Search

Purpose. One of the biggest challenges in cancer immunotherapy is generating robust cancer-specific immunity. This work describes using a biocomplex assembled from a toll-like receptor agonist CpG oligodeoxynucleotide 1826 (CpG) and a pancreatic cancer antigen peptide mesothelin for tuning pancreatic tumor immunity. Methods. This biocomplex was assembled via electrostatic interactions and characterized in size, morphology, zeta potential, and cargo loading. The effect of biocomplex on cell viability and activation of DCs and macrophages were measured by flow cytometry. The production of cytokines (GM-CSF, TNF, and IL-6) was evaluated by using ELISA kits. The effect of biocomplex on tumor cell proliferation was also evaluated by in vivo tumor model. Result. We can modulate the surface charge of the biocomplex by simply varying the ratios of the two components. In cell models, this biocomplex did not impact cell viability in the antigen-presenting cell (i.e., dendritic cell and macrophage)-directed immunity. Moreover, this biocomplex regulated the secretion of tumor-related cytokines (i.e., GM-CSF, TNF, and IL-6) and promoted the activation of immune cell surface markers (i.e., CD80+, CD86+, and CD40+). In the mouse model, the biocomplex inhibited the tumor burden effectively and promoted the production of effector cytokines. Conclusion. The present studies showed that the biocomplex with antigen peptide and toll-like receptor agonist was able to potentiate the antitumor immunity in vivo. This study will help understanding of immunity in pancreatic cancer and developing new immune therapeutic strategies for pancreatic adenocarcinoma.

Growing evidence has shown that some pharmaceutical excipients can act on drug transporters. The present study was aimed at investigating the effects of 13 commonly used excipients on the intestinal absorption of metformin (MTF) and the underlying mechanisms using Caco-2 cells and an ex vivo mouse non-everted gut sac model. First, the uptake of MTF in Caco-2 cells was markedly inhibited by nonionic excipients including Solutol HS 15, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and crospovidone. Second, transport profile studies showed that MTF was taken up via multiple cation-selective transporters, among which a novel pyrilamine-sensitive proton-coupled organic cation (H+/OC+) antiporter played a key role. Third, Solutol HS 15, polysorbate 40, and polysorbate 60 showed cis-inhibitory effects on the uptake of either pyrilamine (prototypical substrate of the pyrilamine-sensitive H+/OC+ antiporter) or 1-methyl-4-phenylpyridinium (substrate of traditional cation-selective transporters including OCTs, MATEs, PMAT, SERT, and THTR-2), indicating that their suppression on MTF uptake is due to the synergistic inhibition toward multiple influx transporters. Finally, the pH-dependent mouse intestinal absorption of MTF was significantly decreased by Solutol HS 15, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and pyrilamine. In conclusion, this study revealed that a novel transport process mediated by the pyrilamine-sensitive H+/OC+ antiporter contributes to the intestinal absorption of MTF in conjunction with the traditional cation-selective transporters. Mechanistic understanding of the interaction of excipients with cation-selective transporters can improve the formulation design and clinical application of cationic drugs.

The study aims to investigate the propylene glycol-based liposomes named ‘proposomes’ in enhancing skin permeation of drugs with different physicochemical properties. Ibuprofen, tofacitinib citrate, rhodamine B, and lidocaine were loaded into proposomes. These drug formulations were analyzed for particle size, zeta potential, polydispersity index, entrapment efficiency, and in vitro skin permeation. The confocal laser scanning microscopy was performed on skin treated with calcein and rhodamine B laden proposomes. The transdermal delivery relative to physicochemical properties of drugs such as logP, melting point, molecular weight, solubility, etc., were analyzed. We tested the safety of the proposomes using reconstructed human skin tissue equivalents, which were fabricated in-house. We also used human cadaver skin samples as a control. The proposomes had an average diameter of 128 to 148 nm. The drug’s entrapment efficiencies were in the range of 42.9–52.7%, translating into the significant enhancement of drug permeation through the skin. The enhancement ratio was 1.4 to 4.0, and linearly correlated to logP, molecular weight, and melting point. Confocal imaging also showed higher skin permeation of calcein and rhodamine B in proposome than in solution. The proposome was found safe for skin application. The enhancement of skin delivery of drugs through proposomes was dependent on the lipophilicity of the drug. The entrapment efficiency was positively correlated with logP of the drug, which led to high drug absorption.

