Dissection of the potential anti-diabetes mechanism of salvianolic acid B by metabolite profiling and network pharmacology.

RATIONALE: Salvianolic acid B (Sal B), the Q-marker in Salvia miltiorrhiza, was proved to present an obvious anti-diabetes effect when treated as a food intake. Until now, the metabolism feature, tissue distribution and anti-diabetes mechanism of Sal B have not been fully elucidated. METHODS: The metabolites of Sal B in rats were profiled using ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry. The potential anti-diabetes mechanism of Sal B was predicted by network pharmacology. RESULTS: A total of 31 metabolites were characterized in rats after ingestion of Sal B at a dosage of 40 mg/kg, including 1 in plasma, 19 in urine, 31 in feces, 0 in heart, 0 in liver, 0 in spleen, 1 in lung, 1 in kidney and 0 in brain. Among them, 18 metabolites were reported for the first time. Phase I reactions of hydrolysis, hydrogenation, dehydroxylation, hydroxylation, decarboxylation and isomerization, and phase II reactions of methylation were found in Sal B. Notably, decarboxylation and dehydroxylation were revealed in Sal B for the first time. The pharmacology network results showed that Sal B and its metabolites could regulate ALB, PLG, ACE, CASP3, MMP9, MMP2, MTOR, etc. The above targets were involved in insulin signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, etc. CONCLUSIONS: The metabolism feature of Sal B in vivo was systematically revealed, and its anti-diabetes mechanism for further pharmacological validations was predicted based on metabolite profiling and network pharmacology for the first time.

Rational formulation engineering of fraxinellone utilizing 6-O-α-D-maltosyl-ß-cyclodextrin for enhanced oral bioavailability and hepatic fibrosis therapy.

Although Fraxinellone (Frax) isolated from Dictamnus albus L. possessed excellent anti-hepatic fibrosis activity, oral administration of Frax suffered from the inefficient therapeutic outcome in vivo due to negligible oral absorption. At present, the oral formulation of Frax is rarely exploited. For rational formulation design, we evaluated preabsorption risks of Frax and found that Frax was rather stable while poorly dissolved in the gastrointestinal tract (78.88 µg/mL), which predominantly limited its oral absorption. Further solubility test revealed the outstanding capacity of cyclodextrin derivatives (CDs) to solubilize Frax (6.8-12.8 mg/mL). This led us to study the inclusion complexes of Frax with a series of CDs and holistically explore their drug delivery performance. Characterization techniques involving 1H-NMR, FT-IR, DSC, PXRD, and molecular docking confirmed the most stable binding interactions when Frax complexed with 6-O-α-D-maltosyl-ß-cyclodextrin (G2-ß-CD-Frax). Notably, G2-ß-CD-Frax exhibited the highest solubilizing capacity, fast dissolution rate, and superior Caco-2 cell internalization with no obvious toxicity. Pharmacokinetic studies demonstrated markedly higher oral bioavailability of G2-ß-CD-Frax (5.8-fold that of free drug) than other Frax-CDs. Further, long-term administration of G2-ß-CD-Frax (5 mg/kg) efficiently inhibited CCl4-induced hepatic fibrosis in the mouse without inducing any toxicity. Our results will inspire the continued advancement of optimal oral Frax formulations for anti-fibrotic therapy.

Pharmacokinetic Characterization and Bioavailability Barrier for the Key Active Components of Botanical Drug Antitumor B (ATB) in Mice for Chemoprevention of Oral Cancer.

This study aims to characterize the pharmacokinetic (PK) profiles and identify important bioavailability barriers and pharmacological pathways of the key active components (KACs) of Antitumor B (ATB), a chemopreventive agent. KACs (matrine, dictamine, fraxinellone, and maackiain) of ATB were confirmed using the antiproliferative assay and COX-2 inhibition activities in oral cancer cells. The observed in vitro activities of KACs were consistent with their cell signaling pathways predicted using the in silico network pharmacology approach. The pharmacokinetics of KACs were determined after i.v., i.p., and p.o. delivery using ATB extract and a mixture of four KACs in mice. Despite good solubilities and permeabilities, poor oral bioavailabilities were estimated for all KACs, mostly because of first-pass metabolism in the liver (for all KACs) and intestines (for matrine and fraxinellone). Multiple-dose PK studies showed 23.2-fold and 8.5-fold accumulation of dictamine and maackiain in the blood, respectively. Moreover, saliva levels of dictamine and matrine were found significantly higher than their blood levels. In conclusion, the systemic bioavailabilities of ATB-KACs were low, but significant levels of dictamine and matrine were found in saliva upon repeated oral administration. Significant salivary concentrations of matrine justified its possible use as a drug-monitoring tool to track patient compliance during chemoprevention trials.

