In Vitro Rat Hepatic Metabolism of n-Alkanes: Nonane, Decane, and Tetradecane

2007 ◽  
Vol 26 (4) ◽  
pp. 325-330 ◽  
Author(s):  
Sathanandam S. Anand ◽  
Jerry L. Campbell ◽  
Jeffrey W. Fisher
Keyword(s):  
Metabolic Rate ◽  
Rate Constants ◽  
Parent Compound ◽  
Liver Microsomes ◽  
Complex Mixture ◽  
Microsomal Protein ◽  
Intrinsic Clearance ◽  
Metabolic Clearance ◽  
Pbpk Models

Jet propellant 8 (JP-8) jet fuel is a complex mixture of aromatic and aliphatic hydrocarbons. The aim of this study was to determine in vitro metabolic rate constants for semivolatile n-alkanes, nonane (C9), decane (C10), and tetradecane (C14), by rat liver microsomal oxidation. The metabolism was assessed by measuring the disappearance of parent compound by gas chromatography. Various concentrations of n-alkanes were incubated with liver microsomes from adult male F-344 rats. Nonlinear kinetic constants for nonane and decane were Vmax (nmol/mg protein/min) = 7.26 ± 0.20 and 2.80 ± 0.35, respectively, and KM ( μM) = 294.83 ± 68.67 and 398.70 ± 42.70, respectively. Metabolic capacity as assessed by intrinsic clearance ( Vmax/ KM) was ~four-fold higher for nonane (0.03 ± 0.005) than for decane (0.007 ± 0.001). There was no appreciable metabolism of tetradecane even with higher microsomal protein concentration and longer incubation time. These results show a negative correlation between metabolic clearance and chain length of n-alkanes. These metabolic rate constants will be used to update existing physiologically based pharmacokinetic (PBPK) models for nonane and decane as part of developing a PBPK model for JP-8.

In Vitro Metabolism of E2, G2: Novel Bile Acid-Coupling Camptothecin Analogues, in Rat Liver Microsomes

2021 ◽  
Vol 16 ◽  
Author(s):  
Xiangli Zhang ◽  
Qin Shen ◽  
Yi Wang ◽  
Leilei Zhou ◽  
Qi Weng ◽  
...  
Keyword(s):  
Bile Acid ◽  
Phase I ◽  
Rat Liver ◽  
Deoxycholic Acid ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Rat Liver Microsomes ◽  
Camptothecin Analogues

Background: E2 (Camptothecin - 20 (S) - O- glycine - deoxycholic acid), and G2 (Camptothecin - 20 (S) - O - acetate - deoxycholic acid) are two novel bile acid-derived camptothecin analogues by introducing deoxycholic acid in 20-position of CPT(camptothecin) with greater anticancer activity and lower systematic toxicity in vivo. Objective: We aimed to investigate the metabolism of E2 and G2 by Rat Liver Microsomes (RLM). Methods: Phase Ⅰ and Phase Ⅱ metabolism of E2 and G2 in rat liver microsomes were performed respectively, and the mixed incubation of phase I and phase Ⅱ metabolism of E2 and G2 was also processed. Metabolites were identified by liquid chromatographic/mass spectrometry. Results: The results showed that phase I metabolism was the major biotransformation route for both E2 and G2. The isoenzyme involved in their metabolism had some difference. The intrinsic clearance of G2 was 174.7mL/min. mg protein, more than three times of that of E2 (51.3 mL/min . mg protein), indicating a greater metabolism stability of E2. 10 metabolites of E2 and 14 metabolites of G2 were detected, including phase I metabolites (mainly via hydroxylations and hydrolysis) and their further glucuronidation products. Conclusion: These findings suggested that E2 and G2 have similar biotransformation pathways except some difference in the hydrolysis ability of the ester bond and amino bond from the parent compounds, which may result in the diversity of their metabolism stability and responsible CYPs(Cytochrome P450 proteins).

scholarly journals In vitro and in vivo evaluation of hypothermia on pharmacokinetics and pharmacodynamics of nimodipine in rabbits

2017 ◽  
Vol 46 (1) ◽  
pp. 335-347 ◽  
Author(s):  
Yu-xing Fei ◽  
Tian-hong Zhang ◽  
Jing Zhao ◽  
He Ren ◽  
Ya-nan Du ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Protein Binding ◽  
Plasma Concentrations ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Pharmacokinetics And Pharmacodynamics ◽  
In Vivo Evaluation

Objective To investigate the effect of hypothermia on the pharmacokinetics and pharmacodynamics of nimodipine in rabbits using in vivo and in vitro methods. Methods Five healthy New Zealand rabbits received a single dose of nimodipine (0.5 mg/kg) intravenously under normothermic and hypothermic conditions. Doppler ultrasound was used to monitor cerebral blood flow, vascular resistance, and heart rate. In vitro evaluations of protein binding, hepatocyte uptake and intrinsic clearance of liver microsomes at different temperatures were also conducted. Results Plasma concentrations of nimodipine were significantly higher in hypothermia than in normothermia. Nimodipine improved cerebral blood flow under both conditions, but had a longer effective duration during the hypothermic period. Low temperature decreased the intrinsic clearance of liver microsomes, with no change in protein binding or hepatocyte uptake of nimodipine. Conclusion Nimodipine is eliminated at a slower rate during hypothermia than during normothermia, mainly due to the decreased activity of cytochrome P450 enzymes. This results in elevated system exposure with little enhancement in pharmacological effect.

