolyzed to M-I during the permeable process in the intestine. The concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration of [(14)C]TAK-475 to rats. The main elimination route of the radioactivity was fecal excretion after oral administration of [(14)C]TAK-475 to rats and dogs, and the absorbed radioactivity was mainly excreted via the bile as M-I in rats. M-I excreted into the bile was partially subjected to enterohepatic circulation. These results suggest that although the BA values of TAK-475 are low, M-I can exert compensatory pharmacological effects in the animals. These pharmacokinetic characteristics in animals were also confirmed in the clinical studies. The evaluation of M-I disposition is important for the pharmacokinetics, pharmacodynamics and toxicity of TAK-475 in animals and humans.

Urinary excretion and metabolism of miglustat and valproate in patients with Niemann-Pick type C1 disease: One- and two-dimensional solution-state (1)H NMR studies.

Niemann-Pick type C1 (NP-C1) disease is a neurodegenerative lysosomal storage disease for which the only approved therapy is miglustat (MGS). In this study we explored the applications and value of both one- and two-dimensional high-resolution NMR analysis strategies to the detection and quantification of MGS and its potential metabolites in urine samples collected from NP-C1 disease patients (n=47), and also applied these techniques to the analysis of the anticonvulsant drug valproate and one of its major metabolites in ca. 30% of these samples (i.e. from those who were also receiving this agent for the control of epileptic seizures). A combination of high-resolution 1D and 2D TOCSY/NOESY techniques confirmed the identity of MGS in the urinary (1)H NMR profiles of NP-C1 patients treated with this agent (n=25), and its quantification was readily achievable via electronic integration of selected 1D resonance intensities. However, this analysis provided little or no evidence for its metabolism in vivo, observations consistent with those acquired in corresponding experiments performed involving an in vitro microsomal system. Contrastingly, the major valproate metabolite 1-O-valproyl-ß-glucuronide was readily detectable and quantifiable in 14/47 of the urine samples investigated, despite some resonance overlap problems (identification of this agent was confirmed by experiments involving equilibration of these samples with ß-glucuronidase, a process liberating free valproate). In order to facilitate and validate the detection of MGS in urine specimens, full assignments of the (1)H NMR spectra of MGS in both buffered aqueous (pH 7.10) and deuterated methanol solvent systems were also made. The pharmacological and bioanalytical significance of data acquired are discussed, with special reference to the advantages offered by high-resolution NMR analysis.

Pharmacokinetics and Urinary Excretion Mechanism of Orteronel (TAK-700), A Novel 17,20-Lyase Inhibitor, in Animals.

Orteronel is newly identified as a selective 17,20-lyase inhibitor for an agent for castration resistant prostate cancer. The absorption and disposition of [(14)C]orteronel were investigated in rats and monkeys. Orteronel was extensively excreted into rat and monkey urine in an unchanged form after oral administration. The unbound based renal clearances in rats and monkeys were greater than the respective glomerular filtration rates (GFR), suggesting that urinary tubular secretion plays an important role in the renal excretion of orteronel. Therefore, the uptake of [(14)C]orteronel was investigated using rat kidney slices to estimate the contribution of carrier-mediated transport on the urinary tubular secretion. The uptake study using rat kidney slices suggested that the transport of orteronel from the blood circulation to the kidney was mediated by a digoxin sensitive transport system represented by Oatp4c1 and non-saturable components. Furthermore, the saturable component accounted for a limited fraction of the total renal uptake by rat kidney slices. These results suggested that non-saturable uptake mainly contributed to the renal excretion of orteronel in rats.

Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat.

Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions.

Elucidating the mechanism of cytochrome P450-mediated pyrimidine ring conversion to pyrazole metabolites with the BACE1 inhibitor GNE-892 in rats.

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a ß-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.

An alternative explanation of hypertension associated with 17α-hydroxylase deficiency syndrome.

