Efficacy of protocol-based pharmacotherapy management on anticoagulation with warfarin for patients with cardiovascular surgery.

WHAT IS KNOWN AND OBJECTIVE: Anticoagulation therapy with warfarin requires periodic monitoring of prothrombin time-international normalized ratio (PT-INR) and adequate dose adjustments based on the data to minimize the risk of bleeding and thromboembolic events. In our hospital, we have developed protocol-based pharmaceutical care, which we called protocol-based pharmacotherapy management (PBPM), for warfarin therapy. The protocol requires pharmacists to manage timing of blood sampling for measuring PT-INR and warfarin dosage determination based on an algorithm. This study evaluated the efficacy of PBPM in warfarin therapy by comparing to conventional pharmaceutical care. METHODS: From October 2013 to June 2015, a total of 134 hospitalized patients who underwent cardiovascular surgeries received post-operative warfarin therapy. The early series of patients received warfarin therapy as the conventional care (control group, n=77), whereas the latter received warfarin therapy based on the PBPM (PBPM group, n=68). These patients formed the cohort of the present study and were retrospectively analysed. RESULTS: The indications for warfarin included aortic valve replacement (n=56), mitral valve replacement (n=4), mitral valve plasty (n=22) and atrial fibrillation (n=29). There were no differences in patients' characteristics between both groups. The percentage time in therapeutic range in the first 10 days was significantly higher in the PBPM group (47.1%) than that in the control group (34.4%, P<.005). The average time to reach the steady state was significantly (P<.005) shorter in the PBPM group compared to the control group (7.3 vs 8.6 days). WHAT IS NEW AND CONCLUSION: Warfarin therapy based on our novel PBPM was clinically safe and resulted in significantly better anticoagulation control compared to conventional care.

Distribution and characterization of anandamide amidohydrolase in mouse brain and liver.

Anandamide (N-Arachidonoylethanolamine) amidohydrolase catalyzing hydrolysis of anandamide was characterized in mice. The enzymatic activity was highest in the liver, followed by the brain and testis. Negligible activity was found in heart, lung and spleen. The activity in brain and liver was mainly localized in the microsomal fractions. Kinetic experiments demonstrated that Km (microM) and Vmax (nmol/min/mg protein) for the brain microsomes were 9.3 and 2.58, respectively, while those for the hepatic microsomes were 180 and 18.9, respectively. The activity in the microsomes from the liver and brain was markedly inhibited by Cu2+, Hg2+, Se4+, phenylmethylsulfonylfluoride and sodium dodecylsulfate. Brain but not hepatic microsomal enzyme activity was inhibited by delta9-tetrahydrocannabinol, cannabidiol and cannabinol. Kinetic parameters demonstrated that the inhibition by the cannabinoids was competitive in nature. Relatively high distribution of the enzyme activity in brain suggests an importance of the enzyme in the central nervous system to regulate the neuromodulatory fatty-acid amides.

Involvement of a novel mouse hepatic microsomal esterase, ES46.5K, in the hydrolysis of phthalate esters.

ES46.5K, a novel esterase from mouse hepatic microsomes (Watanabe K., et al., Biochem. Mol. Biol. Int., 31, 25-30 (1993)), catalyzed hydrolysis of phthalate esters. ES46.5K and mouse hepatic microsomes hydrolyzed diethyl-, dibutyl-, diisobutyl-, dioctyl- and diethylhexyl phthalates, whereas dicyclohexyl- and diphenyl phthalates having ring structure were not hydrolyzed by the enzymes. Vmax (mumol/min/mg protein)/K(m) (microM) ratios of ES46.5K for diethyl-, dibutyl-, diisobutyl-, dioctyl- and diethylhexyl phthalates were 291, 2786, 565, 51 and 57, respectively, while those of microsomes were 0.58, 0.83, 1.71, 0.05 and 1.10, respectively. The hydrolytic activity of ES46.5K was inhibited by diisopropylfluorophosphate and bis-p-nitrophenylphosphate. These results suggest that ES46.5K has high catalytic activity for phthalate esters and some role in the metabolism of phthalate esters in mice.

