Rick Greupink

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1

Organic anion–transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the Vmax for OATP1B1-mediated transport of E217β-G (estradiol 17β-d-glucuronide) was decreased >60%, whereas Km values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (Km 13.1 ± 0.43 μM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC50 values for inhibition of E217β-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

Interaction of fluvastatin with the liver-specific Na+-dependent taurocholate cotransporting polypeptide (NTCP)

It has been reported that polymorphisms in the organic anion transporting polypeptide 1B1 (OATP1B1, SLCO1B1) result in decreased hepatic uptake of simvastatin carboxy acid, the active metabolite of simvastatin. This is not the case for fluvastatin and it has been hypothesized that for this drug other hepatic uptake pathways exist. Here, we studied whether Na(+)-dependent taurocholate co-transporting polypeptide (NTCP, SLC10A1) can be an alternative hepatic uptake route for fluvastatin. Chinese Hamster Ovary cells transfected with human NTCP (CHO-NTCP) were used to investigate the inhibitory effect of fluvastatin and other statins on [(3)H]-taurocholic acid uptake ([(3)H]-TCA). Statin uptake by CHO-NTCP and cryopreserved human hepatocytes was assessed via LC-MS/MS. Fluvastatin appeared to be a potent and competitive inhibitor of [(3)H]-TCA uptake (IC(50) of 40μM), pointing to an interaction at the level of the bile acid binding pocket of NTCP. The inhibitory action of other statins was also studied, which revealed that statin inhibitory potency increased with molecular descriptors of lipophilicity: calculated logP (r(2)=0.82, p=0.034), logD(7.4) (r(2)=0.77, p=0.0001). Studies in CHO-NTCP cells showed that fluvastatin was indeed an NTCP substrate (K(m) 250±30μM, V(max) 1340±50ng/mg total cell protein/min). However, subsequent studies revealed that at clinically relevant plasma concentrations, NTCP contributed minimally to overall accumulation in human hepatocytes. In conclusion, fluvastatin interacts with NTCP at the level of the bile acid binding pocket and is an NTCP substrate. However, under normal conditions, NTCP-mediated uptake of this drug seems not to be a significant hepatocellular uptake pathway.

In silico identification of potential cholestasis-inducing agents via modeling of Na(+)-dependent taurocholate cotransporting polypeptide substrate specificity.

Na(+)-dependent taurocholate cotransporting polypeptide (NTCP, SLC10A1) is the main transporter facilitating the hepatic uptake of bile acids from the circulation. Consequently, the interaction of xenobiotics, including therapeutic drugs, with the bile acid binding pocket of NTCP could lead to impairment of hepatic bile acid uptake. We pursued a 3D-pharmacophore approach to model the NTCP substrate and inhibitor specificity and investigated whether it is possible to identify compounds with intrinsic NTCP inhibitory properties. Based on known endogenous NTCP substrates, a 3D-pharmacophore model was built, which was subsequently used to screen two virtual libraries together containing the structures of 10 million compounds. Studies with Chinese hamster ovary cells overexpressing human NTCP, human hepatocytes, ex vivo perfused rat livers, and bile duct-cannulated rats were conducted to validate the activity of the virtual screening hits. Modeling yielded a 3D-pharmacophore, consisting of two hydrogen bond acceptors and three hydrophobic features. Six out of 10 structurally diverse compounds selected in the first virtual screening procedure significantly inhibited taurocholate uptake in the NTCP overexpressing cells. For the most potent inhibitor identified, an anthraquinone derivative, this finding was confirmed in human hepatocytes and perfused rat livers. Subsequent structure and activity relationship studies with analogs of this derivative indicated that an appropriate distance between hydrogen bond acceptor features and presence of one or two negative charges appear critical for a successful NTCP interaction. In conclusion, pharmacophore modeling was successfully used to identify compounds that inhibit NTCP. Our approach represents an important first step toward the in silico flagging of potential cholestasis-inducing molecules.

Interaction of Digitalis-Like Compounds with P-Glycoprotein

Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12β seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a δ-lactone ring and a sugar moiety at 3β of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5β and 19, whereas inhibition is decreased by those at positions 1β, 11α, 12β, and 16β. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.

