Characterization of phase I and phase II metabolites of hop (Humulus lupulus L.) bitter acids: In vitro and in vivo metabolic profiling by UHPLC-Q-Orbitrap

BACKGROUND 3,4-Methylendioxymethamphetamine (MDMA) is excreted in human urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion. METHODS We used LC–high-resolution MS and GC-MS to reanalyze blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally. We determined median Cmax, tmax, first and last detection times, and total urinary recovery; calculated ratios of sulfates and glucuronides; and performed in vitro–in vivo correlations. RESULTS Phase II metabolites of 3,4-dihydroxymethamphetamine (DHMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substantial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate (R2 = 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA sulfate/glucuronide and DHMA 3-sulfate/4-sulfate ratios for the first 24 h were 2.0 and 5.3, respectively, in accordance with previous in vitro calculations from human liver microsomes and cytosol experiments. CONCLUSIONS Human MDMA urinary metabolites are primarily sulfates and glucuronides, with sulfates present in higher concentrations than glucuronides. This new knowledge may lead to improvements in urine MDMA and metabolite analysis in clinical and forensic toxicology, particularly for the performance of direct urine analysis.

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Effects of Temperature on Metabolism of Benzo[a]pyrene by Toadfish (Opsanus beta) Hepatocytes

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f90-096 ◽

1990 ◽

Vol 47 (4) ◽

pp. 831-837 ◽

Cited By ~ 22

Author(s):

Kenneth A. Gill ◽

Patrick J. Walsh

Keyword(s):

Phase I ◽

Phase Ii ◽

In Vivo Studies ◽

Kinetic Properties ◽

Opsanus Beta ◽

Effects Of Temperature ◽

Lower Temperature ◽

Phase Ii Metabolites

Prior in vivo studies of the effects of temperature on uptake, metabolism and excretion of benzo[a]pyrene (BaP) by the gulf toadfish (Opsanus beta) suggested that metabolism of BaP may be partially temperature insensitive under certain conditions. In the present study, hepatocytes were isolated from toadfish acclimated to 18 or 28 °C, and then cells were exposed to BaP at 18, 23, and 28 °C in vitro to more directly examine the effects of temperature on metabolism. Toadfish hepatocytes metabolized BaP to a variety of Phase I and Phase II metabolites in similar proportions to toadfish in vivo, with the exception that fewer Phase I metabolites were detected. A marked temperature dependence of metabolism of BaP to Phase II metabolites was noted between 18 and 28 °C at near saturating concentration of BaP (13 μg∙mL−1). Warm-acclimated toadfish displayed a lower temperature sensitivity than cold-acclimated toadfish (Q10, the ratio of the rate at T + 10 °C: rate at T = 1.85 and 2.65, respectively). However, at a lower, subsaturating concentration of BaP (5 μg∙mL−1), production of all metabolites showed a marked temperature independence. We speculate that this independence is the result of temperature effects on the kinetic properties of the enzymes of xenobiotic metabolism.

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Use of UPLC-HRMS/MS for In Vitro and In Vivo Metabolite Identification of Three Methylphenidate-derived New Psychoactive Substances

Journal of Analytical Toxicology ◽

10.1093/jat/bkz052 ◽

2019 ◽

Vol 44 (2) ◽

pp. 156-162

Author(s):

Sascha K Manier ◽

Sophia Niedermeier ◽

Jan Schäper ◽

Markus R Meyer

Keyword(s):

