MNTA/M118 – Further to the lack of a drug interaction between M118 and aspirin or Plavix, M118 may have an edge vs LMWH’s such as Lovenox on this score. The FDA label for Lovenox cites a potential drug interaction with aspirin; although Plavix is not mentioned by name, the Lovenox label cites platelet inhibitors as a class:
Unless really needed, agents which may enhance the risk of hemorrhage should be discontinued prior to initiation of Lovenox therapy. These agents include medications such as: anticoagulants, platelet inhibitors including acetylsalicylic acid [aspirin], salicylates, NSAIDs (including ketorolac tromethamine), dipyridamole, or sulfinpyrazone. If co-administration is essential, conduct close clinical and laboratory monitoring. <<
The bigger advantages of M118 vs Lovenox are FIIa inhibition and reversibility (#msg-26897124).
Let’s talk biotech! “The efficient-market hypothesis may be the foremost piece of B.S. ever promulgated in any area of human knowledge!”
We evaluated the effect of the CYP2C19 genotype on the pharmacokinetics and pharmacodynamcis of clopidogrel. Twenty-four subjects were divided into three groups on the basis of their CYP2C19 genotype: homozygous extensive metabolizers (homoEMs, n = 8), heterozygous EMs (heteroEMs, n = 8), and poor metabolizers (PMs, n = 8). After a single 300-mg loading dose of clopidogrel on day 1, followed by a 75-mg daily maintenance dose from days 2 to 7, we measured the plasma levels of clopidogrel and assessed the antiplatelet effect as pharmacodynamics. The mean clopidogrel area under the curve (AUC) for PMs was 1.8- and 2.9-fold higher than that for heteroEMs and homoEMs, respectively (P = 0.013). The mean peak plasma concentration in PMs was 1.8- and 4.7-fold higher than that of heteroEMs and homoEMs, respectively (P = 0.008). PMs exhibited a significantly lower antiplatelet effect than heteroEMs or homoEMs (P < 0.001). From these findings it is clear that the CYP2C19 genotype affects the plasma levels of clopidogrel and modulates the antiplatelet effect of clopidogrel.
Recently, it was estimated that up to 30% of patients do not achieve an adequate antiplatelet effect from clopidogrel, which is probably because of significant interindividual variability in the clopidogrel response. Clopidogrel resistance has been suggested as a possible cause, with putative mechanistic explanations involving drug–drug interactions or genetic polymorphisms in the drug metabolizing enzyme involved in metabolizing clopidogrel.
It is thought that clopidogrel is activated by the hepatic cytochrome P450 3A (CYP3A) to generate an active metabolite. The active metabolite of clopidogrel inhibits adenosine diphosphate (ADP)-induced platelet aggregation through irreversible binding to the platelet P2Y12 receptor. Consistently, coadministration of CYP3A inducer or inhibitor modulates clopidogrel responsiveness in vivo. Interestingly, it has been reported that subjects with a defective CYP2C19 allele (CYP2C19*2) exhibit lower clopidogrel responsiveness than those with a normal CYP2C19 allele. Furthermore, in vitro studies showed that, in addition to CYP3A, CYP2C19 is also involved in the activation of clopidogrel. However, another study has shown that the mutant CYP2C19*2 allele is not associated with clopidogrel responsiveness. CYP2C19 is very polymorphic16 and the polymorphisms are thought to be the main cause for the large differences in the pharmacokinetics of CYP2C19 substrates.
In this study, we analyzed the role of the CYP2C19 genotype in the pharmacokinetics of clopidogrel and evaluated the possibility that genotypic differences cause the observed interindividual variability in clopidogrel responsiveness.
Results
The association between the CYP2C19 genotype and clopidogrel pharmacokinetics The plasma concentration–time profiles for clopidogrel were compared among the CYP2C19 genotype groups after administration of a 300-mg loading dose of clopidogrel (Figure 1); the pharmacokinetic parameters are summarized in Table 1.