Pharmacokinetics of five phthalides in volatile oil of Ligusticum sinense Oliv.cv. Chaxiong, and comparison study on physicochemistry and pharmacokinetics after being formulated into solid dispersion and inclusion compound.

BACKGROUNDS: The dried rhizome of Ligusticum sinense Oliv.cv. Chaxiong has been used to treat cardiovascular and cerebrovascular diseases, atherosclerosis, anemia and stroke. A high purity extract from chaxiong (VOC, brownish yellow oil) was extracted and separated. Its main components were senkyunolide A (SA, 33.81%), N-butylphthalide (NBP, 1.38%), Neocnidilide (NOL, 16.53%), Z-ligustilide (ZL, 38.36%), and butenyl phthalide (BP, 2.48%), respectively. Little is known about the pharmacokinetics of these phthalides in Chaxiong, and different preparations to improve the physicochemistry and pharmacokinetics of VOC have not been investigated. METHODS: At different predetermined time points after oral administration or intravenous administration, the concentrations of SA, NBP, NOL, ZL and BP in the rat plasma were determined using LC-MS/MS, and the main PK parameters were investigated. VOC-P188 solid dispersion and VOC-ß-CD inclusion compound were prepared by melting solvent method and grinding method, respectively. Moreover, the physicochemical properties, dissolution and pharmacokinetics of VOC-P188 solid dispersion and VOC-ß-CD inclusion compound in rats were assessed in comparison to VOC. RESULTS: The absorptions of SA, NBP, NOL, ZL and BP in VOC were rapid after oral administration, and the absolute bioavailability was less than 25%. After the two preparations were prepared, dissolution rate was improved at pH 5.8 phosphate buffer solution. Comparing VOC and physical mixture with the solid dispersion and inclusion compound, it was observed differences occurred in the chemical composition, thermal stability, and morphology. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound had a significantly higher AUC and longer MRT in comparison with VOC. CONCLUSION: SA, NBP, NOL, ZL and BP in VOC from chaxiong possessed poor absolute oral bioavailability. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound could be prospective means for improving oral bioavailability of SA, NBP, NOL, ZL and BP in VOC.

The effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA, salvianolic acid B and ginsenoside Rg1 in Fufang Danshen preparation in rats.

Fufang Danshen preparation (FDP) is consisted of Salviae Miltiorrhizar Radix et Rhizoma (Danshen), Notoginseng Radix et Rhizoma (Sanqi) and Borneolum Syntheticum (borneol). FDP is usually used to treat myocardial ischemia hypoxia, cerebral ischemia and alzheimer's disease, etc. In the treatment of cerebrovascular diseases, borneol is usually used to promote the absorption and distribution of the bioactive components to proper organs, especially to the brain. The purpose of this study is investigating the effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA (TS IIA), salvianolic acid B (SAB) and ginsenoside Rg1 in FDP. Male healthy Sprague-Dawley (SD) rats were given Danshen extracts, Sanqi extracts (Panax notoginsengsaponins) or simultaneously administered Danshenextracts, Sanqi extracts and borneol. Plasma and brain samples were collected at different points in time. The concentration of TS IIA, SAB and Rg1 was determined by UPLC-MS/MS method. The main pharmacokinetics parameters of plasma and brain tissue were calculated by using Phoenix WinNolin 6.1 software. In comparison with Danshen and Sanqi alone, there were significant differences in pharmacokinetic parameters of TS IIA, SAB and Rg1, and the brain distribution of SAB and TS IIA when Danshen, Sanqi and borneol were administrated together. Borneol statistically significant shortened tmax of TS IIA, SAB and Rg1 in plasma and brain, increased the bioavaiability of Rg1, inhibited metabolism of Rg1 and enhanced the transport of TS IIA and SAB to brain. These results indicated that borneol could affect the multiple targets components and produce synergistic effects. Through accelerating the intestinal absorption and brain distribution, borneol caused the effective ingredients of Danshen and Sanqi to play a quicker therapeutic role and improved the therapeutic effect.

