Human S9 Fractions Research Articles

In Vitro and In Vivo Study of the Gastrointestinal Absorption and Metabolisation of Hymenocardine, a Cyclopeptide Alkaloid.

Hymenocardine is a cyclopeptide alkaloid present in the root bark of Hymenocardia acida. In traditional African medicine, the leaves and roots of this plant are used to treat malaria, and moderate in vitro antiplasmodial activity has been reported for hymenocardine. However, in view of its peptide-like nature, potential metabolisation after oral ingestion has to be taken into account when considering in vivo experiments. In this study, the stability and small intestinal absorption of hymenocardine was assessed using an in vitro gastrointestinal dialysis model. In addition, potential liver metabolisation was investigated in vitro by incubation with a human S9 fraction. Moreover, hymenocardine was administered to rats per os, and blood and urine samples were collected until 48 and 24 h after oral administration, respectively. All samples resulting from these three experiments were analyzed by LC-MS. Analysis of the dialysate and retentate, obtained from the gastrointestinal dialysis model, indicated that hymenocardine is absorbed unchanged from the gastrointestinal tract, at least in part. After S9 metabolisation, several metabolites of hymenocardine could be identified, the major ones being formed by the reduction and/or the loss of an N-methyl group. The in vivo study confirmed that hymenocardine is absorbed from the gastrointestinal tract unchanged, since it could be identified in both rat plasma and urine, together with hymenocardinol, its reduction product.

In Vitro Evaluation of Absorption Characteristics of Peramivir for Oral Delivery.

Peramivir is a novel antiviral agent approved for the treatment of severe influenza. However, the development of oral formulation of peramivir has been severely hurdled by poor bioavailability (human, ≤3%). The present work aims to evaluate oral permeability characteristics of peramivir. In vitro gastrointestinal stability, metabolic stability in human intestinal S9 fraction and Caco-2 permeability were performed. The liquid chromatography with tandem mass spectrometric (LC-MS/MS) was used to quantify peramivir in buffer and biological sample. Using GastroPlus™ software, intestinal effective permeability coefficient (P eff) of peramivir was estimated. Our results indicated that peramivir maintained stability in pH 5.5 and 7.4 buffers, fasted state simulated gastric fluid and fasted state simulated intestinal fluid, and human intestinal S9 fractions. The apparent permeability coefficient (P app) values of peramivir (10μM) were 3.29±0.73×10-7cm/s in a Caco-2 cell model. In vivo intestinal effective permeability coefficient (P eff) was estimated to be 0.06×10-4cm/s. Furthermore, co-incubating with cyclosporine, mitoxantrone, rifampicin, or paroxetine, the apical (AP) to basolateral (BL) flux of peramivir decreased (p<0.05). The efflux and influx of peramivir was not significantly affected with co-incubation with verapamil, MK-571, or diclofenac (p>0.05). These results revealed that carrier-mediated transports, including OATP1B (organic anion transport 1B) and OCT1 (organic cation transport 1), might be involved in the absorption of peramivir. In conclusion, our results provide insight into the poor oral bioavailability of peramivir. Peramivir can be classified as a BCS-III (high solubility/low permeability) and BDDCS-III high solubility/poor metabolism) drug. The oral bioavailability of peramivir primarily depends on its permeability across cell membranes. Both of passive and active transports are involved in the permeability of peramivir.

In vitro ocular metabolism and bioactivation of ketoconazole in rat, rabbit and human

Oral ketoconazole is clinically administered for treatment of severe cases for fungal keratitis. Pharmacodynamics and efficacy of oral and topical (ocular) ketoconazole have been explored in rabbit. However, metabolism of ketoconazole in the eye in any species is not well explored in any preclinical species or human. An understanding of ocular drug metabolism in the eye is crucial for ocular therapeutics to facilitate the risk assessment and development of potential drug candidates for the clinic. We aimed to investigate the metabolism of ketoconazole in rat, rabbit and human ocular S9 fractions. Metabolism in liver S9 fractions was also studied for a direct comparison. Eleven putative metabolites were identified in the in vitro incubations. Of these metabolites, six were present in rat ocular S9 whereas eight were present in rabbit and human ocular matrices. Metabolic pathways in rabbit and human ocular fractions suggested the formation of reactive intermediates in rabbit and human liver and ocular S9 incubations, which was confirmed with trapping studies. Herein, we report eight human ocular metabolites of ketoconazole for the first time. To the best of our knowledge, this is the first report of ocular metabolic pathways and ocular bioactivation of ketoconazole in preclinical species and human.