The syndrome of 17α-hydroxylase deficiency is due to the inability to synthesize cortisol and is associated with enhanced secretion of both corticosterone and 11-deoxy-corticosterone (DOC). In humans, corticosterone and its 5α-Ring A-reduced metabolites are excreted via the bile into the intestine and transformed by anaerobic bacteria to 21-dehydroxylated products: 11ß-OH-progesterone or 11ß-OH-(allo)-5α-preganolones (potent inhibitors of 11ß-HSD2 and 11ß-HSD1 dehydrogenase). Neomycin blocks the formation of these steroid metabolites and can blunt the hypertension in rats induced by either ACTH or corticosterone. 3α,5α-Tetrahydro-corticosterone, 11ß-hydroxy-progesterone, and 3α,5α-tetrahydro-11ß-hydroxy-progesterone strongly inhibit 11ß-HSD2 and 11ß-HSD1 dehydrogenase activity; all these compounds are hypertensinogenic when infused in adrenally intact rats. Urine obtained from a patient with 17α-hydroxylase deficiency demonstrated markedly elevated levels of endogenous glycyrrhetinic acid-like factors (GALFs) that inhibit 11ß-HSD2 and 11ß-HSD1 dehydrogenase activity (>300 times greater, and >400 times greater, respectively, than those in normotensive controls). Thus, in addition to DOC, corticosterone and its 5α-pathway products as well as the 11-oxygenated progesterone derivatives may play a previously unrecognized role in the increased Na(+) retention and BP associated with patients with 17α-hydroxylase deficiency.

Single-dose pharmacokinetics of the HCV polymerase inhibitor mericitabine in healthy Caucasian and Japanese subjects.

To investigate the pharmacokinetics of mericitabine in healthy Caucasian and Japanese subjects, healthy Caucasian (n = 32) and Japanese (n = 32) subjects were randomized to receive single 500, 1,000, or 2,000 mg doses of mericitabine or a placebo, after which plasma and urine samples were collected for 72 h. Mericitabine (prodrug), RO4995855 (parent), and RO5012433 (uridine metabolite) concentrations were quantified by tandem mass spectrometry. Pharmacokinetics were estimated by non-compartmental methods, and pharmacokinetic parameters of RO4995855 were normalized by body weight. Exposure to RO4995855 was similar in both populations after administration of mericitabine 500, 1,000, and 2,000 mg. Mean AUCinf of RO4995855 increased in a dose-proportional manner from 28.8 to 52.3, and 113.0 µg·h/mL in Caucasian subjects, and from 32.5 to 57.1 and 119 µg·h/mL in Japanese subjects. A linear relationship was observed between the weight-adjusted dose of mericitabine and Cmax (r(2) = 0.83 and 0.80) and AUC (r(2) = 0.94 and 0.74) for RO4995855 in Caucasian and Japanese subjects, respectively. Mean half-life and renal clearance of RO4995855 were similar and independent of dose in both populations. The results support the use of the same dosing regimens in Caucasian and Asian subjects.

Pharmacokinetics, pharmacodynamics and tolerability of opicapone, a novel catechol-O-methyltransferase inhibitor, in healthy subjects: prediction of slow enzyme-inhibitor complex dissociation of a short-living and very long-acting inhibitor.

