Hideo Naganuma

Platelet inhibitory activity and pharmacokinetics of prasugrel (CS-747) a novel thienopyridine P2Y12 inhibitor: A single ascending dose study in healthy humans

We assessed the tolerability, pharmacodynamics as measured by inhibition of platelet aggregation (IPA), and pharmacokinetics of prasugrel (CS-747, LY640315), a novel thienopyridine antiplatelet agent in healthy volunteers. Twenty-four subjects were randomized into four groups of six in a double-blind, placebo-controlled trial. One subject in each group received placebo and five subjects received prasugrel orally at single doses of 2.5, 10, 30, or 75 mg. The IPA, assessed using 5 and 20 µM ADP, was periodically measured over a 7-day period by light transmission aggregometry. Plasma concentrations for three major metabolites, R-95913, R-106583, and R-100932, were measured. There were no serious adverse events and no clinically significant changes noted in any laboratory or clinical evaluations in any subject. At 1 h after prasugrel 30 and 75 mg, platelet aggregation induced by 20 µM ADP was inhibited by 43.5 ± 7.8 and 43.2 ± 15.7%, respectively, and this inhibition was significantly greater than that following placebo (5.9 ± 3.5%) (P < 0.05 for both doses). The degree of inhibition observed at 2 h was slightly higher with both prasugrel 30 and 75 mg (59.8 ± 9.9 and 57.0 ± 7.2%) and was maintained through the subsequent 22 h. At 24 h, maximal platelet aggregation induced by 20 µM ADP was reduced to ≤39% in all subjects receiving prasugrel 30 mg and to ≤38% in subjects receiving prasugrel 75 mg. Full recovery of platelet aggregation occurred between 48 h and 7 days suggesting irreversible inhibition by prasugrel and/or its metabolites. With prasugrel 2.5 and 10 mg, there was no measurable effect on platelet aggregation throughout the study (P > 0.05 for 2.5 and 10 mg prasugrel vs. placebo). With prasugrel 75 mg at 4 h postdose, there was a significant increase in the mean bleeding time compared to placebo (682 vs. 161 s; P < 0.05). Prasugrel metabolites obeyed linear pharmacokinetics and the three metabolites appeared in the plasma soon after administration, reaching maximum levels at approximately 1 h. In conclusion, prasugrel 30 and 75 mg were well tolerated and achieved a consistently high level of platelet inhibition with a fast onset of action. Errors appear in the original version of this article. These errors have since been rectified, and a corrected PDF is available here

Brain Insulin Impairs Amyloid-β(1-40) Clearance from the Brain

Cerebral amyloid-β peptide (Aβ) clearance plays a key role in determining the brain level of Aβ; however, its mechanism remains unclear. In this study, we investigated cerebral Aβ clearance across the blood-brain barrier (BBB) by using the Brain Efflux Index method. [125I]Aβ(1-40) was eliminated from rat brain to circulating blood with a half-life of 48.8 min and a half-saturation concentration of 8.15 nm. The Aβ(1-40) elimination rate was reduced by 30.5% in 23-month-old rats compared with 7-week-old rats. The intact form of Aβ(1-40) was detected in plasma after intracerebral administration, indicating the occurrence of efflux transport of intact Aβ(1-40). The Aβ(1-40) elimination rate was significantly inhibited by coadministration of 100 μg/ml insulin and 1 mm thiorphan by 44.6 and 34.0%, respectively. The level of intact [125I]Aβ(1-40) in the brain was increased by coadministration of insulin. Among insulin-degrading enzyme inhibitors, bacitracin inhibited the elimination rate, whereas N-ethylmaleimide and metal chelators had no effect. Receptor-associated protein, fucoidan, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile, anti-IGF-I receptor antibody, and l-tyrosine did not affect the Aβ(1-40) elimination rate, suggesting that the relevant receptors or transporters are not likely to be involved in the clearance. In conclusion, the present study has demonstrated the involvement of a proteolytic degradation process and an insulin-sensitive process in cerebral Aβ(1-40) clearance in the rat.

Dose-dependent inhibition of human platelet aggregation by prasugrel and its interaction with aspirin in healthy subjects.

