Mikko T. Holmberg

The surface protease PgtE of Salmonella enterica affects complement activity by proteolytically cleaving C3b, C4b and C5

Complement activity in mammalian serum is fundamentally based on three homologous components C3b, C4b and C5. During systemic infection, the gastrointestinal pathogen Salmonella enterica disseminates within host phagocytic cells but also extracellularly. Consequently, systemic Salmonella transiently confronts the complement system. We show here that the surface protease PgtE of S. enterica proteolytically cleaves C3b, C4b and C5 and that the expression of PgtE enhances bacterial resistance to human serum. Degradation of C3b was further enhanced by PgtE‐mediated plasminogen activation.

Carboxylesterase 1 c.428G>A single nucleotide variation increases the antiplatelet effects of clopidogrel by reducing its hydrolysis in humans

Carboxylesterase 1 (CES1) hydrolyzes the prodrug clopidogrel to an inactive carboxylic acid metabolite. We studied the pharmacokinetics and pharmacodynamics of 600 mg oral clopidogrel in healthy white volunteers, including 10 carriers and 12 noncarriers of CES1 c.428G>A (p.Gly143Glu, rs71647871) single nucleotide variation (SNV). Clopidogrel carboxylic acid to clopidogrel area under the plasma concentration‐time curve from 0 hours to infinity (AUC0–∞) ratio was 53% less in CES1 c.428G>A carriers than in noncarriers (P = 0.009), indicating impaired hydrolysis of clopidogrel. Consequently, the AUC0–∞ of clopidogrel and its active metabolite were 123% (P = 0.004) and 67% (P = 0.009) larger in the c.428G>A carriers than in noncarriers. Consistent with these findings, the average inhibition of P2Y12‐mediated platelet aggregation 0–12 hours after clopidogrel intake was 19 percentage points higher in the c.428G>A carriers than in noncarriers (P = 0.036). In conclusion, the CES1 c.428G>A SNV increases clopidogrel active metabolite concentrations and antiplatelet effects by reducing clopidogrel hydrolysis to inactive metabolites.

Grapefruit Juice Inhibits the Metabolic Activation of Clopidogrel

Cytochrome P450 (CYP) enzymes, including CYP2C19 and CYP3A4, participate in the bioactivation of clopidogrel. Grapefruit juice constituents potently inactivate intestinal CYP3A4 and have been shown to inhibit CYP2C19 as well. In a randomized crossover study, 14 healthy volunteers ingested 200 ml of grapefruit juice or water three times daily for 3 days. On day 3, they ingested a single 600‐mg dose of clopidogrel. Grapefruit juice reduced the peak plasma concentration (Cmax) of the active metabolite of clopidogrel to 13% of the control (range 11–17%, P < 0.001) and the area under the plasma concentration–time curve from 0 to 3 h to 14% (range 12–17%, P < 0.001) of the control, but it had no significant effect on the parent clopidogrel. Moreover, grapefruit juice markedly decreased the platelet‐inhibitory effect of clopidogrel, as assessed with the VerifyNow P2Y12 test in two of the participants. In conclusion, concomitant use of grapefruit juice may impair the efficacy of clopidogrel. Therefore, the use of grapefruit juice is best avoided during clopidogrel therapy.

Regulation of complement classical pathway by association of C4b-binding protein to the surfaces of SK-OV-3 and caov-3 ovarian adenocarcinoma cells

The role of fluid-phase regulators of complement is to inhibit excessive complement activation and maintain homeostasis in blood. By binding to and inactivating complement components on cell surfaces, they can also protect autologous cells from complement-mediated cytotoxicity and phagocytosis. In this study, we wanted to find out whether C4b-binding protein (C4bp), a fluid-phase regulator of the classical complement pathway, could directly bind to cell surfaces in a functionally active form. After screening several malignant cell lines, we observed that the ovarian adenocarcinoma cell lines SK-OV-3, Caov-3, and SW626 were capable of binding C4bp. Binding tests with recombinant deletion mutants suggested that the primary binding site on C4bp is located on the α-chain complement control protein 4 domain. Functional tests showed that tumor cell-bound C4bp retained its cofactor activity for factor I-mediated inactivation of C4b, thus increasing the control of classical complement pathway activation on the surfaces of these cells. These results demonstrate a novel mechanism of complement regulation on cell surfaces, particularly on those of malignant ovarian tumor cells.

