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Ganoderic acid A (GAA) is a lanostane-type triterpenoid, isolated from medicinal fungus Ganoderma lucidum, and possesses multiple bioactivities. In the present study, GAA was sequentially biotransformed by 2 recently discovered Bacillus glycosyltransferases (GT), BtGT_16345 and BsGT110, and the final product was purified and identified as a new compound, GAA-15,26-O-β-diglucoside, which showed 1024-fold aqueous solubility than GAA., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

BACKGROUND Spinal cord injury (SCI) has a high incidence and causes serious harm. Lipoxin A4 (LXA4) receptor agonist BML-111 was reported to regulate inflammation and oxidative stress. The goal of this study was to assess whether BML-111 could protect against SCI by suppressing inflammation and oxidative stress. MATERIAL AND METHODS We developed a rat SCI model, then BML-111 was intraperitoneally injected into SCI rats to observe the BML-111 function. The pathological changes of SCI were observed with hematoxylin and eosin (HE) staining. Motor function of rats were assessed by the modified Tarlov's scale. ELISA was used to assess the changes in levels of TNF-alpha, IL-1ß, and IL-6. Western blot analysis was performed to assess the expressions of TNF-alpha, IL-1ß, IL-6, Bcl2, Bax, and cleaved caspase3 in spinal cord tissue. TOS and TAS in rat serum were detected by xylenol orange method and ABTS method, respectively. The apoptotic cells in spinal cord tissue were observed with TUNEL assay. RESULTS The results indicated that BML-111 effectively improved the SCI and motor function of rats. BML-111 treatment decreased the levels of TNF-alpha, IL-1ß, and IL-6 in serum and spinal cord tissue, as well as decreasing the levels of TOS and TAS and cell apoptosis. CONCLUSIONS BML-111 alleviated inflammation and oxidative stress in SCI rats.

Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15- O -β-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis . Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes ( FQZ25_16345 , FQZ25_19840 , and FQZ25_19010 ) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010 , were successfully cloned and expressed in a soluble form in Escherichia coli , and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15- O -β-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (k cat /K M ) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15- O -glycosylation of GAA and it was also regioselective toward triterpenoid substrates.

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum . Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound ( 1 ), GAA-15- O -β-glucoside, and compound ( 2 ). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15- O -β-glucoside, the chemical structure of compound ( 2 ) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound ( 2 ) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound ( 2 ) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26- O -β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (k cat ) of BsGT110 toward GAA at pH 6 (k cat = 11.2 min -1 ) was 3-fold higher than that at pH 7 (k cat = 3.8 min -1 ), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

Bacillus subtilis ATCC (American type culture collection) 6633 was found to biotransform ganoderic acid A (GAA), which is a major lanostane triterpenoid from the medicinal fungus Ganoderma lucidum . Five glycosyltransferase family 1 (GT1) genes of this bacterium, including two uridine diphosphate-dependent glycosyltransferase (UGT) genes, BsUGT398 and BsUGT489 , were cloned and overexpressed in Escherichia coli . Ultra-performance liquid chromatography confirmed the two purified UGT proteins biotransform ganoderic acid A into a metabolite, while the other three purified GT1 proteins cannot biotransform GAA. The optimal enzyme activities of BsUGT398 and BsUGT489 were at pH 8.0 with 10 mM of magnesium or calcium ion. In addition, no candidates showed biotransformation activity toward antcin K, which is a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea . One biotransformed metabolite from each BsUGT enzyme was then isolated with preparative high-performance liquid chromatography. The isolated metabolite from each BsUGT was identified as ganoderic acid A-15- O -β-glucoside by mass and nuclear magnetic resonance spectroscopy. The two BsUGTs in the present study are the first identified enzymes that catalyze the 15- O -glycosylation of triterpenoids.

Docosapentaenoic acid/docosahexaenoic acid ratio (DPA/DHA ratio) in Schizochytrium was relatively stable. But ideally the ratio of DPA/DHA will vary according to the desired end use. This study reports several ways of modulating the DPA/DHA ratio. Incubation times changed the DPA/DHA ratio, and changes in this ratio were associated with the variations in the saturated fatty acid (SFAs) content. Propionic acid sharply increased the SFAs content in lipids, dramatically decreased the even-chain SFAs content, and reduced the DPA/DHA ratio. Pentanoic acid (C5:0) and heptanoic acid (C7:0) had similar effects as propionic acid, whereas butyric acid (C4:0), hexanoic acid (C6:0), and octanoic acid (C8:0) did not change the fatty acid profile and the DPA/DHA ratio. Transcription analyses show that β-oxidation might be responsible for this phenomenon. Iodoacetamide upregulated polyunsaturated fatty acid (PUFA) synthase genes, reduced the DHA content, and improved the DPA content, causing the DPA/DHA ratio to increase. These results present new insights into the regulation of the DPA/DHA ratio.