Ocular Metabolism of Levobunolol: Historic and Emerging Metabolic Pathways

Although ocular transport and delivery have been well studied, metabolism in the eye is not well documented, even for clinically available medications such as levobunolol, a potent and nonselective β-adrenergic receptor antagonist. Recently, we reported an in vitro methodology that could be used to evaluate ocular metabolism across preclinical species and humans. The current investigation provides detailed in vitro ocular and liver metabolism of levobunolol in rat, rabbit, and human S9 fractions, including the formation of equipotent active metabolite, dihydrolevobunolol, with the help of high-resolution mass spectrometry. 11 of the 16 metabolites of levobunolol identified herein, including a direct acetyl conjugate of levobunolol observed in all ocular and liver fractions, have not been reported in the literature. The study documents the identification of six human ocular metabolites that have never been reported. The current investigation presents evidence for ocular and hepatic metabolism of levobunolol via non-cytochrome P450 pathways, which have not been comprehensively investigated to date. Our results indicated that rat liver S9 and human ocular S9 fractions formed the most metabolites. Furthermore, liver was a poor in vitro surrogate for eye, and rat and rabbit were poor surrogates for human in terms of the rate and extent of levobunolol metabolism.

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

Metabolism of Opicapone, a Novel COMT Inhibitor, in the Rat: Characterization of In Vitro Sulfation

In this study, opicapone, a novel catechol‐O‐methyltransferase (COMT) inhibitor, metabolism was evaluated in the rat and the sulfotransferases (SULTs) responsible for opicapone sulfation were characterized. Opicapone 3‐O‐sulfate was present in monkey, dog, rat and human liver cellular fractions and represented the major metabolite in the rat. Regarding human kidney, liver and small intestine cellular fractions, the later displayed the highest activity towards opicapone sulfation. A systematic analysis of seven recombinant human SULTs, showed that SULT1A1*1 and SULT1A1*2 exhibited the highest catalytic activity for opicapone sulfation. The kinetic analysis of opicapone sulfation revealed that the affinity for the conjugation of opicapone by intestinal S9 fraction was 20‐fold higher than that for the liver S9 fraction. Similar apparent Km values were obtained for SULT1A1 and human liver S9. Opicapone sulfation by human S9 fractions and SULT1A1 isoform was inhibited in a concentration manner by quercetin and 2,6‐dichloro‐4‐nitrophenol, highly selective inhibitors of SULT1A1. Dopamine, the major endogenous substrate of SULT1A3, also inhibited opicapone sulfation, but acetaminophen had no effect. The enhanced catalytic activity for opicapone sulfation and the high level of expression of SULT1A1 in liver together with the similar Km values obtained for SULT1A1 and for human liver S9 fraction, suggest that SULT1A1 may play a major role in opicapone sulfation, though other SULTs, such as SULT1A3, may also be involved in opicapone sulfation, namely at the intestinal level.

In Vitro Acetylation of Seven Anti‐glaucoma Agents in Rat, Rabbit and Human Ocular and Liver S9 Fractions

