Non-clinical characterization of the disposition of EMA401, a novel small molecule angiotensin II type 2 receptor (AT2R) antagonist.

EMA401, (the S-enantiomer of 5-(benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), also known as Olodanrigan, is an orally active selective angiotensin II type 2 receptor (AT2 R) antagonist that is in Phase IIb clinical development as a novel analgesic for the relief of chronic pain. The main purpose of the present work was to investigate the disposition of a single 14 C- labeled EMA401 in non-clinical studies. The in vitro metabolism studies of EMA401 were undertaken to understand the hepatic biotransformation pathways in animal species used in toxicology studies and how they compare to human. Furthermore, investigation of EMA401's PK was carried out in vivo in rats. The study demonstrates the rapid absorption and distribution of drug-related material mainly to the tissues associated with absorption and elimination (GI tract, liver, and kidney). EMA401was then readily eliminated metabolically via the bile (95% of dose) predominantly in the form of the direct acylglucuronide (40% of dose), which was further hydrolysed by the intestinal flora to the active parent drug. Other metabolic pathways such as dealkylations and hydroxylation were also involved in the elimination of EMA401 to a lesser extent. EMA401 was metabolically unstable in hepatocytes of all species investigated and the key metabolites produced in the in vitro model were also detected in vivo. Independent of the dosing route, the S-enantiomer EMA401 showed a good in vivo chiral stability. Overall, the present study provides the first full characterization of the disposition of EMA401 in preclinical species.

Pharmacokinetics of Tropifexor, a Potent Farnesoid X Receptor Agonist, in Participants With Varying Degrees of Hepatic Impairment.

Tropifexor, a non-bile acid farnesoid X receptor (FXR) agonist, has dose-proportional pharmacokinetics and no obvious major enterohepatic circulation. This open-label study investigated the effect of hepatic impairment (HI), as determined by Child-Pugh grade, on tropifexor's pharmacokinetics, safety, and tolerability following a 200-µg dose in the fasted state. Blood samples were collected through 168 hours after dosing for quantification and plasma protein-binding determination. Total tropifexor exposure was comparable across participants with HI vs those with normal hepatic function. Tropifexor was highly protein bound (>99%) in human plasma across participants of all groups. The average unbound fractions (percentage free) were 0.14% in participants with normal hepatic function and mild HI, which increased to 0.17% and 0.24% in participants with moderate and severe HI, respectively. Similar unbound drug exposure was noted in participants with mild HI and normal hepatic function. Participants with moderate HI (N = 8) had a 1.6-fold increase in unbound exposure (area under the plasma concentration-time curve from time 0 to infinity [AUCinf,u ]) and a 1.3-fold increase in maximal exposure (Cmax,u ) vs those with normal hepatic function (geometric mean ratio: AUCinf,u , 1.64 [90%CI, 1.25-2.16]; Cmax,u , 1.30 [90%CI, 0.96-1.76]). Participants with severe HI (N = 8) had a 1.6-fold increase in AUCinf,u (1.61 [90%CI, 1.04-2.49]) and comparable Cmax,u (1.02 [90%CI, 0.60-1.72]) compared to participants with normal hepatic function. Tropifexor was well tolerated. The relative insensitivity of tropifexor to HI offers the potential to treat patients with severe liver disease without dose adjustment.

Plasma Protein Binding of Highly Bound Drugs Determined With Equilibrium Gel Filtration of Nonradiolabeled Compounds and LC-MS/MS Detection.

Accurate determination of the free fraction of a drug in plasma can be challenging when it falls below 1% and even more so when below 0.1%. Equilibrium dialysis with diluted plasma has been used to determine unbound fraction below 1%, but some analytes are not amenable to this method. One robust alternative for accurately measuring very highly bound compounds is equilibrium gel filtration; however, radiolabeled compounds have been used with this technique to quantify the low analyte concentrations. This report examined results obtained using radiolabeled compounds with liquid scintillation detection and those obtained using their nonradiolabeled analogs with liquid chromatography-tandem mass spectrometry detection. The 2 methods provided comparable results over the range of 0.005%-4% free, with a slope of 1.0 and a R2 = 0.93. These results demonstrate that equilibrium gel filtration with liquid chromatography-tandem mass spectrometry detection can be used earlier in the drug discovery process to determine the unbound fraction of highly bound drugs and may help obviate the need for radiolabeled compound.