A rather new approach in the treatment of long-chain fatty acid oxidation disorders is represented by triheptanoin, a triglyceride with three medium-odd-chain heptanoic acids (C7), due to its anaplerotic potential. We here investigate the effects of a 1-year triheptanoin-based diet on the clinical phenotype of very long-chain-acyl-CoA-dehydrogenase-deficient (VLCAD -/- ) mice. The cardiac function was assessed in VLCAD -/- mice by in vivo MRI. Metabolic adaptations were identified by the expression of genes regulating energy metabolism and anaplerotic processes using real-time PCR, and the results were correlated with the measurement of the glycolytic enzymes pyruvate dehydrogenase and pyruvate kinase. Finally, the intrahepatic lipid accumulation and oxidative stress in response to the long-term triheptanoin diet were assessed. Triheptanoin was not able to prevent the development of systolic dysfunction in VLCAD -/- mice despite an upregulation of cardiac glucose oxidation. Strikingly, the anaplerotic effects of triheptanoin were restricted to the liver. Despite this, the hepatic lipic content was increased upon triheptanoin supplementation. Our data demonstrate that the concept of anaplerosis does not apply to all tissues equally., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-sn-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 μM and 0.18 μM, respectively (n = 3, and p = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion and tube formation by human umbilical vein endothelial cells. OHdiA monoamide epitopes are only slightly less potent than CEP epitopes that contribute to the pathological angiogenesis of age-related macular degeneration and tumor growth.

Background: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS)., Results: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification., Conclusion: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Background: Atorvastatin is a potent inhibitor of the mevalonate pathway and widely used as a hypolipidemic drug. Some epidemiological studies and animal experiments indicate that the long-term use of atorvastatin and structurally related drugs might be associated with a reduced risk of developing hepatocellular carcinoma (HCC), the most common hepatocellular malignancy in humans. However, the potential of atorvastatin to inhibit HCC formation is controversially discussed., Methods: Hepatocellular tumors were chemically induced by treatment of C3H/He mice with 10 μg/g body weight N-nitrosodiethylamine and the ability of atorvastatin to interfere with tumor formation was investigated by treatment of mice with 0.1% atorvastatin in the diet for 6 months. Tumor size and tumor multiplicity were analyzed, as were tissue levels of cholesterol and atorvastatin., Results: Atorvastatin treatment efficiently reduced serum cholesterol levels. However, the growth of tumors driven by activated MAPK (mitogen-activated protein kinase) signaling was not attenuated by the presence of the drug, as evidenced by a lack of reduction of tumor volume or tumor multiplicity by atorvastatin. Levels of the atorvastatin uptake transporters Oatp1a4 and Oatp1b2 were down-regulated at the mRNA and protein levels in chemically induced mouse liver tumors, but without striking effects on atorvastatin concentrations in the tumor tissue., Conclusion: In summary, the present data provide substantial evidence that atorvastatin does not beneficially influence tumor growth in mouse liver and thereby challenge the hypothesis that statin use might protect against hepatocellular cancer.

Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CLupt) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CLupt in 12 human liver samples. The impact of inhibition on atorvastatin CLupt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7- to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 ≈ NTCP ≈ OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CLupt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

Atorvastatin is eliminated by CYP3A4 which follows carrier-mediated uptake into hepatocytes by OATP1B1, OATP1B3, and OATP2B1. Multiple clinical studies demonstrated that OATP inhibition by rifampin had a greater impact on atorvastatin systemic concentration than itraconazole-mediated CYP3A4 inhibition. If it is assumed that the blood and hepatocyte compartments are differentiated by the concentration gradient that is established by OATPs, and if the rate of uptake into the hepatocyte is rate-determining to the elimination of atorvastatin from the body, then it is hypothesized that blood concentrations may not necessarily reflect liver concentrations. In wild-type mice, rifampin had a greater effect on systemic exposure of atorvastatin than ketoconazole, as the blood area under the blood concentration-time curve increased 7- and 2-fold, respectively. In contrast, liver concentrations were affected more by ketoconazole than by rifampin, as liver levels increased 21- and 4-fold, respectively. Similarly, in Cyp3a knockout animals, 39-fold increases in liver concentrations were observed despite insignificant changes in the blood area under the blood concentration-time curve. Interestingly, blood and liver levels in Oatp1a/b knockout animals were similar to wild types, suggesting that Oatp1a/b knockout may be necessary but not sufficient to completely describe atorvastatin uptake in mice. Data presented in this work indicate that there is a substantial drug interaction when blocking atorvastatin metabolism, but the effects of this interaction are predominantly manifested in the liver and may not be captured when monitoring changes in the systemic circulation. Consequently, there may be a disconnect when trying to relate blood exposure to instances of hepatotoxicity because a pharmacokinetic-toxicity relationship may not be obvious from blood concentrations.

In this study, a Soluplus-coated colloidal silica nanomatrix (SCCSN) formulation for the entrapment of poorly water-soluble drugs was devised. The maximum supersaturation of the drug-loaded nanomatrix was higher than that of a physical mixture as indicated by the results of in vitro kinetic solubility studies. For atorvastatin calcium, dutasteride, and sorafenib tosylate, there were 2.8-, 326-, and 46.4-fold increases in solubility, respectively. For dutasteride, a promising 4.7-fold increase for in vivo oral drug absorption in the entrapped nanomatrix was observed as compared to the free physical mixture, supported by statistical significance testing of pharmacokinetic parameters.