Mass Spectrometry ◽

Phase I ◽

Phase Ii ◽

Male Rats ◽

New Psychoactive Substances ◽

Psychoactive Substances ◽

Screening Procedures ◽

Phase Ii Metabolites

Abstract The distribution of so-called new psychoactive substances (NPS) as substitute for common drug of abuse was steadily increasing in the last years, but knowledge about their toxicodynamic and toxicokinetic properties is lacking. However, a comprehensive knowledge of their toxicokinetics, particularly their metabolism, is crucial for developing reliable screening procedures and to verify their intake, e.g., in case of intoxications. The aim of this study was therefore to tentatively identify the metabolites of the methylphenidate-derived NPS isopropylphenidate (isopropyl 2-phenyl-2-(2-piperidyl) acetate, IPH), 4-fluoromethylphenidate (methyl 2-(4-fluorophenyl)-2-(piperidin-2-yl) acetate, 4-FMPH) and 3,4-dichloromethylphenidate (methyl 2-(3,4-dichlorophenyl)-2-(piperidin-2-yl) acetate, 3,4-CTMP) using different in vivo and in vitro techniques and ultra-high performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS/MS). Urine samples of male rats were analyzed, and the transfer to human metabolism was done by using pooled human S9 fraction (pS9), which contains the microsomal fraction of liver homogenisate as well as its cytosol. UHPLC-HRMS/MS analysis of rat urine revealed 17 metabolites for IPH (14 phase I and 3 phase II metabolites), 13 metabolites were found for 4-FMPH (12 phase I metabolites and 1 phase II metabolite) and 7 phase I metabolites and no phase II metabolites were found for 3,4-CTMP. pS9 incubations additionally indicated that all investigated substances were primarily hydrolyzed, resulting in the corresponding carboxy metabolites. Finally, these carboxy metabolites should be used as additional analytical targets besides the parent compounds for comprehensive mass spectrometry–based screening procedures.

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Effects of Ofloxacin Administration on the Reliability of Urine Glucose Testing

Annals of Pharmacotherapy ◽

10.1177/106002809603000506 ◽

1996 ◽

Vol 30 (5) ◽

pp. 469-472

Author(s):

Tsong-Mei Tsai ◽

Brian F Shea ◽

Paul F Souney ◽

Fred G Volinsky ◽

Joseph M Scavone ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Tertiary Care ◽

Urine Samples ◽

Care Hospital ◽

Open Label ◽

Urine Glucose ◽

Glucose Testing

OBJECTIVE: TO study the effects of ofloxacin on the reliability of urine glucose testing. DESIGN: Open-label, nonrandomized. SETTING: A university-affiliated tertiary care hospital, ambulatory clinic. PARTICIPANTS: Ten healthy volunteers (8 men and 2 women) aged 22-39 years. MAIN OUTCOME MEASURES: Phase I (in vitro) involved the addition of selected amounts of ofloxacin to a set of standard 50-mL urine samples prepared to simulate glycosuria. Phase II (in vivo) involved the oral administration of ofloxacin 400 mg to 10 subjects. Urine was collected: (1) immediately predose, (2) pooled 0–4 hours postdose, and (3) pooled 4–8 hours postdose. Known glucose concentrations were then added to these samples. Clinitest and Diastix tests were performed on all samples. The accuracy of these tests in determining glucose concentrations was compared among urine samples taken before and after ofloxacin dosing. RESULTS: None of the ofloxacin concentrations in phase I (0,25,50, 100, 200,400, and 800 μg/mL) influenced these testing methods at the urine glucose concentrations of 0.0%, 0.5%, 1%, and 2%. Likewise, the accuracy of these two tests was unaffected by ofloxacin administration in phase II. CONCLUSIONS: In single-dose administration, ofloxacin does not interfere with Clinitest or Diastix for determining urine glucose concentrations. Supported by a grant from the RW Johnson Pharmaceutical Research Institute. Presented in abstract form at the American College of Clinical Pharmacy 1994 Winter Practice and Research Forum, February 6–9, 1994, San Diego. CA.

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Gas Chromatographic Determination of Poly chlorinated Biphenyls (as Aroclor 1254) in Serum: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/72.4.649 ◽

1989 ◽

Vol 72 (4) ◽

pp. 649-659

Author(s):

Virlyn W Burse ◽

Margaret P Korver ◽

Larry L Needham ◽

Chester R Lapeza ◽

Elizabeth L Chester R ◽

...

