Potent S-enantiomer Research Articles

The Influence of CYP2C9 and VKORC1 Gene Polymorphisms on the Response to Warfarin in Egyptians

Warfarin is the most commonly used drug for chronic prevention of thromboembolic events, it also ranks high among drugs that cause serious adverse events. The variability in dose requirements has been attributed to inter-individual differences in medical, personal, and genetic factor. Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin by catalyzing the conversion of the pharmacologically more potent S-enantiomer to its inactive metabolites. Warfarin exerts its effect by inhibition of vitamin K epoxide reductase. This protein is encoded by vitamin K epoxide reductase complex subunit 1 gene (VKORC1). The current study aimed to investigate the pharmacogenetic effect of CYP2C9 and VKORC1 gene polymorphisms on the patients response to warfarin. One hundred cases starting warfarin treatment and 20 healthy controls were enrolled. The mean daily dose of warfarin was calculated from patient's medical records. For each patient, less than 10% variability in warfarin dose and a target international normalized ratio (INR) within the therapeutic target range were required for at least 3months for one of the following indications (deep vein thrombosis, pulmonary embolism, cerebrovascular stroke and myocardial infarction) prior to inclusion in the study. Tetraprimer amplification refractory mutation system PCR was performed to determine CYP2C9*2, CYP2C9*3, and the VKORC1 1639 G>A genetic polymorphisms. Plasma warfarin determination was performed using rapid fluorometric assay. Plasma warfarin concentration ranged from 2.19 to 10.98μg/ml with a median 3.52μg/ml. Supratherpeutic INR was observed in 11% of the cases. Thromboembolic complications occurred in 7% of the cases and 8% of cases experienced major bleeding. High maintenance dose (>7mg/day) was associated with the combined non VKORC1*2 and homozygous wild type CYP2C9 (CYP2C9*1*1) alleles, while low maintenance dose was associated with the Variant (AG+AA)/Wild (*1/*1). (p value <0.001). CYP2C9 variant was a risk factor for supratherapeutic INR in the multivariate logistic regression model. Thromboembolic complication and incidence of supratherapeutic INR were observed in patients carrying combined VKORC1 Variant (AG+AA) and CYP2C9 Variant (*2/*3). Data from our study suggest that together with clinical factors, VKORC1 and CYP2C9 polymorphisms are important contributors to warfarin dosing and may help predict adverse effects in Egyptian patients.

CYP2C9 Genotype-Dependent Warfarin Pharmacokinetics: Impact of CYP2C9 Genotype on R- and S-Warfarin and Their Oxidative Metabolites.

Multiple factors can impact warfarin therapy, including genetic variations in the drug-metabolizing enzyme cytochrome P450 2C9 (CYP2C9). Compared with individuals with the wild-type allele, CYP2C9*1, carriers of the common *3 variant have significantly impaired CYP2C9 metabolism. Genetic variations in CYP2C9, the primary enzyme governing the metabolic clearance of the more potent S-enantiomer of the racemic anticoagulant warfarin, may impact warfarin-drug interactions. To establish a baseline for such studies, plasma and urine concentrations of R- and S-warfarin and 10 warfarin metabolites were monitored for up to 360 hours following a 10-mg warfarin dose in healthy subjects with 4 different CYP2C9 genotypes: CYP2C9*1/*1 (n = 8), CYP2C9*1/*3 (n = 9), CYP2C9*2/*3 (n = 3), and CYP2C9*3/*3 (n = 4). Plasma clearance of S-warfarin, but not R-warfarin, decreased multiexponentially and in a CYP2C9 gene-dependent manner: 56%, 70%, and 75% for CYP2C9*1/*3, CYP2C9*2/*3, and CYP2C9*3/*3 genotypes, respectively, compared with CYP2C9*1/*1, resulting in pronounced differences in the S:R ratio that identified warfarin-sensitive genotypes. CYP2C9 was the primary P450 enzyme contributing to S-warfarin metabolism and a minor contributor to R-warfarin metabolism. In the presence of a defective CYP2C9 allele, switching of warfarin metabolism to other oxidative pathways and P450 enzymes for the metabolic elimination of S-warfarin was not observed. The 10-hydroxywarfarin metabolites, whose detailed pharmacokinetics are reported for the first time, exhibited a prolonged half-life with no evidence of renal excretion and displayed elimination rate-limited kinetics. Understanding the impact of CYP2C9 genetics on warfarin pharmacokinetics lays the foundation for future genotype-dependent warfarin-drug interaction studies.

