Contact lenses for pravastatin delivery to eye segments: Design and in vitro-in vivo correlations.

Oral administration of cholesterol-lowering statins, HMG-CoA reductase inhibitors, is associated with beneficial effects on eye conditions. This work aims to design contact lenses (CLs) that can sustainedly deliver pravastatin and thus improve the ocular efficacy while avoiding systemic side effects of statins. Bioinspired hydrogels were prepared with monomers that resemble hydrophobic (ethylene glycol phenyl ether methacrylate) and amino (2-aminoethyl methacrylamide hydrochloride) functionalities of the active site of HMG-CoA. Best performing CLs loaded >6 mg/g, in vitro fulfilled the release demands for daily wearing, and showed anti-inflammatory activity (lowering TNF-α). High hydrostatic pressure sterilization preserved the stability of both the drug and the hydrogel network. Ex vivo tests revealed the ability of pravastatin to accumulate in cornea and sclera and to penetrate through transscleral route. In vivo tests (rabbits) confirmed that, compared to eye drops and for the same dose, CLs provided significantly higher pravastatin levels in tear fluid within 1 to 7 h of wearing. Moreover, after 8 h wearing pravastatin was present in cornea, sclera, aqueous humour and vitreous humour. Strong correlations between percentages of drug released in vitro and in vivo were found. Effects of volume and proteins on release rate and Levy plots were identified.

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin.

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.

Evaluation of Hepatic Uptake of OATP1B Substrates by Short Term-Cultured Plated Human Hepatocytes: Comparison With Isolated Suspended Hepatocytes.

Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PSinf) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PSinf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (Kp,uu). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and Kp,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding.

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma.

Generating accurate in vitro data is crucial for in vitro to in vivo extrapolation and pharmacokinetic predictions. The use of human embryonic kidney (HEK) 293 cells overexpressing organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 in protein-free buffer and 100% human plasma incubations was explored for the uptake of four OATP substrates: pravastatin, rosuvastatin, repaglinide, and pitavastatin. Differences were observed for each parameter [unbound Michaelis constant (K m,u), V max, intrinsic clearance (CLint), and unbound passive diffusion Pdif,u] obtained from the buffer and plasma incubations in both cells, and the fold differences were greatest for the highly protein bound compounds. The fold change in K m,u values ranged from 1.91 to 619, and the fold change in V max values ranged from 1.22 to 97.4. As a result, in both cells, the CLint values generated in the plasma incubations were higher by 0.762- to 31.7-fold than the values generated in the protein-free buffer. The passive diffusion was also higher in the plasma incubations for all four compounds, with a fold difference range of 1.73-23.4. These shifts in the presence and absence of human plasma suggest that plasma proteins may play a role in both the active uptake and passive diffusion processes. The results also support the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters. The addition of plasma to incubations leading to higher CLint values for transporter substrates helps mitigate the underprediction commonly noted with in vitro to in vivo extrapolation. SIGNIFICANCE STATEMENT: The current investigation brings a new perspective on how to mitigate the underprediction commonly noted with in vitro to in vivo extrapolation for OATP substrates by using HEK293 cells overexpressing OATP1B1 and OATP1B3. It also supports the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.

Functional characterization for polymorphic organic anion transporting polypeptides (OATP/SLCO1B1, 1B3, 2B1) of monkeys recombinantly expressed with various OATP probes.

The hepatic uptake of clinical drugs mediated by human hepatic organic anion transporting polypeptides (OATP/SLCO) has been reported extensively. In this study, hepatic uptake by recombinantly expressed monkey OATP1B1, OATP1B3 and OATP2B1 was investigated using three human OATP1B1 and OATP1B3 substrates (pitavastatin, pravastatin and rosuvastatin) and one OATP1B3 substrate (telmisartan), as the governmental drug interaction guidelines recommend, and seven reported clinical drugs. The uptake of known human probes into recombinant OATP-expressing cells was significantly greater than that of mock cells. Consequently, pitavastatin, pravastatin and rosuvastatin were suggested to be substrates of recombinant monkey OATP1B1 and OATP1B3, and telmisartan was suggested to be a substrate of recombinant monkey OATP1B3, in a manner similar to human OATPs. In contrast, atorvastatin, bosentan, etoposide, fexofenadine, fluvastatin, glibenclamide and simeprevir were broadly transported by recombinant monkey OATP1B1, OATP1B3 and OATP2B1. Furthermore, some of the 16 non-synonymous monkey OATP1B1 variants found in 64 cynomolgus and 32 rhesus monkeys mediated up to a 1.6-fold [3 H]pitavastatin uptake (with low Michaelis constant values) in comparison with the wild type under the present conditions. Despite sequences of monkey recombinant OATPs not being totally reflective of those of human OATPs, our results collectively suggested that OATP1B1, OATP1B3 or OATP2B1 in monkeys could mediate roughly a similar hepatic uptake of various OATP probes. Recombinant monkey OATPs would be good experimental tools for in vitro hepatic uptake in cell systems.

