Aldehyde oxidase 1 activity and protein expression in human, rabbit, and pig ocular tissues.

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species.

Extrahepatic in vitro metabolism of peptides; comparison of human kidney and intestinal S9 fraction, human plasma and proximal tubule cells, using cyclosporine A, leuprorelin, and cetrorelix as model compounds.

Peptide therapeutics showcase number of advantages compared to the traditional small molecule drugs, e,g. they usually have higher affinity to target and lower toxicity profiles. Endogenous peptides are mostly cleared from the body through renal clearance or proteolytic hydrolysis. As a part of drug discovery, metabolite identification is an important part in their development to identify metabolic hot spots and to further improve their stability. As the catabolism of the peptides and peptide-like drugs is often considered to be extrahepatic, the use of in vitro systems derived from these organs might be beneficial. In this study, multiple extrahepatic metabolic systems were evaluated for the applicability for peptide metabolism studies. Three peptide drugs (leuprorelin, cetrorelix, cyclosporin) were incubated in kidney and intestinal S9 fraction ( ± NADPH), fresh plasma (anticoagulants EDTA and heparin separately), and plated proximal tubule cells. Additionally, leuprorelin was also incubated with human kidney microsomes and cytosol to further investigate the NADPH-dependent metabolism detected in kidney S9 fraction. Both substrate disappearance and metabolite formation were monitored, using UPLC/HR-MS analysis of the collected samples.Overall, the largest number of metabolites was formed in the incubation with kidney S9 fraction, followed by intestinal S9, while incubations with proximal tubule cells produced lower number of metabolites All investigated peptides were stable in plasma and only a few metabolites were detected, likely because the studied peptide drugs have been optimized to be stable in plasma. Leuprorelin showed NADPH-dependent metabolite formation in kidney S9 fraction, while the metabolism of cetrorelix was more NADPH independent. As expected, formation of cytochrome P450 (CYP) catalyzed metabolism of cyclosporine was not observed with the employed extrahepatic systems. The NADPH-dependent metabolism of leuprorelin was detected also in the incubation with kidney cytosol, but not with kidney microsomes, and was thus not caused by CYPs or FMOs, but with cytosolic NADPH-dependent drug metabolizing enzymes. These enzymes could, in principle, activate the amide bond via reductive or oxidative metabolism outside the amide bond. The identity of the involved drug metabolizing enzymes in this process is still unknown.

Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues.

As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye. SIGNIFICANCE STATEMENT: Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.

Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism.

BACKGROUND AND PURPOSE: Many drugs and environmental contaminants induce hypercholesterolemia and promote the risk of atherosclerotic cardiovascular disease. We tested the hypothesis that pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, regulates the level of circulating atherogenic lipids in humans and utilized mouse experiments to identify the mechanisms involved. EXPERIMENTAL APPROACH: We performed serum NMR metabolomics in healthy volunteers administered rifampicin, a prototypical human PXR ligand or placebo in a crossover setting. We used high-fat diet fed wild-type and PXR knockout mice to investigate the mechanisms mediating the PXR-induced alterations in cholesterol homeostasis. KEY RESULTS: Activation of PXR induced cholesterogenesis both in pre-clinical and clinical settings. In human volunteers, rifampicin increased intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and total cholesterol and lathosterol-cholesterol ratio, a marker of cholesterol synthesis, suggesting increased cholesterol synthesis. Experiments in mice indicated that PXR activation causes widespread induction of the cholesterol synthesis genes including the rate-limiting Hmgcr and upregulates the intermediates in the Kandutsch-Russell cholesterol synthesis pathway in the liver. Additionally, PXR activation induced plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of hepatic LDL uptake, in both mice and humans. We propose that these effects were mediated through increased proteolytic activation of sterol regulatory element-binding protein 2 (SREBP2) in response to PXR activation. CONCLUSION AND IMPLICATIONS: PXR activation induces cholesterol synthesis, elevating LDL and total cholesterol in humans. The PXR-SREBP2 pathway is a novel regulator of the cholesterol and PCSK9 synthesis and a molecular mechanism for drug- and chemical-induced hypercholesterolemia.

Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues.

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (d-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Hepatic in vitro metabolism of peptides; Comparison of human liver S9, hepatocytes and Upcyte hepatocytes with cyclosporine A, leuprorelin, desmopressin and cetrorelix as model compounds.

The number of approved peptide therapeutics has increased significantly in recent years. Peptide therapeutics have many advances over small molecule drugs, such as higher affinity to target and lower toxicity profiles. Although peptide-like drugs are mainly metabolized/catabolized in the body for smaller peptides and amino acids, metabolite identification still has an essential part of in their development, especially if their structure contains modified amino acids, and also to identify the metabolic soft spots enabling modification to more stable sequence. The use of human derived in vitro systems is an important tool when investigating metabolism of peptide drugs, and comparison of results by various hepatic systems was investigated here. Peptides were incubated in several different in vitro human liver-derived subcellular and cellular incubation systems, i.e. liver S9 fraction, suspended cryo-preserved human primary hepatocytes and plated Upcyte hepatocytes. Samples were collected at different time points and analysed by UPLC/HR-MS-method developed for the purpose. Both substrate disappearance and metabolite formation were monitored, and the systems were compared. S9 fraction formed the highest number of metabolites for leuprorelin and cetrorelix, while for desmopressin and cyclosporin, primary hepatocytes and liver S9 produced similar metabolite profiles. Interestingly, not only cyclosporin, but also leuprorelin and cetrorelix showed metabolites whose formation was CYP (NADPH) dependent in liver S9. For leuprorelin and cetrorelix, the metabolites that showed NADPH dependency with liver S9, were not detected with hepatocytes, even though for leuprorelin these reactions played a major role in liver S9. The hydrolytic metabolic reactions were very similar between liver S9 and hepatocytes, i.e. the metabolite profiles in hepatocytes matched better with liver S9 profiles without NADPH, which may be caused by cell uptake rate limitation with hepatocytes, or then hydrolytic processes are more stressed in peptide metabolism with hepatocytes, in comparison to CYP-mediated processes.

4ß-Hydroxycholesterol Signals From the Liver to Regulate Peripheral Cholesterol Transporters.

Activation of pregnane X receptor (PXR) elevates circulating 4ß-hydroxycholesterol (4ßHC), an agonist of liver X receptor (LXR). PXR may also regulate 25-hydroxycholesterol and 27-hydroxycholesterol. Our aim was to elucidate the roles of PXR and oxysterols in the regulation of cholesterol transporters. We measured oxysterols in serum of volunteers dosed with PXR agonist rifampicin 600 mg/day versus placebo for a week and analyzed the expression of cholesterol transporters in mononuclear cells. The effect of 4ßHC on the transport of cholesterol and the expression of cholesterol transporters was studied in human primary monocyte-derived macrophages and foam cells in vitro. The expression of cholesterol transporters was measured also in rat tissues after dosing with a PXR agonist. The levels of 4ßHC were elevated, while 25-hydroxycholesterol and 27-hydroxycholesterol remained unchanged in volunteers dosed with rifampicin. The expression of ATP binding cassette transporter A1 (ABCA1) was induced in human mononuclear cells in vivo. The influx of cholesterol was repressed by 4ßHC, as was the expression of influx transporter lectin-like oxidized LDL receptor-1 in vitro. The cholesterol efflux and the expression of efflux transporters ABCA1 and ABCG1 were induced. The expression of inducible degrader of the LDL receptor was induced. In rats, PXR agonist increased circulating 4ßHC and expression of LXR targets in peripheral tissues, especially ABCA1 and ABCG1 in heart. In conclusion, PXR activation-elevated 4ßHC is a signaling molecule that represses cholesterol influx and induces efflux. The PXR-4ßHC-LXR pathway could link the hepatic xenobiotic exposure and the regulation of cholesterol transport in peripheral tissues.

In vitro modulation of cytochrome P450 isozymes and pharmacokinetics of caffeine by extracts of Hibiscus sabdariffa Linn calyx.