The Biopharmaceutics Drug Disposition Classification System (BDDCS) predicts intestinal transporter effects to be clinically insignificant following oral dosing for highly soluble and highly permeable/metabolized drugs (class 1 drugs). We investigated the effect of inhibiting P-glycoprotein (P-gp) on the in vitro rat intestinal permeability (Papp) and metabolism of the class 1 drug verapamil. Jejunal segments from Sprague-Dawley rats fasted overnight were mounted in Ussing chambers filled with 10 mL of Krebs-Ringer buffer (KRB). For P-gp inhibition studies, GG918 0.5 μM was added to the KRB solution. The experiment started by the addition of verapamil (1 or 10 μM) to either apical or basolateral sides. Samples from verapamil donor and receiver compartments were collected at 30 s and 0.166, 0.5, 1, 1.83 and 3 h after the start of the experiment. Analysis of verapamil and its major metabolite, norverapamil, in the samples and intracellularly at 3 h was performed by HPLC. The same experiment was repeated with norverapamil 10 μM (verapamil metabolite), digoxin 100 nM (positive control for P-gp activity) and atorvastatin 1 and 10 μM (example of a class 2 drug). For 1 μM verapamil, efflux ratio (B to A Papp/A to B Papp) was 4.6 and markedly decreased by GG918 (efflux ratio = 1.1). For 10 μM verapamil efflux ratio was 4.1 (control) vs 1.8 (GG918), comparable to the change seen for digoxin 100 nM with an efflux ratio of 3.6 (control) vs 1.6 (with GG918) and atorvastatin (efflux ratio of 5.2 and 3.0 for atorvastatin 1.0 and 10 μM, respectively, changed to 1.0 and 0.65 with GG918). The changes observed in the norverapamil 10 μM experiment were also significant, where efflux ratio decreased from 13.5 (control) to 1.5 (GG918). The extraction ratio (ER) of 10 μM verapamil to norverapamil decreased from 0.41 after an apical dose to 0.21 after a basolateral dose, but was unaffected by the incubation with GG918. The results suggest that P-gp inhibition has an effect on class 1 drug verapamil and class 2 drug atorvastatin Papp in the rat intestine. Moreover, a stronger P-gp effect on the Papp of the more polar norverapamil metabolite was observed. Papp changes caused by the P-gp inhibitor GG918 do not affect the extent of verapamil metabolism.

Ultrahigh-performance liquid chromatography coupled with high-mass-accuracy tandem mass spectrometry (UHPLC-MS-MS) has been used for elucidation of the structures of oxidation products of atorvastatin (AT), one of the most popular commercially available drugs. The purpose of the study was identification of AT metabolites in rat hepatocytes and comparison with electrochemically generated oxidation products. AT was incubated with rat hepatocytes for 24 h. Electrochemical oxidation of AT was performed by use of a three-electrode off-line system with a glassy carbon working electrode. Three supporting electrolytes (0.1 mol L(-1) H2SO4, 0.1 mol L(-1) HCl, and 0.1 mol L(-1) NaCl) were tested, and dependence on pH was also investigated. AT undergoes oxidation by a single irreversible process at approximately +1.0 V vs. Ag/AgCl electrode. The results obtained revealed a simple and relatively fast way of determining the type of oxidation and its position, on the basis of characteristic neutral losses (NLs) and fragment ions. Unfortunately, different products were obtained by electrochemical oxidation and biotransformation of AT. High-mass-accuracy measurement combined with different UHPLC-MS-MS scans, for example reconstructed ion-current chromatograms, constant neutral loss chromatograms, or exact mass filtering, enable rapid identification of drug-related compounds. β-Oxidation, aromatic hydroxylation of the phenylaminocarbonyl group, sulfation, AT lactone and glycol formation were observed in rat biotransformation samples. In contrast, a variety of oxidation reactions on the conjugated skeleton of isopropyl substituent of AT were identified as products of electrolysis.

We tested the hypothesis that atorvastatin active metabolite (ATM), on the basis of its distinct structural features and potent antioxidant activity, preferentially inhibits lipid oxidation in human small dense low-density lipoprotein (sdLDL) and other small lipid vesicles. LDL, sdLDL, and various subfractions were isolated from human plasma by sequential ultracentrifugation, treated with ATM, atorvastatin, pravastatin, rosuvastatin, or simvastatin and were subjected to copper-induced oxidation. Lipid oxidation was measured spectrophotometrically as a function of thiobarbituric acid reactive substances formation. Similar analyses were performed in reconstituted lipid vesicles enriched in polyunsaturated fatty acids and prepared at various sizes. ATM was found to inhibit sdLDL oxidation in a dose-dependent manner. The antioxidant effects of ATM in sdLDL were 1.5 and 4.7 times greater (P < 0.001) than those observed in large buoyant LDL and very low-density lipoprotein subfractions, respectively. ATM had similar dose- and size-dependent effects in reconstituted lipid vesicles. None of these effects were reproduced by atorvastatin (parent) or any of the other statins examined in this study. These data suggest that ATM interacts with sdLDL in a specific manner that also confers preferential resistance to oxidative stress. Such interactions may reduce sdLDL atherogenicity and improve clinical outcomes in patients with cardiovascular disease.