Figure 1.Mean plasma concentrations of clopidogrel after the loading dose (300 mg), in relation to, CYP2C19 genotype. Data are expressed as mean values s.e.m. homoEMs, homozygous EMs; heteroEMs, heterozygous EMs; PMs, poor metabolizers.
Table 1 - Mean (s.d.) pharmacokinetic parameters of clopidogrel after a single loading dose of 300-mg clopidogrel according to the CYP2C19 genotype.Full table (6K)
Poor metabolizers (PMs) showed higher plasma concentrations of clopidogrel than heterozygous extensive metabolizers (heteroEMs) did, and homozygous EMs (homoEMs) had the lowest plasma levels (Figure 1). The mean area under the curve (AUC) of PMs was 1.8- and 2.9-fold higher than that of heteroEMs and homoEMs, respectively (P = 0.013). The mean Cmax (peak plasma concentration) of PMs was also 1.8- and 4.7-fold higher than that of heteroEMs and homoEMs, respectively (P = 0.008) (Table 1 and Figure 2).
Figure 2.Comparisons of the pharmacokinetics ((a) Cmax and (b) AUC) and the inhibition of platelet aggregation ((c) Emax(0–24) and (d) AUEC(0–24)) in relation to the CYP2C19 genotype. Results are mean values s.e.m. (n = 8 for each group) AUC, area under the curve; AUEC, area under the time–effect curve; Cmax, peak plasma concentration; Emax, maximal antiplatelet effect; homoEMs, homozygous EMs; heteroEMs, heterozygous EMs; NS, not significant; PMs, poor metabolizers.
Association between the CYP2C19 genotype and IPA of clopidogrel For a pharmacodynamic assessment of clopidogrel, the inhibitory effects on ADP-induced platelet aggregation were evaluated according to the CYP2C19 genotype.
The predose platelet aggregation values induced by 5 mol/l ADP in homoEMs (73.1 5.6%), heteroEMs (72.7 10.6%), and PMs (74.7 5.3%) were not statistically different (P = 0.846). The mean IPA profiles (determined using 5 mol/l ADP) obtained during the study period, in relation to the CYP2C19 genotype, are shown in Figure 3. Throughout the study period, PMs showed far lower IPA levels than heteroEMs did, and homoEMs had the highest levels. After a 300-mg loading dose of clopidogrel, the mean maximal IPA value (Emax(0–24)) of PMs was 31 and 40% lower than that of heteroEMs and homoEMs, respectively (P = 0.0001) (Table 2 and Figure 2c). Consistently, the area under the effect–time curve (AUEC)(0–24) of PMs was 40 and 50% lower than that of heteroEMs and homoEMs, respectively (Figure 2d, P = 0.0006). During the maintenance dosing period, the Emax(0–168) of PMs was 30 and 37% lower than that of heteroEMs and homoEMs (P = 0.0001), respectively, and the AUEC(0–168) of PMs was also 37 and 61% lower than that of heteroEMs and homoEMs (P < 0.0001), respectively (Table 2).
Figure 3.Mean percentage change in IPA (inhibition of platelet aggregation) after a single 300-mg loading dose of clopidogrel on day 1, followed by a 75-mg daily maintenance dose from days 2 to 7, in relation to CYP2C19 genotype. Data are expressed as mean values s.e.m. homoEMs, homozygous EMs; heteroEMs, heterozygous EMs; PMs, poor metabolizers.
Table 2 - Mean pharmacodynamic parameters for the assessment of the inhibition of platelet aggregation of clopidogrel after a single 300-mg loading dose of clopidogrel on day 1, followed by a 75-mg daily maintenance dose from day 2 to 7 according to the CYP2C19 genotype.
Association between clopidogrel pharmacokinetics and its IPA When we assessed whether there was a correlation between the clopidogrel pharmacokinetics and the IPA values, we observed a significant but negative correlation between them (Figure 4).