A matrix metalloproteinases (MMPs) triggered multifunctional micellar system loading traditional chemotherapeutic agent paclitaxel (PTX) and marimastat (MATT), an inhibition of matrix metalloproteinases, was established to prevent tumor growth and metastasis. The micelles were self-assembled by the conjugate synthesized with an MMPs sensitive peptide as the bridge between PTX and poly(ethylene glycol) (PEG). 4T1 cell line derived from a murine breast tumor was selected as the cell model due to its high metastasis. The cytotoxicity assay and cell apoptosis analysis revealed that the sensitive micelles increased the toxicity and cell apoptosis compared with the insensitive micelles. In addition, this system also exhibited high penetration ability in tumor spheroids and significant invasion inhibition with the method of Transwell invasion assay. In the BALB/c mice bearing 4T1 tumors, this system inhibited the growth of the metastatic tumor and prevented the incidence of lung metastasis with low systemic toxicity. And the expression of MMP-2 and MMP-9, which are important in the process of tumor metastasis, was down-regulated. Generally, the data obtained from the in vitro and in vivo studies confirmed that the codelivery of PTX and MATT by the MMPs sensitive micelles achieved not only significant antitumor effect but also obvious inhibition effect of tumor metastasis, which are the leading cause of cancer deaths. This functional particle system may provide a promising strategy for metastatic breast cancer therapy.

Multidrug resistance (MDR) is a common intractable barrier in success of clinical cancer chemotherapy. Codelivery of two drugs using nanocarriers is a commonly used approach to treat the MDR cancer. However, the drug payload in the conventional nanocarriers is low and thus compromises the treatment outcomes. Disulfiram (DSF) is promising to reverse MDR and increases the sensitivity of cancer cells to chemotherapy. While, paclitaxel (PTX) is one of the frequently used anticancer drug. Here, by using a drug-delivering-drug (DDD) strategy based on nanocrystals, hybrid PTX-DSF nanocrystals (PTX-DSF Ns) were developed for codelivery of PTX and DSF to reverse MDR in cancer. The 160-nm PTX-DSF Ns with rod-like morphology had drug-loading up to 43% at mass ratio of 5:1. Interestingly, the nanoparticles entered cells via caveolar endocytosis. By reducing intracellular ATP level and GST activity, PTX-DSF Ns killed the Taxol resistant A549 cells with higher efficiency than PTX alone, exhibiting as 6-fold increase of apoptosis in MDR tumor. The nanoparticles circulated in blood over time, accumulated in tumor efficiently and reduced the tumor volume by 12-fold in MDR tumor-bearing BALB/c nude mice and allowed 12-fold apoptosis in tumor. Additionally, the immunohistochemical examination demonstrated the safety of the nanoparticles. Overall, the DDD strategy-based PTX-DSF Ns have promising potential for the treatment of MDR cancer.