Predicting drug pharmacokinetics in humans from in vitro metabolism studies

2001 ◽  
Vol 29 (2) ◽  
pp. 135-139 ◽  
Author(s):  
D. F. McGinnity ◽  
R. J. Riley
Keyword(s):  
Specific Binding ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
In Vitro Assays ◽  
Recombinant Enzymes ◽  
Pharmacokinetic Properties ◽  
Ic50 Values ◽  
Drug Oxidation ◽  
Cyp Inhibition

The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC50 values determined for a series of test compounds in HLM and recombinant CYPs were similar (r2 = 0.9, P < 0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Clint) and HLM CIint (r2 = 0.8, P< 0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on Km and Ki determinations. There were clear correlations between binding and lipophilicity (logD7.4) for a selection of bases (r2 = 0.98, P < 0.001) and acids (r2 = 0.79, P < 0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for ‘frontline’ predictive human metabolism studies in early drug discovery.

scholarly journals Interaction of Rifampin and Darunavir-Ritonavir or Darunavir-Cobicistat In Vitro

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Owain Roberts ◽  
Saye Khoo ◽  
Andrew Owen ◽  
Marco Siccardi
Keyword(s):  
High Performance ◽  
Linear Regression Analysis ◽  
Intrinsic Clearance ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Pbpk Models ◽  
Physiologically Based Pharmacokinetic ◽  
Dosing Regimens

ABSTRACT Treatment of HIV-infected patients coinfected with Mycobacterium tuberculosis is challenging due to drug-drug interactions (DDIs) between antiretrovirals (ARVs) and antituberculosis (anti-TB) drugs. The aim of this study was to quantify the effect of cobicistat (COBI) or ritonavir (RTV) in modulating DDIs between darunavir (DRV) and rifampin (RIF) in a human hepatocyte-based in vitro model. Human primary hepatocyte cultures were incubated with RIF alone or in combination with either COBI or RTV for 3 days, followed by coincubation with DRV for 1 h. The resultant DRV concentrations were quantified by high-performance liquid chromatography with UV detection, and the apparent intrinsic clearance (CLint.app.) of DRV was calculated. Both RTV and COBI lowered the RIF-induced increases in CLint.app. in a concentration-dependent manner. Linear regression analysis showed that log10 RTV and log10 COBI concentrations were associated with the percent inhibition of RIF-induced elevations in DRV CLint.app., where β was equal to −234 (95% confidence interval [CI] = −275 to −193; P < 0.0001) and −73 (95% CI = −89 to −57; P < 0.0001), respectively. RTV was more effective in lowering 10 μM RIF-induced elevations in DRV CLint.app. (half-maximal [50%] inhibitory concentration [IC50] = 0.025 μM) than COBI (IC50 = 0.223 μM). Incubation of either RTV or COBI in combination with RIF was sufficient to overcome RIF-induced elevations in DRV CLint.app., with RTV being more potent than COBI. These data provide the first in vitro experimental insight into DDIs between RIF and COBI-boosted or RTV-boosted DRV and will be useful to inform physiologically based pharmacokinetic (PBPK) models to aid in optimizing dosing regimens for the treatment of patients coinfected with HIV and M. tuberculosis.

scholarly journals Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos

2019 ◽  
Vol 20 (7) ◽  
pp. 1712 ◽  
Author(s):  
Arianna Giusti ◽  
Xuan-Bac Nguyen ◽  
Stanislav Kislyuk ◽  
Mélanie Mignot ◽  
Cecilia Ranieri ◽  
...  
Keyword(s):  
High Performance ◽  
Safety Assessment ◽  
Parent Compound ◽  
Liver Microsomes ◽  
Toxicity Testing ◽  
Rat Liver Microsomes ◽  
Metabolic Conversion ◽  
Toxic Potential

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at −20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau’s medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.

scholarly journals An in vitro toolbox to accelerate anti-malarial drug discovery and development

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Susan A. Charman ◽  
Alice Andreu ◽  
Helena Barker ◽  
Scott Blundell ◽  
Anna Campbell ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Human Liver ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Target Product ◽  
Experimental Conditions ◽  
Data Set ◽  
Vitro Data ◽  
In Vitro Data

Abstract Background Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. Methods Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC–MS/MS. Results Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. Conclusions This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug–drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

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