Figure 4.Correlation between individual pharmacokinetic parameters ((a,c) Cmax, (b,d) AUC) of clopidogrel and its inhibition of platelet aggregation, assessed by Emax (during (a) 24 h and (c) during 168 h), and AUEC ((b) during 24 h and (d) during 168 h). AUC, area under the curve; AUEC, area under the time – effect curve; Cmax, peak plasma concentration; Emax, maximal antiplatelet effect.
Association between CYP2C19 metabolic activity and IPA by clopidogrel
We questioned whether the IPA by clopidogrel was directly related to CYP2C19 metabolic activity. Baseline CYP2C19 showed two clusters between EM and PM groups largely and was randomly distributed in each group, as measured by omeprazole hydroxylation. CYP2C19 metabolic activity was not related to the baseline IPA values in this population (r = 0.1066, P = 0.62). After clopidogrel dosing, there was a significant and negative correlation between the Emax and the AUEC of IPA values, and also between the Emax and the extent of omeprazole hydroxylation (i.e., CYP2C19 metabolic activity) (P < 0.0001) (Figure 5).
Figure 5.Correlation between CYP2C19 metabolic activity (measured by omeprazole hydroxylation) and the values of (a) Emax and (b) AUEC relating to the inhibition of ADP-induced platelet aggregation after clopidogrel dosing. AUEC, area under the time–effect curve; Emax, maximal antiplatelet effect.
Discussion
It is now widely accepted that there is marked interindividual variability in the antiplatelet effect of clopidogrel treatment. Available data show that 4–30% of patients who receive the conventional dose of clopidogrel do not display an adequate antiplatelet response; that is, they display clopidogrel resistance. Despite the many mechanisms that have been postulated to explain the variability in individual responsiveness to clopidogrel, the mechanisms are not fully elucidated and are multifactorial. In this study, we determined that the CYP2C19 genotype is a major determinant of interindividual variability in clopidogrel responsiveness. It is believed that CYP3A is mainly involved in converting clopidogrel into an active metabolite, which irreversibly blocks the platelet P2Y12 receptor. Therefore, CYP3A genetic polymorphisms or drug interaction with CYP3A modulators could be factors causing interindividual variability in clopidogrel responsiveness. Earlier studies, therefore, have focused on uncovering the role of CYP3A in the antiplatelet effect produced by clopidogrel. Recently, in vitro findings showed that CYP2C19 is also involved in the metabolism of clopidogrel. Furthermore, there have recently been several reports that a defective CYP2C19 genotype (i.e., CYP2C19*2) contributes to the variability in clopidogrel responsiveness. On the other hand, Fontana et al. reported the contradictory finding that the CYP2C19*2 allele is not associated with clopidogrel responsiveness. However, because that study was carried out with too small a number of PMs, it had the limitation of being unable to find a statistical significance between EMs and PMs, and this might have caused the inconsistency in its findings. Even though there are subtle differences between homoEMs and heteroEMs in the pharmacokinetics of CYP2C19 substrates, both groups are classified as EMs, and they show higher metabolic activity than PMs do. Therefore, a comparison of clopidogrel responsiveness between homoEMs and heteroEMs provides limited information. As a result, many pharmacogenetic studies related to CYP2C19 are performed by classifying subjects only as PMs or EMs (comprised of heteroEMs and homoEMs). When we assessed the differences in clopidogrel responsiveness among homoEMs, heteroEMS, and PMs, PMs showed a significantly lower antiplatelet effect than EMs. However, as with the results for other CYP2C19 substrates, although there were differences between homoEMs and heteroEM in the pharmacokinetics and antiplatelet effects of clopidogrel, none of these differences were statistically significant. The plasma levels of clopidogrel were highest in PMs, followed by heteroEMs, and then homoEMs. Therefore, the results of this study strongly suggest that a defective CYP2C19 enzyme function associated with polymorphic CYP2C19 genotypes is a major source of interindividual variability in clopidogrel responsiveness.