BACKGROUND AND OBJECTIVES: Opicapone is a novel catechol-O-methyltransferase (COMT) inhibitor. The purpose of this study was to evaluate the tolerability, pharmacokinetics (including the effect of food) and pharmacodynamics (effect on COMT activity) following single oral doses of opicapone in young healthy male volunteers. METHODS: Single rising oral doses of opicapone (10, 25, 50, 100, 200, 400, 800 and 1,200 mg) were administered to eight groups of eight subjects per group (two subjects randomized to placebo and six subjects to opicapone), under a double-blind, randomized, placebo-controlled design. In an additional group of 12 subjects, a 50 mg single dose of opicapone was administered on two occasions, once having fasted overnight and once with a high-fat high-calorie meal. RESULTS: Opicapone was well tolerated at all doses tested. The extent of systemic exposure (area under the plasma concentration-time curve and maximum plasma concentration) to opicapone and metabolites increased in an approximately dose-proportional manner and showed a decrease following concomitant ingestion of a high-fat high-calorie meal. The apparent terminal elimination half-life of opicapone was 0.8-3.2 h. Sulphation appeared to be the main metabolic pathway for opicapone, and both opicapone and the main sulphated metabolite are likely excreted by the biliary route. Maximum COMT inhibition by opicapone was dose dependent, ranged from 36.1% (10 mg) to 100% (200 mg and above), and reached statistical significance at all doses tested. The long duration of COMT inhibition by opicapone, however, tended to be independent from the dose taken. The observed half-life of opicapone-induced COMT inhibition in human erythrocytes was 61.6 h (standard deviation [SD] = 37.6 h), which reflects an underlying dissociative process with a kinetic rate constant of 3.1 × 10(-6) s(-1) (SD = 1.9 × 10(-6) s(-1)). Such a process compares well to the estimated dissociation rate constant (k(off)) of the COMT-opicapone molecular complex (k(off) = 1.9 × 10(-6) s(-1)). CONCLUSIONS: Opicapone was well-tolerated and presented dose-proportional kinetics. Opicapone demonstrated marked and sustained inhibition of erythrocyte soluble COMT activity. Based on the observation that the half-life of COMT inhibition is independent of the dose and that it reflects an underlying kinetic process that is consistent with the k(off) value of the COMT-opicapone complex, we propose that the sustained COMT inhibition, far beyond the observable point of clearance of circulating drug, is due to the long residence time of the reversible complex formed between COMT and opicapone. Globally, these promising results provide a basis for further clinical development of opicapone.

Regiospecific and stereospecific triangulation of the structures of metabolites formed by sequential metabolism at multiple prochiral centers.

Structures of in vivo secondary metabolites of a norbornane-containing drug candidate with multiple prochiral centers were triangulated, in a regio- and stereospecific fashion, using in vitro metabolism data from synthetic primary metabolites and in vivo metabolism data from the separate administration of a radiolabeled primary metabolite, [(14)C]-(S)-2-((1R,2S,4R,5S)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (M1). A mass balance study on the 11ß hydroxysteroid dehydrogenase type 1 enzyme inhibitor [(14)C]-(S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221) in rats was dosed at 2 mg/kg. Radioactivity was excreted mainly in urine. Metabolites of AMG 221 were quantified by high-performance liquid chromatography with radiometric detection and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS revealed at least 38 metabolites. Seven monohydroxylated metabolites mediated formation of the other 31 metabolites. Twenty-eight metabolites were identified regio- and stereo-specifically. Little parent drug was observed in urine or feces. Monohydroxy metabolite M1 was the major metabolite comprising 17 to 24% of excreted dose, and seven monohydroxy metabolites comprised 29 (male) and 37% (female) of dose. Of 11 quantifiable isobaric dihydroxy metabolites that comprised 8.3 (male) and 24% (female) of dose, 10 were identified regio- and stereospecifically by triangulation. A single trihydroxy metabolite comprised approximately 10% of dose. Complex secondary metabolism of drugs with multiple prochiral centers can be elucidated in a regio- and stereospecific fashion without NMR through synthesis and in vitro and in vivo studies on the metabolism of chiral primary oxidation products.

A nonclinical safety and pharmacokinetic evaluation of N6022: a first-in-class S-nitrosoglutathione reductase inhibitor for the treatment of asthma.

S-nitrosoglutathione reductase is the primary enzyme responsible for the metabolism of S-nitrosoglutathione (GSNO), the body's main source of bioavailable nitric oxide. Through its catabolic activity, GSNO reductase (GSNOR) plays a central role in regulating endogenous S-nitrosothiol levels and protein S-nitrosation-based signaling. By inhibiting GSNOR, we aim to increase pulmonary GSNO and induce bronchodilation while reducing inflammation in lung diseases such as asthma. To support the clinical development of N6022, a first-in-class GSNOR inhibitor, a 14-day toxicology study was conducted. Sprague-Dawley rats were given 2, 10 or 50 mg/kg/day N6022 via IV administration. N6022 was well tolerated at all doses and no biologically significant adverse findings were noted in the study up to 10 mg/kg/day. N6022-related study findings were limited to the high dose group. One male rat had mild hepatocellular necrosis with accompanying increases in ALT and AST and several male animals had histological lung assessments with a slight increase in foreign body granulomas. Systemic exposure was greater in males than females and saturation of plasma clearance was observed in both sexes in the high dose group. Liver was identified as the major organ of elimination. Mechanistic studies showed dose-dependent effects on the integrity of a rat hepatoma cell line.