Oleic acid is a pharmaceutical excipient and has been widely used in many dosage forms. It remains unclear in terms of the fatty acids (FAs) profile. In this study, a sensitive and direct method based on high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was developed to study the compositions of oleic acid. The chromatographic conditions were optimized to achieve good separation and high sensitivity. The components of oleic acid were identified by ion trap/time of flight mass spectrometry (MS-IT-TOF). Twenty-seven FAs were identified based on the exact mass-to-charge ratio and fragments, among which 13 FAs were confirmed with the reference standards. Nine FAs in the oleic acid samples including oleic acid, linolenic acid, myristic acid, palmitoleic acid, linoleic acid, palmitic acid, stearic acid, arachidic acid and behenic acid were simultaneously determined by the developed HPLC-CAD, which showed good linearity with r2>0.999. The limit of detection (LOD) and limit of quantification (LOQ) of 9 FAs were 0.006-0.1 μg mL-1 and 0.032-0.22 μg mL-1, respectively. The components with concentration level not less than 0.03 % (referring to the sample concentration of 1.0 mg mL-1) can be quantified. The mean recovery values of 9 FAs ranged from 96.5%-103.6% at three concentration levels of 80 %, 100 % and 120 %. The repeatability and intermediate precision were less than 5.0 % for oleic acid and components with concentration levels more than 0.05 %. In contrast to the conventional pre-column derivatization gas chromatography (GC), HPLC-CAD could unbiasedly and directly detect more components, especially the FAs with long carbon chains. Overall, the developed novel HPLC-CAD method can ameliorate the deficiency of the indirect GC method recorded in current pharmacopeias, thus having great potential for the comprehensive understanding and quality control of oleic acid.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing content of melanin, with efficacy up to more than 50% upon 16 weeks of twice daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and large molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. We also tested the effects of chemical penetration enhancers and microneedles to deliver two peptides through skin. Enhanced skin permeation was found using an in vitro human skin permeation model. Moreover, we examined peptide retention of different formulations in skin. Our data showed that palm-peptides in microneedle patch was the most effective.

The objective of this work was to investigate the capacity of mogroside V (MOG-V), a food additive, as a novel carrier to improve the bioavailability and liver distribution of silybin (SLY). Solid dispersion particles (SDPs) of SLY/MOG-V were prepared utilizing the solvent evaporation method. The physicochemical characterizations of SDPs were evaluated by using dynamic light scattering (DLS), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) measurements. DLS results demonstrated the formation of nanoparticles (206 nm) of SDPs in water. DSC and PXRD analysis revealed that SLY was in amorphous form or molecularly dispersed in SDPs. SDPs also exhibited a major increase in both dissolution rate and saturation solubility, as evidenced by a 1931-fold improvement (2201 μg/mL) in solubility compared with pure SLY (1.14 μg/mL). The pharmacokinetic study in rats showed that oral absorption of SLY/MOG-V SDPs was dramatically increased. The mean value of AUC until 12 h for SLY/MOG-V SDPs (27,481 ng·min/mL) was 24.5-fold higher than that of pure SLY (1122 ng·min/mL). In vivo tissue distribution experiment in mice confirmed that the major distribution tissue was changed from lungs to liver after SLY was loaded into MOG-V. In addition, even orally administrated to mice at a high dose (4.2 g/kg), MOG-V exhibited no undesirable effect on the plasma glucose concentrations. Thus, MOG-V may have the applicability to serve as an ideal excipient for solubilization or as a novel liver targeting carrier for the delivery of SLY.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing melanin content, with efficacy up to more than 50% upon 16 weeks of twice-daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and high molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. Molecular docking results showed that the two peptides exhibited similar biological activity towards tyrosinase. We also tested the effect of chemical penetration enhancers and microneedles to deliver the two peptides into and through skin, using an in vitro human skin permeation method. It was shown that the palm-peptide achieved the best skin retention owing to the increased lipophilicity. In addition, skin permeation of the palm-peptides was enhanced by the chemical skin penetration enhancers, namely, oleic acid and menthol. Skin permeation of the native peptide was enhanced by the microneedle patch but not the chemical skin penetration enhancers. Cutaneous absorption of the palm-peptides was estimated to have achieved its therapeutic concentration within skin. The combinatory approach of using molecular modification, chemical penetration enhancement, and microneedle patch proves to be useful to enhanceskin permeation of the peptides.

Ethnopharmacological relevance The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. Aim of the study: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). Materials and methods The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. Results In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. Conclusions A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.