Ocular N‐acetyltransferase(NAT) research has been limited to probe substrate activity in specific tissues from rat or rabbit, but not for a model that represents the whole eye. The comparison of acetylation as a biotransformation pathway between preclinical species and human has not been well documented. We aimed to understand functional ocular NAT activity across rat, rabbit, and human ocular S9 fractions in comparison with liver S9 fractions for seven clinically approved anti‐glaucoma compounds. Specific probe substrates were examined in human ocular, liver, and kidney S9 fractions to asses human NAT1 and 2 activities. High resolution mass spectrometry was utilized to characterize the acetyl conjugate(s) for each anti‐glaucoma agent in ocular and liver S9 fractions. Direct acetyl conjugates were detected across all species in ocular and liver fractions for betaxolol, carteolol and levobunolol, indicating that these compounds are non‐specific NAT substrates. Oxidation and O‐acetylation of levobunolol was detected in rat and rabbit liver S9 only. Species‐ and organ‐specific metabolic differences were observed for metoprolol, metipranolol, propranolol, and timolol. Neither ocular nor liver fractions from rat or rabbit were useful to predict human ocular acetylation in vitro. Ocular and renal S9 fractions did not catalyze the acetylation of sulfamethazine, a NAT2 probe substrate, contrary to the observation in liver S9. The data suggests a strong role of NAT1 in human ocular S9 fractions. This is the first report of direct acetyl conjugates for the b‐adrenergic receptor antagonist class of anti‐glaucoma agents in rat, rabbit and human ocular S9s.

Assessment of Active Metabolites of Levobunolol and Betaxolol in Rat, Rabbit and Human Ocular and Liver S9 Fractions

Little is known about ocular metabolism and formation of pharmacologically active metabolites for drugs dosed topically to the eye. We devised a simple in vitro approach coupled with high resolution mass spectrometry to measure the rate and extent of ocular metabolism for comparison with liver. Levobunolol and betaxolol are antagonists of β‐adrenergic receptors and have been clinically utilized for treatment of primary open‐angle glaucoma and/or ocular hypertension. Levobunolol has been reported to be metabolized to an equipotent active metabolite, dihydrolevobunolol, in plasma and urine of mice, rat, dog, and human. Hydroxybetaxolol, an active metabolite of betaxolol, has been reported in the liver. We aimed to understand whether the eye has functional metabolic activity and to understand the extent of formation of two historically reported in vivo active metabolites, one each for levobunolol and betaxolol, across rat (R), rabbit (Rbt) and human (H) ocular S9 (OS9) fractions and liver S9 (LS9) fractions. Based on high resolution mass spectrometry M11 was identified as the active dihydroxylevobunolol while M2 was identified as the active hydroxybetaxolol. The in vitro data shows that betaxolol's M2 was formed in the eye across preclinical species and human. M2 pharmacological activity for antagonism toward β1‐ or β2‐receptors has not been assessed to date. This is the first report of hydroxybetaxolol being formed in the eye. Results from levobunolol and betaxolol metabolism studies point toward species differences in conversion of levobunolol and betaxolol to their respective active metabolites and strongly indicate that LS9 fractions are a poor surrogate for assessment of active metabolites formed in the eye.

Sulfonation of curcuminoids: characterization and contribution of individual SULT enzymes.

Poor oral bioavailability of curcuminoids limited their various applications, and one of the main reasons is their rapid metabolism in vivo. Sulfonation via sulfotransferases (SULTs) is an important metabolic pathway for such compounds. The objective of this study is to determine the SULT-isoform-specific metabolic fingerprint, tissue-specific rate, and reaction kinetic profiles to describe the characterization and contribution of curcuminoids sulfonation. Sulfonation of curcuminoids was investigated by using nine expressed SULT isoforms and four pooled human tissue S9 fractions. The results showed that human small intestine is the predominant tissue responsible for sulfonation of curcuminoids. SULT1A3 is a major isoform catalyzing sulfonation of curcumin and demethoxycurcumin, but not for bisdemethoxycurcumin. SULT1B1 is only responsible for sulfonation of curcumin. Although SULT1C4 and 1E1 could highly catalyze the sulfate conjugations toward all the three compounds, the correlativities with human small intestine S9 fractions were much weaker (R(2) = 0.100-0.482). Almost all the kinetic profiles of the SULT isoforms for curcuminoids exhibited substrate inhibition kinetics. This investigation contributed to elucidate the SULT-mediated metabolism and detoxication of curcuminoids at molecular levels and in different organs.