Specific expression of ES46.5K, a novel microsomal esterase, in the mouse liver and its catalytic activity for xenobiotic amide and esters.

ES46.5K, a novel esterase, was found in mouse hepatic microsomes. The enzyme catalyzed hydrolysis of 2-acetylaminofluorene and cannabinoid esters. In latter case, the enzyme regioselectively hydrolyzed acetates of 11-hydroxy-delta8-tetrahydrocannabinol. Western immunoblotting analysis demonstrated that none of immuno-reactive proteins against ES46.5K were present in hepatic microsomes from rats, guinea-pigs, monkeys and humans. Rabbit hepatic microsomes contained an immuno-reactive protein, although molecular weight of the protein was rather high (50 kDa) by SDS-PAGE. Esterase activity stained after PAGE demonstrated that ES46.5K retained at origin. Hepatic microsomes of above animal species contained several activity bands on the PAGE, while only mouse hepatic microsomes exhibited significant activity at origin. In mice, liver was only an organ containing ES46.5K by analyzing Western immunoblotting. These results indicate that distribution of ES46.5K is quite different from known carboxylesterases, and suggest that the enzyme has some role in the biotransformation of xenobiotic amide and esters.

Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids.

Mouse brain microsomes contain an amidase that catalyzes the hydrolysis of N-arachidonylethanolamide to arachidonic acid and ethanolamine. The enzymatic activity is dependent on the protein concentration of the microsomes and observed over a wide range of pH, 7.4 to 9.5. Kinetic analysis indicated that K(m) (microM) and Vmax (nmol/min/mg protein) were 17.7 +/- 4.1 and 1.81 +/- 0.32, respectively. Cannabidiol (CBD), cannabinol (CBN) and delta 9-tetrahydrocannabinol (delta 9-THC) significantly inhibited the hydrolysis of the amide by mouse brain microsomes. At a concentration of 160 microM, the inhibitory potency decreased in the following order, CBD (66%) > CBN (46%) > delta 9-THC (31%).

3,4,5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, is a substrate for microsomal aldehyde oxygenase in the mouse liver.

3,4,5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, was oxidized to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. The reaction was NADPH-dependent, and inhibited by SKF 525-A, metyrapone and disulfiram. A P450 isozyme in mouse hepatic microsomes, P450 MUT-2 (CYP2C29), catalyzed the reaction (0.96 nmol/min/nmol P450) in which NADPH and NADPH-cytochrome c reductase were essential for the catalytic activity. The reaction was confirmed to be an oxygenation since molecular oxygen was incorporated into the carboxylic acid metabolite formed under oxygen-18 gas by GC-MS analysis. By addition of antibody against CYP2C29 to the microsomes (3.2 mg/mg microsomal protein) the MALDO activity was inhibited by 35% of the control value with preimmune serum, suggesting that CYP2C29 or an immunologically-related isozyme(s) plays a major role in the NADPH-dependent oxidation of 3,4,5-trimethoxyphenylacetaldehyde to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. Pharmacological experiments on mescaline and its deaminated metabolites using mice indicated that the metabolites were much less active or were inactive in cataleptogenic effect and pentobarbital-induced sleep prolongation as compared with the parent compound.

Purification and characterization of a novel 46.5-kilodalton esterase from mouse hepatic microsomes.

A novel membrane-bound esterase was purified from mouse hepatic microsomes. The purified protein (ES46.5K) showed a single protein-staining band on sodium dodecyl sulfate-polyacrylamide gel with a minimum molecular weight of 46.5 Kdalton. ES46.5K possessed esterase activity toward 11-acetoxy-delta 8-tetrahydrocannabinol (11-OAc-delta 8-THC) (27.1 mumol/min/mg protein) and p-nitrophenylacetate (119 mumol/min/mg protein), and these activities were 38 and 47 times, respectively, as high as those of microsomes. The N-terminal amino acid sequence of the protein was as follows: G-K-T-I-S-L-L-I-S-V-V-L-V-A-Y-Y-L-Y-I. This sequence has no homology to those of the known carboxylesterases, indicating that this enzyme is a novel type of esterase bound to the microsomal membrane.