WBQ5187, a Multitarget Directed Agent, Ameliorates Cognitive Impairment in a Transgenic Mouse Model of Alzheimer's Disease and Modulates Cerebral ß-Amyloid, Gliosis, cAMP Levels, and Neurodegeneration.

Previously, we designed, synthesized, and evaluated a series of quinolone-benzofuran derivatives as multitargeted anti-Alzheimer's disease (anti-AD) compounds, and we discovered that WBQ5187 possesses superior anti-AD bioactivity. In this work, we investigated the pharmacokinetics of this new molecule, as well as its therapeutic efficacy in restoring cognition and neuropathology, in the APP/PS1 mouse model of AD. Pharmacokinetic analyses demonstrated that WBQ5187 possessed rational oral bioavailability, metabolic stability, and excellent blood-brain barrier (BBB) permeability. Pharmacodynamics studies indicated that a 12-week treatment with the lead compound at doses of 40 mg/kg or higher significantly enhanced the learning and memory performance of the APP/PS1 transgenic mice, and the effect was more potent than that of clioquinol (CQ). Furthermore, WBQ5187 notably reduced cerebral ß-amyloid pathology, gliosis, and neuronal cell loss and increased the levels of cAMP in the hippocampus of these mice. The surrogate measures of emesis indicated that WBQ5187 had no effect at its cognitive effective doses. Overall, our results demonstrated that this compound markedly improves cognitive and spatial memory functions in AD mice and represents a promising pharmaceutical agent with potential for the treatment of AD.

Brain targeting of Baicalin and Salvianolic acid B combination by OX26 functionalized nanostructured lipid carriers.

In order to deliver Salvianolic acid B (Sal B) and Baicalin (BA) to the brain tissue to repair neuron damage and improve cerebral ischemia-reperfusion injury (IRI), in our previous study, a nanostructured lipid carrier (NLC) containing BA and Sal B, and modified by the transferrin receptor monoclonal antibody OX26 (OX26-BA/Sal B-NLC) was constructed. The present study is to evaluate its in vitro release behavior, in vitro and in vivo targeting ability, in vitro pharmacodynamics and brain pharmacokinetics. The results showed that the release mechanism of the formulation was in line with the Weibull model release equation. The in-vitro and in-vivo targeting ability study exhibited that OX26 modified formulations was obviously higher than that of non-modified and solution groups. The results of in vitro preliminary study to investigate the protective effect of OX26-BA/Sal B-NLC on oxygen-glucose deprivation/reperfusion injured cells showed that it could decrease the injury. Furthermore, the results of brain microdialysis study showed that the OX26-modified preparation group could significantly increase the content of BA in the brain. In the solution group and the unmodified group, Sal B can only be detected at few time points, while OX26-modified BA/Sal B-NLC could be detected within 4 h. These results indicating that OX26-modified NLC can promote the brain delivery of Sal B and BA combination.

Immunomodulatory and Metabolic Changes after Gnetin-C Supplementation in Humans.