Abstract 3357: CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide.

Abstract IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition compared to the R-enantiomer (Wnendt et al., 1996; Moreira et al., 1993). We have demonstrated that S-lenalidomide is more potent than racemic or R-lenalidomide in biological activities that are believed to be important for clinical efficacy of lenalidomide. Due to facile in vivo conversion, isolated S-enantiomers of IMiDs have not been developed clinically. Lenalidomide (Revlimid®) is a racemic mixture of S- and R-enantiomers that interconvert through epimerization. Revlimid is labeled for the treatment of 5q-myelodysplastic syndromes (MDS) and multiple myeloma. CTP-221 is a deuterium-modified analog of S-lenalidomide containing deuterium atoms at key positions including lenalidomide's chiral center. Deuterium modification has the potential, albeit unpredictably, to alter the metabolic fate and hence the pharmacokinetic disposition of drugs, especially those that are biotransformed via the cleavage of carbon-hydrogen bonds (Fisher et al, 2006). The effect of deuterium modification on the epimerization rate and pharmacokinetic profile of CTP-221 was investigated in vitro and in vivo in mice, rats and monkeys. The rates of epimerization of CTP-221 and S-lenalidomide were compared in vitro in whole blood from mouse, rat, monkey, and human. It was found that CTP-221 was 2- to 3-fold more stable to epimerization than S-lenalidomide in the four species. To compare the in vivo pharmacokinetic profiles of CTP-221 and racemic lenalidomide, the compounds (10 mg/kg, PO) were administered to mice, rats and monkeys. In all three species, the exposure (AUC) of S- and R-lenalidomide following administration of racemic lenalidomide was about 50-57% and 43-50%, respectively, of the sum of the AUC of the individual enantiomers. However, when CTP-221 was administered, the AUC of CTP-221 and the deuterated R-enantiomers formed in vivo were about 96-99% and 1-4% respectively, of the sum of the AUC of the two enantiomers. Thus, CTP-221 epimerizes to a minimal extent in vivo and its administration provides exposure to very low levels of the R-enantiomer. In conclusion, the stabilization of CTP-221 via deuterium substitution resulted in maximal exposure to the more potent S-enantiomer and minimal exposure to the R-enantiomer. As a result, CTP-221 has the potential for improved potency and therapeutic index in comparison to racemic lenalidomide. Citation Format: Vinita Uttamsingh, Richard Gallegos, Changfu Cheng, Ara Aslanian, Julie Fields Liu, Roger Tung, Lijun Wu. CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2013-3357

Differential adaptation of REM sleep latency, intermediate stage and theta power effects of escitalopram after chronic treatment

The effects of the widely used selective serotonin reuptake inhibitor (SSRI) antidepressants on sleep have been intensively investigated. However, only a few animal studies examined the effect of escitalopram, the more potent S-enantiomer of citalopram, and conclusions of these studies on sleep architecture are limited due to the experimental design. Here, we investigate the acute (2 and 10 mg/kg, i.p. injected at the beginning of the passive phase) or chronic (10 mg/kg/day for 21 days, by osmotic minipumps) effects of escitalopram on the sleep and quantitative electroencephalogram (EEG) of Wistar rats. The first 3 h of EEG recording was analyzed at the beginning of passive phase, immediately after injections. The acutely injected 2 and 10 mg/kg and the chronically administered 10 mg/kg/day escitalopram caused an approximately three, six and twofold increases in rapid eye movement sleep (REMS) latency, respectively. Acute 2-mg/kg escitalopram reduced REMS, but increased intermediate stage of sleep (IS) while the 10 mg/kg reduced both. We also observed some increase in light slow wave sleep and passive wake parallel with a decrease in deep slow wave sleep and theta power in both active wake and REMS after acute dosing. Following chronic treatment, only the increase in REMS latency remained significant compared to control animals. In conclusion, adaptive changes in the effects of escitalopram, which occur after 3 weeks of treatment, suggest desensitization in the function of 5-HT(1A) and 5-HT(1B) receptors.

