Human Liver Microsomal Preparations Research Articles

Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine demethylation in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism

Structure-Dependent Toxicokinetics of Selected Pyrrolizidine Alkaloids In Vitro

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.

A unique sequential C-S or N-S inductive cleavage and retro-Diels-Alder fragmentation mechanism for alkylsulfonyl piperidine- and piperazine-containing compounds.

BI 605906 undergoes a collision-induced dissociation (CID) fragmentation resulting in the loss of methylsulfinic acid and butadiene to produce a corresponding imine. The fragmentation is hypothesized to occur via inductive cleavage of the C-S bond, generating a six-membered cyclic ene, followed by the retro-Diels-Alder (RDA) reaction. The aim of this study was to provide mechanistic evidence for the proposed fragmentation by investigating the CID spectra of BI 605906 and other alkylsulfonyl piperidine- and piperazine-containing compounds. The positive electrospray ionization tandem mass spectrometric (ESI+ -MS/MS) fragmentations of BI 605906, D9 -BI 605906, GK02935, GK02942, ketoconazole, terazosin, and homopiperazine were investigated. Additionally, incubations of BI 605906 and GK02942 in human liver microsomes (HLM) preparations were conducted. Metabolite identification experiments were performed following these incubations to investigate corresponding in vitro metabolism. BI 605906, D9 -BI 605906, GK02935, and GK02942 demonstrated the same fragmentation pattern by generating a respective imine ion, supporting the hypothesized inductive cleavage and subsequent RDA mechanism. Ketoconazole and terazosin, which contain either an N-acetyl or tetrahydrofuranyl piperazine group, respectively, did not demonstrate this mechanism, notably because they do not have the alkylsulfonyl moiety as a good leaving group. Although homopiperazine contains an arylsulfonyl diazepane group, and the initial step produced an unsaturated diazepane ring, the subsequent RDA reaction was unable to proceed due to the absence of a six-membered cyclic ene intermediate. Additionally, we identified oxidative metabolites of BI 605906 and GK02942 in HLM incubations utilizing the proposed fragmentation pattern. In the mass spectrometer, compounds containing alkylsulfonyl piperidine or piperazine groups can undergo inductive cleavage, leading to a six-membered cyclic ene intermediate. This intermediate will then form a corresponding imine ion via the RDA reaction. A practical application of this work is to utilize this fragmentation for elucidating structures of metabolites arising from parent compounds containing alkylsulfonyl piperidine or piperazine moieties.

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450–P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Crosstalk between CYP2E1 and CYP3A enzymes and its Possible Involvement in Alcohol‐Drug Interactions.