Pharmacokinetic-pharmacodynamic interaction between nebicapone, a novel catechol-o-methyltransferase inhibitor, and controlled-release levodopa/carbidopa 200 mg/50 mg : randomized, double-blind, placebo-controlled, crossover study in healthy subjects.

BACKGROUND AND OBJECTIVES: Levodopa is the most effective symptomatic treatment for Parkinson's disease (PD), but its use is often associated with development of motor complications. These adverse responses to fluctuations in dopaminergic stimulation can be reduced by concomitant administration of a catechol-O-methyltransferase (COMT) inhibitor. Nebicapone is a new COMT inhibitor currently being developed for use as an adjunct to levodopa/dopa decarboxylase inhibitor in the treatment of PD. This article aimed to investigate the effect of single oral doses (50 mg, 100 mg and 200 mg) of nebicapone on levodopa pharmacokinetics and erythrocyte-soluble COMT (S-COMT) activity when coadministered with a single dose of controlled-release (CR) levodopa/carbidopa 200 mg/50 mg (Sinemet((R)) CR 200/50) in healthy subjects (n = 16). METHODS: This was a randomized, double-blind, placebo-controlled, four-way crossover study in healthy subjects, with at least 5 days of washout between treatment periods. RESULTS: There was a dose-dependent and significant increase in levodopa extent of exposure (area under the plasma concentration-time curve from time zero to infinity [AUC(infinity)]) without a significant change in peak exposure (maximum plasma concentration; [C(max)]). Using placebo as a reference, levodopa geometric mean ratios (GMRs) and 90% CIs following nebicapone 50 mg, 100 mg and 200 mg were, respectively, 1.13 (0.98, 1.30), 1.04 (0.90, 1.19) and 1.10 (0.96, 1.27) for C(max) and 1.26 (1.16, 1.34), 1.37 (1.27, 1.75) and 1.47 (1.42, 1.65) for AUC(infinity). For 3-O-methyldopa (3-OMD), the GMRs and 90% CIs were, respectively, 0.61 (0.55, 0.67), 0.45 (0.41, 0.50) and 0.33 (0.30, 0.36) for C(max) and 0.69 (0.61, 0.78), 0.53 (0.41, 0.61) and 0.41 (0.37, 0.47) for AUC(infinity). Nebicapone dose dependently and significantly decreased COMT activity. Maximum COMT inhibition occurred at 1.5-2.4 hours post-dose and ranged from 56% to 73% with nebicapone 50 mg and 200 mg, respectively. There was a good correlation between plasma concentrations of nebicapone and inhibition of S-COMT activity. Treatments were well tolerated. CONCLUSION: Following concomitant administration with levodopa/carbidopa CR 200 mg/50 mg, single doses of nebicapone 50 mg, 100 mg and 200 mg significantly and dose-dependently inhibited S-COMT activity, increased systemic exposure to levodopa, and reduced 3-OMD formation.

Programmed cell death induces male sterility in Actinidia deliciosa female flowers.

The importance of programmed cell death (PCD) during the life cycle of plants is well established, although the underlying molecular mechanisms are still poorly defined. An emerging system for the study of PCD during development in plants is that of sex organ abortion. In this work we investigate the degeneration of microspores in the anthers of Actinidia deliciosa female flowers. The kiwifruit, A. deliciosa, is a dioecious species native to China. Pollen development in female flowers is equivalent to pollen development in the male flowers, until the microspores are released from the tetrads. At this time the first differences appear, and include the condensation and shrinkage of the cytoplasm, blebbing of the plasma membrane and of the nuclear envelope, and condensation of chromatin. However, at the time these events are occurring, all other cellular organelles, including mitochondria, have their structures well preserved. Fragmentation of DNA was detected in situ by the TUNEL procedure, which involves the end labeling of the DNA fragments by terminal deoxynucleotidyl transferase with UTP conjugated to a detectable marker. This assay confirmed the morphological characterization of PCD in this system.