Background: The role of tumor necrosis factor-α (TNF-α), Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and mitochondrial Permeability Transition Pore in atorvastatin-induced cardioprotection were examined in human myocardium, in vitro., Methods: Isometric force of contraction of human right atrial trabeculae was recorded during 30-min hypoxia and 60-min reoxygenation (control) and in the presence of atorvastatin (0.1 µM, 1 µM, 10 µM). In early reoxygenation, the TNF-α inhibitor, AG490 (inhibitor of JAK/STAT), or atractyloside (mitochondrial Permeability Transition Pore opener), were administered. Cyclosporine A (inhibitor of mitochondrial Permeability Transition Pore opening) was administered during the first minute of reoxygenation alone or in presence of atorvastatin and TNF-α inhibitor or AG490. The force of contraction (percentage of baseline) at the end of reoxygenation period was compared (mean ± SD; n = 6 in each group). Protein expression of JAK/STAT pathway was measured using Western immunoblotting., Results: Atorvastatin 0.1 µM (70 ± 9%), 1 µM (85 ± 5%), 10 µM (89 ± 5%), and Cyclosporine A (87 ± 10%) improved the recovery of force of contraction at the end of reoxygenation, as compared with control (50 ± 3%). Atorvastatin 1 µM (4.64 ± 2.90 ng · ml(-1) · g(-1) of tissue) decreased the release of troponin Ic after hypoxia-reoxygenation (control: 26.34 ± 19.30 ng · ml(-1) · g(-1); P < 0.001). The enhanced recovery of force of contraction after atorvastatin administration was abolished by TNF-α inhibitor (53 ± 8%), AG490 (56 ± 7%), atractyloside (48 ± 8%). Cyclosporine A restored the atorvastatin-induced cardioprotection abolished by TNF-α inhibitor (87 ± 6%) and AG490 (83 ± 9%). Atorvastatin significantly increased the phosphorylation of JAK-2 and STAT-3, TNF-α inhibitor abolished the enhanced phosphorylation of JAK-2 and STAT-3 by atorvastatin., Conclusions: Atorvastatin-induced cardioprotection involved the inhibition of the mitochondrial Permeability Transition Pore opening via the activation of TNF-α and the JAK/STAT pathway in early reoxygenation.

1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells "as fresh" for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation. 2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix. 3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7.

The population pharmacokinetics (PPK) of atorvastatin and its principal active metabolite, o-hydroxyatorvastatin, were described in 6-17 years old pediatric hypercholesterolemia patients with a 2-compartment model for both parent and metabolite. Informative prior distributions on selected parameters, based on adult data, were required to stabilize the model and were implemented using a Bayesian penalty term on the likelihood function in the nonlinear mixed effects model (NONMEM VI with PRIOR). Concentrations below the limit of quantitation were treated as censored data using a conditional likelihood function. Atorvastatin apparent oral clearance (CL/F) was described as a function of body weight using an allometric equation. Based on the final model, the typical CL/F estimates for a Tanner Stage 1 patient (35 kg weight) and Tanner Stage ≥2 (50 kg weight), would be 553 and 543 L/hour, respectively. When scaled allometrically, CL/F was similar to values reported for adults. Variability in atorvastatin PK was primarily affected by weight., (© The Author(s) 2013.)

1. In this manuscript we describe a non-radioactive, high-throughput method to evaluate hepatic uptake using cryopreserved hepatocytes. We have validated the uptake of pravastatin with different amounts of hepatocytes and the impact of the oil layer used in separation. The time- and concentration-dependent uptake profiles of several anionic and cationic charged drugs were evaluated. The results with our method compare favourably with the literature for pravastatin, atorvastatin and estrone 3-sulfate. 2. Two approaches for kinetic determination (temperature difference and fitting the linear and non-saturable passive diffusion rate in the equation, i.e. V = (V(max) × S)/(K(m) + S) + P(dif) × S) have been evaluated. Kinetic studies indicate that the different approaches for determining passive diffusion can affect K(m) and V(max), but not the clearance of active uptake (V(max)/K(m)). 3. Using pravastatin as a probe substrate, species differences were observed in the organic anion-transporting polypeptide (OATP) 1B1 and 1B3 activities. Plasma protein significantly reduced the uptake of atorvastatin, but not pravastatin. 4. Our data suggests that evaluation of the role of active uptake in hepatic clearance in humans should consider the relative ratio of active uptake to passive diffusion, species differences and plasma protein binding when applying in vitro uptake data.

This study provides data on the relationship between flavour deterioration and the volatile compound profile of semi-ripened pork salami kept under retail conditions for up to 150 days. The flavour of salami deteriorated for 120 days, resulting in rancidity and a loss of acceptability. TBARS increased from 0.16 to 0.57 MDA/kg. The flavour changes during the shelf life of salami were monitored from changes in the volatile profile. The retailing time influenced (p<0.05) the level of 27 of the 30 headspace volatiles determined by SPME-GC/MS. Flavour deterioration was associated with the loss and/or degradation of volatiles resulting from spices and microbial activities, and the formation of volatiles from lipid oxidation. The levels of 2-heptenal and methyl esters of heptanoic, pentanoic and hexanoic acids were the best discriminators of storage time, and therefore seem to be promising as marker compounds of flavour deterioration and acceptability., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Statins are indicated for prevention of atherosclerotic cardiovascular disease. Metabolism of certain statins involves the cytochrome P450 3A (CYP3A) enzymes, and CYP3A4*22 significantly influences the dose needed for achieving optimal lipid control for atorvastatin, simvastatin, and lovastatin. CYP3A4/5 combined genotype approaches have proved useful in some studies involving CYP3A substrates. We intend to compare a combined genotype analysis to our previously reported single gene CYP3A4 analysis., Methods: A total of 235 patients receiving stable statin doses were genotyped and grouped by CYP3A4/5 status., Results: The number and demographic composition of the patients categorized into the combined genotype groups were consistent with those reported for other cohorts. Dose requirement was significantly associated with the ordered combined-genotype grouping; median daily doses were nearly 40% greater for CYP3A4/5 intermediate metabolizers compared with poor metabolizers, and median daily doses were nearly double for extensive metabolizers compared with poor metabolizers. The combined-genotype approach, however, did not improve the genotype-dosage correlation p-values when compared with the previously-reported analysis; values changed from 0.129 to 0.166, 0.036 to 0.185, and 0.014 to 0.044 for atorvastatin, simvastatin, and the combined statin analysis, respectively., Conclusions: The previously-reported single-gene approach was superior for predicting statin dose requirement in this cohort.