Gnetin-C is a naturally occurring stilbene derived from the seeds of Gnetum gnemon L., an edible plant native to Southeast Asia that is called melinjo. Although the biological properties and safety of G. gnemon extract, which contains nearly 3% Gnetin-C, have been confirmed in various human studies, whether or not pure Gnetin-C is safe for humans is unclear at present. We conducted a randomized, double-blind, placebo-controlled trial. Healthy subjects were randomly divided into two groups. The interventional group (n = 6) was given Gnetin-C, and the control group (n = 6) was provided a placebo, for 14 days. Lipid profiles, biomarkers of oxidative stress and circulating blood cells were assessed before and after the intervention. All subjects completed the study, with no side effects reported across the study duration. Gnetin-C supplementation demonstrated a statistically significant increase in the absolute number of circulating natural killer (NK) cells expressing the activating receptors NKG2D and NKp46. NK cells derived from subjects who received Gnetin-C for two weeks showed higher cytotoxicity against K562 target cells than those before receiving Gnetin-C. In addition, Gnetin-C also resulted in a significant decrease in the absolute neutrophil count in the blood compared with the placebo. Furthermore, Gnetin-C significantly reduced the levels of uric acid, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total adiponectin, and high-molecular-weight adiponectin. These data indicate that Gnetin-C has biological effects of enhancing the NK activity on circulating human immune cells. The immunomodulatory effects are consistent with a putative improvement in cancer immunosurveillance via the upregulation of the NKG2D receptor. The study was registered with UMIN-CTR, number 000030364, on 12 December 2017.

Simultaneous quantification of ligustilide, dl-3-n-butylphthalide and senkyunolide A in rat plasma by GC-MS and its application to comparative pharmacokinetic studies of Rhizoma Chuanxiong extract alone and Baizhi Chuanxiong Decoction.

The herb couple has special clinical significance in reducing the toxicity and increasing the efficacy of drugs. The combination of Radix Angelicae Dahuricae (Baizhi, BZ) and Rhizoma Chuanxiong (ChuanXiong, CX) is a traditional herb couple. The combination performs better than the CX extract alone in the treatment of migraine and has been used for thousands of years. However, the specific compatibility mechanisms are still unclear. Ligustilide, dl-3-n-butylphthalide and senkyunolide A are the major active ingredients in CX and BZ-CX decoction. However, a comprehensive study of the pharmacokinetics of CX has not been carried out. A gas chromatography-mass spectroscopy (GC-MS) method with high selectivity, sensitivity and accuracy was developed. An SH-Rxi-5Sil (30 m × 0.25 mm i.d., and 0.25 µm film thickness) column was employed in the GC separation. Selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were used to validate the current GC-MS method. Using the validated method, this is the first time to study on the comparative pharmacokinetics of ligustilide, dl-3-n-butylphthalide and senkyunolide A from CX alone and BZ-CX decoction in rat plasma. The pharmacokinetic parameters (Cmax , Tmax , T1/2 , AUC0-t , AUC0-∞ and CLz/F) of all of the detected ingredients showed significant differences between the two groups (P < 0.05). The results are helpful for further investigation of the compatibility mechanism of BZ-CX decoction.

Pharmacokinetics of salvianolic acid B, rosmarinic acid and Danshensu in rat after pulmonary administration of Salvia miltiorrhiza polyphenolic acid solution.

A sensitive and accurate LC-MS/MS method was established for quantifying salvianolic acid B (Sal B), rosmarinic acid (Ros A) and Danshensu (DA) in rat plasma. Salvia miltiorrhiza polyphenolic acid (SMPA), active water-soluble ingredients isolated and purified from Salvia miltiorrhiza Bge included Sal B, Ros A and DA. The pharmacokinetic analysis of Sal B, Ros A and DA after pulmonary administration of SMPA solution to rat was performed by LC-MS/MS. Results from the pharmacokinetic studies showed that the peak concentration of DA was 21.85 ± 6.43 and 65.39 ± 3.83 ng/mL after pulmonary and intravenous administration, respectively. DA was not detected at 2 h after administration. The absolute bioavailabilities of Sal B and Ros A were respectively 50.37 ± 27.04 and 89.63 ± 12.16% after pulmonary administration of 10 mg/kg SMPA solution in rats. The absolute bioavailability of Sal B increased at least 10-fold after pulmonary administration, compared with oral administration. It was concluded that the newly established LC-MS/MS method was suitable for describing the pharmacokinetic characteristics of Sal B, Ros A and DA in rat after pulmonary administration of SMPA solution. The data from this study will provide a preclinical insight into the feasibility of pulmonary administration of SMPA.

Development of sustained-release pellets to modulate the in vivo processes of the main active components of Danshen: A pharmacokinetic and pharmacodynamic evaluation.