The core of the drug‐metabolizing ensemble in human liver is formed by the ensemble of different P450 enzymes co‐localized in the membrane of endoplasmic reticulum (ER). There is an emerging recognition of the fact that multiple P450 species interact with each other with the formation of heteromeric complexes, where the catalytic properties of the individual enzymes are modified. Functional consequences of these interactions are likely to be responsible for the lack of a straight correlation between the composition of the P450 ensemble and the profile of drug metabolism. Of particular importance are the changes in P450‐P450 crosstalk induced by alcohol consumption. Although the multi‐fold increase in the content of CYP2E1 in liver of alcohol consumers is well documented, the involvement of CYP2E1 in alcohol‐drug interactions is considered insignificant due to a minor role of this enzyme in drug metabolism. However, the effects of induction of CYP2E1 may also involve its effects on the functional properties of other drug‐metabolizing enzymes.In order to probe the crosstalk between CYP2E1 and other P450 species and elucidate its role in alcohol‐drug interactions we established a model of alcohol‐induced increase in CYP2E1 content that implements incorporation of purified CYP2E1 into human liver microsomes (HLM). The adopted CYP2E1 becomes a fully‐functional member of the drug‐metabolizing ensemble and interacts with other P450 enzymes with the formation of heteromeric complexes (Davydova et al. 2019, https://doi.org/10.1101/685545). Investigating the impact of CYP2E1 on CYP3A enzymes, we found that the enrichment of HLM with CYP2E1 results in an increased rate of metabolism of 7‐benzyloxyquinoline (BQ) and eliminates the homotropic cooperativity observed with this CYP3A‐specific substrate. To probe the physiological relevance of these effects we compared three pulled preparations of HLM from normal donors (N‐HLM) with a preparation obtained from heavy alcohol consumers (A‐HLM). The content of major P450 species in the samples was characterized with mass‐spectroscopic determination of their specific peptides. The content of CYP2E1 in N‐HLM samples was from 2 to 5 lower than that found in A‐HLM. In contrast, the content of CYP3A in A‐HLM was the lowest among all four preparations. Despite the lower content of CYP3A, A‐HLM exhibited much higher rate of metabolism and lower homotropic cooperativity with BQ, similar to what is observed in CYP2E1‐enriched N‐HLM. Incorporation of additional CYP2E1 into A‐HLM results in a further increase in the rate of BQ metabolism that makes it an order of magnitude higher than in the N‐HLM sample with a similar CYP3A content. Importantly, the CYP2E1‐dependent activation observed with BQ, a highly specific substrate of CYP3A, is not observable with 7‐dimethylamino‐4‐trifluoromethyl coumarin (Coumarin 152), a substrate preferentially metabolized by CYP3A, but capable of high‐affinity interactions with several other P450 species. This observation corroborates our hypothesis that the mechanisms of substrate‐dependent activation of a particular P450 enzyme through P450‐P450 crosstalk involve a reorganization of P450 heterooligomers induced by the interactions of the target enzyme with its specific substrate.Support or Funding InformationThis research was supported by the grant R21‐AA024548 from NIH.

Liquid chromatography-tandem mass spectrometry metabolic profiling of nazartinib reveals the formation of unexpected reactive metabolites.

Nazartinib (EGF816, NZB) is a promising third-generation human epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. This novel irreversible mutant-selective EGFR inhibitor targets EGFR containing both the resistance mutation (T790M) and the activating mutations (L858R and Del19), while it does not affect wild-type EGFR. However, the metabolic pathway and bioactivation mechanisms of NZB are still unexplored. Thus, using liquid chromatography–tandem mass spectrometry, we screened for products of NZB metabolism formed in vitro by human liver microsomal preparations and investigated the formation of reactive intermediates using potassium cyanide as a nucleophile trap. Unexpectedly, the azepane ring was not bioactivated. Instead, the carbon atom between the aliphatic linear tertiary amine and electron-withdrawing system (butenoyl amide group) was bioactivated, generating iminium intermediates as reactive species. Six NZB phase I metabolites, formed by hydroxylation, oxidation and N-demethylation, were characterized. Moreover, two reactive iminium ions were characterized and their corresponding bioactivation mechanisms were proposed. Based on our results, we speculate that bioactivation of NZB can be blocked by small sterically hindering groups, isosteric replacement or a spacer. This approach might reduce the toxicity of NZB by avoiding the generation of reactive species.

Atropselective Oxidation of 2,2',3,3',4,6'-Hexachlorobiphenyl (PCB 132) to Hydroxylated Metabolites by Human Liver Microsomes and Its Implications for PCB 132 Neurotoxicity.

