org 69554  (Addgene inc)

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: A, PHT is metabolized to a mixture of (S)- and (R)-p-HPPH by CYP2C9 and CYP2C19. CYP2C9 has a major contribution to this enantioselective metabolism and preferentially forms (S)-p-HPPH with a urinary (S)/(R)-p-HPPH ratio of 40:1 compared with CYP2C19 that forms both enantiomers equally and gives a ratio of 1:1. B, putative TF and nuclear receptor binding sites in or near the -2663delTG and -3089G>A are shown. YY1 and Nrf2 binding sites are underlined and bolded. A CYP2C9 promoter map indicates the location of these polymorphisms and known CAR/PXR binding sites (−2898, −1839, and −1818) (▴).

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Binding Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: Association of CYP2C9 variants with PHT maintenance dose

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques:

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: CYP2C9*1B haplotype is associated with PHT maintenance dose in patients with epilepsy. The box plot shows only those patients found to be noncarriers of the reduced-function CYP2C9*2 and CYP2C9*3 alleles. A linear trend of decreased PHT dose requirements with increasing number of CYP2C9*1B alleles was observed. ∗, p = 0.017 versus CYP2C9*1/*1; ∗∗, p = 0.060 versus CYP2C9*1/*1B.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques:

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: Association of CYP2C9 variants with PHT serum levels normalized to PHT maintenance dose

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques:

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: A, effect of -3089G>A on warfarin dosing in a cohort of 46 Chinese patients on warfarin therapy after excluding samples with CYP2C9*2 and *3 and VKORC1 variant alleles. B, CYP2C9 mRNA expression in 28 human livers analyzed by quantitative real-time PCR. y-axis depicts mRNA expression each liver (normalized to its GAPDH and this value depicted relative to the normalized value obtained for one liver arbitrarily set at 100%). The x-axis represents visual genotyping results for each polymorphism. Light-gray squares represent homozygous major, dark-gray represent heterozygous, and black represents homozygous minor alleles. Box plot in inset depicts the effect of -3089G>A on mRNA expression after excluding CYP2C9*2 and *3 samples. C, effect of -3089G>A on the in vitro formation of (S)- and (R)-p-HPPH in 14 human liver microsome samples after excluding samples with CYP2C9*2 and *3. Group differences were analyzed nonparametrically using the Wilcoxon rank-sum test to compare two genotypes with the phenotype. Box plots indicate second and third quartiles. The bold line within the box represents median and whiskers represent the range after excluding the outliers.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Variant Assay, Expressing, Real-time Polymerase Chain Reaction, In Vitro

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: Effect of individual CYP2C9 promoter polymorphisms on basal and PHT-inducible promoter activity. HepG2 cells were transfected with CYP2C9 reporters -3089A and -3089G (A), and -2663TG and -2663delTG (B) with or without cotransfected PXR, CAR, YY1, or NRF2 expression plasmids and luciferase expression measured after treatment with vehicle or PHT for 24 h. Luciferase activities were normalized to β-galactosidase. The relative luciferase normalized activity in vehicle-treated cells were each set as one, and the fold change in relative luciferase activity with respect to vector (w.r.t.) was graphed relative to this baseline. Values represent the mean ± S.D. measured in triplicate. p values indicate where there is a significant difference between the fold change in the variant allele compared with the corresponding wild-type allele for the identical condition. ∗, p = 0.05; ∗∗, p = 0.02; ∗∗∗, p = 0.03; #, p = 0.007; ##, p = 0.0005; ###, p = 0.005.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Variant Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: The CYP2C9 variant haplotype shows diminished responsiveness to PHT induction in vitro and in vivo. A–C, HepG2 cells were transfected with CYP2C9 WT and VAR-HAP reporters +/ PXR, YY1, or Nrf2 expression plasmids, and luciferase expression measured after treatment with vehicle or PHT for 24 h. Results were analyzed and graphed as in the legend to Fig. 4. p values indicate significant differences between the fold change in the VAR-HAP compared with the corresponding WT-HAP for the identical condition, ∗, p = 0.04; ∗∗, p = 0.02; #, p = 0.007; ##, p = 0.005; ###, p = 0.002.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Variant Assay, In Vitro, In Vivo, Transfection, Expressing, Luciferase

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: EMSA of transcription factor binding to the wild-type and polymorphic CYP2C9 YY1 promoter elements. 32P-labeled oligonucleotides representing the -3089G and A were incubated with products from in vitro synthesis with the empty expression vector (pcDNA3), or synthesized PXR:RXR, CAR:RXR, and YY1 proteins in the absence (no competitor) or presence of increasing amounts of unlabeled oligonucleotide cold competitor (CC). After electrophoresis, complex formation was assessed by a phosphoimager. Lanes 17, 18, and 19 represent binding of YY1, CAR, and PXR to positive control (+ve Ctrl) oligonucleotides, respectively. Sequences for all oligonucleotides are provided in Supplemental Table 1.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Binding Assay, Labeling, Incubation, In Vitro, Expressing, Plasmid Preparation, Synthesized, Electrophoresis, Positive Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: CYP2C9 * 1B Promoter Polymorphisms, in Linkage with CYP2C19 * 2 , Affect Phenytoin Autoinduction of Clearance and Maintenance Dose S⃞

doi: 10.1124/jpet.109.161026

Figure Lengend Snippet: Effect of CYP2C9 promoter genotypes on CYP expression in human hepatocytes treated with PHT. Primary human hepatocytes were treated with vehicle or PHT for 48 h, RNA extracted and analyzed by quantitative real-time PCR. CYP2C9 and CYP2C18 like Ex1/ 2C9 Ex2 chimeric transcripts were amplified from cDNA using specific primers as shown. The fold change of each mRNA after PHT treatment is depicted on the y-axis and CYP2C9-3089G>A genotypes on the x-axis. Group differences were analyzed nonparametrically using the Wilcoxon rank-sum test as described in Fig. 3 legend.

Article Snippet: Cloning of the −5470/+25 CYP2C9 promoter luciferase reporter plasmid in the pGL3-basic vector (Promega) has been described previously ( Al-Dosari et al., 2006 ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Amplification