Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.

Pharmacokinetic study of Ninjin'yoeito: Absorption and brain distribution of Ninjin'yoeito ingredients in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ninjin'yoeito (NYT), a Japanese traditional Kampo medicine, has been reported to exert various clinical benefits such as relief from fatigue, malaise, anorexia, frailty, sarcopenia, and cognitive dysfunction. Recently, some review articles described the pharmacological effects of NYT and additionally indicated the possibility that multiple ingredients in NYT contribute to these effects. However, pharmacokinetic data on the ingredients are essential in addition to data on their pharmacological activities to accurately determine the active ingredients in NYT. AIM OF THE STUDY: This study assessed the in vivo pharmacokinetics of NYT using mice. MATERIALS AND METHODS: Target liquid chromatography-mass spectrometry (LC-MS) and wide target LC-MS or LC-tandem MS of NYT ingredients in plasma and the brain after oral administration of NYT were performed. Imaging MS was performed to investigate the detailed brain distributions of NYT ingredients. RESULTS: The concentrations of 13 ingredients in plasma and schizandrin in the brain were quantified via target LC-MS, and the wide target analysis illustrated that several ingredients are absorbed into blood and transported into the brain. Imaging MS revealed that schizandrin was homogenously dispersed in the NYT-treated mouse brain. CONCLUSION: These results should be useful for clarifying the active ingredients of NYT and their mechanisms of actions.

Bioorthogonal Pretargeting Strategy for Anchoring Activatable Photosensitizers on Plasma Membranes for Effective Photodynamic Therapy.

Developing novel activatable photosensitizers with excellent plasma membrane targeting ability is urgently needed for smart photodynamic therapy (PDT). Herein, a tumor acidity-activatable photosensitizer combined with a two-step bioorthogonal pretargeting strategy to anchor photosensitizers on the plasma membrane for effective PDT is developed. Briefly, artificial receptors are first anchored on the cell plasma membrane using cell-labeling agents (Az-NPs) via the enhanced permeability and retention effect to achieve the tumor cell labeling. Then, pH-sensitive nanoparticles (S-NPs) modified with dibenzocyclooctyne (DBCO) and chlorin e6 (Ce6) accumulate in tumor tissue and disassemble upon protonation of their tertiary amines in response to the acidic tumor environment, exposing the contained DBCO and Ce6. The selective, highly specific click reactions between DBCO and azide groups enable Ce6 to be anchored on the tumor cell surface. Upon laser irradiation, the cell membrane is severely damaged by the cytotoxic reactive oxygen species, resulting in remarkable cellular apoptosis. Taken together, the membrane-localized PDT by our bioorthogonal pretargeting strategy to anchor activatable photosensitizers on the plasma membrane provides a simple but effective method for enhancing the therapeutic efficacy of photosensitizers in anticancer therapy.

Dual-responsive click-crosslinked micelles designed for enhanced chemotherapy for solid tumors.

The design of multiple stimuli-responsive, stable polymeric drug carriers is key for efficient drug release against solid tumors. Herein, core-crosslinked micelles were readily prepared from a pair of redox/pH-sensitive clickable copolymers. The two copolymers comprised the same poly(ethylene glycol) (PEG)-poly(ε-benzyloxycarbonyl-l-lysine) (PZLL) block but with either disulfide-linked azadibenzocyclooctyne (DBCO) or azide (AZ) group-tagged branched polyethylenimine (BPEI, 1.8 kDa). The data showed that an equivalent of the two copolymers could self-assemble into nanosized micelles with the crosslinked core via the DBCO-AZ click chemistry. The click-crosslinked micelles showed excellent size stability under multiple dilutions but destabilization in an acidic or reductive environment. Besides, they could load doxorubicin (DOX), an anticancer drug, and mediate slow drug release in a neutral environment but sufficient drug unloading under acidic plus reductive conditions. In vitro, DOX-loaded crosslinked micelles led to higher DOX accumulation in the cellular nucleus in comparison with non-crosslinked micelles from the PEG-PZLL-BPEI copolymer (PP), thus causing more marked cytotoxicity in SKOV-3 cells. In vivo, DOX-loaded crosslinked micelles caused significant growth inhibition of SKOV-3 tumors xenografted in BALB/c nude mice, and showed superior anticancer efficacy to non-crosslinked PP micelles. Chemotherapy with core-crosslinked micelles had no adverse side effects on the health (serum levels and body weight) of the mice. This study highlights the design of clickable block copolymers to easily construct core-crosslinked and multiple stimuli-responsive micelles for enhanced anticancer therapy.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Efficacy of human-simulated bronchopulmonary exposures of cefepime, zidebactam and the combination (WCK 5222) against MDR Pseudomonas aeruginosa in a neutropenic murine pneumonia model.