The cover image is based on the Original Article Keratinocytes maintain compartmentalization between dermal papilla and fibroblasts in 3D heterotypic tri‐cultures by Justin J. Y. Tan et al., DOI 10.1111/cpr.12668. [Image: see text]

Objectives Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle‐like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle‐related cells. Materials and methods Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three‐dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two‐dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. Results The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core‐shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell‐cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. Conclusions Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function., Cultures comprising of dermal papilla cells, keratinocytes, and human dermal fibroblasts were prepared from sequential seeding into hydrogel microwells, in order to reproduce the spatial orientation of hair follicular cells in vivo. This work demonstrated the importance of keratinocytes in maintaining cellular compartmentalization and dermal papilla aggregation, which is necessary for hair follicle development and function.

Zhi-Zi-Da-Huang decoction (ZZDHD) has been used for treatment of alcoholic liver disease in China for thousands of years. In order to reveal the dynamic biotransformation of the decoction in vivo, a high-throughout, sensitive and special method based on high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (HPLC–DAD–TOF/MS) and high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC–QqQ/MS) was developed and validated. 25 parent compounds and 28 metabolites were characterized, among which, two metabolites were found for the first time and tentatively identified by neutral-loss scan and product-ion scan. All the compounds were assigned to iridoids, flavones, anthraquinones, coumarin or p-coumaric acid, and their biotransformation pathways were found to involve glucuronidation, sulfation, reduction and ring cleavage. Glucuronidation occurred as a major metabolic pathway of genipin and flavanone and the conjugates could be detected almost during the whole sampling duration. To compounds such as anthraquinones, coumarin and p-coumaric acid, sulfation is the only transformation pathway and the metabolites were found at 0–12, 4–18, 4–48 h respectively after administration. Reduction and/or ring cleavage of genipin glucuronide and naringin were also observed obviously. The phenomena that parts of parent compounds and metabolites were able to be detected even 48 h after administration implied that the accumulating effect of these constituents in vivo would happen and the potential toxicity of the decoction might appear if multiple dosing is adopted. The strategy used in this paper was proved helpful to offer important information for the clinical safe use of ZZDHD.

(5R)-5-Hydroxytriptolide (LLDT-8), a novel triptolide derivative, will proceed to phase II clinical trials for the treatment of rheumatoid arthritis and cancer. However, the selection of disease and patients is largely limited by the testis toxicity, yet toxicity mechanisms are still poorly understood. In this study, LLDT-8 dose and time-dependently decreased the testes weight, germinal cell layers and induced abnormal spermatid development. Analysis of the germ cell-specific marker showed that spermatocytes were more sensitive to LLDT-8, which was confirmed by the in vitro sensitivity assay with spermatocyte-like GC-2spd and sertoli-like TM4 cells. In GC-2spd, LLDT-8 induced G1/S arrest and apoptosis. MAPK activity screening identified that TGF-β activated kinase 1 (Tak1) is critical in LLDT-8 induced apoptosis. LLDT-8 reduced the Tak1 protein and dephosphorylated Tak1 at Ser412 in GC-2spd and the testes, but not in TM4. RNAi mediated depletion or pharmacologic inhibition of Tak1 induced apoptosis in GC-2spd. Meanwhile, activating Tak1 rescued up to 50% of the GC-2spd cells from the apoptosis induced by LLDT-8. Altogether, our study firstly revealed the important role of Tak1 in the survival of spermatocytes, and dephosphorylation of Tak1 at Ser412 may contribute to the spermatocyte-specific testis toxicity induced by LLDT-8.

Transdermal drug delivery is widely investigated as an alternative drug administration route to oral delivery and hypodermic injections. Owing to the availability of human skin samples, in vitro tests are used to predict the in vivo delivery of transdermal drugs. The most widely used validation method is skin permeation using diffusion cells. Traditional diffusion cells, however, are capacious and often require large amounts of skin sample and drugs, which is undesirable, given the scarcity of new drug entities and the limitation of skin sample supply. In this study, we fabricated miniaturized multichannel devices (MCDs) by 3D printing, to minimize the use of skin and drug samples. The MCDs were compared with conventional static diffusion cells and achieved comparable drug permeation profiles. The finite element method-based simulation revealed the efficient carry-off of permeated ingredients by the multichannel devices, and a critical role of distance between the buffer stream and skin sample in determining the flow velocity inside the chamber. The results support these devices as qualified alternatives to Franz cells for in vitro permeation studies using biomembranes, with reduced use of skin and drug samples.