The Presence of a Transporter-Induced Protein Binding Shift: A New Explanation for Protein-Facilitated Uptake and Improvement for In Vitro-In Vivo Extrapolation.

Accurately predicting hepatic clearance is an integral part of the drug-development process, and yet current in vitro to in vivo (IVIVE) extrapolation methods yield poor predictions, particularly for highly protein-bound transporter substrates. Explanations for error include inaccuracies in protein-binding measurements and the lack of recognition of protein-facilitated uptake, where both unbound and bound drug may be cleared, violating the principles of the widely accepted free drug theory. A new explanation for protein-facilitated uptake is proposed here, called a transporter-induced protein binding shift High-affinity binding to cell-membrane proteins may change the equilibrium of the nonspecific binding between drugs and plasma proteins, leading to greater cellular uptake and clearance than currently predicted. The uptake of two lower protein-binding organic anion transporting polypeptide substrates (pravastatin and rosuvastatin) and two higher binding substrates (atorvastatin and pitavastatin) were measured in rat hepatocytes in incubations with protein-free buffer versus 100% plasma. Decreased unbound K m values and increased intrinsic clearance values were seen in the plasma incubations for the highly bound compounds, supporting the new hypothesis and mitigating the IVIVE underprediction previously seen for highly bound transporter substrates.

Role of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin.

The expression and activity of human placental transporters during pregnancy could be altered by several factors including pathological changes associated with preeclampsia. The aims of this study were to identify the placental efflux transporters involved in the bio-disposition of pravastatin, determine the protein expression of these transporters and their encoding genes as well as the activity of pravastatin uptake in placentas obtained from patients with preeclampsia. ATP-dependent uptake of [3H]-pravastatin by trophoblast tissue apical and basal membrane vesicles exhibited sigmoidal kinetics. The curved shapes of Eadie-Hofstee plots indicate that more than one placental transporter are involved in the uptake of pravastatin. ATP-dependent uptake of [3H]-pravastatin into vesicles expressing MRP1-5, BCRP, and P-gp, as well as the results of inhibition studies suggest that BCRP and MRP1 are the major placental efflux transporters responsible for the in vitro uptake of pravastatin. Compared to placentas from healthy pregnancies, preeclamptic placentas had increased number of syncytial knots with increased expression of BCRP in their apical membrane and increased expression of MRP1 in the cytoplasm of the syncytiotrophoblast and in cytoplasm of syncytial knots. There was a concomitant increase in ABCC1 but not in ABCG2 gene expressions in preeclamptic placentas. ATP-dependent uptake of [3H]-pravastatin by vesicles prepared from apical membranes of preeclamptic placentas was similar to the uptake by vesicles prepared from placentas obtained after uncomplicated pregnancies (13.9 ±â€¯6.5 vs 14.1 ±â€¯5.8 pmol·mg protein-1 min-1). The transporter-specific changes in the expression of BCRP and MRP1 in preeclamptic placentas did not affect the efflux activity of transporters localized on the apical membrane of the syncytiotrophoblast.

[Regulation of pravastatin on long-chain fatty acid oxidative enzyme in pre-eclampsia-like mouse model].