Effect of rutin on warfarin anticoagulation and pharmacokinetics of warfarin enantiomers in rats

The effects of the flavonoid rutin on the anticoagulant activity of oral warfarin and the protein binding and pharmacokinetics of its enantiomers were investigated in rats. A single dose of racemic warfarin, 1.5 mg/kg, was administered orally to rats either alone or on day 5 of an 8-day oral regimen of rutin, 1 g/kg daily. Rutin reduced the anticoagulant effect of racemic warfarin, evident as a 31% reduction in the area under the prothrombin complex activty-time curve (P < 0.05). Rutin had no apparent effect on pre-treatment baseline blood coagulation. It enhanced the in-vitro serum protein binding of S- and R-warfarin (reflected by 40% and 26% reductions in unbound fraction, respectively), and thus restricted distribution by 33 and 21%, respectively. Treatment with rutin significantly decreased the elimination half-life of S-warfarin by 37% as a result of the 69% increase in unbound clearance of the S-enantiomer. This effect was attributed to a significant 77% increase in the unbound formation clearance of the overall oxidative and reductive metabolites, and an increase in the unbound renal clearance of the more potent S-enantiomer of warfarin. Concurrent rutin administration is likely to reduce the anticoagulant effect of racemic warfarin, reflecting a significant decrease in the elimination half-life of the more potent S-enantiomer.

Effect of rutin on warfarin anticoagulation and pharmacokinetics of warfarin enantiomers in rats

Abstract Objectives The effects of the flavonoid rutin on the anticoagulant activity of oral warfarin and the protein binding and pharmacokinetics of its enantiomers were investigated in rats. Methods A single dose of racemic warfarin, 1.5 mg/kg, was administered orally to rats either alone or on day 5 of an 8-day oral regimen of rutin, 1 g/kg daily. Results Rutin reduced the anticoagulant effect of racemic warfarin, evident as a 31% reduction in the area under the prothrombin complex activty–time curve (P &amp;lt; 0.05). Key findings Rutin had no apparent effect on pre-treatment baseline blood coagulation. It enhanced the in-vitro serum protein binding of S- and R-warfarin (reflected by 40% and 26% reductions in unbound fraction, respectively), and thus restricted distribution by 33 and 21%, respectively. Treatment with rutin significantly decreased the elimination half-life of S-warfarin by 37% as a result of the 69% increase in unbound clearance of the S-enantiomer. This effect was attributed to a significant 77% increase in the unbound formation clearance of the overall oxidative and reductive metabolites, and an increase in the unbound renal clearance of the more potent S-enantiomer of warfarin. Conclusions Concurrent rutin administration is likely to reduce the anticoagulant effect of racemic warfarin, reflecting a significant decrease in the elimination half-life of the more potent S-enantiomer.

Crystal Structure of the Peroxisome Proliferator-Activated Receptor γ (PPARγ) Ligand Binding Domain Complexed with a Novel Partial Agonist: A New Region of the Hydrophobic Pocket Could Be Exploited for Drug Design

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

Comparative Pharmacokinetics of Vitamin K Antagonists

Vitamin K antagonists belong to the group of most frequently used drugs worldwide. They are used for long-term anticoagulation therapy, and exhibit their anticoagulant effect by inhibition of vitamin K epoxide reductase. Each drug exists in two different enantiomeric forms and is administered orally as a racemate. The use of vitamin K antagonists is complicated by a narrow therapeutic index and an unpredictable dose-response relationship, giving rise to frequent bleeding complications or insufficient anticoagulation. These large dose response variations are markedly influenced by pharmacokinetic aspects that are determined by genetic, environmental and possibly other yet unknown factors. Previous knowledge in this regard principally referred to warfarin. Cytochrome P450 (CYP) 2C9 has clearly been established as the predominant catalyst responsible for the metabolism of its more potent S-enantiomer. More recently, CYP2C9 has also been reported to catalyse the hydroxylation of phenprocoumon and acenocoumarol. However, the relative importance of CYP2C9 for the clearance of each anticoagulant substantially differs. Overall, the CYP2C9 isoenzyme appears to be most important for the clearance of warfarin, followed by acenocoumarol and, lastly, phenprocoumon. The less important role of CYP2C9 for the clearance of phenprocoumon is due to the involvement of CYP3A4 as an additional catalyst of phenprocoumon hydroxylation and significant excretion of unchanged drug in bile and urine, while the elimination of warfarin and acenocoumarol is almost completely by metabolism. Consequently, the effects of CYP2C9 polymorphisms on the pharmacokinetics and anticoagulant response are also least pronounced in the case of phenprocoumon; this drug seems preferable for therapeutic anticoagulation in poor metabolisers of CYP2C9. In addition to these vitamin K antagonists, oral thrombin inhibitors are currently under clinical development for the prevention and treatment of thromboembolism. Of these, ximelagatran has recently gained marketing authorisation in Europe. These novel drugs all feature some major advantages over traditional anticoagulants, including a wide therapeutic interval, the lack of anticoagulant effect monitoring and a low drug-drug interaction potential. However, they are also characterised by some pitfalls. Amendments of traditional anticoagulant therapy, including self-monitoring of international normalised ratio values or prospective genotyping for individual dose-tailoring may contribute to the continuous use of warfarin, phenprocoumon and acenocoumarol in the future.