Pharmacological considerations on the use of antiretrovirals in pregnancy

PURPOSE OF REVIEW Treatment with combination antiretroviral therapy during pregnancy reduces the chance of mother to child transmission of HIV. Physiological changes during pregnancy can lead to lower exposure to antiretrovirals, possibly resulting in virological failure. For most antiretrovirals, data on exposure during pregnancy and transplacental passage are limited. This review summarizes the most recent information on pharmacokinetics (including transplacental passage), efficacy, as well as the safety of antiretrovirals during pregnancy. RECENT FINDINGS Intensive-sampling pharmacokinetic studies as well as observational studies using sparse sampling were performed to explore the exposure to antiretrovirals during pregnancy. Transplacental passage, efficacy (viral load at delivery and infection status of the newborn) and safety information were evaluated for several antiretrovirals. SUMMARY For most nucleoside/nucleotide reverse transcriptase inhibitors and protease inhibitors, recent research shows a decreased exposure during pregnancy. However, the advantage of a general dose increase during pregnancy still remains unclear. For newer compounds and efavirenz, limited or no data on pharmacokinetics during pregnancy or transplacentally are available, while the mechanisms of transplacental passage also remain unknown. For safety reasons, it will be important to monitor pregnancy outcomes in resource-limited settings during the implementation of the WHO guidelines (including the use of efavirenz during pregnancy).

Exploiting transport activity of p-glycoprotein at the blood-brain barrier for the development of peripheral cannabinoid type 1 receptor antagonists.

Although the CB1 receptor antagonist/inverse agonist rimonabant has positive effects on weight loss and cardiometabolic risk factors, neuropsychiatric side effects have prompted researchers to develop peripherally acting derivatives. Here, we investigated for a series of 3,4-diarylpyrazoline CB1 receptor antagonists if transport by the brain efflux transporter P-gp could be used as a selection criterion in the development of such drugs. All 3,4-diarylpyrazolines and rimonabant inhibited P-gp transport activity in membrane vesicles isolated from HEK293 cells overexpressing the transporter, but only the 1,1-dioxo-thiomorpholino analogue 23 exhibited a reduced accumulation (-38 ± 2%) in these cells, which could be completely reversed by the P-gp/BCRP inhibitor elacridar. In addition, 23 appeared to be a BCRP substrate, whereas rimonabant was not. In rats, the in vivo brain/plasma concentration ratio of 23 was significantly lower than for rimonabant (0.4 ± 0.1 vs 6.2 ± 1.6, p < 0.001). Coadministration of elacridar resulted in an 11-fold increase of the brain/plasma ratio for 23 (p < 0.01) and only 1.4-fold for rimonabant (p < 0.05), confirming the involvement of P-gp and possibly BCRP in limiting the brain entrance of 23 in vivo. In conclusion, these data support the conception that efflux via transporters such as P-gp and BCRP can limit the brain penetration of CB1 receptor antagonists, and that this property could be used in the development of peripheral antagonists.

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 β-estradiol 17-β-d-glucuronide (E217βG) 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.

In silico identification and in vitro validation of potential cholestatic compounds through 3D ligand-based pharmacophore modeling of BSEP inhibitors

Drug-induced cholestasis is a frequently observed side effect of drugs and is often caused by an unexpected interaction with the bile salt export pump (BSEP/ABCB11). BSEP is the key membrane transporter responsible for the transport of bile acids from hepatocytes into bile. Here, we developed a pharmacophore model that describes the molecular features of compounds associated with BSEP inhibitory activity. To generate input and validation data sets, in vitro experiments with membrane vesicles overexpressing human BSEP were used to assess the effect of compounds (50 μM) on BSEP-mediated (3)H-taurocholic acid transport. The model contains two hydrogen bond acceptor/anionic features, two hydrogen bond acceptor vector features, four hydrophobic/aromatic features, and exclusion volumes. The pharmacophore was validated against a set of 59 compounds, including registered drugs. The model recognized 9 out of 12 inhibitors (75%), which could not be identified based on general parameters, such as molecular weight or SlogP, alone. Finally, the model was used to screen a virtual compound database. A number of compounds found via virtual screening were tested and displayed statistically significant BSEP inhibition, ranging from 13 ± 1% to 67 ± 7% of control (P < 0.05). In conclusion, we developed and validated a pharmacophore model that describes molecular features found in BSEP inhibitors. The model may be used as an in silico screening tool to identify potentially harmful drug candidates at an early stage in drug development.