Multidimensional LC–MS/MS analysis of liver proteins in rat, mouse and human microsomal and S9 fractions

Liver plays a key role in metabolism and detoxification, therefore analysis of its proteome is relevant for toxicology and drug discovery studies. To optimize for high proteome coverage, protein and peptide-level ion exchange fractionation were assessed using rat liver microsomes and S9 fractions. 2D-(SCX-RP)-LC–MS/MS analysis with peptide fractionation was subsequently employed for rat, mouse and human samples, yielding between 1400 and 1939 identified proteins, 58% of which were shared between species, and with relatively high sequence coverage. This rich dataset is specifically interesting for the toxicology community, and could serve as an excellent source for targeted assay development.

Abstract 3255: Single oral dose pharmacokinetics of cancer chemopreventive phytochemicals from Angelica gigas Nakai in men and women

Abstract The ethanol extracts of the root of Korean herb Angelica gigas Nakai (AGN) have promising anti-cancer, anti-nociceptive and other activities in rodent models. It is currently believed that the pyranocoumarin decursin and its structural isomer decursinol angelate (DA) in such extracts contribute to the in vitro anti-cancer activity. We and others have demonstrated that both decursin and DA were extensively converted to decursinol (DOH) in rodents. However, our in vitro experiments using human and rodent S9 fractions suggested the hypothesis that decursin and/or DA might be metabolized differently in humans. Given the notable difference in the biological activities between decursin/DA and DOH, we conducted a single oral dose pharmacokinetic study of these phytochemicals in healthy volunteers to address a key question for human translatability of preclinical animal studies of decursin, DA or AGN extract. Knowing that chemicals other than decursin and DA in AGN had minimal effect on their absorption and metabolism in rodents, we used an AGN-based dietary supplement as the study drug in compliance with the FDA and institutional requirements. In all, 20 healthy subjects (10 men and 10 women) had completed the study in full. AGN product was very well tolerated by all subjects as evidenced by plasma biochemistry, complete blood cell count and physical examination. Analyzing plasma samples using UHPLC-MS/MS showed that, though humans metabolized DA slightly slower than rodents, both decursin and DA were extensively converted to DOH. Gender and racial/ethnicity do not appear to dramatically affect the PK parameters. Since we show here that humans metabolize decursin and DA in similar pattern with rodents, the anti-cancer efficacy, safety, tissue distribution and pharmacodynamic biomarker data generated in rodent models will likely be relevant for human translation. In spite of the limited number of healthy individuals in the trial, this project demonstrated feasibility for our team to consider translational research in disease-oriented populations. Citation Format: Jinhui Zhang, Li Li, Thomas W. Hale, Wayne Chee, Chengguo Xing, Cheng Jiang, Junxuan Lü. Single oral dose pharmacokinetics of cancer chemopreventive phytochemicals from Angelica gigas Nakai in men and women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3255. doi:10.1158/1538-7445.AM2014-3255

In vitro synthesis and characterisation of three fenoterol sulfoconjugates detected in fenoterol post-administration urine samples

Fenoterol, a fast-acting β2-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for β2-agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono-sulfoconjugates and one bis-sulfoconjugate were synthesised and their structures confirmed by liquid chromatography–high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two monosulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono-sulfoconjugates and a fenoterol bis-sulfoconjugate were detected in urine. This is the first report of the bis-sulfoconjugate.

Presystemic Metabolism of AZ’0908, A Novel mPGES-1 Inhibitor: An In Vitro and In Vivo Cross-Species Comparison