BACKGROUND: Danshen is a first-line traditional Chinese medicine derived from Salvia miltiorrhiza Bunge consisting mainly of tanshinone IIA, tanshinol, protocatechuic aldehyde, and salvianolic acid B, it is widely used to treat cardiovascular diseases based on the synergistic effect of its multiple active components. Recent studies have indicated that the overall effect of traditional Chinese medicine is closely related to the in vivo coexistence of a variety of active components. HYPOTHESIS: The prolongation of the coexistence of the four active components in Danshen in vivo by regulating their pharmacokinetic processes may contribute to better efficiency. METHODS/STUDY DESIGNS: Individual sustained-release pellets of the four main active components in Danshen were respectively prepared according to the optimised formulations developed in our previous studies to modulate their in vivo processes, in which the desired release profiles of each kind of sustained-release pellets for formulation optimisation were calculated based on the point-area deconvolution and circadian rhythm of variant angina. The four kinds of sustained-release pellets were filled into capsules on the basis of the original weight ratio of the four active components in purified Salvia miltiorrhiza extract for further in vitro release and pharmacokinetic and pharmacodynamic investigations. RESULTS: The release behaviours of the combined Danshen capsules composed of the four kinds of sustained-release pellets were evaluated in three media with different pH levels (pH 1.2, 6.8, and pure water). The release profiles of each kind of sustained-release pellets in pH 6.8 PBS and pH 1.2 HCl were similar to the release profile of those in pure water (similarity factors f2 > 50). Pharmacokinetic studies revealed that the four kinds of sustained-release pellets in the combined Danshen capsules possessed the same Tmax and similar and extended MRT. Moreover, pharmacodynamic studies indicated that the combined Danshen capsules had much better anti-angina effects than commercial Danshen capsules according to comprehensive evaluations via electrocardiogram, serum index (CK-MB, cTn-I, ET, and NO), myocardial oxidative damage, and myocardial pathologic biopsy. CONCLUSION: Sustained-release preparations can markedly prolong the in vivo coexistence of multiple components in Danshen to enhance their overall effects, which provides a potent strategy for developing the combination therapy of traditional Chinese medicine.

Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line.

OBJECTIVE: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. METHODS: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3, -8 and -9 were also determined. RESULTS: Psoralidin reduces cell viability greatly in a time dependent manner (64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 µmol/L psoralidin respectively) and up-regulates activities of caspase-3, -8 and -9 in a concentration dependent manner (between 4 to 64 µmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 µmol/L (P<0.05 at 16 and 64 µmol/L). Caspase-3 inhibitor (Ac-DEVD-CHO at concentrations between 10 to 20 µmol/L), p53 inhibitor (pifithrin-α at 5 µmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin. CONCLUSION: The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.

Bone and Joint Tissue Penetration of the Staphylococcus-Selective Antibiotic Afabicin in Patients Undergoing Elective Hip Replacement Surgery.

Afabicin (formerly Debio 1450, AFN-1720) is a prodrug of afabicin desphosphono (Debio 1452, AFN-1252), a novel antibiotic in development which targets the staphylococcal enoyl-acyl carrier protein reductase (FabI) and exhibits selective potent antibacterial activity against staphylococcal species, including methicillin-resistant Staphylococcus aureus As part of clinical development in bone and joint infections, a distribution study in bone was performed in 17 patients who underwent elective hip replacement surgery. Patients received 3 doses of 240 mg afabicin orally (every 12 h) at various time points before surgery. Afabicin desphosphono concentrations were measured by liquid chromatography-tandem mass spectrometry in plasma, cortical bone, cancellous bone, bone marrow, soft tissue, and synovial fluid collected during surgery at 2, 4, 6, or 12 h after the third afabicin dose. The study showed good penetration of afabicin desphosphono into bone tissues, with mean area under the curve ratios for cortical bone-, cancellous bone-, bone marrow-, soft tissue-, and synovial fluid-to-total plasma concentrations of 0.21, 0.40, 0.32, 0.35, and 0.61, respectively. When accounting for the free fraction in plasma (2%) and synovial fluid (9.4%), the mean ratio was 2.88, which is indicative of excellent penetration and which showed that the afabicin desphosphono concentration was beyond the MIC90 of S. aureus over the complete dosing interval. These findings, along with preclinical efficacy data, clinical efficacy data for skin and soft tissue staphylococcal infection, the availability of both intravenous and oral formulations, and potential advantages over broad-spectrum antibiotics for the treatment of staphylococcal bone or joint infections, support the clinical development of afabicin for bone and joint infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02726438.).