Placental transfer of the HIV integrase inhibitor dolutegravir in an ex vivo human cotyledon perfusion model

OBJECTIVES Data on fetal exposure to antiretroviral agents during pregnancy are important to estimate their potential for prevention of mother-to-child transmission (PMTCT) and possible toxicity. For the recently developed HIV integrase inhibitor dolutegravir, clinical data on fetal disposition are not yet available. Dual perfusion of a single placental lobule (cotyledon) provides a useful ex vivo model to predict the in vivo maternal-to-fetal transfer of this drug. The aim of this study was to estimate the transfer of dolutegravir across the human term placenta, using a dual-perfusion cotyledon model. METHODS After cannulation of the cotyledons (n = 6), a fetal circulation of 6 mL/min and maternal circulation of 12 mL/min were initiated. The perfusion medium consisted of Krebs-Henseleit buffer (pH = 7.2-7.4) supplemented with 10.1 mM glucose, 30 g/L human serum albumin and 0.5 mL/L heparin 5000IE. Dolutegravir was administered to the maternal circulation (∼ 4.2 mg/L) and analysed by UPLC-MS/MS. RESULTS After 3 h of perfusion, the mean ± SD fetal-to-maternal (FTM) concentration ratio of dolutegravir was 0.6 ± 0.2 and the mean ± SD concentrations in the maternal and fetal compartments were 2.3 ± 0.4 and 1.3 ± 0.3 mg/L, respectively. CONCLUSIONS Dolutegravir crosses the blood-placental barrier with a mean FTM concentration ratio of 0.6. Compared with other antiretroviral agents, placental transfer of dolutegravir is moderate to high. These data suggest that dolutegravir holds clinical potential for pre-exposure prophylaxis and consequently PMTCT, but also risk of fetal toxicity.

Semi-mechanistic physiologically-based pharmacokinetic modeling of clinical glibenclamide pharmacokinetics and drug-drug-interactions

We studied if the clinical pharmacokinetics and drug-drug interactions (DDIs) of the sulfonylurea-derivative glibenclamide can be simulated via a physiologically-based pharmacokinetic modeling approach. To this end, a glibenclamide PBPK-model was build in Simcyp using in vitro physicochemical and biotransformation data of the drug, and was subsequently optimized using plasma disappearance data observed after i.v. administration. The model was validated against data observed after glibenclamide oral dosing, including DDIs. We found that glibenclamide pharmacokinetics could be adequately modeled if next to CYP metabolism an active hepatic uptake process was assumed. This hepatic uptake process was subsequently included in the model in a non-mechanistic manner. After an oral dose of 0.875 mg predicted Cmax and AUC were 39.7 (95% CI:37.0-42.7)ng/mL and 108 (95% CI: 96.9-120)ng/mLh, respectively, which is in line with observed values of 43.6 (95% CI: 37.7-49.5)ng/mL and 133 (95% CI: 107-159)ng/mLh. For a 1.75 mg oral dose, the predicted and observed values were 82.5 (95% CI:76.6-88.9)ng/mL vs 91.1 (95% CI: 67.9-115.9) for Cmax and 224 (95% CI: 202-248) vs 324 (95% CI: 197-451)ng/mLh for AUC, respectively. The model correctly predicted a decrease in exposure after rifampicin pre-treatment. An increase in glibenclamide exposure after clarithromycin co-treatment was predicted, but the magnitude of the effect was underestimated because part of this DDI is the result of an interaction at the transporter level. Finally, the effects of glibenclamide and fluconazol co-administration were simulated. Our simulations indicated that co-administration of this potent CYP450 inhibitor will profoundly increase glibenclamide exposure, which is in line with clinical observations linking the glibenclamide-fluconazol combination to an increased risk of hypoglycemia. In conclusion, glibenclamide pharmacokinetics and its CYP-mediated DDIs can be simulated via PBPK-modeling. In addition, our data underline the relevance of modeling transporters on a full mechanistic level to further improve pharmacokinetic and DDI predictions of this sulfonylurea-derivative.