Background Hibiscus sabdariffa beverage (HSB) is widely consumed as a medicinal herb and sometimes used concomitantly with drugs. This study evaluated the in vitro inhibitory potential of the aqueous extract of H. sabdariffa calyces (AEHS) on selected cytochrome P450 (CYP) isozymes and the effect of HSB on the pharmacokinetics of caffeine in vivo. Methods In vitro inhibitions of eight major CYP isozymes by AEHS were estimated by monitoring CYP-specific model reactions of 10 CYP probe substrates using N-in-one assay method. Subsequently, an open, randomized, two-period crossover design was used to evaluate the effect of HSB on the pharmacokinetics of single-dose 200 mg caffeine in six healthy human volunteers. Blood samples were obtained at specific times over a 24 h period. Probe drugs and metabolites were analyzed in their respective matrices with ultra-performance liquid chromatography/mass spectrometer/mass spectrometer and reversed-phase high-performance liquid chromatography/ultraviolet detection. Results The H. sabdariffa aqueous extract weakly inhibited the selected CYP isozymes in vitro, with IC50 of >100 µgmL-1 in the order of CYP1A2 > CYP2C8 > CYP2B6 >> CYP2D6 > CYP2C19 > CYP3A4 > CYP2A6 > CYP2C9. HSB decreased terminal t1/2 and Tmax of caffeine by 13.6% and 13.0%, respectively, and increased Cmax by 10.3%. Point estimates of primary pharmacokinetic endpoints, Cmax = 1.142 (90% confidence interval (CI) = 0.882, 1.480) and AUC0-∞ = 0.992 (90% CI = 0.745, 1.320), were outside the 90% CI of 0.8-1.25 bioequivalence limits. Conclusion The aqueous extract of H. sabdariffa weakly inhibited eight CYP isozymes in vitro, but HSB modified the exposure to caffeine in human. Caution should be exercised in administering HSB with caffeine or similar substrates of CYP1A2 until more clinical data are available.

Potential inhibition of major human cytochrome P450 isoenzymes by selected tropical medicinal herbs-Implication for herb-drug interactions.

BACKGROUND: Increasing use of medicinal herbs as nutritional supplements and traditional medicines for the treatment of diabetes, hypertension, hyperlipidemia, and malaria fever with conventional drugs poses possibilities of herb-drug interactions (HDIs). The potential of nine selected widely used tropical medicinal herbs in inhibiting human cytochrome P450 (CYP) isoenzymes was investigated. MATERIALS AND METHODS: In vitro inhibition of eight major CYP isoenzymes by aqueous extracts of Allium sativum, Gongronema latifolium, Moringa oleifera, Musa sapientum, Mangifera indica, Tetracarpidium conophorum, Alstonia boonei, Bauhinia monandra, and Picralima nitida was estimated in human liver microsomes by monitoring twelve probe metabolites of nine probe substrates with UPLC/MS-MS using validated N-in-one assay method. RESULTS: Mangifera indica moderately inhibited CYP2C8, CYP2B6, CYP2D6, CYP1A2, and CYP2C9 with IC 50 values of 37.93, 57.83, 67.39, 54.83, and 107.48 µg/ml, respectively, and Alstonia boonei inhibited CYP2D6 (IC 50 = 77.19 µg/ml). Picralima nitida inhibited CYP3A4 (IC 50 = 45.58 µg/ml) and CYP2C19 (IC 50 = 73.06 µg/ml) moderately but strongly inhibited CYP2D6 (IC 50 = 1.19 µg/ml). Other aqueous extracts of Gongronema latifolium, Bauhinia monandra, and Moringa oleifera showed weak inhibitory activities against CYP1A2. Musa sapientum, Allium sativum, and Tetracarpidium conophorum did not inhibit the CYP isoenzymes investigated. CONCLUSION: Potential for clinically important CYP-metabolism-mediated HDIs is possible for Alstonia boonei, Mangifera indica, and Picralima nitida with drugs metabolized by CYP 2C8, 2B6, 2D6, 1A2, 2C9, 2C19, and 3A4. Inhibition of CYP2D6 by Picralima nitida is of particular concern and needs immediate in vivo investigations.