Pharmacokinetic-pharmacodynamic interaction between BIA 3-202, a novel COMT inhibitor, and levodopa/carbidopa.

This study investigated the tolerability and the pharmacokinetic and pharmacodynamic interactions between single oral administration of BIA 3-202 (50 mg, 100 mg, 200 mg, and 400 mg), a novel catechol-O-methyltransferase (COMT) inhibitor, and standard carbidopa/levodopa 25 mg/100 mg (Sinemet 25/100) in healthy adult volunteers. This was a single-center, double-blind, placebo-controlled, randomized, crossover study with 5 single-dose treatment periods with a washout period of 2 weeks between doses. During each treatment period, a different dose of BIA 3-202 or placebo was administered concomitantly with Sinemet 25/100. Tolerability was assessed by recording adverse events, vital signs, continuous EKG, and clinical laboratory parameters. Pharmacokinetic parameters of levodopa and 3-O-methyl-levodopa (3-OMD) were determined. The activity of soluble COMT in erythrocytes was also measured. Eighteen subjects (10 men and 8 women) participated in the study. The drug combination was well tolerated, with the adverse events reported being transient and generally mild in severity. Mean levodopa Cmax values were attained at 0.8 to 1.8 hours postdose. Thereafter, plasma levodopa levels declined with a mean t1/2 that increased in a manner that depended on the dose of BIA 3-202. The increase in systemic exposure to levodopa (AUC0-infinity) occurred at all doses of BIA 3-202, attaining its maximum at 200 mg BIA 3-202 (95% conficence interval, 1.43-1.73). The mean Cmax and AUC0-infinity values of 3-OMD decreased dose proportionally in BIA 3-202-treated subjects, with differences being statistically significant for all the doses tested. Maximum COMT inhibition occurred between 0.8 and 2.0 hours postdose, and ranged from 56 (50 mg) to 85% (400 mg). Time to return to baseline COMT activity ranged from 6 (50 mg) to 18 hours (400 mg), following the same dose-dependent tendency. In conclusion, the novel COMT inhibitor BIA 3-202 increased the bioavailability of levodopa and reduced the formation of 3-OMD when administered with standard levodopa/carbidopa.

Chronopharmacology of nebicapone, a new catechol-O-methyltransferase inhibitor.

OBJECTIVE: To investigate the chronopharmacology of nebicapone, a new catechol-O-methyltransferase (COMT) inhibitor currently being developed for use as an adjunct to levodopa/carbidopa or levodopa/benserazide in the treatment of Parkinson's disease. METHODS: This was a double-blind, randomised, placebo-controlled, parallel-group study. Eighteen Caucasian subjects were randomly assigned to treatment with either nebicapone 100 mg (n = 6), nebicapone 200 mg (n = 6) or placebo (n = 6) at 4-h intervals for 7 days. First dose occurred at 8:00 AM on day 1 and last dose at 8:00 AM on day 8. Blood samples for the determination of plasma drug concentrations of nebicapone and its glucuronidated and methylated metabolites and for the assay of erythrocyte soluble COMT (S-COMT) activity were taken at frequent times following the first and last doses, and before the 8:00 AM and 8:00 PM doses on days 2-7. RESULTS: Three men and three women in each group participated in the study. Mean +/- SD (range) age of study participants was 23.7 +/- 3.1 (21-28) years in the nebicapone 100 mg group, 22.2 +/- 0.4 (22-23) years in the nebicapone 200 mg group and 24.3 +/- 5.4 (18-32) in the placebo group. A circadian variation in the pre-dose nebicapone and nebicapone-glucuronide plasma concentrations was apparent. Both nebicapone and nebicapone-glucuronide levels were lower before the 8 PM dose in comparison to the 8 AM dose, suggesting that the absorption of nebicapone may follow a circadian variation. S-COMT activity showed no circadian variation in the placebo group. Therefore, the S-COMT activity variation found in nebicapone-treated subjects is considered to be due to changes in plasma concentrations of nebicapone, which is consistent with the fact that the pre-dose S-COMT activity was lower at the time at which nebicapone levels were maximal. Four subjects in the nebicapone 100 mg and placebo groups and six subjects in the nebicapone 200 mg group reported at least one adverse event (AE). All AEs were of mild or moderate intensity. Both nebicapone treatment regimens were subjectively well-tolerated, but a clinically relevant elevation in aspartate transaminase was observed in one subject of each nebicapone group. CONCLUSION: Nebicapone showed chronopharmacology in young Caucasian healthy subjects. The clinical impact of the circadian variation in the nebicapone metabolism and activity in Parkinson's disease patients deserves evaluation as it may have implications for drug prescription by modulating the distribution of the total daily dose along the 24-h scale.