AZ'0908 is a novel microsomal prostaglandin E synthase-1 inhibitor intended for oral administration. Pharmacokinetic experiments in rats showed that bioavailability was much lower than anticipated and increased following pretreatment with the nonspecific cytochrome P450 (CYP) inhibitor 1-aminobenzotriazole, presumably by inhibition of intestinal metabolism. Stability experiments in rat liver and intestinal fractions revealed that the intrinsic clearance (Cl(int)) was much higher in intestinal than in liver microsomes. Caco2 experiments showed that AZ'0908 was a substrate for breast cancer resistance protein. Permeability was generally high and the efflux component was saturable predicting good absorption. The Cl(int) values in human intestinal microsome and S9 fractions were low. A correlation occurred between in vitro intestinal metabolism and in vivo intestinal loss in rats and dogs. Enzyme identification experiments showed that human CYP2J2 was involved in the oxidation of AZ'0908. In rats, the major metabolic enzyme was not identified. However, rat CYP2J2 analogs were not investigated. Intestinal metabolism appeared to be a major occurrence, explaining intestinal loss of AZ'0908 in the rats. In view of good overall permeability, low in vitro intestinal turnover, and relative low intestinal abundance of CYP2J2, we predict that intestinal metabolism of AZ'0908 in human does not exert a major issue.

Radiosynthesis and Preclinical Evaluation of 3′-Aza-2′-[18F]fluorofolic Acid: A Novel PET Radiotracer for Folate Receptor Targeting

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [(18)F]fluoride (integrated approach). 3'-Azafolic acid and its 2'-halogenated derivatives (2'-chloro and 2'-fluoro) were evaluated in vitro to determine their binding affinity. 3'-Aza-2'-[(18)F]fluorofolic acid ([(18)F]6) was obtained, starting from N(2)-acetyl-3'-aza-2'-chlorofolic acid di-tert-butylester (2), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35-127 GBq/μmol. Binding affinity to the FR was high (IC(50) = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 °C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 °C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [(18)F]6 in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new (18)F-labeled 3'-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue.

The stereoselective sulfate conjugation of 4'-methoxyfenoterol stereoisomers by sulfotransferase enzymes.

The presystemic sulfate conjugation of the stereoisomers of 4'-methoxyfenoterol, (R,R')-MF, (S,S')-MF, (R,S')-MF, and (S,R')-MF, was investigated using commercially available human intestinal S9 fractions, a mixture of sulfotransferase (SULT) enzymes. The results indicate that the sulfation was stereospecific and that an S-configuration at the β-OH carbon of the MF molecule enhanced the maximal formation rates with (S,R')-MF (S,S')-MF (R,S')-MF ≈ (R,R')-MF, and competition studies demonstrated that (S,R')-MF is an effective inhibitor of (R,R')-MF sulfation (IC(50) = 60 μM). In addition, the results from a cDNA-expressed human SULT isoform screen indicated that SULT1A1, SULT1A3, and SULT1E1 can mediate the sulfation of all four MF stereoisomers. Previously published molecular models of SULT1A3 and SULT1A1 were used in docking simulations of the MF stereoisomers using Molegro Virtual Docker. The models of the MF-SULT1A3 and MF-SULT1A1 complexes indicate that each of the two chiral centers of MF molecule plays a role in the observed relative stabilities. The observed stereoselectivity is the result of multiple hydrogen bonding interactions and induced conformational changes within the substrate-enzyme complex. In conclusion, the results suggest that a formulation developed from a mixture of (R,R')-MF and (S,R')-MF may increase the oral bioavailability of (R,R')-MF.

Effect of Structural Variation on Aldehyde Oxidase-Catalyzed Oxidation of Zoniporide

Current studies explored the effect of structural changes on the aldehyde oxidase (AO)-mediated metabolism of zoniporide (1). Zoniporide analogs with modifications of the acylguanidine moiety, the cyclopropyl group on the pyrazole ring, and the quinoline ring were studied for their AO-catalyzed metabolism using the human S9 fraction. Analysis of the half-lives suggested that subtle changes in the structure of 1 influenced its metabolism and that the guanidine and the quinoline moieties were prerequisites for AO-catalyzed oxidation to 2-oxozoniporide (M1). In contrast, replacement of the cyclopropyl group with other alkyl groups was tolerated. The effect of structural variation on AO properties was rationalized by docking 1 and its analogs into the human AO homology model. These studies indicated the importance of electrostatic, π-π stacking and hydrophobic interactions of the three motifs with residues in the active site. Differences in substrate properties were also rationalized by comparing their half-lives with cLogD, electrophilicity parameters [electrostatic potential (ESP) charges and energy of lowest unoccupied molecular orbitals (E(LUMO))], and the energies of formation of tetrahedral intermediates (J Med Chem 50:4642-4647, 2007). Whereas the success of energetics in predicting the AO substrate properties of analogs was 87%, the predictive ability of other descriptors was none (cLogD) to 60% (ESP charges and E(LUMO)). Overall, the structure-metabolism relationship could be rationalized using a combination of both the energy calculations and docking studies. This combination method can be incorporated into a strategy for mitigating AO liabilities observed in the lead candidate or studying structure-metabolism relationships of other AO substrates.