Evaluation of the Absorption Behavior of Main Component Compounds of Salt-Fried Herb Ingredients in Qing'e Pills by Using Caco-2 Cell Model.

Qing'e Pills is a Chinese traditional herbal product, which is often used to strengthen muscles and bones in TCM (traditional Chinese Medicine) practice. Its two main component herbs, namely, Cortex Eucommiae and Fructus Psoraleae are both required to be salt-fried according to TCM theory. We have evaluated the effects of salt-frying treated herbs on Caco-2 cell uptake behavior for those active ingredients of Qing'e Pills. By investigating of various variables, including MTT, temperature, inhibitors, pH, salt concentration and herb processing methods, we tried to clarify whether the salt-processing on herbs was necessary or not. Results showed that, compared to other processing methods, the salt-frying process significantly (p < 0.01) enhanced the absorption of effective components of Qing'e Pills. The way that psoralen, isopsoralen, psoralenoside and geniposide acid entered Caco-2 cells at low concentrations was via passive diffusion. These components were not substrates of P-glycoprotein. It demonstrated that the salt-frying process not only enhanced the concentration of active components in herb extract, but also changed their absorption behaviors. Nevertheless, the mechanism of absorption behavior changing needs to be further investigated.

Pharmacokinetics-Based Identification of Potential Therapeutic Phthalides from XueBiJing, a Chinese Herbal Injection Used in Sepsis Management.

XueBiJing, an injectable five-herb preparation, has been incorporated into routine sepsis care in China. Phthalides, originating from XueBiJing's component herbs Ligusticum chuanxiong rhizomes and Angelica sinensis roots, are believed to contribute to its therapeutic effects due to their presence in the preparation and antisepsis-related properties. This investigation aimed to identify potential therapeutic phthalides that are bioavailable to act on XueBiJing's therapeutic targets and that could serve as pharmacokinetic markers to supplement classic biomarkers for sepsis care. Among 10 phthalides detected in XueBiJing, senkyunolides I and G were the major circulating phthalides in human subjects, but their different pharmacokinetics might influence their contribution to XueBiJing's therapeutic action. Senkyunolide I exhibited a large distribution volume (1.32 l/kg) and was moderately bound in plasma (54% unbound), whereas senkyunolide G exhibited a small distribution volume (0.10 l/kg) and was extensively bound in plasma (3% unbound). Clearance of senkyunolide I from the systemic circulation was governed by UGT2B15-mediated hepatic glucuronidation; the resulting electrophilic glucuronides were conjugated with glutathione in the liver. Senkyunolide G was selectively bound to albumin (99%) in human plasma. To our knowledge, the human pharmacokinetic data of XueBiJing's phthalides are reported here for the first time. Based on this investigation and such investigations of the other component herbs, follow-up pharmacodynamic assessments of bioavailable herbal compounds are planned to elucidate XueBiJing's chemical basis responsible for its therapeutic action. Senkyunolides I and G, having the preceding disposition characteristics that could be detectably altered by septic pathophysiology, could serve as pharmacokinetic markers for sepsis care.

A validated LC-MS/MS method for the determination of senkyunolide I in dog plasma and its application to a pharmacokinetic and bioavailability studies.

