Shaokun Pang - Academia.edu (original) (raw)

Papers by Shaokun Pang

Research paper thumbnail of 3-Alpha-hydroxy 21-n-heteroaryl-pregnane derivatives for modulation of brain excitability and a process for the production thereof

Research paper thumbnail of In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22

The AAPS Journal, 2016

In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blend... more In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 μM FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 μM was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the same metabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after β-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.

Research paper thumbnail of Metabolic characterization of AH-7921, a synthetic opioid designer drug: in vitro metabolic stability assessment and metabolite identification, evaluation of in silico prediction, and in vivo confirmation

Drug Testing and Analysis, 2015

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid a... more AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non-fatal and fatal intoxications. To comprehensively study AH-7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH-7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH-7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time-of-flight high-resolution mass spectrometry (MS). High-resolution full scan MS and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot™ (SCIEX) using multiple data processing algorithms. The presence of AH-7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite™ (Molecular Discovery). AH-7921 in vitro half-life was 13.5 ± 0.4 min. We identified 12 AH-7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di-demethylated, N-hydroxylated and glucuronidated, eluted after AH-7921 and was the most abundant metabolite in non-hydrolyzed urine. MetaSite™ correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di-desmethyl AH-7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH-7921 intake. Copyright © 2015 John Wiley & Sons, Ltd.

Research paper thumbnail of High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

Clinical Chemistry, 2015

Despite increasing prevalence of novel psychoactive substances, no human metabolism data are curr... more Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600(+)) to identify optimal metabolite markers after incubating 10 μ mol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids.

Research paper thumbnail of Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry

Forensic Toxicology, 2015

Research paper thumbnail of Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

The AAPS journal, Jan 28, 2015

Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferabl... more Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs' major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF ...

Research paper thumbnail of Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

Bioanalysis, 2014

Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to c... more Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results: With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabo...

Research paper thumbnail of Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS

Bioanalysis, 2014

Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively me... more Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS-8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides...

Research paper thumbnail of High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), using cryopreserved human hepatocytes and assessment of metabolic stability with human liv

Drug Testing and Analysis, 2014

yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal... more yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on Internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolites. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine.

Research paper thumbnail of Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls

Toxicology in Vitro, 2002

The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction... more The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction of human cytochrome P450 1B1 (CYP1B1), an estradiol 4-hydroxylase, were investigated in MDA-MB-231 breast cancer cells. Three independent quantitative assays were used, in which the rates of estrogen metabolism, the levels of the CYP1B1 and CYP1A1 mRNAs, and luciferase activities under the control of the CYP1B1 promoter were measured. Of the congeners investigated, 3,4,4 0 ,5-tetrachlorobiphenyl (PCB 81), 3,3 0 ,4,4 0 ,5-pentachlorobiphenyl (PCB 126), 3,4 0 ,5-trichlorobiphenyl (PCB 39) and 3,3 0 ,4,5-tetrachlorobiphenyl (PCB 78) were the most potent in each assay, causing four to 10-fold increases in response. Exposure to 3,3 0 ,4,4 0 ,5,5 0 -hexachlorobiphenyl (PCB 169) resulted in elevated CYP1B1 mRNA and increased CYP1B1-promoter driven luciferase activity, but caused depressed rather than elevated rates of E 2 metabolism due to inhibition of CYP1B1. The relative magnitudes of CYP1B1 induction by the PCB congeners, as determined by the three assays, were in close agreement, with the exception noted for PCB 169. These results indicate that PCB structure-activity relationships for the induction of human CYP1B1 are similar to those observed for human CYP1A1, but differ somewhat from what has been reported for induction of rat CYP1A1. #

Research paper thumbnail of Simultaneous determination of etoposide and its catechol metabolite in the plasma of pediatric patients by liquid chromatography/tandem mass spectrometry

