An effective QWBA/UHPLC-MS/tissue punch approach: solving a pharmacokinetic issue via quantitative Met-ID

2021 ◽  
Vol 14 ◽  
Author(s):  
József Pánczél ◽  
Manfred Schudok ◽  
Matthias Schiell ◽  
Jens Riedel ◽  
Vilmos Kertesz
Keyword(s):  
High Performance ◽  
Metabolite Identification ◽  
Whole Body ◽  
Pharmacokinetic Data ◽  
Test Compound ◽  
Absolute Quantitation ◽  
Tissue Sections ◽  
Signal Intensities

Background: Methods to provide absolute quantitation of the administered drug and corresponding metabolites in tissue in a spatially resolved manner is a challenging but much needed necessity in pharmaceutical research. Quantitative whole-body autoradiography (QWBA) after a single-dose intravenous (3 mg/kg) and extravascular (30 mg/kg) administrations of an in vitro metabolically stable test compound (structure not reported here) indicated quick tissue distribution and excretion. Objective: Good bioavailability and short in vivo half-lives were determined formerly for the same test compound. For closing gaps in the understanding of pharmacokinetic data and in vitro results, radioactive hot spots on whole-body tissue sections had been profiled. Method: Punches from selected tissue regions containing high radioactivity in the tissue sections previously analyzed by QWBA were extracted by a highly organic solvent and analyzed without any consecutive sample preparation step, applying ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and off-line radioanalysis to maximize signal levels for metabolite identification and profiling. Results: The analysis revealed that the test compound was metabolized intensively by phase I reactions in vivo and the metabolites formed were excreted in bile and in urine. The predominant metabolites showed abundant signal intensities both by MS and by radioanalysis but the MS signal intensities generally underestimated the real abundances of metabolites relative to the unchanged drug. Conclusions: This work illustrates that maximizing the sensitivity of tissue punch radioanalysis and the combination with UHPLC-MS leads to a better insight of pharmacokinetic processes by providing quantitative data with high molecular selectivity.

scholarly journals Evaluation of Marine Diindolinonepyrane in Vitro and in Vivo: Permeability Characterization in Caco-2 Cells Monolayer and Pharmacokinetic Properties in Beagle Dogs

Marine Drugs ◽  
2019 ◽  
Vol 17 (12) ◽  
pp. 651 ◽  
Author(s):  
Ma ◽  
Guo ◽  
Elango ◽  
Bao ◽  
Wu
Keyword(s):  
Liquid Chromatography ◽  
Mass Spectrum ◽  
Tissue Distribution ◽  
High Performance ◽  
Intravenous Route ◽  
Pharmacokinetic Data ◽  
Beagle Dogs ◽  
Total Recovery

A marine fibrinolytic compound was studied for use in thrombolytic therapy. Firstly, the absorption and transportation characteristics of 2,5-BHPA (2,5-BHPA:2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, a novel pyran-isoindolone derivative with bioactivity isolated from a rare marine microorganism in our laboratory) in the human Caco-2 cells monolayer model were investigated. We collected 2,5-BHPA in the cells to calculate the total recovery, and its concentration was analyzed by LC/MS/MS (Liquid Chromatography/ Mass Spectrum/ Mass Spectrum). The results showed that 2,5-BHPA has low permeability and low total recoveries in the Caco-2 cells membrane. Pharmacokinetics and tissue distribution of 2,5-BHPA were investigated in beagle dogs using HPLC (High Performance Liquid Chromatography) after intravenous administration of three different doses (7.5, 5.0, 2.5 mg·kg−1). Pharmacokinetic data indicated that 2,5-BHPA fitted well to a two-compartment model. Elimination half-lives (T1/2) were 49 ± 2, 48 ± 2, and 49 ± 2 min, respectively; the peak concentrations (Cmax) were 56.48 ± 6.23, 48.63 ± 5.53, and 13.64 ± 2.76 μg·mL−1, respectively; clearance rates (CL) were 0.0062 ± 0.0004, 0.0071 ± 0.0008, and 0.0092 ±0.0006 L·min−1·kg−1, respectively; mean retention times (MRT) were 28.17 ± 1.16, 26.23 ± 0.35, and 28.66 ± 0.84 min, respectively. The low penetrability of 2,5-BHPA indicated that the intravenous route of administration is more appropriate than the oral route. Meanwhile, 2,5-BHPA showed a good pharmacokinetic profile in beagle dogs. The tissue distribution showed that 2,5-BHPA could quickly distribute into the heart, intestines, liver, stomach, spleen, lungs, testicles, urine, intestine, kidneys, brain, and feces. The concentration of 2,5-BHPA was higher in the liver and bile. Interestingly, 2,5-BHPA was detected in the brain. Taken together, the above results suggested that our work might be beneficial in the development of agents for thrombolytic treatment.