Polychlorinated biphenyls (PCBs) have been associated with neurodevelopmental disorders. Several neurotoxic congeners display axial chirality and atropselectively affect cellular targets implicated in PCB neurotoxicity. Only limited information is available regarding the atropselective metabolism of these congeners in humans and their atropselective effects on neurotoxic outcomes. Here we investigate the hypothesis that the oxidation of 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) by human liver microsomes (HLMs) and their effects on dopaminergic cells in culture are atropselective. Racemic PCB 132 was incubated with pooled or single donor HLMs, and levels and enantiomeric fractions of PCB 132 and its metabolites were determined gas chromatographically. The major metabolite was either 2,2',3,4,4',6'-hexachlorobiphenyl-3'-ol (3'-140), a 1,2-shift product, or 2,2',3,3',4,6'-hexachlorobiphenyl-5'-ol (5'-132). The PCB 132 metabolite profiles displayed interindividual differences and depended on the PCB 132 atropisomer. Computational studies suggested that 3'-140 is formed via a 3,4-arene oxide intermediate. The second eluting atropisomer of PCB 132, first eluting atropisomer of 3'-140, and second eluting atropisomer of 5'-132 were enriched in all HLM incubations. Enantiomeric fractions of the PCB 132 metabolites differed only slightly between the single donor HLM preparations investigated. Reactive oxygen species and levels of dopamine and its metabolites were not significantly altered after a 24 h exposure of dopaminergic cells to pure PCB 132 atropisomers. These findings suggest that there are interindividual differences in the atropselective biotransformation of PCB 132 to its metabolites in humans; however, the resulting atropisomeric enrichment of PCB 132 is unlikely to affect neurotoxic outcomes associated with the endpoints investigated in the study.

Highly Variable Expression of CYP1A1 in Human Liver and Impact on Pharmacokinetics of Riociguat and Granisetron in Humans.

Cytochrome P450s (CYPs), a superfamily of enzymes, are involved in the biotransformation of endogenous and xenobiotic chemicals and mainly responsible for the metabolic clearance of widely prescribed drugs. Out of the 57 human isoforms, only a few, most notably CYP3A4, are considered to be important in this process. CYP1A1, one of the three isoforms of the CYP1 family, is widely believed to play an important role in the metabolism and activation of numerous procarcinogens, e.g., polyaromatic hydrocarbons (PAHs) or aromatic amines. It is also known that CYP1A1 is highly inducible by endogenous and exogenous factors, e.g., PAHs. However, CYP1A1 has not been considered to play a significant role in the metabolic clearance of drugs, since this isoform has been detected only in extrahepatic tissues in small amounts. In contrast to conventional wisdom, we herein demonstrate the expression of CYP1A1 protein in human liver microsomal preparations. The expression levels of CYP1A1 were quantified by Western blot and LC/MS analyses and corresponded well with enzymatic activities of highly selective CYP1A1 reactions. In a panel of 29 individual liver microsomal preparations, highly variable and substantial expression levels (up to ∼10 pmol/mg) were measured. Together with the high selectivity and especially the high metabolic efficiency of CYP1A1 shown for granisetron and riociguat, it is demonstrated that CYP1A1 plays an important role in the metabolic clearance of these drugs and is responsible for the clinically observed interindividual variability in their pharmacokinetics. Therefore, the importance of CYP1A1 in drug discovery and development needs to be reconsidered.

Resveratrol glucuronidation in vitro: potential implications of inhibition by probenecid.

Resveratrol is a naturally occurring antioxidant with therapeutic potential in prevention and treatment of neoplastic disease and other human disorders. However, net clearance of resveratrol in humans is very high, mainly due to glucuronide conjugation. This leads to extensive presystemic extraction and low plasma concentrations after oral dosage. The present study evaluated the effect of probenecid, an inhibitor of glucuronide conjugation, on resveratrol metabolism invitro. Biotransformation of resveratrol to its 3-O-glucuronide and 4'-O-glucuronide conjugates was studied invitro using human liver microsomal preparations. The mechanism and inhibitory potency of probenecid were evaluated based on a mixed competitive-noncompetitive inhibition model. Probenecid inhibition of resveratrol 3-O-glucuronidation was predominantly noncompetitive, with an inhibition constant (Ki ) averaging 3.1mm. The ratio of invivo maximum concentration of probenecid [I] during usual clinical use to the invitro Ki value ([I]/Ki ) exceeds the boundary value of 0.1, used by regulatory agencies to identify the possibility of clinical drug interactions. This finding, together with the known property of probenecid as an inhibitor of glucuronide conjugation in humans, suggests that probenecid could serve as a pharmacokinetic boosting agent to enhance systemic exposure to resveratrol in humans.

Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib.