In addition, in this study, PMs showed lower IPA values than heteroEMs and homoEMs did. The Emax and AUEC values of PMs were substantially lower than those of heteroEMs and homoEMs, thereby suggesting that the antiplatelet effect of clopidogrel is lower in PMs than in EMs. Furthermore, there was a significant negative correlation between the IPA values and pharmacokinetic parameters of clopidogrel. Taken together, our findings strongly suggest that clopidogrel is extensively metabolized by CYP2C19 in humans, and therefore a lower (i.e., heteroEMs) or nonfunctional (i.e., PMs) CYP2C19 enzyme should have resulted in elevated plasma levels of clopidogrel, but a reduced formation of active metabolite of clopidogrel, which results in a decreased antiplatelet effect. Patients treated with omeprazole, an inhibitor of CYP2C19, had significantly higher intraplatelet vasodilator-stimulated phosphoprotein values than the untreated subjects did. This indicates that omeprazole reduces the clopidogrel responsiveness by inhibiting CYP2C19 metabolic activity, thereby decreasing CYP2C19-mediated metabolism of clopidogrel into its active metabolites.26
When we evaluated whether the difference in CYP2C19 metabolic activity was related to clopidogrel metabolism, we found that CYP2C19 metabolic activity, as assessed by omeprazole hydroxylation, strongly correlated with IPA values of clopidogrel. Furthermore, the inverse correlation was also observed; the lower the CYP2C19 activity, the higher were the plasma clopidogrel concentrations observed. This suggests that metabolic conversion of clopidogrel into the active metabolite might be related to CYP2C19 activity. On the basis of our findings, we propose a possible mechanism to explain clopidogrel resistance: a reduction in CYP2C19 metabolic activity related to CYP2C19 polymorphism.
Our data reveal that the CYP2C19 genotype is a major determinant of interindividual variability in the response to clopidogrel, and that the PM genotype can be classified as clopidogrel nonresponders. In this study, we calculated IPA values so as to adjust the variability of the baseline values. Even though the existing definitions of poor response to the antiplatelet effect of clopidogrel are empirical, Weerakkody et al. classified the poor clopidogrel responder as one with an IPA value <25% at 24 h after 300-mg loading dose of clopidogrel, as per the Bayesian classification. When we assessed the frequency of occurrence of poor responders according to this classification after the clopidogrel loading dose,28 we found five poor responders in the eight PM subjects but no poor responder in the homoEM and heteroEM groups. The observed mean (s.d.) IPA values at 24 h after the clopidogrel loading dose were 22.9 13.4% for PMs, 45.5 9.7% for heteroEMs, and 51.4 10.7% for homoEMs (Figure 3). By increasing either the loading dose (>300 mg) or the maintenance dose (>75 mg), it should be possible to increase clopidogrel responsiveness in PMs.
The distribution of EM and PM CYP2C19 genotype groups encompassed a wide range of ethnicities. Even though nongenetic factors such as enzyme inhibition and induction, age, and liver function can also modulate CYP2C19 activity, the plasma levels of many drugs, including proton-pump inhibitors, can be modulated by the CYP2C19 genotype, and we therefore recommend adjusting the drug regimens according to the CYP2C19 genotype. It has been reported that a 600-mg loading dose (as compared with 300 mg) resulted in faster and more profound platelet inhibition in patients undergoing coronary stenting.30 Additionally, poor responsiveness was significantly lower after a 600-mg loading dose as compared to the conventional 300-mg dose.