scholarly journals Metabolite identification, tissue distribution, excretion and preclinical pharmacokinetic studies of ET-26-HCl, a new analogue of etomidate

2020 ◽  
Vol 7 (2) ◽  
pp. 191666
Author(s):  
Lu Yu ◽  
Xu Chen ◽  
Wen Sheng Zhang ◽  
Liang Zheng ◽  
Wen Wen Xu ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Intravenous Administration ◽  
High Performance ◽  
Anaesthetic Agent ◽  
Parent Compound ◽  
Liver Microsomes ◽  
Metabolite Identification ◽  
Pharmacokinetic Studies

ET-26-HCl, a novel anaesthetic agent with promising pharmacological properties, lacks extensive studies on pharmacokinetics and disposition in vitro and in vivo . In this study, we investigated the metabolic stability, metabolite production and plasma protein binding (PPB) of ET-26-HCl along with its tissue distribution, excretion and pharmacokinetics in animals after intravenous administration. Ultra-high performance liquid chromatography–tandem quadrupole time-of-flight mass spectrometry identified a total of eight new metabolites after ET-26-HCl biotransformation in liver microsomes from different species. A hypothetical cytochrome P450-metabolic pathway including dehydrogenation, hydroxylation and demethylation was proposed. The PPB rate was highest in mouse and lowest in human. After intravenous administration, ET-26-HCl distributed rapidly to all tissues in rats and beagle dogs, with the highest concentrations in fat and liver. High concentrations of ET-26-acid, a major hydroxylation metabolite of ET-26-HCl, were found in liver, plasma and kidney. Almost complete clearance of ET-26-HCl from plasma occurred within 4 h after administration. Only a small fraction of the parent compound and its acid form were excreted via the urine and faeces. Taken together, the results added to a better understanding of the metabolic and pharmacokinetic properties of ET-26-HCl, which may contribute to the further development of this drug.

scholarly journals Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS

Molecules ◽  
2019 ◽  
Vol 24 (14) ◽  
pp. 2658 ◽  
Author(s):  
Luya Li ◽  
Yuting Chen ◽  
Xue Feng ◽  
Jintuo Yin ◽  
Shenghao Li ◽  
...  
Keyword(s):  
High Performance ◽  
Liver Microsomes ◽  
Intestinal Flora ◽  
Metabolite Identification ◽  
Rat Plasma ◽  
Rat Liver Microsomes ◽  
Online Data ◽  
Tof Ms

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Characterization of Casearin X Metabolism by Rat and Human Liver Microsomes

Planta Medica ◽  
2018 ◽  
Vol 85 (04) ◽  
pp. 282-291 ◽  
Author(s):  
Rodrigo Moreira da Silva ◽  
Cristiane de Gaitani ◽  
Lucas Marques ◽  
Karina Fraige Baraco ◽  
Alberto Cavalheiro ◽  
...  
Keyword(s):  
Human Liver ◽  
High Performance ◽  
Liver Microsomes ◽  
Hepatic Clearance ◽  
Human Liver Microsomes ◽  
European Medicines Agency ◽  
Antitumor Action ◽  
Pharmacokinetic Data

AbstractCasearin X (CAS X) is the major clerodane diterpene isolated from the leaves of Casearia sylvestris and has been extensively studied due to its powerful cytotoxic activity at low concentrations. Promising results for in vivo antitumor action have also been described when CAS X was administered intraperitoneally in mice. Conversely, loss of activity was observed when orally administered. Since the advancement of natural products as drug candidates requires satisfactory bioavailability for their pharmacological effect, this work aimed to characterize the CAS X metabolism by employing an in vitro microsomal model for the prediction of preclinical pharmacokinetic data. Rat and human liver microsomes were used to assess species differences. A high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for the quantification of CAS X in microsomes was developed and validated according to European Medicines Agency guidelines. CAS X was demonstrated to be a substrate for carboxylesterases via hydrolysis reaction, with a Michaelis-Menten kinetic profile. The enzyme kinetic parameters were determined, and the intrinsic clearance was 1.7-fold higher in humans than in rats. The hepatic clearance was estimated by in vitro-in vivo extrapolation, resulting in more than 90% of the hepatic blood flow for both species. A qualitative study was also carried out for the metabolite identification by mass spectrometry and indicated the formation of the inactive metabolite CAS X dialdehyde. These findings demonstrate that CAS X is susceptible to first-pass metabolism and is a substrate for specific carboxylesterases expressed in liver, which may contribute to a reduction in antitumor activity when administered by the oral route.