Objective: To investigate the modulation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) expression by pravastatin in pre-eclampsia-like mouse model. Methods: C57BL/6J mice were randomly injected with N-nitro-L-arginine methyl ester (L-NAME) as pre-eclampsia-like model group (PE) or saline as normal pregnancy control group (Con) respectively, from gestational the 7th to 18th day. For each group, pravastatin (PE+Pra, Con+Pra group) or saline (PE+N, Con+N Group) was given from the 8th to 18th day of gestation, respectively. Liver and placenta of pregnant mice were collected on gestational day 18. The LCHAD protein expression and mRNA levels of liver and placenta were detected through western blot, immunohistochemistry and real-time quantitative PCR. Results: (1) The average arterial pressure of pregnant mice increased gradually from the 8th to 18th day in PE+N group, but decreased in PE+Pra group from gestational 10th day, 24 hour urinary protein levels in PE+N group [(1 494 ± 201) µg] were significantly higher than that in Con+N group [(935±128) µg, P<0.01], and also higher than that in PE+Pra group [(981±116) µg, P<0.01].(2) The results of western blot: the expression of LCHAD was significantly lower in PE+N group (liver: 0.64±0.11, placenta: 0.48±0.06) than that in Con+N group (liver: 1.06±0.10, placenta: 0.60±0.10), and lower than that in PE+Pra group (liver: 0.99±0.04, placenta: 0.60±0.08; all P<0.01).(3)The results of real-time quantitative PCR: the levels of LCHAD mRNA in liver and placenta in PE+N group (liver: 0.621±0.128, placenta: 0.646±0.129) were significantly decreased compared with Con+N group (liver: 1.007±0.130, placenta: 1.004±0.103; all P<0.01), but there was no significant difference between PE+Pra group (liver: 0.693±0.678, placenta: 0.662±0.183; P>0.05). (4) LCHAD protein was expressed widely and evenly in liver. The expression in placental cytotrophoblast and syncytial trophoblast cells located in outer layer of villous in labyrinth layer was the most. The expression of LCHAD was significantly lower in PE+N group (liver: 0.062±0.016, placenta: 0.147±0.018) than that in Con+N group (liver: 0.126±0.013, placenta: 0.183±0.024), and lower than that in PE+Pra group (liver: 0.111±0.017, placenta: 0.174±0.027; all P<0.05). Conclusion: Pravastatin could upregulate the LCHAD protein expression of liver and placenta in the pre-eclampsia-like mouse, which may be a mechanism to improve the clinical manifestations of pre-eclampsia.

A Clinical Quantitative Evaluation of Hepatobiliary Transport of [11C]Dehydropravastatin in Humans Using Positron Emission Tomography.

Various positron emission tomography (PET) probes have been developed to assess in vivo activities in humans of drug transporters, which aid in the prediction of pharmacokinetic properties of drugs and the impact of drug-drug interactions. We developed a new PET probe, sodium (3R, 5R)-3, 5-dihydroxy-7-((1S, 2S, 6S, 8S)-6-hydroxy-2-methyl-8- ((1-[11C]-(E)-2-methyl-but-2-enoyl) oxy) -1, 2, 6, 7, 8, 8a-hexahydronaphthalen-1-yl) heptanoate ([11C]DPV), and demonstrated its usefulness for the quantitative investigation of Oatps (gene symbol SLCO) and Mrp2 (gene symbol ABCC2) in rats. To further analyze the species differences and verify the pharmacokinetic parameters in humans, serial PET scanning of the abdominal region with [11C]DPV was performed in six healthy volunteers with and without an OATP1Bs and MRP2 inhibitor, rifampicin (600 mg, oral), in a crossover fashion. After intravenous injection, [11C]DPV rapidly distributed to the liver and kidney followed by secretion into the bile and urine. Rifampicin significantly reduced the liver distribution of [11C]DPV 3-fold, resulting in a 7.5-fold reduced amount of excretion into the bile and the delayed elimination of [11C]DPV from the blood circulation. The hepatic uptake clearance (CLuptake, liver) and canalicular efflux clearance (CLint, bile) of [11C]DPV (544 ± 204 and 10.2 ± 3.5 µl/min per gram liver, respectively) in humans were lower than the previously reported corresponding parameters in rats (1800 and 298 µl/min per gram liver, respectively) (Shingaki et al., 2013). Furthermore, rifampicin treatment significantly reduced CLuptake, liver and CLint, bile by 58% and 44%, respectively. These results suggest that PET imaging with [11C]DPV is an effective tool for quantitatively characterizing the OATP1Bs and MRP2 functions in the human hepatobiliary transport system.

Investigation of the Importance of Multidrug Resistance-Associated Protein 4 (Mrp4/Abcc4) in the Active Efflux of Anionic Drugs Across the Blood-Brain Barrier.