Discovery of a long-acting, peripherally selective inhibitor of catechol-O-methyltransferase.

Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a 1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide (BIA 9-1067)) was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation as an adjunct to L-Dopa therapy of Parkinson's disease.

Species differences in pharmacokinetic and pharmacodynamic properties of nebicapone.

The present study was designed to characterize pharmacodynamic and pharmacokinetic properties of nebicapone in rats and mice. Upon oral administration of nebicapone the extent of mouse liver catechol-O-methyltransferase (COMT) inhibition is half that in the rat. Nebicapone was rapidly absorbed reaching plasma C(max) within 30min and being completely eliminated by 8h. Nebicapone was metabolized mainly by glucuronidation and methylation in both species, but rat had an extra major metabolite, resulting from sulphation. Administration of nebicapone by the intraperitoneal route significantly increased compound AUC in the rat while in the mouse a significant increase in AUC of metabolites was observed. These results show that nebicapone exhibited marked species differences in bioavailability and metabolic profile. Evaluation of COMT activity in rat and mice liver homogenates revealed that both had similar methylation efficiencies (K(cat) values, respectively 7.3 and 6.4min(-1)), but rat had twice active enzyme units as the mouse (molar equivalency respectively 150 and 83). Furthermore, nebicapone inhibited rat liver COMT with a lower K(i) than mouse liver COMT (respectively 0.2nM vs. 1.2nM). In conclusion, the results from the present study show that mice and rats respond differently to COMT inhibition by nebicapone. The more pronounced inhibitory effects of nebicapone in the rat may be related to an enhanced oral availability and less pronounced metabolism of nebicapone in this specie, but also concerned with the predominant expression of S-COMT over MB-COMT, the latter of which is less sensitive to inhibition by nebicapone than the former.

Pharmacokinetic-pharmacodynamic interaction between nebicapone and controlled-release levodopa/benserazide: a single-center, Phase I, double-blind, randomized, placebo-controlled, four-way crossover study in healthy subjects.