Sunitinib is a multitargeted tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity. The mechanisms of this toxicity are unknown. We hypothesized that sunitinib undergoes metabolic activation to form chemically reactive, potentially toxic metabolites which may contribute to development of sunitinib-induced hepatotoxicity. The purpose of this study was to define the role of cytochrome P450 (P450) enzymes in sunitinib bioactivation. Metabolic incubations were performed using individual recombinant P450s, human liver microsomal fractions, and P450-selective chemical inhibitors. Glutathione (GSH) and dansylated GSH were used as trapping agents to detect reactive metabolite formation. Sunitinib metabolites were analyzed by liquid chromatography-tandem mass spectrometry. A putative quinoneimine-GSH conjugate (M5) of sunitinib was detected from trapping studies with GSH and dansyl-GSH in human liver microsomal incubations, and M5 was formed in an NADPH-dependent manner. Recombinant P450 1A2 generated the highest levels of defluorinated sunitinib (M3) and M5, with less formation by P450 3A4 and 2D6. P450 3A4 was the major enzyme forming the primary active metabolite N-desethylsunitinib (M1). In human liver microsomal incubations, P450 3A inhibitor ketoconazole reduced formation of M1 by 88%, while P450 1A2 inhibitor furafylline decreased generation of M5 by 62% compared to control levels. P450 2D6 and P450 3A inhibition also decreased M5 by 54 and 52%, respectively, compared to control. In kinetic assays, recombinant P450 1A2 showed greater efficiency for generation of M3 and M5 compared to that of P450 3A4 and 2D6. Moreover, M5 formation was 2.7-fold more efficient in human liver microsomal preparations from an individual donor with high P450 1A2 activity compared to a donor with low P450 1A2 activity. Collectively, these data suggest that P450 1A2 and 3A4 contribute to oxidative defluorination of sunitinib to generate a reactive, potentially toxic quinoneimine. Factors that alter P450 1A2 and 3A activity may affect patient risk for sunitinib toxicity.

Towards a System Approach to the Human Cytochrome P450 Ensemble: A New Strategy for Studying the Network of P450‐P450 Interactions in Human Liver Microsomes

The composition of the human cytochrome P450 ensemble is commonly recognized as a key factor determining the interindividual variability in drug metabolism. However, recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a direct correlation between the composition of the cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of interactions between multiple cytochrome P450 species co‐localized in the microsomal membrane. A change in the content of any particular P450 enzyme alters the landscape of protein‐protein interactions and thus affects drug metabolism in a complex, hard‐to‐predict manner. Therefore, a systematic exploration of the network of P450‐P450 interactions and identification of their functional consequences represents an issue of emerging importance for revealing the interrelationships between the profile of P450 expression and the profile of drug metabolism. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into the microsomes. Here we demonstrate high potential of this approach for creating preparations of HLM with altered composition of the P450 ensemble. An important characteristic of this strategy is its adaptability for incorporating genetically or chemically modified proteins. This includes the incorporation of proteins bearing fluorescent probes and light‐activating crosslinkers. Therefore, this strategy may be used for monitoring protein‐protein interactions in HLM with a variety of biophysical techniques, as well as for searching the partners of P450 protein‐protein interactions with the technique of molecular fishing.Here we report the use of our new approach for studying the effect of enriching the HLM with ethanol‐inducible CYP2E1, one of the the most populated P450 species in human liver. Incorporation of CYP2E1 into HLM results in an increase in the rate of metabolism of CYP2E1‐specific substrates, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Our studies with a series of substrates of drug‐metabolizing P450s showed that CYP2E1 incorporation results in pronounced alterations in the metabolism of substrates of several other P450 species present in HLM. In particular, we observed over 2‐fold increase in the rate of metabolism and a substantial decrease in the apparent KM observed with 3‐cyano‐7‐etoxycoumarin (CEC), the substrate of CYP1A2 and CYP2C19. This finding may indicate that the increase in CYP2E1 concentration results in redirecting the main route of CEC metabolism from CYP2C19 to CYP1A2, which is characterized with lower KM and higher turnover numbers with this substrate. Similarly, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax, observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5.Support or Funding InformationThis research was supported by grant R01‐GM114369 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacokinetic profile of PBRM in rodents, a first selective covalent inhibitor of 17β-HSD1 for breast cancer and endometriosis treatments