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitative analysis of 1,2:5,6-dianhydrogalactitol (DAG) in mouse plasma and tissues. Sodium diethyldithiocarbamate (DDTC) was used as the derivatization reagent to improve its LC-MS/MS behavior. Analytes were separated on a Welch Ultimate XB-CN column with a mobile phase consisting of acetonitrile and 0.1% formic acid solution (65:35). The MS analysis was conducted by positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity (r2 > 0.9958) was observed over the concentration range of 1–1000 ng/mL in plasma and tissue homogenates (brain, liver, heart, spleen, lung and kidney). The intra- and inter-batch precision and accuracy of DAG in plasma and brain samples were all within the acceptable limits. The extraction recovery was stable and no significant matrix effects were observed. The method was successfully applied to study the pharmacokinetic and tissue distribution of DAG in mice after intravenous administration. DAG could cross the blood-brain barrier and had limited liver distribution. Rat primary hepatocytes in vitro experiments demonstrated that DAG had a safe profile in liver.

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N-oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC-MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C18 column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500-5000 ng/mL for CBZ and 0.525-2100 ng/mL for CBN. The extraction recoveries were constant and the intra- and inter-batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague-Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose-proportional increases in exposure.

Patrinia scabra Bunge has long been used in clinic as a traditional Chinese medicine for treating leukemia and cancer and regulating host immune response. Despite their wide use in China, no report on system analysis on their chemical constituents is available so far. The current study was designed to profile the fingerprint of ethyl acetate extract of it, and in addition, to characterize the major fingerprint peaks and determine their quantity. Therefore, a detailed gradient high-performance liquid chromatography was described to separate more than 30 compounds with satisfactory resolution in P. scabra Bunge. Based on the chromatograms of 10 batches samples, a typical high-performance liquid chromatographic (HPLC) fingerprint was established with 23 chromatographic peaks being assigned as common fingerprint peaks. Furthermore, a quadrupole time of flight mass spectrometry (Q-TOF/MS) was coupled for the characterization of major compound. As (+)-nortrachelogenin was the most predominant compound in P. sca...

Triptolide has been intensively studied in numerous preclinical and clinical assessments for immunosuppressive and anti-tumor activities. However, further clinical use is limited by the cumulative toxicity of triptolide in the testis and the mechanisms are poorly understood. In this study, we found significant triptolide accumulation in the testis, and further investigated the role of efflux transporters in its accumulation and toxicity. Chronic administration of triptolide induced time- and dose-dependent testicular injury and resulted in the accumulation of triptolide in the liver and testis, but not in the plasma. Using transporter-expressed cells, triptolide efflux was found in BCRP-expressing cells, which could be blocked by novobiocin (an inhibitor of BCRP) in accumulation assays. Triptolide also displayed apically directed transport across BCRP-expressing cell layers in transwell assays, strongly supporting that triptolide is a substrate of BCRP. Bcrp knockout mice (Bcrp−/−) were further used to examine the effects of triptolide. Knockout of Bcrp aggravated triptolide-induced testicular injury and increased the testis content and testis to plasma ratio of triptolide in Bcrp−/− mice. Notably, triptolide decreased the transcript and protein levels of Bcrp in the testis, which may be due to the downregulation of RNA polymerase II. In conclusion, as a substrate of BCRP, triptolide decreased the expression of Bcrp and RNA polymerase II in the testis, and further increased the testis content and enhanced its testicular toxicity, which contributes to the cumulative toxicity of triptolide in the testis.