Our study has several potential limitations. First, we recruited healthy male subjects with different CYP2C19 genotypes in order to exclude other possible factors that could affect the clopidogrel responsiveness. Patients treated with clopidogrel are more likely to have also been medicated with other drugs such as aspirin. Therefore, further clinical studies are necessary to assess the role of the CYP2C19 genotype on clopidogrel responsiveness. Second, we did not measure the plasma levels of the active metabolite of clopidogrel. Therefore, even though we identified a relationship between the pharmacokinetics of clopidogrel and IPA values, it is necessary to determine whether the plasma levels of the active metabolite are also related to the IPA values. However, because the active metabolite is chemically unstable and labile, we are limited in our ability to practically and quantitatively detect it in biological samples. Third, we did not consider the metabolic activity of polymorphic CYP3A, including CYP3A4 and CYP3A5. However, although previous studies have shown that CYP3A5*3 is a highly polymorphic enzyme, the genotypic differences do not produce differences in plasma levels or clopidogrel responsiveness. Like CYP3A5, CYP3A4 is also very polymorphic. However, unlike the CYP3A5 gene, there is no evidence of a "null" allele for CYP3A4. While more than 30 single-nucleotide polymorphisms have been identified in the CYP3A4 gene,35 the allele frequencies are very low, making it unlikely that genotypic differences in CYP3A4 account for interindividual variability in clopidogrel metabolism.
In summary, the CYP2C19 genotype affects the plasma levels of clopidogrel and clopidogrel responsiveness. Therefore, polymorphic CYP2C19 genotypes appear to be major determinants of interindividual variability in clopidogrel responsiveness.
Methods Subjects and study design. The study protocol was approved by the Ethical Committee of Anam Hospital, Korea University College of Medicine, Korea, and all subjects provided written informed consent. The 24 healthy subjects selected for this study were classified into groups according to their CYP2C19 genotype: homozygous extensive metabolizers (homoEMs, n = 8), heterozygous EMs (heteroEMs, n = 8), and poor metabolizers (PMs, n = 8).16
The mean age of our subjects was 24.0 2.4 years (range 20–29 years) and the mean weight was 69.0 6.3 kg (range 58–83 kg). There were no significant differences among the three CYP2C19 genotypes (homoEMs, heteroEMs, and PMs) with respect to age (23.5 2.9, 24.3 1.7, and 24.1 2.8 years, respectively) or body weight (66.6 5.6, 73.0 6.3, and 67.3 5.6 kg, respectively). The subjects were examined by physicians and were deemed to be healthy, on the basis of the results of a detailed physical examination, 12-lead electrocardiography, and laboratory tests. Subjects were excluded if they had a history or evidence of hepatic, renal, gastrointestinal, or hematologic abnormalities, any other acute or chronic disease, or an allergy to any drugs. None of the subjects smoked tobacco or used any continuous medication. No medications, herbal medicine, alcohol, citrus juice, or beverages containing caffeine were permitted for 10 days prior to the study and for the duration of the study.
Study procedures. All subjects received a single 300-mg loading dose of clopidogrel on day 1, followed by a 75-mg daily maintenance dose from days 2 to 7. Blood samples were collected to measure plasma concentrations of clopidogrel after the loading dose (300 mg) at 0 (predose), 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h. Blood samples were also collected to measure ADP-induced platelet aggregation during treatment, at day 1 (before the loading dose, and at 2, 6, and 12 h after the dose), and at days 2, 3, 5, and 7 (before the maintenance dose), and at 24, 72, 120, and 216 h after the last dose, administered at day 7.
