Simultaneous pharmacokinetics and stability studies of physalins in rat plasma and intestinal bacteria culture media using liquid chromatography with mass spectrometry.

Physalins are the major steroidal constituent of Physalis plants and display a range of biological activities. For this study, a rapid and sensitive high-performance liquid chromatography with triple quadrupole mass spectrometry method was developed for the simultaneous quantification of six physalins. Specifically, it was for the quantification of physalin A, physalin B, physalin D, physalin G, 4,7-didehydroneophysalin B, and isophysalin B in rat plasma and rat intestinal bacteria. After a solid-phase extraction, analytes and internal standards (prednisolone) were separated on a Shield reverse-phase C18 column (measuring 3 mm × 150 mm with an internal diameter of 3.5 µm) and determined using multiple reactions in a monitoring mode with a positive-ion electrospray ionization source. The mobile phase was a mixture of 0.1% formic acid in water (A) and acetonitrile (B) and was used at a flow rate of 0.6 mL/min. The intra- and interday precisions were within 15% with accuracies ranging from 86.2 to 114%. The method was validated and successfully applied to pharmacokinetics and stability studies of six physalins in rat plasma and rat intestinal bacteria, respectively. The results showed that physalin B and isophysalin B could not be absorbed by rats, and rat intestinal bacteria could quickly transform physalins.

Identification of absorbed constituents and in vivo metabolites in rats after oral administration of Physalis alkekengi var. franchetii by ultra-high-pressure liquid chromatography quadrupole time-of-flight mass spectrometry.

The calyces of Physalis alkekengi var. franchetii (Chinese Lantern, JDL) are well-known as traditional Chinese medicine owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of JDL and their metabolites in vivo are still unclear to date. In this paper, an ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) method was established to identify absorbed constituents and in vivo metabolites in rat biological fluids after oral administration of JDL. Based on the proposed strategy, 33 compounds were observed in dosed rat biosamples. Twelve of 33 compounds were indicated as prototype components of JDL, and 21 compounds were predicted to be metabolites of JDL. Finally, the metabolic pathways were proposed, which were glucuronidation, sulfation, methylation and dehydroxylation for flavonoid constituents and sulfonation and hydroxylation for physalin consitituents. This is the first systematic study on the absorbed constituents and metabolic profiling of JDL and will provide a useful template for screening and characterizing the ingredients and metabolites of traditional Chinese medicine.

Identification and quantitative analysis of physalin D and its metabolites in rat urine and feces by liquid chromatography with triple quadrupole time-of-flight mass spectrometry.

Physalin D is known to show extensive bioactivities. However, no excretion study has elucidated the excretion of physalin D and its metabolites. This study investigates the excretion of physalin D and its metabolites in rats. Metabolites in rat urine and feces were separated and identified by liquid chromatography with triple quadrupole time-of-flight mass spectrometry. Furthermore, a validated high-performance liquid chromatography with tandem mass spectrometry method was developed to quantify physalin D, physalin D glucuronide, and physalin D sulfate in rat feces and urine after the intragastric administration of physalin D. The analyte showed good linearity over a wide concentration range (r > 0.995), and the lower limit of quantification was 0.0532 µg/mL and 0.226 µg/g for urine and feces, respectively. Nine metabolites, including five phase I and four phase II metabolites, were identified and clarified after dosing in vivo. Only 4.0% of the gavaged dose, including physalin D and its phase II metabolites, was excreted in urine, whereas 10.8% was found in feces in the unchanged form. The results indicate that the extensive and rapid metabolism may be the main factors leading to the short half-life of physalin D. These results can provide a basis for further studies on the structural modification and pharmacology of physalin D.

LC-MS/MS method for simultaneous determination of flavonoids and physalins in rat plasma: Application to pharmacokinetic study after oral administration of Physalis alkekengi var. franchetii (Chinese lantern) extract.

A rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of luteolin, luteolin-7-O-ß-D-glucopyranoside, physalin A, physalin D and physalin L in rat plasma. Scutellarein and dexamethasone were used as the internal standards (IS). Plasma samples were prepared by liquid-liquid extraction with ethyl acetate. The five constituents were separated on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm). A gradient elution procedure was used with acetonitrile (A)-0.1% aqueous formic acid (B). Mass spectrometric detection was performed in negative ion multiple reaction monitoring mode with an electrospray ionization (ESI) source. This method showed good linearity (r2 > 0.997) over a concentration range of 2.0-500 ng/mL with a lower limit of quantification of 2.0 ng/mL for all five compounds. The inter- and intra-day accuracy ranged from 91.7 to 104%, and precisions (RSD) were <6.46% for all analytes. The extraction recoveries of all analytes were >85%. This validated method was successfully applied for the first time to the pharmacokinetic study of five ingredients after oral administration of 70% ethanol extract of Chinese lantern in rats.

In vivo pharmacokinetics of and tissue distribution study of physalin B after intravenous administration in rats by liquid chromatography with tandem mass spectrometry.

A rapid and sensitive liquid chromatography tandem mass spectrometry quantitative analysis method was established for the pharmacokinetics and tissue distribution study of physalin B in rat. Physalin B and physalin H (internal standard, IS) were separated on an Agilent Eclips XDB C8 column. MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration ranges of 22.6-22600 ng/mL for heart and lung and 4.52-4520 ng/mL for other tissues. The intra- and inter-day precisions (RSD) were ≤9.23 and ≤12.51%, respectively, with accuracy (%) in the range of 88.07-113.2%. A pharmacokinetic study showed that physalin B has a long dwell time with a half-life of 321.2 ± 29.5 min and clearance of 175.4 ± 25.7 mL/min/kg after intravenous administration. Additionally, physalin B showed a wide tissue distribution with a special higher penetration in lung. The data presented in this study could provide useful information for the further study of physalin B. Copyright © 2016 John Wiley & Sons, Ltd.

Chronic administration of cholesterol oximes in mice increases transcription of cytoprotective genes and improves transcriptome alterations induced by alpha-synuclein overexpression in nigrostriatal dopaminergic neurons.

Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson's disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4months of age, approximately 10months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased alpha-synuclein aggregates in the substantia nigra; this effect, not seen with TRO40303 was inconsistent and may represent a protective mechanism as in other neurodegenerative diseases. Overall, the results suggest that cholesterol oximes, while not improving early effects of alpha-synuclein overexpression on motor behavior or pathology, may ameliorate the function and resilience of dopaminergic neurons in vivo and support further studies of neuroprotection in models with dopaminergic cell loss.

TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition.

3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.

TRO40303 Ameliorates Alcohol-Induced Pancreatitis Through Reduction of Fatty Acid Ethyl Ester-Induced Mitochondrial Injury and Necrotic Cell Death.

OBJECTIVES: Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). METHODS: Changes in mitochondrial membrane potential (Δψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. RESULTS: TRO40303 prevented loss of Δψm and necrosis induced by 100 µM palmitoleic acid ethyl ester or 500 µM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. CONCLUSIONS: TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.

Synthesis, estrogen receptor binding, and tissue distribution of [18F]fluorodoisynolic acids.