Specific pharmacokinetic aspects of the urinary tract.

This chapter reviews the evidence for "specific" pharmacokinetics playing a role in currently marketed drugs intended to treat lower urinary tract (LUT) symptoms. Principles of drug targeting include intrinsic properties of drugs or organs as well as drug formulations to modify drug release or to create confinement of drug presence. Prodrugs and specific formulations to deliver high drug concentrations at the site(s) of action as well as other ways to manipulate drug distribution to achieve enrichment in target tissues are considered. In overactive bladder (OAB), specific formulations for oxybutynin have been introduced to reduce the level of side effects of the active drug. Extended release tablet formulations and a topical gel formulation have been introduced, with efficacy similar to immediate release (IR) tablets, but with a reduction in anticholinergic adverse effects. However, these modifications have not led to outstanding performance parameters compared to other anticholinergic drugs marketed as IR formulations. Urinary excretion is discussed as potential mechanism for targeting LUT symptoms, but no strong indications appear to exist that this mechanism would contribute for currently available drugs. Intravesical administration of drugs is not a preferred option and only considered for drugs like botulinum toxin, where the inconvenient application compensates for a reasonable degree of long-term efficacy in severe refractory OAB. Alpha acid glycoprotein binding is discussed as a potential factor to influence drug tissue distribution, and it is concluded that there is reasonable evidence that for tamsulosin this mechanism is responsible for the difference in free fraction of the drug observed in plasma and prostate, which could contribute to its relative absence of blood pressure effects in patients with LUT symptoms related to benign prostate hyperplasia (LUTS-BPH). The principle of irreversible inhibition of type II 5α-reductase as a tool to develop drugs to reduce prostatic levels of dihydrotestosterone is employed by both dutasteride and finasteride for treatment of LUTS-BPH. Of the mechanisms discussed, the principles employed for the 5α-reductase blockers and tamsulosin in this respect can be considered relatively specific for its urological indication.

Safety, tolerability, and pharmacokinetics of eliglustat tartrate (Genz-112638) after single doses, multiple doses, and food in healthy volunteers.

Three phase 1 studies of eliglustat tartrate (Genz-112638), an oral inhibitor of glucosylceramide synthase under development for treating Gaucher disease type 1 (GD1), evaluated the safety, tolerability, and pharmacokinetics in healthy volunteers after escalating single doses (n = 99), escalating multiple doses (n = 36), and food (n = 24). Eliglustat tartrate was well tolerated at single doses ≤ 20 mg/kg and multiple doses ≤ 200 mg bid, with 50 mg bid producing plasma concentrations in the predicted therapeutic range. No serious adverse events occurred. Mild to moderate events of nausea, dizziness, and vomiting increased in frequency with escalating single and multiple doses. Single doses ≥ 10 mg/kg caused mild increases in electrocardiogram PR, QRS, and QT/QTc intervals. Single-dose pharmacokinetics showed dose linearity but not proportionality. Maximum plasma concentrations occurred at ~2 hours, followed by a monophasic decline with a ~6-hour terminal half-life. Unchanged drug in 8-hour urine collections was <1.5% of administered doses. Food did not significantly affect the rate or extent of absorption. Multiple-dose pharmacokinetics was nonlinear, showing higher than expected plasma drug concentrations. Steady state was reached ~60 hours after bid dosing. Higher drug exposure occurred in slower CYP2D6 metabolizers. Based on favorable results in healthy participants, a phase 2 trial of eliglustat tartrate was initiated in GD1 patients.