The importance of multidrug resistance-associated protein 4 (Mrp4/Abcc4) in limiting the penetration of Mrp4 substrate compounds into the central nervous system across the blood-brain barrier was investigated using Mrp4-/- mice. Significant adenosine triphosphate-dependent uptake by MRP4 was observed for ochratoxin A, pitavastatin, raltitrexed (Km = 43.7 µM), pravastatin, cyclic guanosine monophosphate, 2,4-dichlorophenoxyacetate, and urate. The defect in the Mrp4 gene did not affect the brain-to-plasma ratio (Kp,brain) of quinidine and dantrolene. Following intravenous infusion in wild-type and Mrp4-/- mice, the plasma concentrations of the tested compounds (cefazolin, cefmetazole, ciprofloxacin, cyclophosphamide, furosemide, hydrochlorothiazide, methotrexate, pitavastatin, pravastatin, and raltitrexed) were identical; however, Mrp4-/- mice showed a significantly higher (1.9- to 2.5-fold) Kp,brain than wild-type mice for methotrexate, raltitrexed, and cyclophosphamide. GF120918, a dual inhibitor of P-gp and Bcrp, significantly decreased Kp,cortex and Kp,cerebellum only in Mrp4-/- mice. Methotrexate and raltitrexed are also substrates of multispecific organic anion transporters such as Oatp1a4 and Oat3. GF120918 showed an inhibition potency against Oatp1a4, but not against Oat3. These results suggest that Mrp4 limits the penetration of methotrexate and raltitrexed into the brain across the blood-brain barrier, which is likely to be facilitated by some uptake transporters.

Comparison of Methods for Estimating Unbound Intracellular-to-Medium Concentration Ratios in Rat and Human Hepatocytes Using Statins.

It is essential to estimate concentrations of unbound drugs inside the hepatocytes to predict hepatic clearance, efficacy, and toxicity of the drugs. The present study was undertaken to compare predictability of the unbound hepatocyte-to-medium concentration ratios (Kp,uu) by two methods based on the steady-state cell-to-medium total concentration ratios at 37°C and on ice (Kp,uu,ss) and based on their initial uptake rates (Kp,uu,V0). Poorly metabolized statins were used as test drugs because of their concentrative uptake via organic anion-transporting polypeptides. Kp,uu,ss values of these statins provided less interexperimental variation than the Kp,uu,V0 values, because only data at longer time are required for Kp,uu,ss Kp,uu,V0 values for pitavastatin, rosuvastatin, and pravastatin were 1.2- to 5.1-fold Kp,uu,ss in rat hepatocytes; Kp,uu,V0 values in human hepatocytes also tended to be larger than corresponding Kp,uu,ss To explain these discrepancies, theoretical values of Kp,uu,ss and Kp,uu,V0 were compared with true Kp,uu (Kp,uu,true), considering the inside-negative membrane potential and ionization of the drugs in hepatocytes and medium. Membrane potentials were approximately -30 mV in human hepatocytes at 37°C and almost abolished on ice. Theoretical equations considering the membrane potentials indicate that Kp,uu,ss values for the statins are 0.85- to 1.2-fold Kp,uu,true, whereas Kp,uu,V0 values are 2.2- to 3.1-fold Kp,uu,true, depending on the ratio of the passive permeability of the ionized to nonionized forms. In conclusion, Kp,uu,ss values of anions are similar to Kp,uu,true when the inside-negative membrane potential is considered. This suggests that Kp,uu,ss is preferable for estimating the concentration of unbound drugs inside the hepatocytes.

Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469) from Streptomyces avermitilis.

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The Km and kcat values for compactin were 39.1 ± 8.8 µM and 1.12 ± 0.09 min-1, respectively. The kcat/Km value for compactin (0.029 min-1·µM-1) was lower than that for diclofenac (0.114 min-1·µM-1). Spectroscopic analysis showed that CYP105D7 binds to compactin with a Kd value of 17.5 ± 3.6 µM. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

Interaction of different statins with model membranes by NMR data.

Hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins reduce the amount of low-density lipoprotein (LDL) cholesterol, which is known as a well-established risk factor for atherosclerosis. Despite the fact that statins have a common pharmacologic target essential to sterol biosynthesis, their efficacy, safety, and potential non-LDL actions vary significantly for different statins. There is a hypothesis that pharmacological features of statins depend on their location in cell membrane, but to the present day there is a lack of information in literature on interactions of statins with the surface of the cell membrane in liquid media. The results of NMR experiments showed that all studied statins form intermolecular complexes with models of cell membranes (dodecylphosphocholine micelles) in water solution. Locations of pravastatin, simvastatin, fluvastatin and cerivastatin on model membranes were established by NOESY NMR data. Distinctions in their positions can explain differences in pharmacological properties of studied compounds.

Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin.