Ganoderic acid A and α- and β-D-glucan content were compared among morphologically different basidiocarps of the medicinal mushroom Ganoderma lucidum. Ginkgo leaf-shaped basidiocarps gradually hardened from the base to the pileus and accumulated a higher amount of bioactive components than normal (kidney-shaped) and antler/deer horn-shaped basidiocarps. In the normal G. lucidum stipe, the outer context contained the highest amount of α- and β-D-glucan (approximately 55%) and the highest amount of ganoderic acid A (approximately 0.3%). Ginkgo leaf-shaped G. lucidum had a large area of outer layer and stout outer context, which contributed to their high α- and β-D-glucan and ganoderic acid A content.

Case Description: A 75-year-old man developed rhabdomyolysis and acute renal failure during atorvastatin therapy. All medications were discontinued and the patient was treated with intermittent hemodialysis throughout the course of hospitalization. After four weeks, patient's kidney function tests and serum myoglobin levels decreased to normal values and muscle weakness gradually disappeared. Genotyping results showed that the patient had a single-nucleotide polymorphism within genes encoding the organic anion-transporting polypeptide 1B1 and ATP binding cassette sub-family B member 1, which predisposed him for statin-induced myopathy. He was also a poor metabolizer of cytochrome P450 2C19. Concomitant therapy with pantoprazole could have resulted in the inhibition of cytochrome P450 3A4-mediated metabolism of atorvastatin and contributed to the development of rhabdomyolysis., Conclusion: The case illustrates the clinical relevance and relationship between pharmacogenetic and pharmacokinetic factors in the development of statin-induced myopathy.

Previously, the steroid hormone progesterone has been demonstrated to stimulate OATP2B1-mediated transport of estrone-3-sulphate (E(1)S), dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PS), which may influence the uptake of precursor molecules for steroid hormone synthesis. However, it is unclear whether OATP2B1 drug substrates like atorvastatin or glibenclamide are also affected by this phenomenon. In addition, it has not been studied so far if this stimulatory effect is specific for OATP2B1. To address these questions, we examined the influence of progesterone on OATP2B1-mediated atorvastatin and glibenclamide uptake and studied the impact of steroid hormones on the transport activity of OATP1A2, OATP1B1 and OATP1B3. Comparison of the substrate spectrum of the investigated OATPs revealed that DHEAS and atorvastatin are substrates of all transporters, while E(1)S was only significantly transported by OATP1A2, OATP2B1 and OATP1B1. Glibenclamide uptake was limited to OATP1A2, OATP1B1 and OATP2'B1. Subsequent interaction studies indicated that progesterone only increases OATP2B1-mediated E(1)S and DHEAS transport, whereas uptake of BSP, atorvastatin and glibenclamide was either inhibited or not affected. Moreover, the steroid hormone effect was specific for OATP2B1; neither OATP1B1, OATP1B3 nor OATP1A2 function was stimulated in the presence of progesterone. Similar to progesterone, the glucocorticoide dexamethasone stimulated OATP2B1-mediated transport of E(1)S and DHEAS (EC(50) for E(1)S: 10.2 ± 5.6 μM and 17.9 ± 15.4 μM for DHEAS). In conclusion, our data demonstrate that among the tested compounds the stimulatory effect of progesterone is specific for OATP2B1 and restricted to sulphated steroids like E(1)S and DHEAS while the OATP-mediated drug transport is not enhanced., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The purpose of this study was to develop a new therapeutic approach for atorvastatin (ATV) adopting nanostructured polymeric micelles for its controlled delivery to the cancer cells. Amphiphilic block copolymers of stearyl chitosan (SC) and sulfated stearyl chitosan (S-SC) that could self assemble to form polymeric micelles with different degree of substitution (DS) were synthesized and characterized. The synthesized chitosan derivatives were able to self assemble and form micelles encapsulating ATV with critical micellar concentrations ranging from 6.9 to 21μg/ml, drug-loading ranging from 40% to 84.1% and encapsulation efficiency ranging from 10.4% to 35%. ATV caused a significant decrease in particle size and zeta potential of both SC and S-SC micelles. Micelles encapsulating ATV exhibited a sustained release and more cytotoxic activity against MCF 7 and HCT 116 cell lines than ATV alone. The 50% cellular growth inhibition (IC50%) of the drug decreased from 10.4 to 3.7 in case of MCF 7 and from 9.4 to 3.4 in case of HCT 116 after its loading in micelles. These results indicate that SC ATV polymeric micelles can be considered as a promising system for site specific controlled delivery of ATV to tumor cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This research investigates the environmental behavior of two widely prescribed cholesterol-lowering statin drugs that are expected to be present at significant concentrations in wastewater influents, namely: atorvastatin and simvastatin. Batch biodegradation experiments suggest that both statins are well degraded during secondary treatment, and removal rates exhibit a substrate-enhancement model reflecting elements of both first-order behavior and cometabolism. Resulting biodegradation parameters are used in conjunction with literature sorption parameters to construct a mass-balance model of statin concentrations during conventional treatment. Model results exhibit excellent accuracy compared to measurements from a medium-sized WWTP in the Southeastern USA. Influent concentrations of 1.56 μg L(-1) and 1.23 μg L(-1) were measured for atorvastatin and simvastatin. Results also suggest that 85-90% of each drug is removed during conventional treatment, with sorption accounting for less than 10% of overall removal. Expected effluent concentrations are orders of magnitude less than previously reported ecotoxicity thresholds for both drugs. Overall, results suggest statin active ingredients do not pose a significant environmental threat. It is recommended that future work characterize the fate of statin metabolites and that the same mass-balance modeling approach be used to assess other highly-prescribed pharmaceutical drugs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Interindividual variability in cytochrome P450 3A4 (CYP3A4) is believed to be largely heritable; however, predictive genetic factors have remained scarce. Using a candidate-gene approach in a human liver bank, we identified single-nucleotide polymorphisms (SNPs) in the Ah-receptor nuclear translocator (ARNT), glucocorticoid receptor (GR), progesterone receptor membrane component 2 (PGRMC2), and peroxisome proliferator-activated receptor-α (PPARA) that are associated with CYP3A4 phenotype. Validation in atorvastatin-treated volunteers confirmed a decrease in atorvastatin-2-hydroxylation in carriers of PPARA SNP rs4253728. Homozygous carriers expressed significantly less PPAR-α protein in the liver. Moreover, shRNA-mediated PPARA gene knockdown in primary human hepatocytes decreased expression levels of the PPAR-α target ACOX1 and of CYP3A4 by more than 50%. In conclusion, this study identified novel genetic determinants of CYP3A4 that, together with nongenetic factors, explained 52, 55, and 33% of hepatic CYP3A4 mRNA, protein, and atorvastatin-2-hydroxylase activity, respectively. These findings have implications for variability in response to drug substrates of CYP3A4.