Androgenetic alopecia (AGA) is characterized by a progressive and patterned transformation of thick, pigmented terminal scalp hairs into short, hypo-pigmented vellus-like hairs. The use of Minoxidil and Finasteride to treat AGA are often associated with complications in safety and efficacy. However, herbal remedies are deemed to have lesser side effects in many societies. This study aims to identify potential hair growth properties of individual compounds from a Chinese proprietary medicine known as Yangxue Shengfa capsule (YSC), used in China for many years for improving AGA. Six marker compounds, including 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), Chlorogenic acid, Emodin, Ferulic acid, Isoimperatorin, and Paeoniflorin were used for simultaneous HPLC quantification and anti-AGA in-vitro screening. Simultaneous quantification of these components was performed on 75% (v/v) methanol extracts of YSC, using a Welch Ultimate XB-C18 column and gradient elution. Five compounds significantly promoted cell proliferation in cultured immortalized human Dermal Papilla Cells (DPC). Multiple genes associated with the progression of AGA, including IGF-1, DKK-1, and TGF-β1, were found to be regulated by some of these compounds. Interestingly, Ferulic acid and Emodin demonstrated good pharmacological properties against AGA, thereby concluding the potential of these bioactives to be used in the treatment against AGA.

1. The objective was to investigate the underlying mechanism of the stereoselectivity in the metabolism of rhynchophylline (RIN) and isorhynchophylline (IRN) epimers in rat liver microsomes (RLM). 2. After incubation, eight metabolites of RIN (M1-5) and IRN (M6-8) reacted at A- and C-ring were identified using LC-Q-TOF/MS. Metabolic pathways included oxidation, hydroxylation, N-oxidation and dehydrogenation. In addition, hydroxylation at A-ring was the major metabolic pathway for RIN whereas the oxidation at C-ring was the major one for IRN. 3. Enzyme kinetics showed that the intrinsic clearance (CLint) for IRN elimination was 1.9-fold higher than RIN and the degradation half-life (T1/2) of RIN was 4.7-fold higher than that of IRN, indicating IRN was more favorable to be metabolized than RIN in RLM. 4. Data from chemical inhibition study demonstrated CYP3A was the predominant isoform involved in the metabolic elimination of both epimers, as well as the formation of M1-8. 5. In conclusion, data revealed that due to the spatial configurations at C-7 position, RIN and IRN epimers possessed different hepatic metabolic pathways and elimination rates which were mainly mediated by CYP3A.

The chemical stability of cabozantinib (CBZ) was investigated using a novel stability-indicating LC method. Forced degradation of CBZ was carried out under acidic, basic, thermal, oxidative and photolytic stress conditions. Hydrolysis and oxidation were the primary pathways for this compound and three major unknown degradation products were characterized by LC/TOF-MS and LC-MS/MS. The major oxidative degradation product was isolated by preparative LC and identified by UV, HRMS and NMR techniques to be N-{4-[(N-oxide-6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N'-(4-fluorophenyl)-cyclopropane-1,1-dicarboxamide. The developed method was validated as per ICH guidelines and then successfully applied to investigate the degradation kinetics of CBZ. Degradation of CBZ followed first-order kinetics under all experimental conditions. A V-shaped pH-rate profile over the pH range 2-10 was observed with maximum stability at pH 6. The effect of temperature on the rate of CBZ degradation was characterized using the Arrhenius equation. The activation energy for hydrolysis was 57.31kJmol(-1) in alkaline solution.

Water quality assessment is an essential part of water resources management. Different methods, including the single factor (SF) method and multivariate analysis, employed in assessing water quality can give different results. In this study, SF methods and multivariate analysis – including discriminant analysis (DA) and the distance function model (DFM) – were used to assess the water quality of Xinlicheng Reservoir in Northeast China, and the advantages and disadvantages for each method are demonstrated. Results from the SF method show water quality to be Class 4, while results from the DA and DFM methods show it to be Class 3, according to the Surface Water Standard in China. The concentration of total nitrogen is most polluted amongst all the evaluating factors (as a result of human activities), leading to a Class 4 result with the SF method. The alteration of an individual factor has little impact on the results of the DA and DFM methods, since they integrate the features of the all of the evaluating factors. Because anthropogenic pollution generally alters only a few evaluating factors, DA and the DFM are more useful for polluted water quality assessment than SF is. But DA and DFM methods should be used with sufficient consideration of their mathematical requirements to prevent misinterpretation of the results.