Itraconazole, a P-glycoprotein and CYP3A4 inhibitor, markedly raises the plasma concentrations and enhances the renin-inhibiting effect of aliskiren.

In a randomized crossover study, 11 healthy volunteers took 100 mg (first dose 200 mg) of the antifungal drug itraconazole, a P-glycoprotein and CYP3A4 inhibitor, or placebo twice daily for 5 days. On day 3, they ingested a single 150-mg dose of aliskiren, a renin inhibitor used in the treatment of hypertension. Itraconazole raised the peak plasma aliskiren concentration 5.8-fold (range, 1.1- to 24.3-fold; P < .001) and the area under the plasma aliskiren concentration-time curve 6.5-fold (range, 2.6- to 20.5-fold; P < .001) but had no significant effect on aliskiren elimination half-life. Itraconazole increased the amount of aliskiren excreted into the urine during 12 hours 8.0-fold (P < .001) and its renal clearance 1.2-fold (P = .042). Plasma renin activity 24 hours after aliskiren intake was 68% lower during the itraconazole phase than during the placebo phase (P = .011). In conclusion, itraconazole markedly raises the plasma concentrations and enhances the renin-inhibiting effect of aliskiren. The interaction is probably mainly explained by inhibition of the P-glycoprotein-mediated efflux of aliskiren in the small intestine, with a minor contribution from inhibition of CYP3A4. Concomitant use of aliskiren and itraconazole is best avoided.

Simultaneous quantitative determination of N(G),N(G)-dimethyl-L-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry.

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine. OBJECTIVES: The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 µL of human plasma, serum or urine. METHODS: The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection. RESULTS: The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 µM for all compounds in water, plasma and serum and 0.1 µM in urine. The LOQ was 0.05 µM for ADMA, SDMA, NMMA and H-Arg and 0.5 µM for Arg and Cit in water, plasma and serum; while in urine was 0.1 µM for ADMA, SDMA, NMMA and H-Arg and 0.5 µM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M±SD) 0.56±0.10 µM and 0.84±0.21 µM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54±0.18 µM and 1.14±0.36 µM), while in neonatal urine ADMA was 11.98±7.13 µmol mmol(-1) creatinine. CONCLUSIONS: Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Metabolism, excretion, and pharmacokinetics of [14C]INCB018424, a selective Janus tyrosine kinase 1/2 inhibitor, in humans.

The metabolism, excretion, and pharmacokinetics of 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (INCB018424), a potent, selective inhibitor of Janus tyrosine kinase1/2 and the first investigational drug of its class in phase III studies for the treatment of myelofibrosis, were investigated in healthy human subjects given a single oral 25-mg dose of [(14)C]INCB018424 as an oral solution. INCB018424 and total radioactivity were absorbed rapidly (mean time to reach the maximal drug concentration <1 h), declining in a monophasic or biphasic fashion (mean t(1/2) of 2.32 and 5.81 h, respectively). Recovery of administered radioactivity was fairly rapid (>70% within 24 h postdose) with 74 and 22% recovered in urine and feces, respectively. Parent compound was the predominant entity in the circulation, representing 58 to 74% of the total radioactivity up to 6 h postdose, indicating that the overall circulating metabolite burden was low (<50% of parent). Two metabolite peaks in plasma (M18 and a peak containing M16/M27, both hydroxylations on the cyclopentyl moiety) were identified as major (30 and 14% of parent based on area under the curve from 0 to 24 h). The exposures of other circulating INCB018424-related peaks were <10% of parent, consisting of mono- and dihydroxylated metabolites. The profiles in urine and feces consisted of hydroxyl and oxo metabolites and subsequent glucuronide conjugates with parent drug accounting for <1% of the excreted dose, strongly suggesting that after an oral dose, INCB018424 was >95% absorbed. In healthy subjects administered daily oral doses of unlabeled INCB018424, there were minimal differences in parent and metabolite concentrations between day 1 and day 10, indicating a lack of accumulation of parent or metabolites between single and multiple dosing.