OBJECTIVES: WCK 5222 combines cefepime with zidebactam, a ß-lactam enhancer that binds PBP2 and inhibits class A and C ß-lactamases. The efficacy of human-simulated bronchopulmonary exposures of WCK 5222 against MDR Pseudomonas aeruginosa was investigated in a neutropenic murine pneumonia model. METHODS: Nineteen MDR isolates of P. aeruginosa (cefepime MICs ≥64 mg/L) were studied. MICs of zidebactam and WCK 5222 ranged from 4 to 512 mg/L and from 4 to 32 mg/L, respectively. Dosing regimens of cefepime and zidebactam alone and in combination that achieved epithelial lining fluid (ELF) exposures in mice approximating human ELF exposures after doses of 2 g of cefepime/1 g of zidebactam every 8 h (1 h infusion) were utilized; controls were vehicle-dosed. Lungs were intranasally inoculated with 107-108 cfu/mL bacterial suspensions. Mice were dosed subcutaneously 2 h after inoculation for 24 h, then lungs were harvested. RESULTS: In vitro MIC was predictive of in vivo response to WCK 5222 treatment. Mean±SD changes in bacterial density at 24 h compared with 0 h controls (6.72±0.50 log10 cfu/lungs) for 13 isolates with WCK 5222 MICs ≤16 mg/L were 1.17±1.00, -0.99±1.45 and -2.21±0.79 log10 cfu/lungs for cefepime, zidebactam and WCK 5222, respectively. Against these isolates, zidebactam yielded >1 log10 cfu/lungs reductions in 8/13, while activity was enhanced with WCK 5222, producing >2 log10 cfu/lungs reductions in 10/13 and >1 log10 cfu/lungs reductions in 12/13. Among isolates with WCK 5222 MICs of 32 mg/L, five out of six showed a bacteriostatic response. CONCLUSIONS: Human-simulated bronchopulmonary exposure of WCK 5222 is effective against MDR P. aeruginosa at MIC ≤16 mg/L in a murine pneumonia model. These data support the clinical development of WCK 5222 for pseudomonal lung infections.

Pharmacokinetics and liver uptake of three Schisandra lignans in rats after oral administration of liposome encapsulating ß-cyclodextrin inclusion compound of Schisandra extract.

Schisandra chinensis fructus (SCF) is widely used traditional Chinese medicine, which possesses hepato-protective potential. Schisandrin (SD), schisantherin (ST), and γ-schizandrin (SZ) are the major bioactive lignans. The main problem associated with the major bioactive lignans oral administration is low oral bioavailability due to the lignans' poor aqueous solubility and taste. The aim of the present research work was to develop liposome (SCL) encapsulated ß-cyclodextrin (ß-CD) inclusion complex loaded with SCF extract (SCF-E). The SD, ST, and SZ were selected as effective candidates to perform comparisons of liver targeting among the solution (SES), ß-cyclodextrin inclusion compound (SCF-E-ß-CD), liposome (SEL), and SCL of SCF-E to characterize the pharmacokinetic behaviors and liver targeting in rats. The ß-CD inclusion complex (SCF-E-ß-CD) was used to improve the solubility. The concentrations were determined using high-performance liquid chromatography (HPLC) and analyzed by DAS3.0. The pharmacokinetic results indicate that the plasma concentration-time courses were fitted well to the one-compartment model with the first weighing factor. The half-life period (t1/2) and area under the concentration-time curve (AUC) of the three components in SCL were the largest. The SCL exhibit a relatively high liver targeting effect. The results would be helpful for guiding the clinical application of this herbal medicine.