Journal of Mass Spectrometry, 2001

The anticancer drug etoposide is associated with leukemias with MLL gene translocations and other... more The anticancer drug etoposide is associated with leukemias with MLL gene translocations and other translocations as a treatment complication. The genotype of cytochrome P450 3A4 (CYP3A4), which converts etoposide to its catechol metabolite, influences the risk. In order to perform pharmacokinetic studies aimed at further elucidation of the translocation mechanism, we have developed and validated a liquid chromatography/electrospray/tandem mass spectrometry assay for the simultaneous analysis of etoposide and its catechol metabolite in human plasma. The etoposide analog teniposide was used as the internal standard. Liquid chromatography was performed on a YMC ODS-AQ column. Simultaneous determination of etoposide and its catechol metabolite was achieved using a small volume of plasma, so that the method is suitable for pediatric patients. The limits of detection were 200 ng ml(-1) etoposide and 10 ng ml(-1) catechol metabolite in human plasma and 25 ng ml(-1) etoposide and 2.5 ng ml(-1) catechol metabolite in protein-free plasma, respectively. Acceptable precision and accuracy were obtained for concentrations in the calibration curve ranges 0.2--100 microg ml(-1) etoposide and 10--5000 ng ml(-1) catechol metabolite in human plasma. Acceptable precision and accuracy for protein-free human plasma in the range 25--15 000 ng ml(-1) etoposide and 2.5--1500 ng ml(-1) etoposide catechol were also achieved. This method was selective and sensitive enough for the simultaneous quantitation of etoposide and its catechol as a total and protein-free fraction in small plasma volumes from pediatric cancer patients receiving etoposide chemotherapy. A pharmacokinetic model has been developed for future studies in large populations.

Research paper thumbnail of Characterization of an Etoposide-Glutathione Conjugate Derived from Metabolic Activation by Human Cytochrome P450

Current Drug Metabolism, 2006

Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also k... more Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH-6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.

Research paper thumbnail of First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

Research paper thumbnail of SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells

Carcinogenesis, 2000

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estrad... more In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

Research paper thumbnail of Etoposide Metabolites Enhance DNA Topoisomerase II Cleavage near Leukemia-Associated MLL Translocation Breakpoints †

Biochemistry, 2001

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by e... more Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIR, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

Research paper thumbnail of Inductive and inhibitory effects of non-ortho-substituted polychlorinated biphenyls on estrogen metabolism and human cytochromes P450 1A1 and 1B1

Biochemical Pharmacology, 1999

The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytoch... more The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytochrome P450 1A1 (CYP1A1), a 17beta-estradiol (E2) 2-hydroxylase, and P450 1B1 (CYP1B1), an E2 4-hydroxylase, were investigated in HepG2 and MCF-7 cells. Elevated rates of 2- and 4-methoxyestradiol (2- and 4-MeOE2) formation in PCB-treated cultures were measured as activities of CYP1A1 and CYP1B1, respectively. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,4',5-trichlorobiphenyl (PCB 39) caused marked stimulation of E2 metabolism in both cell lines. Northern blot analyses confirmed that exposure of MCF-7 cells to PCBs 81, 126, and 39 caused highly elevated levels of the CYP1A1 and CYP1B1 mRNAs. Exposure of MCF-7 cells to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated levels of the CYP1A1 and CYP1B1 mRNAs, but did not cause elevated rates of E2 metabolism; rather, 4-MeOE2 production was depressed to below control levels in PCB 169-treated cultures. PCB 169 also inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced 4-MeOE2 and, to a lesser extent, 2-MeOE2 production in MCF-7 cells, as did PCB 126 and several other congeners. In microsomal assays, inhibition of cDNA-expressed human CYP1B1 by PCBs 169 and 126 was demonstrated. These studies with one subgroup of PCBs, the non-ortho-substituted congeners, underscore the complexity and diversity of effects of PCBs, as individual congeners were found both to induce expression and to inhibit activity of human CYP1B1 and CYP1A1.

Research paper thumbnail of Metabolism of synthetic cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human hepatocyte incubation and high-resolution mass spectrometry