The aims of this open-label, randomized, dose-escalation pharmacodynamic study of prasugrel, an orally active antiplatelet agent, were to assess its interaction with aspirin (ASA, 325 mg) in healthy subjects after a loading dose (LD) and subsequent 5 days of once-daily maintenance doses (MD) of prasugrel or the active comparator, clopidogrel. We measured platelet aggregation induced by ADP, collagen, and TRAP and compared effects on maximal and residual platelet aggregation responses. On a background of ASA, subjects were randomly assigned to 1 of 4 prasugrel treatment groups (LD/MD in mg: 20/5, 30/7.5, 40/10, or 60/15; n = 8/group) or to clopidogrel 300 mg LD/75 mg MD (n = 11). Prasugrel dose-dependently inhibited ADP-induced platelet aggregation and exhibited higher levels of platelet inhibition than clopidogrel or ASA alone. Prasugrel plus ASA resulted in additive inhibition of collagen- and TRAP-induced platelet aggregation. Although inhibition of residual aggregation was greater than inhibition of maximal aggregation, values were highly correlated. The safety and tolerability of prasugrel plus ASA were also monitored. Within the limitations of the study, prasugrel was found to be well tolerated when dosed as LD followed by MD in the presence of ASA and provided greater platelet inhibition than ASA alone.

Greater inhibition of platelet aggregation and reduced response variability with prasugrel versus clopidogrel : An integrated analysis

Multiple studies report response variability to a 300-mg clopidogrel loading dose (LD). Pooled platelet aggregometry data compared responses (change in maximal platelet aggregation [ΔMPA] or inhibition of platelet aggregation [IPA]) to clopidogrel 300-mg (n = 131) or prasugrel 60-mg (n = 109) LDs. Poor responder rates were determined using empiric criteria (IPA < 10% and ΔMPA < 10% for 20 µM and 5 µM adenosine diphosphate [ADP]) and Bayesian model-based criteria (IPA < 20% and ΔMPA < 15% for 20 µM ADP; IPA < 25% and ΔMPA < 20% for 5 µM ADP). Prasugrel achieved greater ΔMPA and IPA from 2 to 24 hours post-LD (P < .001). For 20 µM ADP, poor responder rates for clopidogrel ranged from 17% to 43%; no prasugrel poor responders were observed. Regardless of the criterion, prasugrel 60 mg achieved greater IPA and fewer poor responders than the clopidogrel 300-mg LD.

Clopidogrel poor responders: an objective definition based on Bayesian classification.

Existing definitions of poor response to the antiplatelet effect of clopidogrel are empiric. Bayesian classification theory is widely used to classify subjects into non-overlapping groups based on observed responses. The purpose of this analysis is to objectively define pharmacodynamic poor responders to clopidogrel using Bayesian classification methodology. An integrated database of turbidometric platelet aggregometry data (5 and 20 µM ADP) was analyzed from 112 healthy subjects who participated in three Phase 1 clinical pharmacology studies. Change in maximum platelet aggregation (ΔMPA) from baseline and percent inhibition of platelet aggregation (IPA) were evaluated at both 4–5 and 24 hours after a clopidogrel 300 mg loading dose (LD). Clopidogrel poor responders were predefined as having a response similar to that of drug-free baseline, derived from multiple MPA values prior to clopidogrel administration. The model identified a clopidogrel poor responder as a subject who failed to achieve and maintain an IPA ≥ 25% (or ΔMPA ≥ 20%) with 5 µM ADP at 4–5 and 24 hours after dosing or who failed to achieve and maintain an IPA ≥ 20% (or ΔMPA ≥ 15%) with 20 µM ADP at 4–5 and 24 hours after dosing. Application of these thresholds indicated that, depending on the concentration of ADP used, 25% to 45% of subjects were classified as clopidogrel poor responders. Using thresholds from the published literature resulted in 17% to 56% of subjects being classified as poor responders. Objective thresholds for pharmacodynamic poor responders to clopidogrel should consider the concentration of the agonist used and may help assess the consistency of pharmacodynamic response to novel ADP receptor antagonists.