Senkyunolide I is one of the major bioactive components in the herbal medicine Ligusticum chuanxiong. The aim of this study was to develop and validate a fast, simple and sensitive LC-MS/MS method for the determination of senkyunolide I in dog plasma. The plasma samples were processed with acetonitrile and separated on a Waters Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm). The mobile phase consisted of 0.1% formic acid aqueous and acetonitrile was delivered at a flow rate of 0.3 mL min-1 . The detection was achieved in the positive selected reaction monitoring mode with precursor-to-product transitions at m/z 225.1 → 161.1 for senkyunolide I and at m/z 349.1 → 305.1 for an internal standard. The assay was linear over the tested concentration range, from 0.5 ng mL-1 to 1000 ng mL-1 , with a correlation coefficient >0.9992. The mean extraction recovery from dog plasma was within the range of 85.78-93.25%, while the matrix effect of the analyte was within the range of 98.23-108.89%. The intra- and inter-day precisions (RSD) were <12.12% and the accuracy (RR) ranged from 98.89% to 104.24%. The validated assay was successfully applied to pharmacokinetic and bioavailability studies of senkyunolide I in dogs. The results demonstrated that (a) senkyunolide I showed short elimination half-life (<1 h) in dog, (b) its oral bioavailability was >40% and (c) senkyunolide I showed dose-independent pharmacokinetic profiles in dog plasma over the dose range of 1-50 mg kg-1 .

Discovery of EBI-1051: A novel and orally efficacious MEK inhibitor with benzofuran scaffold.

A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.

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.

Determination of quantitative retention-activity relationships between pharmacokinetic parameters and biological effectiveness fingerprints of Salvia miltiorrhiza constituents using biopartitioning and microemulsion high-performance liquid chromatography.

In this study, we analyzed danshen (Salvia miltiorrhiza) constituents using biopartitioning and microemulsion high-performance liquid chromatography (MELC). The quantitative retention-activity relationships (QRARs) of the constituents were established to model their pharmacokinetic (PK) parameters and chromatographic retention data, and generate their biological effectiveness fingerprints. A high-performance liquid chromatography (HPLC) method was established to determine the abundance of the extracted danshen constituents, such as sodium danshensu, rosmarinic acid, salvianolic acid B, protocatechuic aldehyde, cryptotanshinone, and tanshinone IIA. And another HPLC protocol was established to determine the abundance of those constituents in rat plasma samples. An experimental model was built in Sprague Dawley (SD) rats, and calculated the corresponding PK parameterst with 3P97 software package. Thirty-five model drugs were selected to test the PK parameter prediction capacities of the various MELC systems and to optimize the chromatographic protocols. QRARs and generated PK fingerprints were established. The test included water/oil-soluble danshen constituents and the prediction capacity of the regression model was validated. The results showed that the model had good predictability.

The effect of deep eutectic solvent on the pharmacokinetics of salvianolic acid B in rats and its acute toxicity test.

Deep eutectic solvent (DES), the benign green solvent with uniquely physical properties, has been widely applied in various fields. Our previous study indicated that DES could improve the stability and extraction efficiency of salvianolic acid B (SAB). In this work, with SAB as a model drug, the feasibility of DES as a drug carrier for oral preparation was investigated by evaluating the influence of DES on the pharmacokinetics of SAB and the toxicity of DES. Acute oral toxicity test illustrated that choline chloride-glycerol (ChCl-GL, molar ratio 1:2) was non-toxic with the median lethal dose of 7733mg/kg. To comparison the difference of pharmacokinetics between SAB dissolved in ChCl-GL (1:2) and in water, a rapid and sensitive ultra-performance liquid chromatography coupled with mass spectrum was established to determine SAB and its metabolites in rat plasma. The method validation was also tested for the specificity, linearity (r2>0.9980 over two orders of magnitude), precision (intra-day relative standard deviation (RSD)<2.73% and inter-day RSD<7.72%), extraction recovery (70.96-80.78%) and stability under three different situations. Compared to water, the pharmacokinetic parameters clarified that ChCl-GL (1:2) could promote the absorption of SAB, the peak concentration (Cmax) of 0.308±0.020mg/L was slightly higher than 0.277±0.024mg/L (SAB dissolved in water), and the peak time (Tmax) was significantly decreased from 30min (SAB dissolved in water) to 20min. There was no significant difference on the metabolites between SAB dissolved in ChCl-GL (1:2) and in water. This is the first report on the pharmacokinetic study of DES as a candidate of drug carrier, and the results provide a meaningful basis for the application of DES in pharmaceutical preparation.