In the current work, we describe the identification and characterization of the first plasmid-encoded P450 (CYP107DY1) from a Bacillus species. The recombinant CYP107DY1 exhibits characteristic P450 absolute and reduced CO-bound difference spectra. Reconstitution with different redox systems revealed the autologous one, consisting of BmCPR and Fdx2, as the most effective one. Screening of a library of 18 pharmaceutically relevant compounds displayed activity towards mevastatin to produce pravastatin. Pravastatin is an important therapeutic drug to treat hypercholesterolemia, which was described to be produced by oxyfunctionlization of mevastatin (compactin) by members of CYP105 family. The hydroxylation at C6 of mevastatin was also suggested by docking this compound into a computer model created for CYP107DY1. Moreover, in view of the biotechnological application, CYP107DY1 as well as its redox partners (BmCPR and Fdx2) were successfully utilized to establish an E. coli based whole-cell system for an efficient biotransformation of mevastatin. The in vitro and in vivo application of the CYP07DY1 also offers the possibility for the screening of more substrates, which could open up further biotechnological usage of this enzyme.

Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors.

Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.

Quantitative determination of pravastatin and its metabolite 3α-hydroxy pravastatin in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α-hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one-step sample preparation by liquid-liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α-hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α-hydroxy pravastatin. The relative deviation of this method was <10% for intra- and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia.

Functional Characterization of Carrier-Mediated Transport of Pravastatin across the Blood-Retinal Barrier in Rats.

Systemically administered pravastatin effectively treats diabetic retinopathy without central nervous system side effects. The efflux transport mechanism of pravastatin from the brain has already been clarified. In this study, the influx of pravastatin across the blood-retinal and blood-brain barriers (BRB and BBB) and the efflux of pravastatin from the retina were investigated using rats. Pravastatin influx (blood-to-tissues) was assessed using the retinal and brain uptake index (RUI and BUI) methods, and microdialysis was performed to investigate the efflux (retina-to-blood) transport of pravastatin. The RUI and BUI values for [(3)H]pravastatin were lower than those expected based on its lipophilicity, suggesting that the influx transport across the BRB and BBB was less than the reverse-direction transport. The RUI and BUI values for [(3)H]pravastatin were significantly decreased by pravastatin, digoxin, and probenecid, indicating that pravastatin undergoes carrier-mediated influx transport in the blood-to-tissues direction across the BRB and BBB. After intravitreal injection, [(3)H]pravastatin and the bulk flow marker [(14)C]d-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [(3)H]pravastatin during the terminal phase was 1.66-fold greater than that of [(14)C]d-mannitol. Efflux transport was reduced in the retinal presence of pravastatin, digoxin, and benzylpenicillin, suggesting that pravastatin is transported via efflux transporters. In conclusion, pravastatin is transported across the BRB via uptake and efflux transporters in both the blood-to-retina and retina-to-blood directions, and the retina-to-blood transporters are dominant, based on the lower values of the RUI compared with the values expected from the lipophilicity.

[Plasma concentrations of anionic uremic toxins in hemodialysis patients and their effects on protein binding of pravastatin].

Uremic toxins (UTs) accumulate in the body of hemodialysis patients. UTs often exert unfavorable effects on patients and cause significant interactions with clinically relevant drugs. In this study, we assayed plasma concentrations of three typical anionic UTs, indoxyl sulfate (IS), 3-indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), in 20 hemodialysis patients and 5 healthy volunteers. Moreover, the effects of these anionic UTs on the binding of pravastatin to human serum albumin (HSA) were also evaluated. CMPF concentrations in the plasma of patients were unchanged before and after dialysis (63.0 ± 6.3 µM and 65.1 ± 6.7 µM, respectively), and these values were about 5-fold greater compared with those in healthy volunteers. Although dialysis decreased the plasma IS concentration from 157.9 ± 19.9 µM to 103.8 ± 13.3 µM, the value after hemodialysis was still ca. 27-fold greater than that in healthy volunteers. IA concentrations before and after hemodialysis were almost identical to those in healthy volunteers. There were no significant differences in the plasma concentrations of the three anionic UTs between male and female patients. The magnitude of protein binding was in the order CMPF>IS>IA, indicating that hemodialysis clearance of these anionic UTs was dependent on their protein binding capacities. The ability of IS to reduce pravastatin binding to HSA was much greater than that of CMPF. The present results suggest that statins bind to site II on HSA, and that their binding is modulated by IS when elevated in hemodialysis patients.