The development of a covalent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a promising approach for the treatment of hormone-dependent breast cancer and endometriosis. After reporting the steroid derivative PBRM as a first potent covalent inhibitor of 17β-HSD1 without estrogenic activity, we are now interested in studying its pharmaceutical behavior. The metabolism study in a human liver microsomal preparation showed a gradual transformation of PBRM into PBRM-O, an oxidized ketonic form of PBRM at position C17. Interestingly, PBRM-O also inhibits 17β-HSD1 and is not estrogenic in estrogen-sensitive T-47D cells. However, when PBRM was injected subcutaneously (sc) in mice, a very small proportion of PBRM-O was measured in a 24 h-time course experiment. A pharmacokinetic study in mice revealed suitable values for half-life (T1/2 = 3.4 h), clearance (CL = 2088 mL/h kg), distribution volume (Vz = 10.3 L/kg) and absolute bioavailability (F = 65%) when PBRM was injected sc at 14.7 mg/kg. A good F value of 33% was also obtained when PBRM was given orally. A tritiated version of PBRM, 3H-PBRM, was synthesized and used for an in vivo biodistribution study that showed its gradual accumulation in various mouse tissues (peak at 6 h) followed by elimination until complete disappearance after 72 h. Elimination was found to occur in feces (93%) and urine (7%) as revealed by a mass balance experiment. PBRM was also evaluated for its toxicity in mice and it was found to be very well tolerated after weekly sc administration (30–405 mg/kg for 8 weeks) or by po administration (300–900 mg/kg for 4 weeks). Overall, these experiments represent important steps in the preclinical characterization of the pharmaceutical behavior of PBRM, as well as for its translation to clinical trials.

Human plasma and urinary metabolic profiles of trimethylamine and trimethylamine <i>N</i>-oxide extrapolated using a simple physiologically based pharmacokinetic model

Trimethylamine, a dietary- and medicinal carnitine-derived amine, is extensively metabolized by liver to non-malodorous trimethylamine N-oxide. Although trimethylamine and trimethylamine N-oxide under daily dietary consumption or carnitine treatment are generally regarded as nontoxic, they have been, and remain, of toxicological and clinical interest because of their potential association with atherosclerosis. The aim of the current study was to model the pharmacokinetics of trimethylamine after oral administration of trimethylamine in humans and compare the results with reported measured values. Adjusted biomonitoring equivalents from rat studies based on reported plasma concentrations were scaled to human equivalents using known species allometric scaling factors. In vitro metabolic clearance data were obtained using rat and human liver microsomal preparations. Renal clearances in humans for trimethylamine and trimethylamine N-oxide were calculated with a clearance concept approach using reported 24-hr urinary excretion rates and assumed areas under plasma concentration curves. The resulting modeled plasma and urinary concentration curves by simple physiologically based pharmacokinetic models (or semi-physiological pharmacokinetic models) were consistent with reported concentrations. This study provides important information to help simulate human plasma levels of trimethylamine and trimethylamine N-oxide in trimethylamine loading tests and during treatment with prescribed medicinal l-carnitine, showing the similar range as that resulting from daily dietary foodstuff consumption along with little toxicological impacts. The present models could estimate relationship between plasma and urine concentrations of trimethylamine or trimethylamine N-oxide and the daily oral doses by both forward and reverse dosimetry from viewpoint of human risk assessment.

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) Is Atropselectively Metabolized to para-Hydroxylated Metabolites by Human Liver Microsomes.

Exposure to neurotoxic, chiral PCBs has been associated with neurodevelopmental disorders, but their metabolism in humans remains unexplored. We investigated the enantioselective metabolism of PCB 95 by human liver microsomes (HLMs) to potentially neurotoxic, hydroxylated metabolites (OH-PCBs). OH-PCB profiles formed in experiments with HLMs differed from metabolite profiles reported for rodent species. The second eluting atropisomer of 2,2′,3,5′,6-pentachlorobiphenyl-4′-ol, the major metabolite, was preferentially formed by all HLM preparations investigated. Differences in metabolite formation rates were observed with single donor HLMs. The metabolism of PCBs and its role in PCB-mediated neurodevelopmental disorders need to be further characterized.