Tetrazine- trans-Cyclooctene Chemistry Applied to Fabricate Self-Assembled Fluorescent and Radioactive Nanoparticles for in Vivo Dual Mode Imaging.

Multimodal imaging agents combine two or more imaging modalities into one probe. Self-assembling fluorescent nanoparticles are a promising class of modular multimodal imaging probes as they can allow easy blending of imaging and targeting modalities. Our group recently developed a class of self-assembling and intrinsically fluorescent small molecule-based nanoparticles (SMNPs) with excellent optical properties. In this article, we describe the efficient radiolabeling of these SMNPs via a two-step bioconjugation strategy involving the inverse-electron-demand Diels-Alder ligation between a tetrazine (Tz)-tagged radiolabel and a trans-cyclooctene (TCO)-tagged fluorescent small molecule building block of the SMNPs. Studies in mice revealed that the SMNPs are well tolerated and could be monitored by both radioactivity and fluorescence, thereby demonstrating the potential of SMNPs in optical and dual-mode imaging in vivo. The work also testifies to the utility of the Tz-TCO conjugation chemistry for the labeling of self-assembled nanoparticles.

The Novel ß-Lactam Enhancer Zidebactam Augments the In Vivo Pharmacodynamic Activity of Cefepime in a Neutropenic Mouse Lung Acinetobacter baumannii Infection Model.

WCK 5222 is a combination of cefepime and the high-affinity PBP2-binding ß-lactam enhancer zidebactam. The cefepime-zidebactam combination is active against multidrug-resistant Gram-negative bacteria, including carbapenemase-expressing Acinetobacter baumannii The mechanism of action of the combination involves concurrent multiple penicillin binding protein inhibition, leading to the enhanced bactericidal action of cefepime. The aim of the present study was to assess the impact of the zidebactam-mediated enhanced in vitro bactericidal action in modulating the percentage of the time that the free drug concentration remains above the MIC (percent fT>MIC) for cefepime required for the in vivo killing of A. baumannii Cefepime and cefepime-zidebactam MICs were comparable and ranged from 2 to 16 mg/liter for the A. baumannii strains (n = 5) employed in the study. Time-kill studies revealed the improved killing of these strains by the cefepime-zidebactam combination compared to that by the constituents alone. Employing a neutropenic mouse lung infection model, exposure-response analyses for all the A. baumannii strains showed that the cefepime fT>MIC required for 1-log10 kill was 38.9%. In the presence of a noneffective dose of zidebactam, the cefepime fT>MIC requirement dropped significantly to 15.5%, but it still rendered a 1-log10 kill effect. Thus, zidebactam mediated the improvement in cefepime's bactericidal effect observed in time-kill studies, manifested in vivo through the lowering of cefepime's pharmacodynamic requirement. This is a first-ever study demonstrating a ß-lactam enhancer role of zidebactam that helps augment the in vivo activity of cefepime by reducing the magnitude of its pharmacodynamically relevant exposures against A. baumannii.

Pharmacokinetics and distribution of schisandrol A and its major metabolites in rats.

1. Schizandrol A is an active component in schisandra, also the representative component for the identification of schisandra. 2. A rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-QTOF/MS) was developed to investigate the pharmacokinetics of schizandrol A after its intragastric administration (50 mg/kg) in rats. 3. Schizandrol A was rapidly absorbed (T max = 2.07 h), with a longer duration (t 1/2 = 9.48 h) and larger apparent volume of distribution (Vz/F = 111.81 l/kg) in rats. Schizandrol A can be detected in main organs and the order of its distribution was in the liver > kidney > heart > spleen > brain, particularly higher in the liver. 4. Five schizandrol A metabolites were identified, including 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, hydroxyl-schizandrin, demethoxy-schizandrin, 2, 3-demethyl-8(R)-hydroxyl-schizandrin, indicating that the hydroxylation and demethylation may be the major metabolic way of schizandrol A. 5. This study defined the pharmacokinetic characteristics of schizandrol A in vivo, and the RRLC-QTOF/MS is more sensitive and less limited by conditions, and needs less samples, which may be a useful resource for the further research and development of schisandrol A.