Background and Purpose: Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR)., Experimental Approach: Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation., Key Results: All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors., Conclusions and Implications: Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Objective: Interactions between coadministered drugs may unfavorably affect pharmacokinetics. This study evaluated whether fimasartan, an angiotensin receptor II antagonist, affected the pharmacokinetics of atorvastatin., Methods: A randomized, open-label, 2-period, 2-sequence, crossover, multiple-dosing study was conducted with 24 healthy male volunteers. Twelve subjects received 80-mg atorvastatin once daily for 7 days; later, they received 80-mg atorvastatin with 240-mg fimasartan for 7 days. Twelve other subjects received the same drugs in the opposite sequence. Blood samples were collected scheduled intervals for 24 hours after the last dosing to determine plasma concentrations of atorvastatin acid, atorvastatin lactone, 2-hydroxy atorvastatin acid, and 2-hydroxy atorvastatin lactone., Results: Compared with atorvastatin alone, coadministration of fimasartan and atorvastatin increased the atorvastatin acid mean (95% confidence interval) maximum concentration (Cmax,ss) by 1.89-fold (1.49-2.39) and the area under the concentration curve (AUCτ,ss) by 1.19-fold (0.96-1.48). Fimasartan also increased the mean 2-hydroxy atorvastatin acid Cmax,ss and AUCτ,ss by 2.45-fold (1.80-3.35) and 1.42-fold (1.09-1.85), respectively. The Cmax,ss and AUCτ,ss of the lactone forms of atorvastatin showed smaller changes than those observed for the acidic forms., Conclusion: We showed that fimasartan raised plasma atorvastatin concentrations. In vitro tests suggested that this effect may have been mediated by fimasartan inhibition of organic anion-transporting polypeptide 1B1.

Telaprevir is a hepatitis C virus protease inhibitor that is both a substrate and an inhibitor of CYP3A. Amlodipine and atorvastatin are both substrates of CYP3A and are among the drugs most frequently used by patients with hepatitis C. This study was conducted to examine the effect of telaprevir on atorvastatin and amlodipine pharmacokinetics (PK). This was an open-label, single sequence, nonrandomized study involving 21 healthy male and female volunteers. A coformulation of 5 mg amlodipine and 20 mg atorvastatin was administered on day 1. Telaprevir was taken with food as a 750-mg dose every 8 h from day 11 until day 26, and a single dose of the amlodipine-atorvastatin combination was readministered on day 17. Plasma samples were collected for determination of the PK of telaprevir, amlodipine, atorvastatin, ortho-hydroxy atorvastatin, and para-hydroxy atorvastatin. When administration with telaprevir was compared with administration without telaprevir, the least-square mean ratios (90% confidence limits) for amlodipine were 1.27 (1.21, 1.33) for the maximum drug concentration in serum (C(max)) and 2.79 (2.58, 3.01) for the area under the concentration-time curve from 0 h to infinity (AUC(0-∞)); for atorvastatin, they were 10.6 (8.74, 12.9) for the C(max) and 7.88 (6.84, 9.07) for the AUC(0-∞). Telaprevir significantly increased exposure to amlodipine and atorvastatin, consistent with the inhibitory effect of telaprevir on the CYP3A-mediated metabolism of these agents.

Clearance of atorvastatin occurs through hepatic uptake by organic anion transporting polypeptides (OATPs) and subsequent metabolism by cytochrome P450 (CYP) 3A4. To demonstrate the relative importance of OATPs and CYP3A4 in the hepatic elimination of atorvastatin in vivo, a clinical cassette microdose study was performed. A cocktail consisting of a microdose of atorvastatin along with probe substrates for OATPs (pravastatin) and CYP3A4 (midazolam) was orally administered to eight healthy volunteers. The pharmacokinetics of this cocktail was observed at baseline, after an oral dose of 600 mg rifampicin (an inhibitor of OATPs), and after an intravenous dose of 200 mg itraconazole (a CYP3A4 inhibitor). Rifampicin increased the pravastatin dose-normalized area under the plasma concentration-time curve (AUC) (4.6-fold), and itraconazole significantly increased the midazolam dose-normalized AUC (1.7-fold). The atorvastatin dose-normalized AUC increased 12-fold when coadministered with rifampicin but did not change when coadministered with itraconazole. These results indicate that hepatic uptake via OATPs makes the dominant contribution to the hepatic elimination of atorvastatin at a subtherapeutic microdose.

