ABSTRACT:Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4*2 (S222P), *7 (G56D), *16 (T185S), and *18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V max and increased K m relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V max values for MDZ and NIF (60 and 84%, respectively) and a higher K m (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V max values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V max/Km . In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.The CYP3A subfamily of cytochrome P450 enzymes comprises approximately 30 to 60% of total cytochrome P450 in human liver (Shimada et al., 1994). The CYP3A enzymes are responsible for the metabolism of more than 50% of clinically used drugs and also some steroids and environmental chemicals (Li et al., 1995;Evans and Relling, 1999;Guengerich, 1999). Four human CYP3A enzymes-CYP3A4, CYP3A5, CYP3A7, and CYP3A43-have been identified, and CYP3A4 is regarded as the most dominant CYP3A enzyme in the liver and small intestine (Wrighton et al., 1990;Shimada et al., 1994;Hustert et al., 2001;Koch et al., 2002). It has been reported that CYP3A4 expression shows large interindividual variation (Guengerich, 1999;Ozdemir et al., 2000;Lin et al., 2002) and that these variations can lead to different responses to human drugs that are substrates for CYP3A4. Because the expression of CYP3A4 and CYP3A5 can be induced by pregnane X receptor, constitutive androstane receptor, and glucuronide receptor ligands, both environmental and genetic factors can influence the CYP3A activity; however, the genetic contribution has been estimated to be larger (Ozdemir et al., 2000), suggesting that polymorphisms in CYP3A4 may predict CYP3A phenotype, at least in part. Genetic analysis is needed to understand interindividual variability. Extensive studies searching allelic variants in the coding regions of CYP3A4 have been done, and currently 20 different CYP3A4 variant proteins have been described, some of them representing proteins of either decreased or increased activity. Those polymorphisms are reported on the Hum...

Intragastric administration of rikkunshito stimulates gastrointestinal contractions in the interdigestive state through cholinergic neurons and 5-HT type 3 receptors. Moreover, rikkunshito increases plasma acylated ghrelin levels. Rikkunshito may alleviate gastrointestinal disorders through its prokinetic effects.

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1 This study investigated a local effect of cooling on the plantar skin blood flow (PSBF) of tetrodotoxin-treated rats by laser-Doppler flowmetry. 2 When the air temperature around the left foot was locally cooled from 25 to 101C, the PSBF of the left foot decreased. 3 The response was inhibited by the a-adrenoceptor antagonist phentolamine, the a 1 -adrenoceptor antagonist bunazosin, the a 2 -adrenoceptor antagonist RS79948, and bretylium and guanethidine that inhibit noradrenaline release from sympathetic nerves. Adrenalectomy of the rats did not affect the cooling-induced response. 4 The P2 purinoceptor antagonists suramin and PPADS also significantly suppressed the coolinginduced reduction of PSBF. However, the inhibitory effect of PPADS on the cooling-induced response was abolished after the treatment with phentolamine. Intra-arterial injections of ATPgS, a stable P2 purinoceptor agonist, at 251C caused a transient decrease in PSBF in a dose-dependent manner, which was significantly inhibited by phentolamine and guanethidine. 5 These results suggest a novel mechanism for local cooling-induced reduction of skin blood flow in vivo; moderate cooling of the skin induces the release of ATP, which stimulates presynaptic P2 purinoceptors on sympathetic nerve terminals and facilitates the release of noradrenaline, thereby causing contractions of skin blood vessels via the activation of a 1 -and a 2 -adrenoceptors.

Background: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (−1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. Methods: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 −1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. Results: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. Conclusions: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2–based diagnostics have key advantages.