Keyword(s):

Phase I ◽

Silica Gel ◽

Phase Ii ◽

Collaborative Study ◽

Chlorinated Hydrocarbons ◽

Aroclor 1254 ◽

Relative Standard

Abstract A gas chromatographic-electron capture detection method for determining the concentration of polychlorinated biphenyls (PCBs) as Aroclor 1254 (AR 1254) in serum was evaluated through a 2-phase collaborative study. In Phase I, each collaborator's lot of Woelm silica gel (70-150 mesh) was evaluated for elution and recovery of AR 1254, which had been added in vitro at 25 ng/mL to a serum extract. In Phase II, each collaborator analyzed a series of bovine serum samples that contained the following: (1) in vitro-spiked AR 1254; (2) in vivo AR 1254 and 8 in vitro-spiked chlorinated hydrocarbons; (3) in vivo AR 1254 only; (4) 8 in vitro-spiked chlorinated hydrocarbons only; and (5) neither AR 1254 nor chlorinated hydrocarbons above the detection limit of the method. In Phase I, the average recovery of AR 1254 from silica gel for the 6 collaborators was 87.9 ± 15.44% (mean ± 1 SD; N = 18; range = 52.3-105.8%). In Phase II, the analysis of in vitro spikes of AR 1254 in serum at 8.58,16.8, 41.8, and 84.3 ppb gave mean (X) interlaboratory recoveries of 89.0, 83.3, 79.4, and 76.9%, respectively, with within-laboratory (repeatability) relative standard deviations (RSDr) of 18.8, 20.5, 10.2, and 14.1%, respectively, and among-laboratory (reproducibility) relative standard deviations (RSDR) of 21.5, 21.1, 14.6, and 20.8%, respectively. The determination of in vivo AR 1254 in samples containing approximately 10, 25, 50, and 100 ng/mL of AR 1254 resulted in interlaboratory means of 10,22,39, and 79 ng/mL, respectively, with RSDr = 6.7,9.7,6.4, and 5.8%, respectively, and RSDR = 20.6,16.0, 10.9, and 10.3%, respectively. The precision of the method for incurred AR 1254 showed a maximum RSDr of less than 10% and a maximum RSDR of less than 21% for a concentration range of 10-100 ng/mL. The accuracy of the method as demonstrated by the mean recovery of in vitro-spiked AR 1254 over a concentration range of 8.58-84.3 ng/mL was 82.2%. The method has been approved interim official first action.

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Novel organic arsenic molecule darinaparsin: Development of IV and oral forms

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.8501 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 8501-8501 ◽

Cited By ~ 2

Author(s):