Background: Myopathic complaints are common in the general population and are more frequent with increasing age. When myopathic symptoms arise in a patient treated with a HMG-CoA reductase inhibitor (statin), it is always a question of whether the symptoms are due to statin-induced myopathy (SIM) or not (non-SIM). Diagnosis of SIM is not as straightforward as previously thought, because the most commonly used biomarker, serum creatine kinase, shows low specificity and selectivity, except in serious cases of rhabdomyolysis. There is a definite need for a novel biomarker for SIM., Objective: Based on a previous study reporting an altered metabolic profile with increased systemic exposure to the suspected muscle-toxic metabolite atorvastatin lactone in patients with SIM compared with healthy controls, this study aimed to explore the use of atorvastatin metabolite measurements to diagnose muscular complaints during statin treatment as being either SIM or non-SIM. PATIENTS, SETTING, AND STUDY DESIGN: Fifty-three patients with self-reported myopathic symptoms during atorvastatin treatment were recruited from our outpatient clinic. The symptoms were clinically evaluated as being SIM or non-SIM, on the basis of atorvastatin re-challenge testing. Atorvastatin and its metabolites were measured at steady state in all patients and compared with the clinical evaluation to see if this could predict the outcome and hence be suitable as a diagnostic tool for SIM., Main Outcome Measure: This was an exploratory study to investigate the proportion of patients correctly diagnosed by different metabolite cut-off ratios., Results: With a cut-off ratio set at 1.1 for the atorvastatin lactone to atorvastatin acid ratio, 15 of 28 SIM patients (sensitivity of 54%) and 20 of 24 non-SIM patients (specificity of 83%) were correctly diagnosed. This corresponds to a positive predictive value of 79% and a negative predictive value of 61% (p = 0.006)., Conclusion: The present study confirms an altered metabolic pattern of atorvastatin in patients with SIM and substantiates a central role of the lactone forms of statins in future investigations of statin myotoxicity. The atorvastatin lactone to acid ratio seems to be a valuable supportive diagnostic tool with high specificity and moderate sensitivity, adding to ordinary clinical evaluations when diagnosing SIM.

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 μM, 0.53 ± 0.2 μM and 2.2 ± 0.4 μM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.

A selective, sensitive, and fast high performance liquid chromatography (HPLC) method with mass spectrometric (MS) detection mode has been developed and validated completely in human plasma. Atorvastatin (ATO), p-hydroxy atorvastatin (p-HATO), o-hydroxy atorvastatin (o-HATO) and internal standard (IS) are extracted from human plasma via solid phase extraction (SPE) technique. After elution, the solution is evaporated, then reconstituted with 250 µL of Mobile Phase and analyzed using HPLC/MS/MS system. An isocratic mode is used to separate interference peaks using a Symmetry C-18, 75 × 4.6 mm ID, 3.5 µ, column. The m/z of ATO, o-HATO and p-HATO are 559.2/440.2, 575.3/440.4 and 575.0/440.4 respectively. Linearity ranges are 0.05 to 252.92 ng/mL for ATO, p-HATO and o-HATO respectively. Calibration functions, lower limit of quantitation (LLOQ), stability, intra- and inter-day reproducibility, accuracy, and recovery are estimated. This method is free from matrix effects and any abnormal ionization. This method was successfully applied to a single dose 80 mg tablet bioequivalence (BE) study of Atorvastatin. Copyright © 2011 John Wiley & Sons, Ltd., (Copyright © 2011 John Wiley & Sons, Ltd.)

Atorvastatin belongs to the group of lipid-lowering drugs known as statins. They significantly reduce the levels of total cholesterol, low-density cholesterol and plasma triglycerides therefore they are widely used in the treatment of hypercholesterolemia. Recently developed methods for the determination of atorvastatin and its metabolites in plasma used SPE (solid phase extraction) or LLE (liquid-liquid extraction) as the sample preparation step. However, both procedures are quite time-consuming and need relatively high volume of solvent/sample, which is impractical for the routine analyses of many biological samples. The aim of this work was to develop and validate more suitable sample preparation method for the determination of atorvastatin and its metabolites in biological samples using MEPS (microextraction by packed sorbent). The optimal conditions of MEPS extraction were using C8 sorbent and only 50 μl of the sample. The analytes were eluted by 100 μl of the mixture of acetonitrile:0.1 M ammonium acetate pH 4.5 (95:5, v:v). The analytical method was validated and demonstrated good linearity (r(2)>0.9990), recovery (89-115%) and intra-day precision (RSD<10%). Total time of the sample preparation was three times shorter (7 min) compared to SPE. The volume of sample was twenty times lower and the volume of solvents about ten times lower compared to SPE. Combination of fast MEPS method together with quick UHPLC-MS/MS was used for the determination of atorvastatin and its two metabolites in serum obtained from patients with familiar hypercholesterolemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models., Results: In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics., Conclusions: A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R(2) ≥ 0.9975, n = 3) over the concentration range of 0.05-100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6-111%. Intra- and inter-run mean percent accuracy were between 85-115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at -80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.

Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate is a potential intermediate for the synthesis of HMG-CoA reductase inhibitor (atorvastatin) that can lower the cholesterol level in human blood. In this study, in order to synthesize ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate by bioreduction, the yeast strains in our lab were screened. Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate was found to be produced efficiently from ethyl 5-(1,3-dioxoisoindolin-2-yl)-3-oxopentanoate by Pichia pastoris X-33. The effects of initial substrate concentration, reaction time, co-substrate, amount of yeast cells, pH, as well as the temperature on the yield and enantiomeric excesses (e.e. value) of product were examined in mono-phase system. The optimal reaction conditions are as fallows: substrate concentration 7 g/L, cell concentration 120 g/L, glucose concentration 120 g/L, pH 6.5, temperature 35 degrees C, reaction time 12 h, and the yield 93.12% with the high e.e. value of 98.55%.

Objective: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance., Methods: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates., Results: We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile&#95;Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant., Conclusion: OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs-Ringer bicarbonate buffer only, 0-1mM [3,4-(13)C(2)]-4-hydroxynonanoate, and 0-2mM [5,6,7-(13)C(3)]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-(13)C(3)]heptanoate perfusates., (2010 Elsevier Inc. All rights reserved.)

Background: Using our statin analysis method, it was possible to uncover a significant drop in statin levels (atorvastatin, simvastatin, and metabolites) after extracorporeal LDL-cholesterol elimination (EE) in severe familial hypercholesterolemia (FH). The purpose of this work was to identify the mechanism underlying this drop and its clinical significance as well as to propose measures to optimize a pharmacotherapeutical regimen that can prevent the loss of statins., Methods: Ultra High Performance Liquid Chromatography (UHPLC) connected to the triple quadrupole MS/MS system was used. Patients. A group of long-term treated patients (3-12 years of treatment) with severe FH (12 patients) and treated regularly by LDL-apheresis (immunoadsorption) or haemorheopheresis (cascade filtration) were included in this study., Results: After EE, the level of statins and their metabolites decreased (atorvastatin before/after LDL-apheresis: 8.83/3.46 nmol/l; before/after haemorheopheresis: 37.02/18.94 nmol/l). A specific loss was found (concentration of atorvastatin for LDL-apheresis/haemorheopheresis: 0.28/3.04 nmol/l in washing fluids; 11.07 nmol/l in filters). To prevent substantial loss of statin concentrations, a pharmacotherapeutic regimen with a longer time interval between the dose of statins and EE is recommended (15 hours)., Conclusions: A specific loss of statins was found in adsorbent columns and filters. The decrease can be prevented by the suggested dosage scheme.

The purpose of this study was to investigate the effect of genetic variations in organic anion-transporting polypeptide 1B1 (OATP1B1) and Na(+)/taurocholate co-transporting polypeptide (NTCP) on the uptake of various statins having different affinities for these transporters. The functional activities and simultaneous expression of NTCP and OATP1B1 were confirmed by the uptake of taurocholate and estrone-3-sulphate as representative substrates for NTCP and OATP1B1, respectively, and by an immunofluorescence analysis. The substrate specificities of NTCP and OATP1B1 for statins and the effects of genetic variations on the uptake of rosuvastatin, pitavastatin, and atorvastatin were measured. Based on the K(m) values and intrinsic clearances of the three statins, pitavastatin was taken up more efficiently than rosuvastatin and atorvastatin by OATP1B1. Consequently, the cellular accumulation of pitavastatin was modulated according to the genetic variation of OATP1B1 (OATP1B1*15), rather than NTCP*2. In contrast, NTCP*2 displayed greater transport of atorvastatin and rosuvastatin, compared with NTCP wild type. Thus, the measurements of decreased rosuvastatin and atorvastatin transport by OATP1B1*15 were confounded by the presence of NTCP and its genetic variant, NTCP*2. In conclusion, the functional consequences of genetic variants of NTCP and OATP1B1 may be different for various statins, depending on the substrate specificity of the OATP1B1 and NTCP transporters.

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (V(max)/K(m)) that were lowered considerably (by 84-60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased K(m) with or without decreased V(max) values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, K(m) values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but V(max) values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34-52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.

The influence of the phenolic hydroxyl group of ortho-hydroxy atorvastatin metabolite (Ato-OH) on the photochemistry of atorvastatin (Ato) has been evaluated by steady and time-resolved experiments. Direct excitation of Ato and Ato-OH led to phenanthrene-like intermediate formation, being ∼30% for Ato-OH less efficient than that for its parent compound in methanol. Both, Ato and Ato-OH are able to quench benzophenone (E(T)∼69 kcal mol(-1)) and xanthone (E(T)∼74 kcal mol(-1)) triplet excited state with rate constants close to diffusion limit control which suggest energy transfer mechanism is taking place. In fact, lower triplet energies ∼63 kcal mol(-1) and π,π* character, were confirmed by DFT calculations for both compounds. Interestingly, only Ato-OH can act as a hydrogen donor towards triplet benzil excited state (E(T)∼ 54 kcal mol(-1)) due to the presence of the phenolic hydroxyl group. Nevertheless, the presence of this group in Ato-OH does not modify to a significant degree the compound reactivity toward singlet oxygen. The importance of triplet energy transfer in biological systems to form Ato and Ato-OH triplet excited state as well as the hydrogen donor capacity of Ato-OH toward triplet excited state are discussed in the present communication.