Interaction of deoxyschizandrin and schizandrin B with liver uptake transporters OATP1B1 and OATP1B3.

1. Deoxyschizandrin and schizandrin B have diverse pharmacological effects, including hepatoprotective activity. We aim to study their hepatic uptake and their effects on the hepatic uptake of other clinical drugs mediated by OATP1B1 and OATP1B3. 2. Deoxyschizandrin exhibited a high affinity for OATP1B1 with Km of 17.61 ± 0.43 µM but a low affinity for OATP1B3. Similarly, schizandrin B also showed a strong affinity for OATP1B1 with Km of 18.45 ± 1.23 µM but a weak affinity for OATP1B3. 3. Atorvastatin and rifampicin could inhibit the uptake of deoxyschizandrin and schizandrin B mediated by OATP1B1. 4. Intriguingly, both deoxyschizandrin and schizandrin B significantly promoted the uptake of atorvastatin (with EC50 of 50.58 ± 8.08 and 24.70 ± 5.82 µM, respectively) and rosuvastatin (with EC50 of 13.46 ± 2.70 and 8.99 ± 4.73 µM, respectively) mediated by OATP1B1. Deoxyschizandrin could markedly promote the uptake of fluvastatin but inhibit the uptake of sodium taurocholate (TCNa) mediated by OATP1B1. 5. The promotion on hepatic uptake of statins mediated by OATP1B1 might lead to enhanced efficacy of cholesterol lowering and reduced risk of myopathy for hyperlipidemia patients when given statins together with deoxyschizandrin or schizandrin B.

Single-Center Evaluation of the Pharmacokinetics of WCK 5222 (Cefepime-Zidebactam Combination) in Subjects with Renal Impairment.

WCK 5222 is a novel ß-lactam-ß-lactam-enhancer combination of cefepime (FEP) and zidebactam (ZID). ZID is a novel ß-lactam enhancer with a dual action of binding to Gram-negative penicillin-binding protein 2 (PBP2) and ß-lactamase inhibition. WCK 5222 is being developed as a new therapeutic option for the treatment of complicated multidrug-resistant Gram-negative pathogen infections. We investigated the effect of renal impairment on the pharmacokinetics (PK) and safety of WCK 5222 in 48 subjects based on Cockcroft-Gault-estimated creatinine clearance (CLCR). We enrolled mild (n = 6; CLCR, 60 to <90 ml/min), moderate (n = 6; CLCR, 30 to <60 ml/min), and severe (n = 6; CLCR, <30 ml/min; not on dialysis) impairment, end-stage renal disease (ESRD) on hemodialysis (HD) (n = 6), and matched normal controls (n = 24; CLCR, ≥90 ml/min). Healthy control subjects and mild and moderate renal impairment subjects received a single 60-min intravenous (i.v.) infusion of 3 g WCK 5222 (2 g FEP/1 g ZID); severe renal impairment and HD subjects received a single 60-min i.v. infusion of 1.5 g WCK 5222 (1 g FEP plus 0.5 g ZID). Body and renal clearance decreased, and plasma half-life (t1/2) and the area under the concentration-time curve from time zero extrapolated to infinity (AUC0-∞ [h µg/ml]) increased in a graded relationship with severity of renal impairment for both FEP and ZID. Our findings suggest that dose adjustments for WCK 5222 will be required according to the degree of renal impairment. Overall, WCK 5222 (FEP-ZID) was found to be safe and well tolerated in subjects with normal and impaired renal function. (This study has been registered at ClinicalTrials.gov under identifier NCT02942810.).