YiGan San (YGS) has long been used in traditional Japanese and Chinese folk medicine and serves as a potent and novel therapeutic agent to treat Alzheimer's disease. In the present study, a rapid and sensitive method based on HPLC coupled with diode-array detection and quadrupole TOF MS (Q-TOF-MS) was designed to reveal the chemical constituents of YGS. Thirty-six compounds were identified and assigned in YGS, including 14 alkaloids, nine γ-lactones, six flavonoids, three triterpenoid saponinares, two small molecular organic acids, and two other types of compounds. In addition, the accurate fragment weight and MS/MS fragmentation reactions of a subtype indole alkaloid in Uncariae ramulus cum uncis were summarized for the first time to realize rapid identification without reference substances. For the first time, 11 major constituents were comprehensively quantified with a HPLC coupled with triple-quadrupole MS method. A three-section switch was used to realize such multicomponent identification. The contents of saikosaponin B2 and isoliquiritin, which produce anti-inflammatory and antidepressant-like effects, were extremely different, up to 700 times, in two sources of YGS. The developed qualitative and quantitative method was proved to be precise, accurate, and reproducible.

Uncaria rhynchophylla (UR) is a species of Uncaria that is distributed mainly in China and Japan. In this study, the chemical constituents, including alkaloids, flavonoids, and quinic acids, in UR have been systematically identified and quantified by a developed method of high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF-MS). Tetracyclic monoterpenoid oxindole alkaloids (TMOAs) are characteristic compounds in this herb, and their fragmentations in Q-TOF-MS have been investigated. Diagnostic fragmentation ions (DFIs) were first delineated for isorhynchophylline-type (7S, C20-ethyl) and corynoxeine-type (7R, C20-vinyl) TMOAs, and these were used for identification of these alkaloids in UR. In this study, a total of 29 compounds, comprising 18 alkaloids, six flavonoids, and five quinic acids, were identified. Among them, there are four novel TMOAs, named as 22-O-β-glucopyranosyl isorhynchophyllic acid (10), 22-O-β-glucopyranosyl rhynchophyllic acid (11), 9-hydroxy isocorynoxeine (16), and 9-hydroxy corynoxeine (20), which have not been reported previously. Furthermore, eight marker compounds, namely chlorogenic acid (3), catechin (8), epicatechin (9), isocorynoxeine (24), rhynchophylline (25), isorhynchophylline (27), vincoside lactam (28), and corynoxeine (29), have been simultaneously quantified. The developed method has been validated and successfully applied to analyze three samples of UR from Jiangxi Province. The contents of the marker compounds have been detected and compared.

YK-1101, with its structure as S-((E)-4-((7S,10S,Z)-4-ethylidene-7-isopropyl-2,5,8,12-tetraoxo -9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-en-1-yl) ethanethioate, is synthesized as a potential histone deacetylase inhibitor. Its quality and stability under various stress conditions are not fully understood. In this study, a high performance liquid chromatographic (HPLC) method was established and validated for the analysis of YK-1101 bulk drug samples. The chromatographic separation was performed on a C18 column with acetonitrile and water as mobile phase in a gradient elution. Based on the established method, the stability studies of YK-1101 under various stress conditions were carried out. YK-1101 was shown to undergo degradation under basic and acidic stress conditions, while it was stable under oxidative, photolytic and thermal conditions. In addition, a time of flight mass spectrometer (TOF/MS) was coupled to HPLC for the characterization of major degradation products produced under basic and acidic stress conditions. Their degradation pathways were also discussed. Keywords: YK-1101, Degradation products, Stress study, Liquid chromatography, Time of flight mass spectrometer