I. Lossos ◽

M. D. Craig ◽

M. S. Tallman ◽

R. V. Boccia ◽

P. R. Conkling ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Phase Ii Study ◽

Complete Response ◽

Median Number ◽

Two Phase ◽

Phase I Studies ◽

Advanced Malignancies

8501 Background: Darinaparsin (ZIO-101) is a novel organic arsenical active against diverse cancers in vitro, and in vivo. Darinaparsin i.v. activity in lymphoma is being evaluated in a phase II study. Darinaparsin is orally bioavailable; the oral form is being investigated in two phase I studies in patients with advanced malignancies. Methods: Phase II trial is being conducted in patients diagnosed with advanced lymphomas who had ≥ 1 prior therapy. Patients receive 300 mg/m2/day of darinaparsin i.v. for 5 consecutive days every 28 days. Efficacy and safety are evaluated by standard criteria. Phase I oral dose escalation studies are being conducted in patients with advanced malignancies and explore safety, MTD, DLTs and preliminary efficacy of continuous and intermittent dosing schedules. Starting continuous dose is 100 mg BID for 3 weeks with 1 week rest, starting intermittent dose is 300 mg twice weekly for 3 weeks followed by 1 week rest. Results: The phase II study has accrued 28 lymphoma patients (21 non-Hodgkin's, 7 Hodgkin's); median age at baseline 61 years, ECOG ≤2, median number of prior therapies 3. Seventeen subjects have received at least 2 cycles of darinaparsin and are evaluable for efficacy. Of these, 1 subject (PTCL) has achieved a complete response, 3 - partial responses (2 marginal zone, 1 Hodgkin's), and 4 stable disease (2 PTCL, 1 DLBCL, 1 Hodgkin's). A total of 63 cycles of darinaparsin have been administered to subjects with lymphoma. No Gr. 3 or higher drug-related AEs were reported. Two SAEs were considered possibly drug-related (fall; neutropenic fever). Phase I studies accrued 35 patients; median age at baseline 58 years, ECOG ≤2, median number of prior therapies 3. Predominant tumor types include: colorectal (17), pancreatic (3), NHL (3). Current darinaparsin dose levels: continuous 200 mg BID, 2× weekly 900 mg. Of 18 patients evaluable for efficacy, 10 demonstrate SD ≥ 3 cycles. Oral darinaparsin bioavailability is 58%. Drug-related AEs include nausea/vomiting, fatigue, decreased appetite/anorexia. Conclusions: Darinaparsin is active in heavily pretreated patients with advanced lymphoma and has been very well tolerated. Oral darinaparsin is also well tolerated, and shows early activity. [Table: see text]

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ME-143, a novel inhibitor of tumor-specific NADH oxidase (tNOX): Results from a first-in-human phase I study.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.3067 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 3067-3067

Author(s):

Carla Kurkjian ◽

Shubham Pant ◽

Howard A. Burris ◽

Johanna C. Bendell ◽

Suzanne Fields Jones ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Phase I Study ◽

Nadh Oxidase ◽

Testicular Atrophy ◽

Pharmacokinetic Analysis ◽

In Vivo Models ◽

Human Dose

3067 Background: ME-143, a second generation isoflavone-derived compound, specifically binds tNOX, shifting the ceramide-S1P equilibrium and resulting in prompt apoptosis via pleotrophic caspase activation. A first generation compound, phenoxodiol (PXD), showed promising phase II activity with cisplatin in ovarian cancer. ME-143 is broadly active against human cancers in both in vitro and in vivo models, with IC50’s 1-2 logs lower than PXD. Toxicology studies up to 140mg/kg (human dose equivalent ~23mg/kg) showed no STD level. The only significant findings were dose dependent hypospermia and testicular atrophy in rats. We report clinical and PK results from the first-in-human phase I study. Methods: A 3+3 dose escalation design was used with 4 dose cohorts: 2.5, 5, 10, and 20mg/kg IV over 30 minutes weekly times 3, followed by a 1 week break, and then continuous weekly dosing in patients with advanced solid tumors. Dense PK sampling was performed at 0, 5, 10, 20, 30, 60, 90, 120, 180, 240, 300, 360 minutes, and 24 hours post-infusion day 1 and 15 of the first treatment cycle. Results: To date, 9 patients have been enrolled, 3 in each of the first 3 cohorts. Median time on treatment is 56 days (range 6 to 62). Five patients have discontinued protocol therapy, all due to PD. No DLT’s have been observed. Related AE’s include grade 1: myalgia (1), anorexia (1), fatigue (2), headache (1), diarrhea (1), vomiting (1) and grade 2: fatigue (1). Preliminary PK analyses in the first 2 cohorts is shown in table below. Conclusions: ME-143 appears to be well tolerated when administered IV. Preliminary PK data suggest that drug levels achieve target concentration extrapolated from pre-clinical studies (AUC0-t ~10mg*hr/mL) and exceed levels obtained with IV PXD in the phase II study (AUC0-t ~2mg*hr/mL) . Updated clinical data from all planned dose cohorts, including the final pharmacokinetic analysis and planned phase II dose, will be presented. [Table: see text]

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