Identification of selected in vitro generated phase-I metabolites of the steroidal selective androgen receptor modulator MK-0773 for doping control purposes.

Research into developing anabolic agents for various therapeutic purposes has been pursued for decades. As the clinical utility of anabolic-androgenic steroids has been found to be limited because of their lack of tissue selectivity and associated off-target effects, alternative drug entities have been designed and are commonly referred to as selective androgen receptor modulators (SARMs). While most of these SARMs are of nonsteroidal structure, the drug candidate MK-0773 comprises a 4-aza-steroidal nucleus. Besides the intended therapeutic use, SARMs have been found to be illicitly distributed and misused as doping agents in sport, necessitating frequently updated doping control analytical assays. As steroidal compounds reportedly undergo considerable metabolic transformations, the phase-I metabolism of MK-0773 was simulated using human liver microsomal (HLM) preparations and electrochemical conversion. Subsequently, major metabolic products were identified and characterized employing liquid chromatography-high-resolution/high- accuracy tandem mass spectrometry with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) as well as nuclear magnetic resonance (NMR) spectroscopy. MK-0773 produced numerous phase-I metabolites under the chosen in vitro incubation reactions, mostly resulting from mono- and bisoxygenation of the steroid. HLM yielded at least 10 monooxygenated species, while electrochemistry-based experiments resulted predominantly in three monohydroxylated metabolites. Elemental composition data and product ion mass spectra were generated for these analytes, ESI/APCI measurements corroborated the formation of at least two N-oxygenated metabolites, and NMR data obtained from electrochemistry-derived products supported structures suggested for three monohydroxylated compounds. Hereby, the hydroxylation of the A-ring located N- bound methyl group was found to be of particular intensity. In the absence of controlled elimination studies, the produced information enables the implementation of new target analytes into routine doping controls and expands the focus of anti-doping efforts concerning this new anabolic agent.

Comparative Metabolism of Batracylin (NSC 320846) and N-acetylbatracylin (NSC 611001) Using Human, Dog, and Rat Preparations In Vitro.

Batracylin is a heterocyclic arylamine topoisomerase inhibitor with preclinical anticancer activity. Marked species differences in sensitivity to the toxicity of batracylin were observed and attributed to differential formation of N-acetylbatracylin by N-acetyltransferase. A Phase I trial of batracylin in cancer patients with slow acetylator genotypes identified a dose-limiting toxicity of hemorrhagic cystitis. To further explore the metabolism of batracylin and N-acetylbatracylin across species, detailed studies using human, rat, and dog liver microsomal and hepatocyte preparations were conducted. Batracylin or N-acetylbatracylin was incubated with microsomes and hepatocytes from human, rat, and dog liver and with CYP-expressing human and rat microsomes. Substrates and metabolites were analyzed by HPLC with diode array, fluorescence, radiochemical, or mass spectrometric detection. Covalent binding of radiolabeled batracylin and N-acetylbatracylin to protein and DNA was measured in 3-methylcholanthrene-induced rat, human, and dog liver microsomes, and with recombinant human cytochromes P450. In microsomal preparations, loss of batracylin was accompanied by formation of one hydroxylated metabolite in human liver microsomes and five hydroxylated metabolites in rat liver microsomes. Six mono- or di-hydroxy-N-acetylbatracylin metabolites were found in incubations of this compound with 3MC rat liver microsomes. Hydroxylation sites were identified for some of the metabolites using deuterated substrates. Incubation with recombinant cytochromes P450 identified rCYP1A1, rCYP1A2, hCYP1A1 and hCYP1B1 as the major CYP isoforms that metabolize batracylin and N-acetylbatracylin. Glucuronide conjugates of batracylin were also identified in hepatocyte incubations. NADPH-dependent covalent binding to protein and DNA was detected in all batracylin and most N-acetylbatracylin preparations evaluated. Microsomal metabolism of batracylin and N-acetylbatracylin results in multiple hydroxylated products (including possible hydroxylamines) and glutathione conjugates. Incubation of batracylin with hepatocytes resulted in production primarily of glucuronides and other conjugates. There was no clear distinction in the metabolism of batracylin and N-acetylbatracylin across species that would explain the differential toxicity.