Cyp2c19 genotyping. Genomic DNA was extracted from peripheral whole blood using a Qiagen DNA extraction kit (Qiagen, Hilden, Germany). For each sample, the CYP2C19 genotype was identified using the PCR–restriction fragment length polymorphism method, as described earlier.24,36 The patients were divided into three groups according to their CYP2C19 genotype: (homoEMs (CYP2C19*1/*1 (n = 8)), heteroEMs (CYP2C19*1/*2 (n = 6) and CYP2C19*1/*3 (n = 2)), and PMs (CYP2C19*2/*2 (n = 6) and CYP2C19*2/*3 (n = 2)).16
Determination of plasma concentrations of clopidogrel. Plasma concentrations of clopidogrel were determined using high-performance liquid chromatography–tandem mass spectrometry, as described earlier, with a slight modification.37 Clopidogrel was extracted from the isolated plasma samples with diethyl ether/n -hexane (8:2, vol/vol) after the addition of the internal standard (ticlopidine). After being vortexed, the mixture was centrifuged and frozen at -70°C for 15 min. The organic phases were transferred to new glass tubes and then evaporated to dryness under nitrogen gas. The residue was reconstituted in 0.1 ml of 70% acetonitrile, and 20 l was injected for analysis. Chromatography was performed using a Thermo Hypersil Gold column (150 2.1 mm2, 5.0-m particle size; Thermo Fisher Scientific, Waltham, MA) at room temperature. The mobile phase consisted of 1 mmol/l ammonium acetate in 90% acetonitrile. For clopidogrel and ticlopidine, the precursor-to-product ion reactions monitored were m/z 322212 and 264125, respectively. The lower limit of quantification for clopidogrel was 0.01 ng/ml. The interday and intraday precisions for all analyses were <12%.
Measurement of platelet aggregation. Platelet aggregation was measured using a Chrono-log Lumi-Aggregometer (model 700–4DR; Chrono-log, Havertown, PA) equipped with an AggroLink software package and using a turbimetric method, as earlier described, with a modification.38,39 In brief, after inverting the Vacutainer tube three to five times for gentle mixing, platelet-rich plasma and platelet-poor plasma were prepared by differential centrifugation. The platelet-rich plasma (0.5 ml) was incubated at 37°C in the aggregometer for 2 min, followed by the addition of ADP (5 mol/l) with continuous stirring at 1,200 r.p.m. Platelet aggregation was recorded for up to 5 min and expressed as the maximal percentage change of light transmission from baseline using platelet-poor plasma as a reference. The IPA value was calculated from the observed maximal platelet aggregation (MPA) at each scheduled time-point for each treatment as2,20
Pharmacokinetic and pharmacodynamic evaluations. WinNonlin Professional version 5.2 software (Pharsight, Mountain View, CA) was used for the pharmacokinetic and pharmacodynamic analyses. We analyzed the plasma concentrations of clopidogrel by noncompartmental analysis. The Cmax and the time to reach Cmax (tmax) were estimated directly from the observed plasma concentration–time data. The area under the plasma concentration–time curve from time 0 to 24 h (AUCall) after the loading dose was calculated using the linear trapezoidal rule.
The pharmacodynamic effect of clopidogrel, that is, the IPA, was expressed as the percentage change from baseline platelet aggregation until 336 h after dosing. The AUEC for the IPA of clopidogrel was calculated from the time vs. IPA value curve, using the linear trapezoidal rule as described earlier.2,20 The maximal percentage change (Emax) and the time to reach Emax (Tmax) were determined for individual subjects.
Measurement of CYP2C19 metabolic activity. We used omeprazole hydroxylation to evaluate the relationship between clopidogrel responsiveness and CYP2C19 metabolic activity. For measuring the CYP2C19 metabolic activities of the subjects, each subject was given a single oral dose of 20 mg omeprazole with 240 ml of water after an overnight fast, and was asked to avoid water and food for 3 h after taking the drug. Three hours after the dose was given, 5 ml of venous blood was collected from each subject. The plasma levels of omeprazole and its metabolite, 5-hydroxyomeprazole, were determined using high-performance liquid chromatography as described elsewhere CYP2C19 metabolic activity was calculated.
Statistical analyses. Data are expressed as mean values s.d. unless otherwise indicated, and differences of P < 0.05 were considered statistically significant. Statistical comparisons of pharmacokinetic and pharmacodynamic parameters among the CYP2C19 genotypes were made using a one-way analysis of variance or Kruskal–Wallis test after a normality test (Shapiro–Wilk test; SAS univariate procedure), followed by the post hoc Bonferroni test for multiple comparisons. Statistical analyses were performed using the statistical software package, SAS version 9.1.3 (SAS Institute, Cary, NC). The authors had full access to the data and take full responsibility for the integrity of the data.
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