Grapefruit juice markedly increases the plasma concentrations and antiplatelet effects of ticagrelor in healthy subjects

AIM This study examined the effects of grapefruit juice on the new P2Y12 inhibitor ticagrelor, which is a substrate of CYP3A4 and P-glycoprotein. METHODS In a randomized crossover study, 10 healthy volunteers ingested 200 ml of grapefruit juice or water thrice daily for 4 days. On day 3, they ingested a single 90 mg dose of ticagrelor. RESULTS Grapefruit juice increased ticagrelor geometric mean peak plasma concentration (Cmax ) to 165% (95% confidence interval 147, 184%) and area under the concentration-time curve (AUC(0,∞)) to 221% of control (95% confidence interval 200, 245%). The Cmax and AUC(0,34 h) (P < 0.05) but not the AUC(0,∞) of the active metabolite C12490XX were decreased significantly. Grapefruit juice had a minor effect on ticagrelor elimination half-life prolonging it from 6.7 to 7.2 h (P = 0.036). In good correlation with the elevated plasma ticagrelor concentrations, grapefruit juice enhanced the antiplatelet effect of ticagrelor, assessed with VerifyNow® and Multiplate® methods, and postponed the recovery of platelet reactivity. CONCLUSIONS Grapefruit juice increased ticagrelor exposure by more than two-fold, leading to an enhanced and prolonged ticagrelor antiplatelet effect. The grapefruit juice-ticagrelor interaction seems clinically important and indicates the significance of intestinal metabolism to ticagrelor pharmacokinetics.

Effect of carboxylesterase 1 c.428G > A single nucleotide variation on the pharmacokinetics of quinapril and enalapril

AIM The aim of the present study was to investigate the effects of the carboxylesterase 1 (CES1) c.428G > A (p.G143E, rs71647871) single nucleotide variation (SNV) on the pharmacokinetics of quinapril and enalapril in a prospective genotype panel study in healthy volunteers. METHODS In a fixed-order crossover study, 10 healthy volunteers with the CES1 c.428G/A genotype and 12 with the c.428G/G genotype ingested a single 10 mg dose of quinapril and enalapril with a washout period of at least 1 week. Plasma concentrations of quinapril and quinaprilat were measured for up to 24 h and those of enalapril and enalaprilat for up to 48 h. Their excretion into the urine was measured from 0 h to 12 h. RESULTS The area under the plasma concentration-time curve from 0 h to infinity (AUC0-∞) of active enalaprilat was 20% lower in subjects with the CES1 c.428G/A genotype than in those with the c.428G/G genotype (95% confidence interval of geometric mean ratio 0.64, 1.00; P = 0.049). The amount of enalaprilat excreted into the urine was 35% smaller in subjects with the CES1 c.428G/A genotype than in those with the c.428G/G genotype (P = 0.044). The CES1 genotype had no significant effect on the enalaprilat to enalapril AUC0-∞ ratio or on any other pharmacokinetic or pharmacodynamic parameters of enalapril or enalaprilat. The CES1 genotype had no significant effect on the pharmacokinetic or pharmacodynamic parameters of quinapril. CONCLUSIONS The CES1 c.428G > A SNV decreased enalaprilat concentrations, probably by reducing the hydrolysis of enalapril, but had no observable effect on the pharmacokinetics of quinapril.

Effect of grapefruit juice on the bioactivation of prasugrel

AIMS The P2Y12 inhibitor prasugrel is a prodrug, which is activated after its initial hydrolysis partly by cytochrome P450 (CYP) 3A4. Grapefruit juice, a strong inactivator of intestinal CYP3A4, greatly reduces the activation and antiplatelet effects of clopidogrel. The aim of this study was to investigate the effects of grapefruit juice on prasugrel. METHODS In a randomized crossover study, seven healthy volunteers ingested 200 ml of grapefruit juice or water three times daily for 4 days. On day 3, they ingested a single 10 mg dose of prasugrel with an additional 200 ml of grapefruit juice or water. Plasma concentrations of prasugrel metabolites and the antiplatelet effect were measured. RESULTS Grapefruit juice increased the geometric mean area under the plasma concentration-time curve (AUC(0-∞)) of the primary, inactive metabolite of prasugrel to 164% of the control value (95% confidence interval 122-220%, P = 0.008), without a significant effect on its peak plasma concentration (C(max)). The C(max) and AUC(0-∞) of the secondary, active metabolite were decreased to 51% (95% confidence interval 32-84%, P = 0.017) and 74% of the control value (95% confidence interval 60-91%, P = 0.014) by grapefruit juice (P < 0.05). The average platelet inhibition, assessed with the VerifyNow® method at 0-24 h after prasugrel intake, was 5 percentage points (95% confidence interval 1-10 percentage points) lower in the grapefruit juice phase than in the water phase (P = 0.034). CONCLUSIONS Grapefruit juice reduces the bioactivation of prasugrel, but this has only a limited effect on the antiplatelet effect of prasugrel.