Analytical and Bioanalytical Chemistry, 2014

Background PB-22 (1-pentyl-8-quinolinyl ester-1H-indole-3carboxylic acid) and 5F-PB-22 (1-(5-fluo... more Background PB-22 (1-pentyl-8-quinolinyl ester-1H-indole-3carboxylic acid) and 5F-PB-22 (1-(5-fluoropentyl)-8quinolinyl ester-1H-indole-3-carboxylic acid) are new synthetic cannabinoids with a quinoline substructure and the first marketed substances with an ester bond linkage. No human metabolism data are currently available, making it difficult to document PB-22 and 5F-PB-22 intake from urine analysis, and complicating assessment of the drugs' pharmacodynamic and toxicological properties. Methods We incubated 10 μmol/l PB-22 and 5F-PB-22 with pooled cryopreserved human hepatocytes up to 3 h and analyzed samples on a TripleTOF 5600+ high-resolution mass spectrometer. Data were acquired via TOF scan, followed by information-dependent acquisition triggered product ion scans with mass defect filtering (MDF). The accurate mass full scan MS and MS/MS metabolite datasets were analyzed with multiple data processing techniques, including MDF, neutral loss and product ion filtering. Results The predominant metabolic pathway for PB-22 and 5F-PB-22 was ester hydrolysis yielding a wide variety of (5-fluoro)pentylindole-3-carboxylic acid metabolites. Twenty metabolites for PB-22 and 22 metabolites for 5F-PB-22 were identified, with the majority generated by oxidation with or without glucuronidation. For 5F-PB-22, oxidative defluorination occurred forming PB-22 metabolites. Both compounds underwent epoxide formation followed by internal hydrolysis and also produced a cysteine conjugate. Conclusion Human hepatic metabolic profiles were generated for PB-22 and 5F-PB-22. Pentylindole-3-carboxylic acid, hydroxypentyl-PB-22 and PB-22 pentanoic acid for PB-22, and 5′-fluoropentylindole-3-carboxylic acid, PB-22 pentanoic acid and the hydroxy-5F-PB-22 metabolite with oxidation at the quinoline system for 5F-PB-22 are likely the best targets to incorporate into analytical methods for urine to document PB-22 and 5F-PB-22 intake.

Research paper thumbnail of First Characterization of AKB-48 Metabolism, a Novel Synthetic Cannabinoid, Using Human Hepatocytes and High-Resolution Mass Spectrometry

The AAPS Journal, 2013

Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, ne... more Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB 1 receptor binding affinity than CB 2 . These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono-and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

Research paper thumbnail of Plasma Etoposide Catechol Increases in Pediatric Patients Undergoing Multiple-Day Chemotherapy with Etoposide

Clinical Cancer Research, 2004

The purpose of this research was to determine inter-and intrapatient differences in the pharmacok... more The purpose of this research was to determine inter-and intrapatient differences in the pharmacokinetic profiles of etoposide and its genotoxic catechol metabolite during conventional multiple-day dosing of etoposide in pediatric patients.

Research paper thumbnail of 3-Alpha-hydroxy 21-n-heteroaryl-pregnane derivatives for modulation of brain excitability and a process for the production thereof

Research paper thumbnail of In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22

The AAPS Journal, 2016

In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blend... more In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 μM FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 μM was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the same metabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after β-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.

Research paper thumbnail of Metabolic characterization of AH-7921, a synthetic opioid designer drug: in vitro metabolic stability assessment and metabolite identification, evaluation of in silico prediction, and in vivo confirmation

Drug Testing and Analysis, 2015

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid a... more AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non-fatal and fatal intoxications. To comprehensively study AH-7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH-7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH-7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time-of-flight high-resolution mass spectrometry (MS). High-resolution full scan MS and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot™ (SCIEX) using multiple data processing algorithms. The presence of AH-7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite™ (Molecular Discovery). AH-7921 in vitro half-life was 13.5 ± 0.4 min. We identified 12 AH-7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di-demethylated, N-hydroxylated and glucuronidated, eluted after AH-7921 and was the most abundant metabolite in non-hydrolyzed urine. MetaSite™ correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di-desmethyl AH-7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH-7921 intake. Copyright © 2015 John Wiley & Sons, Ltd.

Research paper thumbnail of High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

Clinical Chemistry, 2015

Despite increasing prevalence of novel psychoactive substances, no human metabolism data are curr... more Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600(+)) to identify optimal metabolite markers after incubating 10 μ mol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids.

Research paper thumbnail of Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry

Forensic Toxicology, 2015

Research paper thumbnail of Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

The AAPS journal, Jan 28, 2015

Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferabl... more Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs' major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF ...

Research paper thumbnail of Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

Bioanalysis, 2014

Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to c... more Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results: With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabo...

Research paper thumbnail of Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS

Bioanalysis, 2014

Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively me... more Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS-8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides...

Research paper thumbnail of High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), using cryopreserved human hepatocytes and assessment of metabolic stability with human liv

Drug Testing and Analysis, 2014

yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal... more yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on Internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolites. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine.

