Effect of two dosages of sodium chloride intake on the blood pressure response to caffeinated coffee in humans in vivo.

High sodium intake increases cardiovascular risk by increasing blood pressure. The intake of coffee elevates blood pressure acutely. Preclinical evidence shows that this action of caffeine is enhanced by high salt intake. We hypothesised that high sodium intake augments the acute blood pressure response to coffee in humans. A randomised cross-over study (n = 15) was performed comparing the effect of lower (6 g/d; LS) with higher (12 g/d; HS) sodium chloride diet on blood pressure before and 2 h after regular coffee intake. Baseline blood pressure was 115 ± 4/84 ± 2/68 ± 1 during LS and 121 ± 4/89 ± 2/69 ± 1 mmHg during HS (SBP/Mean Arterial Pressure (MAP)/DBP; mean ± SE, p < 0.05 for SBP). During LS, blood pressure increased to 121 ± 4/91 ± 2/73 ± 1 (p < 0.05 for SBP, MAP, DBP versus baseline). HS did not significantly affect the impact of coffee on blood pressure (p > 0.3 for SBP, DBP; p > 0.05 for MAP). Sodium intake does not relevantly modulate the impact of regular coffee consumption on blood pressure.

Proguanil and cycloguanil are organic cation transporter and multidrug and toxin extrusion substrates.

BACKGROUND: Malaria, HIV/AIDS, and tuberculosis endemic areas show considerable geographical overlap, leading to incidence of co-infections. This requires treatment with multiple drugs, potentially causing adverse drug-drug interactions (DDIs). As anti-malarials are generally positively charged at physiological pH, they are likely to interact with human organic cation transporters 1 and 2 (OCT1 and OCT2). These transporters are involved in the uptake of drugs into hepatocytes and proximal tubule cells for subsequent metabolic conversion or elimination. This efflux of cationic drugs from hepatocytes and proximal tubule cells into bile and urine can be mediated by multidrug and toxin extrusion 1 and 2-K (MATE1 and MATE2-K) transporters, respectively. METHODS: Here, the interaction of anti-malarials with these transporters was studied in order to predict potential DDIs. Using baculovirus-transduced HEK293 cells transiently expressing human OCT1, OCT2, MATE1 and MATE2K uptake and inhibition was studied by a range of anti-malarials. RESULTS: Amodiaquine, proguanil, pyrimethamine and quinine were the most potent inhibitors of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) transport, a known substrate of OCT1/2, resulting in half maximal inhibitory concentrations (IC50) of 11, 13, 1.6, and 3.4 µM, respectively. Only quinine had a drug-drug index higher than the cut-off value of 0.1 for OCT2, therefore, in vivo pharmacokinetic studies focusing on DDIs involving this compound and other OCT2-interacting drugs are warranted. Furthermore, proguanil appeared to be a substrate of OCT1 and OCT2 with affinities of 8.1 and 9.0 µM, respectively. Additionally, MATE1 and MATE2-K were identified as putative transport proteins for proguanil. Finally, its metabolite cycloguanil was also identified as an OCT1, OCT2, MATE1 and MATE2-K substrate. CONCLUSION: Anti-malarials can reduce OCT1 and OCT2 transport activity in vitro. Furthermore, proguanil and cycloguanil were found to be substrates of OCT1, OCT2, MATE1 and MATE2-K, highlighting the importance of these transporters in distribution and excretion. As these compounds shares substrate overlap with metformin DDIs can be anticipated during concurrent treatment.

Moxifloxacin Is a Potent In Vitro Inhibitor of OCT- and MATE-Mediated Transport of Metformin and Ethambutol.

It is largely unknown if simultaneous administration of tuberculosis (TB) drugs and metformin leads to drug-drug interactions (DDIs). Disposition of metformin is determined by organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). Thus, any DDIs would primarily be mediated via these transporters. This study aimed to assess the in vitro inhibitory effects of TB drugs (rifampin, isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin, and linezolid) on metformin transport and whether TB drugs are also substrates themselves of OCTs and MATEs. HEK293 cells overexpressing OCT1, OCT2, OCT3, MATE1, and MATE2K were used to study TB drug-mediated inhibition of [14C]metformin uptake and to test if TB drugs are transporter substrates. Metformin uptake was determined by quantifying [14C]metformin radioactivity, and TB drug uptake was analyzed using liquid chromatography-tandem mass spectrometry. DDI indices were calculated (plasma maximum concentrations [Cmax]/50% inhibitory concentrations [IC50]), and based on the literature, a cutoff of >0.1 was assumed to warrant further in vivo investigation. Moxifloxacin was the only TB drug identified as a potent inhibitor (DDI index of >0.1) of MATE1- and MATE2K-mediated metformin transport, with IC50s of 12 µM (95% confidence intervals [CI], 5.1 to 29 µM) and 7.6 µM (95% CI, 0.2 to 242 µM), respectively. Of all TB drugs, only ethambutol appeared to be a substrate of OCT1, OCT2, OCT3, MATE1, and MATE2K. MATE1-mediated ethambutol uptake was inhibited strongly (DDI index of >0.1) by moxifloxacin (IC50, 12 µM [95% CI, 3.4 to 43 µM]). Our findings provide a mechanistic basis for DDI predictions concerning ethambutol. According to international guidelines, an in vivo interaction study is warranted for the observed in vitro interaction between ethambutol and moxifloxacin.