Clopidogrel carboxylic acid glucuronidation is mediated mainly by UGT2B7, UGT2B4 and UGT2B17: Implications for pharmacogenetics and drug-drug interactions

The antiplatelet drug clopidogrel is metabolized to an acyl-β-d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl-β-d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant UGT polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CLint,u of 2.42 and 2.82 µl⋅min−1⋅mg−1, respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CLint,u 0.51 µl⋅min−1⋅mg−1) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl-β-d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the UGT2B17*2 deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl-β-d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC0–4) ratio (P < 0.05). To conclude, clopidogrel carboxylic acid is metabolized mainly by UGT2B7 and UGT2B4 in the liver and by UGT2B17 in the small intestinal wall. The formation of clopidogrel acyl-β-d-glucuronide is impaired in carriers of the UGT2B17 deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel.

CYP3A4*22 Impairs the Elimination of Ticagrelor, But Has No Significant Effect on the Bioactivation of Clopidogrel or Prasugrel

CYP3A enzymes participate in the elimination of ticagrelor and the bioactivation of clopidogrel and prasugrel. We studied the effects of functional CYP3A genetic variants (CYP3A4*22; rs35599367 and CYP3A5*3; rs776746) on the pharmacokinetics and pharmacodynamics of ticagrelor, clopidogrel, and prasugrel. Six healthy volunteers with the CYP3A4*1/*22 and CYP3A5*3/*3 genotype (CYP3A4*22 carriers), eight with the CYP3A4*1/*1 and CYP3A5*1/*3 genotype (CYP3A5 expressors), and 11–13 with the CYP3A4*1/*1 and CYP3A5*3/*3 genotypes (controls) ingested single doses of ticagrelor, clopidogrel, and prasugrel on separate occasions. Ticagrelor area under the plasma concentration‐time curve (AUC) was 89% (P = 0.004) higher in CYP3A4*22 carriers than in controls. CYP3A4*22 carriers also showed more pronounced platelet inhibition at 24 hours after ticagrelor ingestion than the controls (43% vs. 21%; P = 0.029). The CYP3A5 genotype did not affect ticagrelor pharmacokinetics. Neither CYP3A5 nor CYP3A4 genotypes significantly affected prasugrel or clopidogrel. In conclusion, the CYP3A4*22 allele markedly impairs ticagrelor elimination enhancing its antiplatelet effect.

Effects of Genetic Variants on Carboxylesterase 1 Gene Expression, and Clopidogrel Pharmacokinetics and Antiplatelet Effects

Several single nucleotide variations (SNVs) affect carboxylesterase 1 (CES1) activity, but the effects of genetic variants on CES1 gene expression have not been systematically investigated. Therefore, our aim was to investigate effects of genetic variants on CES1 gene expression in two independent whole blood sample cohorts of 192 (discovery) and 88 (replication) healthy volunteers and in a liver sample cohort of 177 patients. Furthermore, we investigated possible effects of the found variants on clopidogrel pharmacokinetics (n = 106) and pharmacodynamics (n = 46) in healthy volunteers, who had ingested a single 300 mg or 600 mg dose of clopidogrel. Using massively parallel sequencing, we discovered two CES1 SNVs, rs12443580 and rs8192935, to be strongly and independently associated with a 39% (p = 4.0 × 10−13) and 31% (p = 2.5 × 10−8) reduction in CES1 whole blood expression per copy of the minor allele. These findings were replicated in the replication cohort. However, these SNVs did not affect CES1 liver expression, or clopidogrel pharmacokinetics or pharmacodynamics. Conversely, the CES1 c.428G>A missense SNV (rs71647871) impaired the hydrolysis of clopidogrel, increased exposure to clopidogrel active metabolite and enhanced its antiplatelet effects. In conclusion, the rs12443580 and rs8192935 variants reduce CES1 expression in whole blood but not in the liver. These tissue‐specific effects may result in substrate‐dependent effects of the two SNVs on CES1‐mediated drug metabolism.