Deoxyschizandrin Loaded Liposomes on the Suppression Lipid Accumulation in 3T3-L1 Adipocytes.

Deoxyschizandrin (DS) is a bioactive benzocyclooctadiene lignan found in the fruit of Schisandra chinensis. However, poor bioavailability and non-specificity of DS frequently caused low therapeutic efficacy. In the present study, DS-liposome (DS-lipo) was implemented to enhance the hepatic targeting and inhibition effects on adipocyte differentiation in 3T3-L1 cells. The formulations enabled encapsulation of as much as 24.14% DS. The DS-lipo prepared was about 73.08 nm, as measured by laser light scattering (LLS) morphology. In the visual field of a scanning electron microscope (SEM), the liposomes were spherical with similar size and uniform dispersion. Fluorescence live imaging study exhibited hepatic targeting of liposomes in vivo. Furthermore, High-Content Analysis (HCS) imaging microassay analyses revealed DS-lipo and DS reduced cytoplasmic lipid droplet in 3T3-L1 adipocytes, with the IC50 value of 8.68 µM and 31.08 µM, respectively. The lipid droplet accumulation inhibition rate of 10 µM DS-lipo was above 90%, which was even superior to the effect of 30 µM DS solution. The current findings suggest that DS-lipo was a therapeutic strategy for alleviating lipid-associated diseases and nonalcoholic fatty liver disease (NAFLD).

Simultaneous UPLC-MS/MS determination of four components of Socheongryong-tang tablet in human plasma: Application to pharmacokinetic study.

The purpose of this study was to develop a method for simultaneous analysis of schizandrin, ephedrine, paeoniflorin, and cinnamic acid as constituents of Socheongryong-tang tablet in human plasma using UPLC-MS/MS. These four components were separated using water containing 0.01% formic acid and methanol as a mobile phase by gradient elution at a flow rate of 0.3 mL/min with a HALO-C18 column (2.1 mm × 100 mm, 2.7 µm particle size). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operated in multiple reaction monitoring mode. Mass transitions were m/z 432.9 → 384.1 for schizandrin, 165.8 → 148.1 for ephedrine, 525.0 → 449.2 for paeoniflorin, 146.8 → 102.9 for cinnamic acid, and 340.0 → 324.0 for papaverine as internal standard. Liquid-liquid extraction and protein precipitation with ethyl acetate-methanol (1:2, v/v) were used to obtain these four components. Chromatograms showed high resolution, sensitivity, and selectivity without interference by plasma constituents. Calibration curves of schizandrin, ephedrine, paeoniflorin, and cinnamic acid in human plasma ranged from 0.02 to 8 ng/mL, 0.5 to 200 ng/mL, 0.2 to 80 ng/mL, and 1 to 400 ng/mL, respectively. Calibration curves of each analyte displayed excellent linearity, with correlation coefficients > 0.99. For all four components, both intra- and inter-day precisions (CV%) were <5.99%. The accuracy was 99.35-103.30% for schizandrin, 98.48-104.38% for ephedrine, 97.06-103.34% for paeoniflorin, and 99.97-104.36% for cinnamic acid. This analytical method developed in this study satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of schizandrin, ephedrine, paeoniflorin, and cinnamic acid after oral administration of Socheongryong-tang tablet to humans.

Simultaneous quantification of Schisandrin B enantiomers in rat plasma by chiral LC-MS/MS: Application in a stereoselective pharmacokinetic study.

Schisandrin B (Sch B) has received much attention owing to its various biological activities. Schisandrin B exists as a racemate in "wuweizi", a traditional Chinese medicine in China. In the present study, a novel chiral LC-MS/MS method was developed for enantioselective separation and determination of Schisandrin B in rat plasma. The plasma samples were prepared by liquid-liquid extraction (LLE). Schisandrol B was used as internal standard. Chiral separation was obtained on a Chiralpak IC column using 0.1% (v/v) formic acid in mixture of methanol and water (90:10, v/v) as a mobile phase. Parameters including the selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability were evaluated. The method described here is simple and reproducible. The lower limit of quantification of 5.0 ng/mL for each Sch B enantiomer permits the use of the method in investigating the stereoselective pharmacokinetics of Sch B. Following racemic Sch B and "wuweizi" extracts, the area under the curve of (8R, 8'S)-Sch B was statistically higher than the one of (8S, 8' R)-Sch B, with a ratio of 1.16-1.40 in three cases. This study firstly reports the development and validation of enantioselective behavior of Sch B in vivo, and provides a reference for clinical practice and encourages further research into Sch B enantioselective metabolism and drug interactions.