Statin Lactonization by Uridine 5'-Diphospho-glucuronosyltransferases (UGTs).

Statins are cholesterol-lowering drugs that have proven to be effective in lowering the risk of major cardiovascular events. Although well tolerated, statin-induced myopathies are the most common side effects. Compared to their pharmacologically active acid form, statin lactones are more potent inducers of toxicity. They can be formed by glucuronidation mediated by uridine 5'-diphospho-glucuronosyltransferases (UGTs), but a systematic characterization of subtype specificity and kinetics of lactonization is lacking. Here, we demonstrate for six clinically relevant statins that only UGT1A1, 1A3, and 2B7 contribute significantly to their lactonization. UGT1A3 appeared to have the highest lactonization capacity with marked differences in statin conversion rates: pitavastatin ≫ atorvastatin > cerivastatin > lovastatin > rosuvastatin (simvastatin not converted). Using in silico modeling we could identify a probable statin interaction region in the UGT binding pocket. Polymorphisms in these regions of UGT1A1, 1A3, and 2B7 may be a contributing factor in statin-induced myopathies, which could be used in personalization of statin therapy with improved safety.

Interaction of digitalis-like compounds with liver uptake transporters NTCP, OATP1B1, and OATP1B3.

Digitalis-like compounds (DLCs) such as digoxin, digitoxin, and ouabain, also known as cardiac glycosides, are among the oldest pharmacological treatments for heart failure. The compounds have a narrow therapeutic window, while at the same time, DLC pharmacokinetics is prone to drug-drug interactions at the transport level. Hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and Na(+)-dependent taurocholate co-transporting polypeptide (NTCP) influence the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. The interaction of digoxin, digitoxin, and ouabain with hepatic uptake transporters has been studied before. However, here, we systematically investigated a much wider range of structurally related DLCs for their capability to inhibit or to be transported by these transporters in order to better understand the relation between the activity and chemical structure of this compound type. We studied the uptake and inhibitory potency of a series of 14 structurally related DLCs in Chinese hamster ovary cells expressing NTCP (CHO-NTCP) and human embryonic kidney cells expressing OATP1B1 and OATP1B3 (HEK-OATP1B1 and HEK-OATP1B3). The inhibitory effect of the DLCs was measured against taurocholic acid (TCA) uptake in CHO-NTCP cells and against uptake of ß-estradiol 17-ß-d-glucuronide (E217ßG) in HEK-OATP1B1 and HEK-OATP1B3 cells. Proscillaridin A was the most effective inhibitor of NTCP-mediated TCA transport (IC50 = 22 µM), whereas digitoxin and digitoxigenin were the most potent inhibitors of OATP1B1 and OAPTP1B3, with IC50 values of 14.2 and 36 µM, respectively. Additionally, we found that the sugar moiety and hydroxyl groups of the DLCs play different roles in their interaction with NTCP, OATP1B1, and OATP1B3. The sugar moiety decreases the inhibition of NTCP and OATP1B3 transport activity, whereas it enhances the inhibitory potency against OATP1B1. Moreover, the hydroxyl group at position 12 reinforces the inhibition of NTCP but decreases the inhibition of OATP1B1 and OATP1B3. To investigate whether DLCs can be translocated, we quantified their uptake in transporter-expressing cells by LC-MS. We demonstrated that convallatoxin, ouabain, dihydroouabain, and ouabagenin are substrates of OATP1B3. No transport was observed for the other compounds in any of the studied transporters. In summary, this work provides a step toward an improved understanding of the interaction of DLCs with three major hepatic uptake transporters. Ultimately, this can be of use in the development of DLCs that are less prone to transporter-mediated drug-drug interactions.

Editor's Highlight: PlacentalDisposition and Effects of Crizotinib: An Ex Vivo Study in the Isolated Dual-Side Perfused Human Cotyledon.