Pharmacokinetics of intra-articular vitamin D analogue calcipotriol in sheep and metabolism in human synovial and mesenchymal stromal cells.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC-MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Quantitative analysis of 4ß- and 4α­hydroxycholesterol in human plasma and serum by UHPLC/ESI-HR-MS.

Cholesterol oxidation product 4ß­hydroxycholesterol (4ß­OHC) may possibly be used as an endogenous biomarker of CYP3A enzyme activity and as CYP3A4 is involved in the metabolism of approximately 50% of the drugs in clinical use, the monitoring of CYP3A activity by 4ß­OHC plasma or serum levels, may be of clinical significance. The plasma and serum concentrations of 4α­hydroxycholesterol (4α­OHC), an isomer of 4ß­OHC, increase during uncontrolled storage conditions and therefore serve as an indicator of proper handling of samples. A sensitive and simple high-throughput method for the simultaneous quantification of both 4α­OHC and 4ß­OHC in human plasma and serum was developed utilizing ultrahigh performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC/ESI-HR-MS). The chromatographic analysis was carried out on a Waters HSS T3 C18 reversed phase column with a mobile phase composed of 0,1% formic acid with 200 mg/l sodium acetate, and methanol. 4ß­OHC and 4α­OHC and also internal standard d7­4ß­OHC were monitored using HR-MS as sodium adducts, which could not be used as a precursor ions in conventional tandem mass spectrometry methods due to their extensive stability in collision for MS/MS. The use of HR-MS detection enabled avoiding laborious sample derivatization, which is required with triple quadrupole mass spectrometer-based methods to achieve adequate analytical sensitivity for 4ß­OHC, as the underivatized molecule is otherwise poorly ionized to other molecular ions than sodium adduct. Chromatographic separation of 4α­OHC and 4ß­OHC was obtained and confirmed with standard samples prepared in blank surrogate matrix. The lower limits of quantitation in the assay were 0.5 ng/ml for 4ß­OHC, and 2 ng/ml for 4α­OHC. Endogenous levels of 4ß­OHC can vary between 10 and 100 ng/ml depending on the possible induction or inhibition of CYP3A4, whereas the levels of 4α­OHC can vary between 5 and 100 ng/ml, depending on the storage conditions of the samples. Thus, the sensitivity of the assay developed allows for the simultaneous measurement of endogenous levels of 4α­OHC and 4ß­OHC cost-effectively and with high throughput. The method was successfully used for the determination of 4ß­OHC and 4α­OHC concentrations in clinical plasma and serum samples collected before and after treatment with a known CYP3A4 inducer rifampicin. The endogenous levels in clinical human samples before treatment varied between 13.4 and 31.9 ng/ml for 4ß­OHC, and between 3.53 and 5.65 ng/ml for 4α­OHC, and a three-fold increase in 4ß­OHC plasma levels was observed after the rifampicin treatment, while 4α­OHC levels remained unaffected.

Bridged Epipolythiodiketopiperazines from Penicillium raciborskii, an Endophytic Fungus of Rhododendron tomentosum Harmaja.

Three new epithiodiketopiperazine natural products [outovirin A (1), outovirin B (2), and outovirin C (3)] resembling the antifungal natural product gliovirin have been identified in extracts of Penicillium raciborskii, an endophytic fungus isolated from Rhododendron tomentosum. The compounds are unusual for their class in that they possess sulfide bridges between α- and ß-carbons rather than the typical α-α bridging. To our knowledge, outovirin A represents the first reported naturally produced epimonothiodiketopiperazine, and antifungal outovirin C is the first reported trisulfide gliovirin-like compound. This report describes the identification and structural elucidation of the compounds by LC-MS/MS and NMR.

Tandem mass spectrometric analysis of S- and N-linked glutathione conjugates of pulegone and menthofuran and identification of P450 enzymes mediating their formation.

RATIONALE: Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. METHODS: (R)-(+)-Pulegone, (S)-(-)-pulegone, and menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. RESULTS: Four GSH conjugates of menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. CONLUSIONS: Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.

Genetically Modified Caco-2 Cells With Improved Cytochrome P450 Metabolic Capacity.

The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells.