Assessment of the near-Infrared Fluorescent Protein FP635 for Dynamic In Vivo Imaging in Models of Adaptive Immune Responses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2436-2436
Author(s):  
Simone S Riedel ◽  
Marco Herold ◽  
Markus Hirschberg ◽  
Christian Brede ◽  
Carina A Baeuerlein ◽  
...  
Keyword(s):  
T Cells ◽  
Near Infrared ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Signal Attenuation ◽  
Tissue Penetration ◽  
Body Imaging ◽  
Signal Intensities

Abstract Abstract 2436 Poster Board II-413 Sensitive in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus–host disease (GVHD) and graft-versus-tumor responses (GVT). Near-infrared (NIF) fluorescent proteins (FP) appear advantageous for deeper tissue penetration due to minimized absorbance by hemoglobin, water and lipids. Therefore we tested whether a recently published NIF FP (FP635, “Katushka”) could serve as a single reporter for whole body and single cell imaging. To compare signal intensities of eGFP and FP635 we generated fluorescent MOSEC cell lines (mouse ovarian cancer), titrated them in vitro and subcutaneously (s.c.) in vivo in Balb/c nu/nu mice. MOSEC FP635 showed twice the signal intensities compared to MOSEC eGFP in vitro by spectral fluorescence imaging (FLI). In vivo the eGFP signal was attenuated >60% in contrast to only 20% for FP635 from subcutaneous sites. However, FP635 signals from deep tissue layers were quenched. To address whether reduced signal attenuation of FP635 may allow sensitive visualization of immune processes by FLI and multiphoton-laser-scanning-microscopy (MPM) we generated transgenic mice in the genetic C57Bl/6 (B6) background, expressing FP635 under the ubiquitin promoter. Transgenic founders were selected upon signal intensities of leukocyte populations measured by flow cytometry in the PerCP channel. Combination of FP635 with colors other than red were possible for multiparameter flow cytometry. Next, eGFP, DsRed and FP635 splenocytes from transgenic donors were titrated as described above. In vitro signal intensities of FP635 splenocytes were >5 times lower compared to the other two FPs. FP635 signal absorption in vivo was low (30%) which is consistent with MOSEC titration results. In vivo DsRed detection was most sensitive and signals were similarly attenuated as FP635 in contrast to eGFP (60%). Subsequently, we aimed to visualize FP635 in a model of GVHD, where alloreactive T cells undergo massive expansion. Balb/c nu/nu mice were lethally irradiated and transplanted with 5×106 B6.WT bone marrow cells plus either 2×107 B6.DsRed+Luciferase+ or 2×107 B6.FP635 splenocytes. Sensitivity for DsRed cell detection was superior over FP635 cells. FP635 signal was only weakly detectable in lymph nodes (LN) by ex vivo FLI, where DsRed signals were detectable at earlier timepoints and LNs were even visualized by in vivo FLI. DsRed+ Luciferase+ double transgenic splenocytes allowed direct comparison of bioluminescence imaging (BLI) to FLI. Timely in vivo visualization of immune cells in deep tissues was feasible only by BLI. After whole body imaging the suitability of FP635 for MPM was checked by co-injecting eGFP B cells and either DsRed or FP635 T cells intravenously into RAG-/- mice. As FP635 is a NIF FP we expected to achieve deeper tissue penetration in hemoglobin rich organs, such as the spleen, in single cell microscopy. After 6 weeks of adoptive cell transfer we imaged spleens by MPM. Tissue penetration depths of DsRed or FP635 T cells were compared to eGFP B cells. No advantage in penetration depth of FP635 over DsRed was measured. Photobleaching is an important factor for microscopy, especially if cells are to be tracked over long time. FP635 transfected 293T cells bleached faster (t1/2=108 sec) than 293T cells transfected with eGFP (t1/2>900 sec) or DsRed (t1/2=411 sec). These experiments indicate that very high expression levels of FP635 need to be achieved for imaging. The signal attenuation of FP635 is low which may increase the sensitivity but in our hands DsRed showed comparable characteristics. Yet, the fast photobleaching of FP635 compared to the broadly established FPs DsRed and eGFP may be disadvantageous for long term microscopic tracking of cells. Our data indicate that BLI is by far superior over FLI in sensitivity and tissue penetration for whole body imaging of immune cells. However, FLI of red or near-infrared clonally selectable tumor cell lines may provide a welcome color addition to study immune cell-tumor interactions in combined models of BLI and FLI. Disclosures: No relevant conflicts of interest to declare.