A Comprehensive Assessment of Repaglinide Metabolic Pathways: Impact of Choice of In Vitro System and Relative Enzyme Contribution to In Vitro Clearance

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound K(m) values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.

Characterization of desoxymethyltestosterone main urinary metabolite produced from cultures of human fresh hepatocytes

Desoxymethyltestosterone (DMT; 17β-hydroxy-17α-methyl-5α-androst-2-ene) is a designer steroid present in hormonal supplements distributed illegally as such or in combination with other steroids, for self-administration. It figures on the list of substances prohibited in sports and its detection in athlete’s urine samples is based upon the presence of the parent compound or the main urinary metabolite, which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17α-methyl-2β,3α,17β-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented.

Nonclinical Pharmacokinetics of Oseltamivir and Oseltamivir Carboxylate in the Central Nervous System

Oseltamivir, a potent and selective inhibitor of influenza A and B virus neuraminidases, is a prodrug that is systemically converted into the active metabolite oseltamivir carboxylate. In light of reported neuropsychiatric events in influenza patients, including some taking oseltamivir, and as part of a full assessment to determine whether oseltamivir could contribute to, or exacerbate, such events, we undertook a series of nonclinical studies. In particular, we investigated (i) the distribution of oseltamivir and oseltamivir carboxylate in the central nervous system of rats after single intravenous doses of oseltamivir and oseltamivir carboxylate and oral doses of oseltamivir, (ii) the active transport of oseltamivir and oseltamivir carboxylate in vitro by transporters located in the blood-brain barrier, and (iii) the extent of local conversion of oseltamivir to oseltamivir carboxylate in brain fractions. In all experiments, results showed that the extent of partitioning of oseltamivir and especially oseltamivir carboxylate to the central nervous system was low. Brain-to-plasma exposure ratios were approximately 0.2 for oseltamivir and 0.01 for oseltamivir carboxylate. Apart from oseltamivir being a good substrate for the P-glycoprotein transporter, no other active transport processes were observed. The conversion of the prodrug to the active metabolite was slow and limited in human and rat brain S9 fractions. Overall, these studies indicate that the potential for oseltamivir and oseltamivir carboxylate to reach the central nervous system in high quantities is low and, together with other analyses and studies, that their involvement in neuropsychiatric events in influenza patients is unlikely.

Pharmacokinetic studies with BLX‐1026, a novel Orexin‐2 receptor agonist with anti‐obesity property, in rats

Orexin receptors have been implicated in non‐exercise activity thermogenesis. Increased levels of Orexin‐2 receptors have been observed in obesity‐resistant rats and decreased Orexin‐A levels observed in obese humans. BLX‐1026 is a water soluble, amino‐acid conjugated small molecule with in vitro Orexin‐2 receptor agonist activity. Phase I metabolism studies conducted with BLX‐1026 in rat, mice, dog, monkey and human liver microsomes and human liver S9 fractions show that it is stable in the liver microsomes but shows a 61% degradation in the human S9 fractions when incubated for 1 hour. In‐vivo pharmacokinetic study with BLX‐1026 was conducted in rats. It was administered at 50, 100 and 200 mg/kg, PO, and 10 mg/kg, IV, to male rats. Blood was collected for 24 hours and urine was collected for 72 hours following its administration. These were analyzed for the parent BLX‐1026 and any metabolites. The results will be presented and discussed.