Sensitive fluorescence labelling for analysis of carboxylic acids with 4-bromomethyl-6,7-methylenedioxycoumarin

Abstract A fluorescence labelling reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC), was synthesized from sesamol and citric acid by Pechman condensation followed by bromination. Nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as a catalyst and were separated by reversed-phase high-performance liquid chromatography (HPLC). Quantitative studies revealed that n -caproic acid was esterifted completely at low temperature and with sufficient reproducibility. The detection limit was just below 15 fmol per injection at a signal-to-noise ratio of 3. The fluorescence quenching of the BrMDC derivative was the lowest in conventional mixed solvent systems in comparison with those of two previously reported coumarin compounds. BrMDC was also applied to the simultaneous analysis of some acidic non-steroidal anti-inflammatry agents by reversed-phase HPLC.

3-Bromomethyl-7-methoxy-1,4-benzoxazin-2-one as a highly sensitive fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography

Abstract A fluorescence labelling reagent, 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB), was synthesized from 2-amino-5-methoxyphenol and ethyl pyruvate by the Wislecenus reaction followed by bromination. Picomole to nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as catalysts. BrMB derivatives of fatty acids were separated on a reversed-phase column and were sensitively detected fluorimetrically at 440 nm with excitation at 345 nm. Quantitative studies revealed that n -caproic acid was esterified completely under mild conditions and with sufficient reproducibility. The detection limit of the BrMB derivative of n -caproic acid was just below 2 fmol.

Design of captopril sustained-release preparation with oily semisolid matrix intended for use in human subjects

Abstract A captopril sustained-release dosage form using oily semisolid matrix (OSSM) has been studied to develop a formulation useful in human treatment. Four OSSMs which had different in vitro dissolution rates were administered to human subjects. The resulting bioavailabilities revealed that the best OSSM suitable for humans had a faster in vitro dissolution rate than that for dogs. Another series of administrations of OSSM also showed that formulated ascorbic acid improved the bioavailability of OSSM, and that the bioavailability was sufficient for a sustained-release dosage form so long as the amount of ascorbic acid in the OSSM was more than 5 times that of the captopril by weight. Pharmacokinetic analysis was performed based on plasma concentrations of captopril in humans ( n = 8) after a single oral administration of conventional tablets or OSSM reformulated for humans. Calculated areas under the curve ( AUC s, 0-∞ h) of plasma captopril concentration were 250.5 for conventional tablets and 283.5 (ng·h/ml) for OSSM. The mean residence times (MRTs) obtained by both formulations were 1.75 h and 3.59 h, respectively. The duration time in which plasma captopril concentration stayed above 50% inhibitory concentration of angiotensin converting enzyme activity was calculated. Total duration time (TDT) per day of conventional tablets (12.5 mg) taken 3 times daily was calculated to be 10.95 h. The TDT of OSSM was 17.00 h when the OSSM (18.75 mg of captopril) was administered twice a day at 12-h intervals. Consequently, OSSM dosed twice a day is expected to show a greater efficiency than conventional tablets taken 3 times daily.

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.

Enantiospecific assay for mammalian carbonyl reductase by liquid chromatography with fluorescence detection

A fluorogenic substrate with an unsymmetrical carbonyl for the sensitive assay of mammalian carbonyl reductase activities, 4-(6-methoxy-2-benzoxazolyl)acetophenone (I), has been prepared. The fluorescence quantum yield of I in acetonitrile is 0.12 at the emission maximum of 448 nm. The corresponding racemic alcohol produced by the chemical reduction of I, (+/-)-sec.-[4-(6-methoxy-2-benzoxazolyl)]phenethyl alcohol (II), exhibits ca. three- to fourteen-fold higher fluorescence at a shorter wavelength emission maximum of 370 nm in conventional solvents. Each enantiomer of II is sufficiently resolved on a chiral cellulose high-performance liquid chromatographic column without derivatization and quantified with high reproducibility. The detection limit for II is 20 fmol per injection at a signal-to-noise ratio of 3. The validity and applicability of I are evaluated with cytosols of mammalian tissues. The optimal pH for metabolic reduction of I in rabbit liver cytosol preparations is 6.2 in the presence of NADPH. The metabolism is proved to be highly stereoselective. The resulting alcohol produced by mammalian tissue preparations, except rabbit kidney, is predominantly of the S-(-)-configuration.