Phytochemical, Analytical and Medicinal Studies of Holoptelea integrifolia Roxb. Planch - A Review

2019 ◽  
Vol 5 (4) ◽  
pp. 270-277 ◽  
Author(s):  
Vijay Kumar ◽  
Simranjeet Singh ◽  
Ragini Bhadouria ◽  
Ravindra Singh ◽  
Om Prakash
Keyword(s):  
Liquid Chromatography ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Biologically Active ◽  
Toxicological Studies ◽  
Wide Range ◽  
Layer Chromatography

Holoptelea integrifolia Roxb. Planch (HI) has been used to treat various ailments including obesity, osteoarthritis, arthritis, inflammation, anemia, diabetes etc. To review the major phytochemicals and medicinal properties of HI, exhaustive bibliographic research was designed by means of various scientific search engines and databases. Only 12 phytochemicals have been reported including biologically active compounds like betulin, betulinic acid, epifriedlin, octacosanol, Friedlin, Holoptelin-A and Holoptelin-B. Analytical methods including the Thin Layer Chromatography (TLC), High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography With Mass Spectral (LC-MS) analysis have been used to analyze the HI. From medicinal potency point of view, these phytochemicals have a wide range of pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. In the current review, it has been noticed that the mechanism of action of HI with biomolecules has not been fully explored. Pharmacology and toxicological studies are very few. This seems a huge literature gap to be fulfilled through the detailed in-vivo and in-vitro studies.

scholarly journals A novel PET tracer 18F-deoxy-thiamine: synthesis, metabolic kinetics, and evaluation on cerebral thiamine metabolism status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Changpeng Wang ◽  
Siwei Zhang ◽  
Yuefei Zou ◽  
Hongzhao Ma ◽  
Donglang Jiang ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
High Accumulation ◽  
Metabolic Kinetics ◽  
Thiamine Metabolism ◽  
Pet Tracer ◽  
The Status ◽  
The Brain

Abstract Background Some neuropsychological diseases are associated with abnormal thiamine metabolism, including Korsakoff–Wernicke syndrome and Alzheimer’s disease. However, in vivo detection of the status of brain thiamine metabolism is still unavailable and needs to be developed. Methods A novel PET tracer of 18F-deoxy-thiamine was synthesized using an automated module via a two-step route. The main quality control parameters, such as specific activity and radiochemical purity, were evaluated by high-performance liquid chromatography (HPLC). Radiochemical concentration was determined by radioactivity calibrator. Metabolic kinetics and the level of 18F-deoxy-thiamine in brains of mice and marmosets were studied by micro-positron emission tomography/computed tomography (PET/CT). In vivo stability, renal excretion rate, and biodistribution of 18F-deoxy-thiamine in the mice were assayed using HPLC and γ-counter, respectively. Also, the correlation between the retention of cerebral 18F-deoxy-thiamine in 60 min after injection as represented by the area under the curve (AUC) and blood thiamine levels was investigated. Results The 18F-deoxy-thiamine was stable both in vitro and in vivo. The uptake and clearance of 18F-deoxy-thiamine were quick in the mice. It reached the max standard uptake value (SUVmax) of 4.61 ± 0.53 in the liver within 1 min, 18.67 ± 7.04 in the kidney within half a minute. The SUV dropped to 0.72 ± 0.05 and 0.77 ± 0.35 after 60 min of injection in the liver and kidney, respectively. After injection, kidney, liver, and pancreas exhibited high accumulation level of 18F-deoxy-thiamine, while brain, muscle, fat, and gonad showed low accumulation concentration, consistent with previous reports on thiamine distribution in mice. Within 90 min after injection, the level of 18F-deoxy-thiamine in the brain of C57BL/6 mice with thiamine deficiency (TD) was 1.9 times higher than that in control mice, and was 3.1 times higher in ICR mice with TD than that in control mice. The AUC of the tracer in the brain of marmosets within 60 min was 29.33 ± 5.15 and negatively correlated with blood thiamine diphosphate levels (r = − 0.985, p = 0.015). Conclusion The 18F-deoxy-thiamine meets the requirements for ideal PET tracer for in vivo detecting the status of cerebral thiamine metabolism.