Doisynolic acids, D-ring seco-steroids derived from alkaline fusion of estrones, are hormonal curiosities: Their binding affinity for the estrogen receptor is low (ca. 1-2% that of estradiol), but their in vivo potency is high and they have a long duration of action. To study the in vivo behavior of the doisynolic acids, we prepared fluorine-substituted analogs of both trans-doisynolic acid (with the natural 14 alpha-hydrogen configuration, trans-FDA) and the more active cis-doisynolic acid (with the unnatural 14 beta-hydrogen configuration, cis-FDA) from estrone and 14 beta-estrone, respectively. Modification of the D-ring haloform cleavage approach of Meyers allowed us to introduce fluorine (or fluorine-18) on the carbon atom derived from C-16 in the estrones. Fluorine substitution had little effect on the estrogen receptor binding affinity of the doisynolic acids. Tissue distribution of the fluorodoisynolic acids (trans-[18F]FDA and cis-[18F]FDA) was unusual and very different from that of typical, high-affinity ligands for the estrogen receptor. At 1-3 h in immature female rats, trans-[18F]FDA shows low and rather nonselective uptake in the principal estrogen target tissue (uterus) and slow clearance. By contrast, cis-[18F]FDA shows high uptake in nearly all tissues, with significant uterine uptake that continues to increase over the 1-6-h period. The uterine uptake of this isomer was blocked at the later times by a sufficiently high dose of unlabeled cis-FDA. After administration of the trans-[18F]FDA, a more polar metabolite slowly accumulates in the blood. The cis-[18F]FDA, however, showed no apparent metabolism, with 84% of the blood activity at 5 h assigned as the unmetabolized radioligand. After 5 h, only limited clearance from blood, liver, and kidneys has occurred. No metabolite from this isomer accumulates in the uterus. Although fluorodoisynolic acids will not be useful breast-tumor imaging agents, their behavior was found to be interesting as it deviates from that of other F-18 estrogens. Further long-term studies of cis-doisynolic acid, labeled with tritium, may be needed to explicate fully its unusual distribution properties and high in vivo activity.

An ultra-pressure liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three physalins in rat plasma and its application to pharmacokinetic study of Physalis alkekengi var. franchetii (Chinese lantern) in rats.

An ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of three major ingredients in Chinese lantern preparations (CLP) in rat plasma. Following extraction by ethyl acetate, the analytes were separated on an Acquity UPLC BEH Shield RP C(18) column using a gradient mobile phase system of acetonitrile-water. Electrospray ionization (ESI) tandem interface was employed prior to mass spectrometric detection. The calibration curves were linear over the range of 5.0-500.0 ng/ml for physalin D, 2.3-230.0 ng/ml for physalin G and 0.71-71.0 ng/ml for 4,7-didehydroneophysalin B. The average extraction recoveries, examined at four concentration levels, carried from 57.1% to 76.9%, and the accuracies ranged from 94.0% to 113.3% with precision (RSD) <15%. The validated method was successfully applied to the determination of the three physalins in rat plasma after intragastric administration of CLP suspension.

Plasma pharmacokinetics and tissue distribution study of physalin D in rats by ultra-pressure liquid chromatography with tandem mass spectrometry.

Physalin D is an important constituent of some traditional Chinese medicines, and has several known bioactivities. An UPLC-MS/MS method for the determination of physalin D in rat plasma and tissues was developed and the pharmacokinetic and tissue distribution characteristics of physalin D after intravenous administrations were investigated. The bio-samples were prepared by a simple protein precipitation, and the separation of physalin D was achieved on a UPLC HSS T3 column with a mobile phase consisting of methanol/acetonitrile (70:30, v/v) and water (containing 0.1% formic acid and 10 mM ammonium acetate) at a flow rate of 0.3 mL/min. The MS/MS detection was carried out by monitoring the fragmentation of m/z 544.9→508.8 for physalin D and m/z 286.7→152.8 for luteolin (internal standard; IS) on a triple-quadrupole mass spectrometer. The total run time was only 3.6 min. The analyte showed good linearity over a wide concentration range (R(2)>0.995) and its lower limit of quantification was 2 ng/mL. The pharmacokinetic study found that physalin D was distributed and eliminated rapidly in rats (t(1/2)<10 min). Tissue distribution showed the highest level was observed in kidney, then in liver, but no physalin D was detected in brain, which indicated that kidney was the major distribution tissue for physalin D in rats and that physalin D does not cross the blood-brain barrier.