Research paper thumbnail of Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls

Toxicology in Vitro, 2002

The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction... more The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction of human cytochrome P450 1B1 (CYP1B1), an estradiol 4-hydroxylase, were investigated in MDA-MB-231 breast cancer cells. Three independent quantitative assays were used, in which the rates of estrogen metabolism, the levels of the CYP1B1 and CYP1A1 mRNAs, and luciferase activities under the control of the CYP1B1 promoter were measured. Of the congeners investigated, 3,4,4 0 ,5-tetrachlorobiphenyl (PCB 81), 3,3 0 ,4,4 0 ,5-pentachlorobiphenyl (PCB 126), 3,4 0 ,5-trichlorobiphenyl (PCB 39) and 3,3 0 ,4,5-tetrachlorobiphenyl (PCB 78) were the most potent in each assay, causing four to 10-fold increases in response. Exposure to 3,3 0 ,4,4 0 ,5,5 0 -hexachlorobiphenyl (PCB 169) resulted in elevated CYP1B1 mRNA and increased CYP1B1-promoter driven luciferase activity, but caused depressed rather than elevated rates of E 2 metabolism due to inhibition of CYP1B1. The relative magnitudes of CYP1B1 induction by the PCB congeners, as determined by the three assays, were in close agreement, with the exception noted for PCB 169. These results indicate that PCB structure-activity relationships for the induction of human CYP1B1 are similar to those observed for human CYP1A1, but differ somewhat from what has been reported for induction of rat CYP1A1. #

Research paper thumbnail of Simultaneous determination of etoposide and its catechol metabolite in the plasma of pediatric patients by liquid chromatography/tandem mass spectrometry

Journal of Mass Spectrometry, 2001

The anticancer drug etoposide is associated with leukemias with MLL gene translocations and other... more The anticancer drug etoposide is associated with leukemias with MLL gene translocations and other translocations as a treatment complication. The genotype of cytochrome P450 3A4 (CYP3A4), which converts etoposide to its catechol metabolite, influences the risk. In order to perform pharmacokinetic studies aimed at further elucidation of the translocation mechanism, we have developed and validated a liquid chromatography/electrospray/tandem mass spectrometry assay for the simultaneous analysis of etoposide and its catechol metabolite in human plasma. The etoposide analog teniposide was used as the internal standard. Liquid chromatography was performed on a YMC ODS-AQ column. Simultaneous determination of etoposide and its catechol metabolite was achieved using a small volume of plasma, so that the method is suitable for pediatric patients. The limits of detection were 200 ng ml(-1) etoposide and 10 ng ml(-1) catechol metabolite in human plasma and 25 ng ml(-1) etoposide and 2.5 ng ml(-1) catechol metabolite in protein-free plasma, respectively. Acceptable precision and accuracy were obtained for concentrations in the calibration curve ranges 0.2--100 microg ml(-1) etoposide and 10--5000 ng ml(-1) catechol metabolite in human plasma. Acceptable precision and accuracy for protein-free human plasma in the range 25--15 000 ng ml(-1) etoposide and 2.5--1500 ng ml(-1) etoposide catechol were also achieved. This method was selective and sensitive enough for the simultaneous quantitation of etoposide and its catechol as a total and protein-free fraction in small plasma volumes from pediatric cancer patients receiving etoposide chemotherapy. A pharmacokinetic model has been developed for future studies in large populations.

Research paper thumbnail of Characterization of an Etoposide-Glutathione Conjugate Derived from Metabolic Activation by Human Cytochrome P450

Current Drug Metabolism, 2006

Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also k... more Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH-6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.

Research paper thumbnail of First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

Research paper thumbnail of SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells

Carcinogenesis, 2000

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estrad... more In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

Research paper thumbnail of Etoposide Metabolites Enhance DNA Topoisomerase II Cleavage near Leukemia-Associated MLL Translocation Breakpoints †

Biochemistry, 2001

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by e... more Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIR, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

Research paper thumbnail of Inductive and inhibitory effects of non-ortho-substituted polychlorinated biphenyls on estrogen metabolism and human cytochromes P450 1A1 and 1B1