Effect of 5-fluorouracil on the anticoagulant activity and the pharmacokinetics of warfarin enantiomers in rats

The interaction between the antineoplastic agent 5-fluorouracil (5-FU) and the oral anticoagulant warfarin enantiomers was investigated in rats. An increase in hypoprothrombinaemic response, assessed by means of percent changes of prothrombin complex activity and clotting factor VII activity, to warfarin, was observed following oral administration of 1.5 mg/kg racemic warfarin to rats during a 8-day intraperitoneal dose regimen of 5-FU (13.3 mg/kg daily). 5-FU had no apparent effect on the baseline blood coagulation, the in vitro rat serum protein binding as well as the absorption and distribution of the S- and R-enantiomers of warfarin in rats. Yet treatment with 5-FU produced a significant decrease in the total serum clearance value of S-warfarin in rats. The decreased total clearance was attributed mainly to a significant decrease in the formation rate of the overall oxidative metabolites of the more potent S-enantiomer of warfarin.

Characterization of R-[ 11C]rolipram for PET imaging of phosphodiesterase-4: in vivo binding, metabolism, and dosimetry studies in rats

The high-affinity phosphodiesterase-4 (PDE4) inhibitor R-rolipram and the less potent S-enantiomer, both labeled with 11C, were evaluated in vivo in rats. Regional brain uptake of R-[ 11C]rolipram was higher than R/S-[ 11C]rolipram, whereas S-[ 11C]rolipram retention subsided rapidly to levels below blood. Binding of R-[ 11C]rolipram was selective for PDE4 over PDE1, since treatment with PDE4 competitors Ro 20–1724, or R-, S- or R/S-rolipram, but not with the PDE1 inhibitor vinpocetine, significantly reduced radioactivity uptake to non-specific levels. R-Rolipram (ED 50 ≅ 0.04 mg/Kg) was approximately 13 fold more potent than S-rolipram at inhibiting R-[ 11C]rolipram binding in all brain regions. Nevertheless, S-[ 11C]rolipram appears to be unsuitable for measuring the non-specific binding of R-[ 11C]rolipram. Only unchanged R-[ 11C]rolipram was detected in rat brain homogenates. Additionally, the estimated absorbed radiation dose extrapolated to humans was low. These results support further investigation of R-[ 11C]rolipram in studying PDE4 in vivo by positron emission tomography imaging.

Genetic association between sensitivity to warfarin and expression of CYP2C9*3.

Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.

Lack of interaction between meloxicam and warfarin in healthy volunteers.

The effect of multiple oral doses of meloxicam 15 mg on the pharmacodynamics and pharmacokinetics of warfarin was investigated in healthy male volunteers. Warfarin was administered in an individualized dose to achieve a stable reduction in prothrombin times calculated as International Normalized Ratio (INR) values. Then INR- and a drug concentration-time profile was determined. For the interaction phase, meloxicam was added for 7 days and then INR measurements and the warfarin drug profiles were repeated for comparison. Overall, warfarin treatment lasted for 30 days. Warfarin and meloxicam were well tolerated by healthy volunteers in this study. Thirteen healthy volunteers with stable INR values entered the interaction phase. Prothrombin times, expressed as mean INR values, were not significantly altered by concomitant meloxicam treatment, being 1.20 for warfarin alone and 1.27 for warfarin with meloxicam cotreatment. R- and S-warfarin pharmacokinetics were similar for both treatments. Geometric mean (% gCV) AUCss values for the more potent S-enantiomer were 5.07 mg.h.l-1 (27.5%) for warfarin alone and 5.64 mg.h.l-1 (28.1%) during the interaction phase. Respective AUCss values for R-warfarin were 7.31 mg.h.l-1 (43.8%) and 7.58 mg.h.l-1 (39.1%). The concomitant administration of the new non-steroidal anti-inflammatory drug (NSAID) meloxicam affected neither the pharmacodynamics nor the pharmacokinetics of a titrated warfarin dose. A combination of both drugs should nevertheless be avoided and, if necessary, INR monitoring is considered mandatory.

Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions.

Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.