Simultaneous determination of zidebactam and cefepime in dog plasma by LC-MS/MS and its application to pre-clinical pharmacokinetic study.

A precise and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of zidebactam (ZID) and cefepime (FEP) in dog plasma. Ceftazidime was used as an internal standard. Protein precipitation method was used as sample preparation approach. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range 0.156-80 µg/mL for ZID and 0.312-160 µg/mL for FEP. The method was validated as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 3.5 min for each sample made it possible to analyze the maximum number of samples per day. The proposed method was successfully applied for pharmacokinetic study in beagle dogs.

Bioorthogonal Masking of Circulating Antibody-TCO Groups Using Tetrazine-Functionalized Dextran Polymers.

Pretargeting strategies have gained popularity for the in vivo imaging and therapy of cancer by combining antibodies with small molecule radioligands. In vivo recombination of both moieties can be achieved using the bioorthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). An issue that arises with pretargeting strategies is that while part of the antibody dose accumulates at antigen-expressing tumor tissue, there is a significant portion of the injected antibody that remains in circulation, causing a reduction in target-to-background ratios. Herein, we report the development of a novel TCO scavenger, the masking agent DP-Tz. DP-Tz is based on Tz-modified dextran polymers (DP, MW = 0.5-2 MDa). Large dextran polymers were reported to exhibit low penetration of tumor vasculature and appeared nontoxic, nonimmunogenic, and easily modifiable. Our newly developed masking agent deactivates the remaining TCO-moieties on the circulating mAbs yet does not impact the tumor uptake of the Tz-radioligand. In pretargeting studies utilizing a 68Ga-labeled tetrazine radioligand ([68Ga]Ga-NOTA-PEG11-tetrazine), DP-Tz constructs (Tz/DP ratios of 62-254) significantly increased TTB ratios from 0.8 ± 0.3 (control cohorts) to up to 5.8 ± 2.3 at 2 h postinjection. Tumor tissue delineation in PET imaging experiments employing DP-Tz is significantly increased compared to control. Uptake values of other significant organs, such as heart, lungs, pancreas, and stomach, were decreased on average by 2-fold when using DP-Tz. Overall, pretargeting experiments utilizing DP-Tz showed significantly improved tumor delineation, enhanced PET image quality, and reduced uptake in vital organs. We believe that this new masking agent is a powerful new addition to the IEDDA-based pretargeting tool box and, due to its properties, an excellent candidate for clinical translation.

Pharmacokinetics of Schizandrin and Its Pharmaceutical Products Assessed Using a Validated LC-MS/MS Method.