Tyrosine kinase inhibitors (TKIs) play an important role in cancer pharmacotherapy, yet there is limited data on their use during pregnancy. We studied placental disposition and placental toxicity of crizotinib, a TKI used to treat nonsmall cell lung cancer. Term placentas were perfused for 3 h with crizotinib (1 µM) using the ex vivo dual-side cotyledon perfusion technique. Interference of TKIs with trophoblast viability was studied using BeWo cells. Expression of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) in placental tissue was assessed by immunohistochemistry and inhibition of these transporters was determined in vitro by transport studies with membrane vesicles overexpressing human P-gp or BCRP. We found that crizotinib rapidly and strongly accumulates in cotyledon perfusion experiments, reaching a concentration of 3.1 ± 0.4 µM in placental tissue. Final drug concentrations in the maternal and foetal reservoirs were 0.2 ± 0.05 and 0.08 ± 0.01 µM, respectively. Furthermore, crizotinib inhibited BeWo cell viability (IC50: 234 nM, 95% CI: 167-328 nM) 10 times more potently than other TKIs tested. In vitro transport studies revealed that crizotinib is a potent inhibitor of the transport activities of BCRP (IC50: 5.7 µM, 95% CI: 2.7-11.8 µM) and P-gp (IC50: 7.8 µM, 95% CI: 3.4-18.0 µM). In conclusion, crizotinib strongly accumulated in placental tissue at clinically relevant concentrations. IC50 values for transporter inhibition and trophoblast cell viability were similar to the tissue concentrations reached, suggesting that crizotinib can inhibit placental BCRP and P-gp function and possibly affect trophoblast viability.

Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay.

Vascular and metabolic effects of the haem oxygenase-1 inducer haem arginate in subjects with the metabolic syndrome: A translational cross-over study.

This translational randomized and vehicle-controlled cross-over study was performed to assess the impact of haem arginate treatment on haem oxygenase-1 induction, endothelial function and insulin sensitivity in subjects with the metabolic syndrome (n = 14). Both treatment periods consisted of 5 days. Haem arginate or vehicle (l-arginine) was administered intravenously on Days 1 and 3. Forearm blood flow in response to acetylcholine and nitroglycerine was measured by venous occlusion plethysmography (Day 3), insulin sensitivity by a hyperinsulinaemic clamp procedure (Day 5). Haem arginate did not improve endothelial function or insulin sensitivity but significantly reduced the vasodilator response to nitroglycerine (p < 0.01). These negative findings are in contrast to the preclinical data, which may be due to short duration of therapy and limited haem oxygenase-1 induction as well as interference by markedly elevated plasma haem levels observed after haem arginate treatment (p < 0.01). Future studies should pay attention to the delicate balance between sufficient dosing and timely normalization of plasma haem levels.

MRP1 mediates folate transport and antifolate sensitivity in Plasmodium falciparum.

Multidrug resistance-associated proteins (MRP) of Plasmodium falciparum have been associated with altered drug sensitivity. Knowledge on MRP substrate specificity is indispensible for the characterization of resistance mechanisms and identifying its physiological roles. An untargeted metabolomics approach detected decreased folate concentrations in red blood cells infected with schizont stage parasites lacking expression of MRP1. Furthermore, a tenfold decrease in sensitivity toward the folate analog methotrexate was detected for parasites lacking MRP1. PfMRP1 is involved in the export of folate from parasites into red blood cells and is therefore a relevant factor for efficient malaria treatment through the folate pathway.

Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.

Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies.

Convallatoxin: a new P-glycoprotein substrate.

Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1±0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp.

The effect of eplerenone on adenosine formation in humans in vivo: a double-blinded randomised controlled study.

BACKGROUND: It has been suggested that mineralocorticoid receptor antagonists have direct cardioprotective properties, because these drugs reduce mortality in patients with heart failure. In murine models of myocardial infarction, mineralocorticoid receptor antagonists reduce infarct size. Using gene deletion and pharmacological approaches, it has been shown that extracellular formation of the endogenous nucleoside adenosine is crucial for this protective effect. We now aim to translate this finding to humans, by investigating the effects of the selective mineralocorticoid receptor antagonist eplerenone on the vasodilator effect of the adenosine uptake inhibitor dipyridamole, which is a well-validated surrogate marker for extracellular adenosine formation. METHODS AND RESULTS: In a randomised, double-blinded, placebo-controlled, cross-over study we measured the forearm blood flow response to the intrabrachial administration of dipyridamole in 14 healthy male subjects before and after treatment with placebo or eplerenone (50 mg bid for 8 days). The forearm blood flow during administration of dipyridamole (10, 30 and 100 µg·min(-1)·dl(-1)) was 1.63 (0.60), 2.13 (1.51) and 2.71 (1.32) ml·dl(-1)·min(-1) during placebo use, versus 2.00 (1.45), 2.68 (1.87) and 3.22 (1.94) ml·dl(-1)·min(-1) during eplerenone treatment (median (interquartile range); P = 0.51). Concomitant administration of the adenosine receptor antagonist caffeine attenuated dipyridamole-induced vasodilation to a similar extent in both groups. The forearm blood flow response to forearm ischemia, as a stimulus for increased formation of adenosine, was similar during both conditions. CONCLUSION: In a dosage of 50 mg bid, eplerenone does not augment extracellular adenosine formation in healthy human subjects. Therefore, it is unlikely that an increased extracellular adenosine formation contributes to the cardioprotective effect of mineralocorticoid receptor antagonists. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01837108.