BACKGROUND: Nebicapone is a reversible catechol-O-methyltransferase (COMT) inhibitor. Coadministration of a COMT inhibitor with levodopa and a dopa-decarboxylase inhibitor (carbidopa or benserazide) increases levodopa exposure and its therapeutic effect. OBJECTIVES: The primary objective of this study was to investigate the effect of nebicapone (50, 100, and 200 mg), compared with placebo, on levodopa pharmacokinetics when coadministered with a single dose of controlled-release levodopa 100 mg/benserazide 25 mg. The secondary objectives were to investigate the effect of nebicapone on the erythrocyte-soluble COMT (S-COMT) activity and on the plasma levels of the levodopa 3-O-methylated metabolite (3-O-methyldopa [3-OMD]). Nebicapone's tolerability was also assessed. METHODS: This was a single-center, Phase I, doubleblind, randomized, placebo-controlled, 4-way crossover study conducted in healthy adult volunteers. Each of the 4 single-dose treatment periods was separated by a washout period of > or = 5 days. During the different treatment periods, subjects received a single dose of controlled-release levodopa 100 mg/benserazide 25 mg concomitantly with nebicapone 50, 100, and 200 mg or placebo. Plasma concentrations of nebicapone, levodopa, and 3-OMD were determined by HPLC. Blood samples (7 mL) for determination of plasma concentrations of levodopa, 3-OMD, and 2258 nebicapone, as well as for the assay of S-COMT activity, were collected in potassium EDTA test tubes at the following times: predose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 hours postdose. S-COMT activity was assessed as the amount of metanephrine formed by the action of S-COMT on an epinephrine substrate. Spontaneously reported clinical adverse events (AEs) were recorded throughout the study. RESULTS: Sixteen subjects (8 females, 8 males; mean [SD] age, 26.13 [6.29] years; weight, 69.4 [12.4] kg; body mass index, 24.0 [3.0] kg/m2) completed the 4 treatment periods and had data available for pharmacokinetic and pharmacodynamic analyses. Compared with placebo, levodopa C(max) increased 25%, 30%, and 34%, and AUC increased 14%, 37%, and 42% after administration of nebicapone 50, 100, and 200 mg, respectively. After administration of nebicapone 50, 100, and 200 mg, 3-OMD C(max) decreased 44%, 57%, and 58%, and 3-OMD AUC(0-infinity) decreased 33%, 37%, and 45%, respectively, compared with placebo. Extent of exposure to levodopa, as assessed by using AUC(0-t), increased with all doses of nebicapone in relationship to placebo, but the difference did not reach statistical significance. This may be related to a relatively high inter-subject variability: %CVs ranged from 48.0% with nebicapone 100 mg to 66.8% with placebo. Maximum S-COMT inhibition by nebicapone occurred at approximately 1.5 hours postdose and ranged from 57% with nebicapone 50 mg to 74% with nebicapone 200 mg. There was an inverse correlation between plasma concentrations of nebicapone and S-COMT activity; T(max) of nebicapone plasma concentrations and time to occurrence of the maximum inhibition of S-COMT activity appeared to correlate. Nineteen AEs were reported; 8 were assessed by the investigator as possibly related to treatment. All AEs were mild in severity. There were no serious AEs or discontinuations due to AEs. No abnormalities in liver enzyme levels were found. CONCLUSIONS: When administered concomitantly with a single dose of controlled-release levodopa 100 mg/benserazide 25 mg, single doses of nebicapone 50, 100, and 200 mg were well tolerated in these healthy adult volunteers, and dose dependently inhibited S-COMT activity and reduced 3-OMD formation compared with placebo. However, there was no significant difference in levodopa bioavailability.

Pharmacokinetic-pharmacodynamic interaction between BIA 3-202, a novel COMT inhibitor, and levodopa/benserazide.

BIA 3-202 is a novel catechol-O-methyltransferase (COMT) inhibitor being developed for use as a levodopa-sparing agent in Parkinson's disease. This study investigated the effect of four single oral doses of BIA 3-202 (50 mg, 100 mg, 200 mg, and 400 mg) compared with placebo on plasma concentrations of levodopa and its metabolite 3- O-methyl-levodopa (3-OMD) and on inhibition of erythrocyte COMT in healthy subjects receiving 100 mg of levodopa and 25 mg of benserazide (Madopar 125). This was a single-centre, double-blind, placebo-controlled, randomised, crossover study with five single-dose treatment periods. The washout period between doses was 2 weeks. On each treatment period, a different dose of BIA 3-202 or placebo was administered concomitantly with Madopar 125. Tolerability was assessed by recording adverse events, vital signs, continuous electrocardiogram and clinical laboratory parameters. In the study, 18 subjects (12 male and 6 female) participated. The drug combination was well tolerated. All doses of BIA 3-202 significantly increased the area under the concentration-time curve (AUC) versus placebo, ranging from 39% (95% confidence intervals, 1.06-1.69) with 50 mg to 80% (95% confidence intervals, 1.42-2.22) with 400 mg. No significant change in mean maximum plasma concentrations (C(max)) of levodopa was found. Mean C(max) and AUC of 3-OMD significantly decreased for all doses tested. BIA 3-202 caused a rapid and reversible inhibition of S-COMT activity, ranging from 57% (50 mg) to 84% (400 mg). In conclusion, the novel COMT inhibitor BIA 3-202 was well tolerated and significantly increased the bioavailability of levodopa and reduced the formation of 3-OMD when administered with standard release levodopa/benserazide.