Schisandra chinensis has been used as an important component in various prescriptions in traditional Chinese medicine and, more recently, in Western-based medicine for its anti-hepatotoxic effect. The aim of this study was to develop a selective, rapid, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for pharmacokinetic studies of schizandrin in rats. Liquid-liquid extraction was used for plasma sample preparation. A UHPLC reverse-phase C18e column (100 mm × 2.1 mm, 2 µm) coupled with a mobile phase of methanol-0.1% formic acid (85:15, v/v) was used for sample separation. A triple quadrupole tandem mass spectrometer was used to detect the analytes in the selected reaction monitoring mode. The linear range of schizandrin in rat plasma was 5.0-1000 ng/mL (r² > 0.999), with a lower limit of quantification of 5 ng/mL. The method was validated with regard to accuracy, intra-day and inter-day precision, linearity, stability, recovery, and matrix effects in rat plasma, which were acceptable according to the biological method validation guidelines developed by the FDA. This method was successfully applied to a pharmacokinetic study after oral administration of 3 g/kg and 10 g/kg of Schisandra chinensis products, which yielded a maximum concentration of schizandrin of 0.08 ± 0.07 and 0.15 ± 0.09 µg/mL, respectively. A parallel study design was used to investigate the oral bioavailability of single compound of schizandrin and the herbal extract, the single compound of pure schizandrin (10 mg/kg, i.v.), pure schizandrin (10 mg/kg, p.o.), and the herbal extract of Schisandra chinensis (3 g/kg and 10 g/kg, p.o.) were given individually. The dose of Schisandra chinensis (3 g/kg) equivalent to schizandrin (5.2 mg/kg); the dose of Schisandra chinensis (10 g/kg) equivalent to schizandrin (17.3 mg/kg). The result demonstrated that the oral bioavailability of schizandrin was approximately 15.56 ± 10.47% in rats, however the oral bioavailability of herbal extract was higher than single compound. The method was successfully applied to the pharmacokinetic study of pure schizandrin after oral administration of its pharmaceutical industry products in rats.

Prediction of Drug-Drug Interaction between Tacrolimus and Principal Ingredients of Wuzhi Capsule in Chinese Healthy Volunteers Using Physiologically-Based Pharmacokinetic Modelling.

Schisantherin A and schisandrin A, the most abundant active ingredients of Wuzhi capsule, are known to inhibit tacrolimus metabolism by inhibiting CYP3A4/5. We aimed to predict the contribution of schisantherin A and schisandrin A to drug-drug interaction (DDI) between Wuzhi capsule and tacrolimus using physiologically-based pharmacokinetic (PBPK) modelling. Firstly, the inhibition mechanism of schisantherin A and schisandrin A on CYP3A4/5 was investigated. Thereafter, PBPK models of schisantherin A, schisandrin A and tacrolimus were established. Finally, tacrolimus pharmacokinetics were evaluated after the combined use with schisantherin A or schisandrin A. The blood area under the curve (AUC) of tacrolimus increased 1.77- and 2.61-fold after a single dose and multiple doses of schisantherin A, respectively. Meanwhile, schisandrin A inhibited tacrolimus metabolism to a smaller extent. Also, it showed that mechanism-based inhibition (MBI) played a more important role in DDI than reversible inhibition after long-term administration, while reversible inhibition was comparable to MBI after single-dose administration. In conclusion, we utilized PBPK modelling to quantify the contribution of schisantherin A and schisandrin A to DDI between tacrolimus and Wuzhi capsule. This may provide more insights for the rational use of this drug combination.

Development and validation of an LC-MS/MS method for the simultaneous quantification of seven constituents in rat plasma and application in a pharmacokinetic study of the Zaoren Anshen prescription.

A sensitive, specific and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of seven constituents of the Zaoren Anshen prescription (ZAP) in rat plasma after oral administration of the ZAP: spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin. The plasma samples and the internal standard (IS) sulfamethoxazole were extracted using acetonitrile. Chromatographic separation was performed with an Agilent HC-C18 column using a gradient elution profile and a mobile phase consisting of 0.01% formic acid in water (A) and acetonitrile (B). The analytes were quantified simultaneously in a single run using an ion trap mass spectrometer operated in the multiple reaction monitoring mode and electrospray ion-source polarity in the positive and negative modes. The calibration curves for spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin were linear over the concentration ranges of 2.90-1160, 2.50-1000, 1.80-720, 0.65-260, 2.50-1000, 8.00-1600 and 1.30-520 ng/mL, respectively. The intra- and inter-day precisions in terms of relative standard deviation were <18.9%, and the accuracies in terms of relative error were within ±14.2%. Consequently, the proposed method was successfully applied to the pharmacokinetic analysis of these seven major active compounds in rats administered ZAP. These results will facilitate research aiming to predict the effectiveness of the optimal dose of ZAP and might be beneficial for the therapeutic use of ZAP in the clinical setting.