Metabolism and metabolite profiles in vitro and in vivo of ospemifene in humans and preclinical species.

BACKGROUND: Metabolite profiles of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, were surveyed as part of its development. METHODS: The pharmacokinetics of ospemifene and its two major, pharmacologically active metabolites 4-hydroxyospemifene and 4'-hydroxyospemifene, was elucidated in studies of volunteer humans given various doses of ospemifene and in experiments of several animal species (rat, mouse, dog, and cynomolgus monkey), which had been used either for pharmacological or toxicological studies of ospemifene. Metabolites produced in in vitro human and animal liver preparations were compared between species and with the metabolite profiles in the in vivo investigations. RESULTS: Considerable interspecies differences were observed in the metabolite profiles and quantities. The major human metabolite, 4-hydroxyospemifene, was produced in substantial amounts both in vitro and in vivo in most animal species, except dog, and thus the exposure to this metabolite seems adequate in the most important toxicology species, the rat and the cynomolgus monkey. 4'-Hydroxyospemifene was equally abundant in vitro and in vivo metabolite in mice and dogs, and consequently, its contribution to the total exposure of ospemifene-related activity would be adequately covered in animal experiments. Other ospemifene metabolites were variably detected in different species, but probably they are not of consequence to pharmacology or toxicology of ospemifene. CONCLUSIONS: Overall, there are quantitative and also some qualitative differences in the metabolism of ospemifene in different species. Generally, in vitro metabolite profiles were predictive for in vivo profiles. The contribution of two major hydroxyl metabolites to activity and toxicity of ospemifene is adequately covered by at least some animal species.

Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-ß-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI).

Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.

The objective was to compare several in vitro human liver-derived subcellular and cellular incubation systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human liver microsomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glutathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using only NADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally different compounds were used as a test set, chosen as either "positive" (ciprofloxacin, clozapine, diclofenac, ethinyl estradiol, pulegone, and ticlopidine) or "negative" (caffeine, citalopram, losartan, montelukast) compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates were observed for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram, or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relative abundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSH conjugates were different for many compounds in cellular subfractions and cell-based systems. Hepatocytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differences were minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactive metabolites do have a high impact on the results, and results between different systems are comparable only qualitatively.

Glutathione trapping of reactive drug metabolites produced by biomimetic metalloporphyrin catalysts.

RATIONALE: Metalloporphyrins can be useful in the production of drug metabolites, as they enable easier production of oxidative metabolites usually produced by the cytochrome P450 enzymes. Our aim was to test metalloporphyrin-based biomimetic oxidation (BMO) methods for production and S-glutathione trapping of reactive drug metabolites in addition to phase I metabolites. METHODS: Clozapine, ticlopidine and citalopram were selected as model compounds. These were incubated with the BMO assay and the incubations were analyzed with high-resolution liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS). Additionally, incubations with human liver S9 fraction were performed to compare the results with the BMO assay. RESULTS: Six glutathione conjugates were identified for clozapine from the S9 incubation, while the BMO assay produced four of these. Four out of the five phase I metabolites produced by S9 were detected using the BMO assay. For ticlopidine, four glutathione conjugates were detected from the S9 incubation, but none of these were observed using the BMO assay. Eight of the nine phase I metabolites produced by S9 incubation were detected in the BMO assay. As expected, no glutathione conjugates were detected for citalopram, and the same three phase I metabolites were detected in both S9 and BMO incubations. CONLUSIONS: Differences in formation of GSH-trapped reactive metabolites by BMO assay between clozapine and ticlopidine are probably due to different reactive intermediates and reaction mechanisms. The reactive intermediate of clozapine, the nitrenium ion was generated, but the reactive intermediates of ticlopidine, S-oxide and epoxide, were not detected from the incubations. However, the results show that for selected cases the use of biomimetic assays can be used to produce high amounts of S-glutathione conjugates identical to those from liver subfraction incubations, on a scale that is relevant for purification and subsequent identification by NMR spectroscopy; which is often difficult using incubations with liver subfractions.

Reactive metabolites in early drug development: predictive in vitro tools.

Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative "negatives" give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.