In vitro Phase I- and Phase II-Drug Metabolism in The Liver of Juvenile and Adult Göttingen Minipigs

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase II-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl-4-hydroxyanisole (BHA; a monofunctional Phase II-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5–9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [14C]-l,4-dichlorobenzene (DCB) and [14C]-1,4-dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a significant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase I-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D7 strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.

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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

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2021.114107 ◽

2021 ◽

pp. 114107

Author(s):

Emanuela Salviati ◽

Eduardo Sommella ◽

Albino Carrizzo ◽

Veronica Di Sarno ◽

Alessia Bertamino ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Metabolic Profiling ◽

Humulus Lupulus ◽

Humulus Lupulus L ◽

Bitter Acids ◽

Phase Ii Metabolites

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Intrathecal Mafosfamide: A Preclinical Pharmacology and Phase I Trial

Journal of Clinical Oncology ◽

10.1200/jco.2005.06.053 ◽

2005 ◽

Vol 23 (7) ◽

pp. 1555-1563 ◽

Cited By ~ 47

Author(s):

Susan M. Blaney ◽

Frank M. Balis ◽

Stacey Berg ◽

Carola A.S. Arndt ◽

Richard Heideman ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Phase I Trial ◽

Neoplastic Meningitis ◽

In Vitro Cytotoxicity ◽

Pharmacokinetic Studies ◽

Preclinical Studies ◽

Phase Ii Trials ◽

Cytotoxicity Studies

Purpose Preclinical studies of mafosfamide, a preactivated cyclophosphamide analog, were performed to define a tolerable and potentially active target concentration for intrathecal (IT) administration. A phase I and pharmacokinetic study of IT mafosfamide was performed to determine a dose for subsequent phase II trials. Patients and Methods In vitro cytotoxicity studies were performed in MCF-7, Molt-4, and rhabdomyosarcoma cell lines. Feasibility and pharmacokinetic studies were performed in nonhuman primates. These preclinical studies were followed by a phase I trial in patients with neoplastic meningitis. There were five dose levels ranging from 1 mg to 6.5 mg. Serial CSF samples were obtained for pharmacokinetic studies in a subset of patients with Ommaya reservoirs. Results The cytotoxic target exposure for mafosfamide was 10 μmol/L. Preclinical studies demonstrated that this concentration could be easily achieved in ventricular CSF after intraventricular dosing. In the phase I clinical trial, headache was the dose-limiting toxicity. Headache was ameliorated at 5 mg by prolonging the infusion rate to 20 minutes, but dose-limiting headache occurred at 6.5 mg dose with prolonged infusion. Ventricular CSF mafosfamide concentrations at 5 mg exceeded target cytotoxic concentrations after an intraventricular dose, but lumbar CSF concentrations 2 hours after the dose were less than 10 μmol/L. Therefore, a strategy to alternate dosing between the intralumbar and intraventricular routes was tested. Seven of 30 registrants who were assessable for response had a partial response, and six had stable disease. Conclusion The recommended phase II dose for IT mafosfamide, administered without concomitant analgesia, is 5 mg over 20 minutes.

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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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Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model

International Journal of Molecular Sciences ◽

10.3390/ijms21113998 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3998 ◽

Cited By ~ 1

Author(s):

Madalena Cipriano ◽

Pedro F Pinheiro ◽

Catarina O Sequeira ◽

Joana S Rodrigues ◽

Nuno G Oliveira ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

3D Model ◽

Toxicological Assessment ◽

Biotransformation Enzymes ◽

Net Flux ◽

Hepatotoxic Drug ◽

Phase I And Ii ◽

3D Spheroids

The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

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