Biochemical Pharmacology, 1999

The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytoch... more The effects of a series of non-ortho-substituted polychlorinated biphenyls (PCBs) on human cytochrome P450 1A1 (CYP1A1), a 17beta-estradiol (E2) 2-hydroxylase, and P450 1B1 (CYP1B1), an E2 4-hydroxylase, were investigated in HepG2 and MCF-7 cells. Elevated rates of 2- and 4-methoxyestradiol (2- and 4-MeOE2) formation in PCB-treated cultures were measured as activities of CYP1A1 and CYP1B1, respectively. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,4',5-trichlorobiphenyl (PCB 39) caused marked stimulation of E2 metabolism in both cell lines. Northern blot analyses confirmed that exposure of MCF-7 cells to PCBs 81, 126, and 39 caused highly elevated levels of the CYP1A1 and CYP1B1 mRNAs. Exposure of MCF-7 cells to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated levels of the CYP1A1 and CYP1B1 mRNAs, but did not cause elevated rates of E2 metabolism; rather, 4-MeOE2 production was depressed to below control levels in PCB 169-treated cultures. PCB 169 also inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced 4-MeOE2 and, to a lesser extent, 2-MeOE2 production in MCF-7 cells, as did PCB 126 and several other congeners. In microsomal assays, inhibition of cDNA-expressed human CYP1B1 by PCBs 169 and 126 was demonstrated. These studies with one subgroup of PCBs, the non-ortho-substituted congeners, underscore the complexity and diversity of effects of PCBs, as individual congeners were found both to induce expression and to inhibit activity of human CYP1B1 and CYP1A1.

Research paper thumbnail of Metabolism of synthetic cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human hepatocyte incubation and high-resolution mass spectrometry

Analytical and Bioanalytical Chemistry, 2014

Background PB-22 (1-pentyl-8-quinolinyl ester-1H-indole-3carboxylic acid) and 5F-PB-22 (1-(5-fluo... more Background PB-22 (1-pentyl-8-quinolinyl ester-1H-indole-3carboxylic acid) and 5F-PB-22 (1-(5-fluoropentyl)-8quinolinyl ester-1H-indole-3-carboxylic acid) are new synthetic cannabinoids with a quinoline substructure and the first marketed substances with an ester bond linkage. No human metabolism data are currently available, making it difficult to document PB-22 and 5F-PB-22 intake from urine analysis, and complicating assessment of the drugs' pharmacodynamic and toxicological properties. Methods We incubated 10 μmol/l PB-22 and 5F-PB-22 with pooled cryopreserved human hepatocytes up to 3 h and analyzed samples on a TripleTOF 5600+ high-resolution mass spectrometer. Data were acquired via TOF scan, followed by information-dependent acquisition triggered product ion scans with mass defect filtering (MDF). The accurate mass full scan MS and MS/MS metabolite datasets were analyzed with multiple data processing techniques, including MDF, neutral loss and product ion filtering. Results The predominant metabolic pathway for PB-22 and 5F-PB-22 was ester hydrolysis yielding a wide variety of (5-fluoro)pentylindole-3-carboxylic acid metabolites. Twenty metabolites for PB-22 and 22 metabolites for 5F-PB-22 were identified, with the majority generated by oxidation with or without glucuronidation. For 5F-PB-22, oxidative defluorination occurred forming PB-22 metabolites. Both compounds underwent epoxide formation followed by internal hydrolysis and also produced a cysteine conjugate. Conclusion Human hepatic metabolic profiles were generated for PB-22 and 5F-PB-22. Pentylindole-3-carboxylic acid, hydroxypentyl-PB-22 and PB-22 pentanoic acid for PB-22, and 5′-fluoropentylindole-3-carboxylic acid, PB-22 pentanoic acid and the hydroxy-5F-PB-22 metabolite with oxidation at the quinoline system for 5F-PB-22 are likely the best targets to incorporate into analytical methods for urine to document PB-22 and 5F-PB-22 intake.

Research paper thumbnail of First Characterization of AKB-48 Metabolism, a Novel Synthetic Cannabinoid, Using Human Hepatocytes and High-Resolution Mass Spectrometry

The AAPS Journal, 2013

Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, ne... more Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB 1 receptor binding affinity than CB 2 . These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono-and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

Research paper thumbnail of Plasma Etoposide Catechol Increases in Pediatric Patients Undergoing Multiple-Day Chemotherapy with Etoposide

Clinical Cancer Research, 2004

The purpose of this research was to determine inter-and intrapatient differences in the pharmacok... more The purpose of this research was to determine inter-and intrapatient differences in the pharmacokinetic profiles of etoposide and its genotoxic catechol metabolite during conventional multiple-day dosing of etoposide in pediatric patients.