Evaluation of possible inhibition of human liver drug metabolizing cytochromes P450 by two new acetylcholinesterase oxime-type reactivators

Two non-symmetric bispyridine oxime – based reactivators of acetylcholinesterase enzyme (AChE), labeled as K027 (1-(4-carbamoylpyridinium)-3-(4-hydroxyiminomethylpyridinium)-propane dibromide) and K203 ((E)-1-(4- carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide) were tested for their potential to inhibit activities of human liver microsomal cytochromes P450 (CYP). Both oximes are very potent reactivators of organophosphate-inhibited AChE.An interaction of both compounds with CYP in human liver microsomal preparation was detected using difference spectroscopy. The compounds were shown to bind to CYP enzymes with spectral binding constants of 5.04 ± 1.79 nM (K027) and 5.2 ± 2.6 nM (K203). Enzymology studies were subsequently performed aimed at determining which of the nine most important CYP involved in drug is affected by this interaction. The results have shown no prominent inhibition of individual CYP activities with either compounds except in the case of CYP2E1 and K203. Diagnostic Dixon plot revealed that K203 acted as an uncompetitive inhibitor of CYP2E1. Inhibition of this activity however is not as prominent as to make a potent drug interaction likely. Hence, the interaction of K027 and K203 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be of prominent clinical importance and both compounds could be safely used in this respect as antidotes with low risk of drug interactions.

Antimalarial activity of 4-amidinoquinoline and 10-amidinobenzonaphthyridine derivatives.

Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug due to its being long-acting and having a high potency against blood stage P. falciparum (Pf). However, serious CNS toxicity of MQ has compromised its clinical value as a prophylaxis drug. Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS toxicity are urgently needed to combat this deadly human disease. In this study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were designed, prepared, and assessed to search for new therapeutic agents to replace CQ and MQ. The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ, and none were toxic in mice up to 160 mpk × 3. The best compound shows IC50 < 1 ng/mL against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K(+) channel blockage testing, negativity in the Ames test, and 5/5 cure @ <15 mpk × 3 in mice infected with Plasmodium berghei. In addition to these desirable pharmacological profiles, compound 13b, one of the most active compounds, is metabolically stable in both human and mouse liver microsomal preparations and has a plasma t(1/2) of 50 h in mice, which made it a good MQ replacement candidate.

PDE7B is involved in nandrolone decanoate hydrolysis in liver cytosol and its transcription is up-regulated by androgens in HepG2.

Most androgenic drugs are available as esters for a prolonged depot action. However, the enzymes involved in the hydrolysis of the esters have not been identified. There is one study indicating that PDE7B may be involved in the activation of testosterone enanthate. The aims are to identify the cellular compartments where the hydrolysis of testosterone enanthate and nandrolone decanoate occurs, and to investigate the involvement of PDE7B in the activation. We also determined if testosterone and nandrolone affect the expression of the PDE7B gene. The hydrolysis studies were performed in isolated human liver cytosolic and microsomal preparations with and without specific PDE7B inhibitor. The gene expression was studied in human hepatoma cells (HepG2) exposed to testosterone and nandrolone. We show that PDE7B serves as a catalyst of the hydrolysis of testosterone enanthate and nandrolone decanoate in liver cytosol. The gene expression of PDE7B was significantly induced 3- and 5- fold after 2 h exposure to 1 μM testosterone enanthate and nandrolone decanoate, respectively. These results show that PDE7B is involved in the activation of esterified nandrolone and testosterone and that the gene expression of PDE7B is induced by supra-physiological concentrations of androgenic drugs.