Use of Toxicokinetics in Risk Assessment Based on In Vitro Data

1993 ◽  
Vol 21 (2) ◽  
pp. 173-180
Author(s):  
Gunnar Johanson
Keyword(s):  
Risk Assessment ◽  
Whole Body ◽  
External Exposure ◽  
Target Dose ◽  
Toxic Response ◽  
Route Of Exposure ◽  
Vitro Data ◽  
In Vitro Data

This presentation addresses some aspects of the methodology, advantages and problems associated with toxicokinetic modelling based on in vitro data. By using toxicokinetic models, particularly physiologically-based ones, it is possible, in principle, to describe whole body toxicokinetics, target doses and toxic effects from in vitro data. Modelling can be divided into three major steps: 1) to relate external exposure (applied dose) of xenobiotic to target dose; 2) to establish the relationship between target dose and effect (in vitro data, e.g. metabolism in microsomes, partitioning in tissue homogenates, and toxicity in cell cultures, are useful in both steps); and 3) to relate external exposure to toxic effect by combining the first two steps. Extrapolations from in vitro to in vivo, between animal and man, and between high and low doses, can easily be carried out by toxicokinetic simulations. In addition, several factors that may affect the toxic response by changing the target dose, such as route of exposure and physical activity, can be studied. New insights concerning the processes involved in toxicity often emerge during the design, refinement and validation of the model. The modelling approach is illustrated by two examples: 1) the carcinogenicity of 1,3-butadiene; and 2) the haematotoxicity of 2-butoxyethanol. Toxicokinetic modelling is an important tool in toxicological risk assessment based on in vitro data. Many factors, some of which can, and should be, studied in vitro, are involved in the expression of toxicity. Successful modelling depends on the identification and quantification of these factors.

scholarly journals Adipocyte PHLPP2 inhibition prevents obesity-induced fatty liver

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
KyeongJin Kim ◽  
Jin Ku Kang ◽  
Young Hoon Jung ◽  
Sang Bae Lee ◽  
Raffaela Rametta ◽  
...  
Keyword(s):  
Fatty Liver ◽  
Whole Body ◽  
Acid Oxidation ◽  
Chow Diet ◽  
Peroxisome Proliferator ◽  
Obese Mice ◽  
Ph Domain ◽  
Peroxisome Proliferator Activated Receptor

AbstractIncreased adiposity confers risk for systemic insulin resistance and type 2 diabetes (T2D), but mechanisms underlying this pathogenic inter-organ crosstalk are incompletely understood. We find PHLPP2 (PH domain and leucine rich repeat protein phosphatase 2), recently identified as the Akt Ser473 phosphatase, to be increased in adipocytes from obese mice. To identify the functional consequence of increased adipocyte PHLPP2 in obese mice, we generated adipocyte-specific PHLPP2 knockout (A-PHLPP2) mice. A-PHLPP2 mice show normal adiposity and glucose metabolism when fed a normal chow diet, but reduced adiposity and improved whole-body glucose tolerance as compared to Cre- controls with high-fat diet (HFD) feeding. Notably, HFD-fed A-PHLPP2 mice show increased HSL phosphorylation, leading to increased lipolysis in vitro and in vivo. Mobilized adipocyte fatty acids are oxidized, leading to increased peroxisome proliferator-activated receptor alpha (PPARα)-dependent adiponectin secretion, which in turn increases hepatic fatty acid oxidation to ameliorate obesity-induced fatty liver. Consistently, adipose PHLPP2 expression is negatively correlated with serum adiponectin levels in obese humans. Overall, these data implicate an adipocyte PHLPP2-HSL-PPARα signaling axis to regulate systemic glucose and lipid homeostasis, and suggest that excess adipocyte PHLPP2 explains decreased adiponectin secretion and downstream metabolic consequence in obesity.

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