A quantitative PET imaging method was used to assess the in vivo kinetics of hepatobiliary and renal excretion of the breast cancer resistance protein (Bcrp) substrate 11C-SC-62807 in mice. Methods: Serial abdominal PET scans were collected in wild-type and Bcrp knockout (Bcrp–/–) mice after intravenous injection of 11C-SC-62807. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after radiotracer administration. Dynamic PET data were analyzed to determine the canalicular and brush-border efflux clearances in the liver and kidney (CLint,bile,liver and CLint,urine,kidney, respectively). Results: SC-62807 is an in vitro substrate of mouse Bcrp and human BCRP. Radioactivity associated with 11C-SC-62807 was predominantly found in the blood, liver, bile, and urine 30 min after administration. Both biliary and urinary excretion of radioactivity was markedly lower in Bcrp–/– mice than in wild-type mice, suggesting greater systemic exposure in Bcrp–/– mice. Both the CLint,bile,liver and the CLint,urine,kidney were significantly lower in Bcrp–/– mice (74% ± 10% and 99% ± 1% lower than controls, respectively). We also found that 11C-SC-62807 is a substrate of the organic anion-transporting polypeptides OATP1B1 and OATP1B3 in vitro. Conclusion: The present study demonstrated that Bcrp plays a significant role in the efflux of 11C-SC-62807 in mouse liver and kidney. We also demonstrated the feasibility of PET using 11C-SC-62807 to study the activity of BCRP in humans.

Current topical treatments using lidocaine (LD) for analgesia have limited applications due to their delayed analgesic actions, resulted from slow drug permeation through skin. The aim of this study is to fabricate a large size microneedle (MN) array patch containing LD, with fast onset of action, for the treatment of acute and chronic pain.The MN patch was developed through photolithography and tested for its mechanical characteristics. In vitro and in vivo skin permeation, plasma pharmacokinetics, histology and skin irritation testing have also been performed for the MN patches.The MN have a mechanical strength of 10-30 N and more than 90% of the microneedles on the patch penetrated skin. It was shown that LD permeated through skin within 5 min of patch application. Subsequently, the in vivo skin permeation study using a porcine model showed that LD administrated by the MN patch was able to achieve the therapeutic level locally within 10 min and sustained for 8 h. It shows most of the drug diffuses perpendicularly against skin, with little lateral diffusion. After skin permeation LD remains within skin and unquantifiable amount of LD was found in the plasma of the pigs. Minor skin irritations were observed after 6 h of microneedle contact. However, the skin irritations resolved within 1 day following the removal of MN patch.The large size MN patches showed fast onset and sustained delivery of LD through skin, potentially useful to increase the application scope of topical LD for pain management.

Although the chemical synthesis of wogonin (5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one) has been established, the impurity profile of this pharmaceutical agent is still not fully understood. In this study, a novel process related impurity present in the synthetic wogonin was isolated by preparative HPLC. Its structure elucidation was unambiguously carried out by NMR, HRMS and IR spectra, and it was characterized as 6-chloro-5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one ( imp-1 ), a new compound which had never been reported before. Moreover, forced degradation studies were also carried out, and wogonin was shown to undergo isomerzation under basic stress condition to form a major degradant, 5,7-dihydroxy-6-methoxy-2-phenyl-4H-chromen-4-one ( imp-2 ) using HPLC–Q-TOF–MS/MS technique. Finally, a selective and simple routine HPLC method was developed for simultaneous quantification of wogonin and the two related compounds. The proposed method is useful for purity control during the process development of wogonin and laboratory-prepared preparations. Their formation pathways were also discussed.

BACKGROUND AND PURPOSE An ATP-binding cassette (ABC) transporter, breast cancer resistance protein (BCRP)/ABCG2, limits oral bioavailability of sulphasalazine. Here we examined the effect of curcumin, the principal curcuminoid of turmeric, on oral bioavailability of microdoses and therapeutic doses of sulphasalazine in humans. EXPERIMENTAL APPROACH Effects of curcumin were measured on the ATP-dependent sulphasalazine uptake by hBCRP-expressing membrane vesicles and on oral bioavailability of sulphasalazine in wild-type and Bcrp(-/-) mice. Eight healthy Japanese subjects received an oral dose of sulphasalazine suspension (100 µg) or tablets (2 g) alone or after curcumin tablets (2 g). Uptake of sulphasalazine was studied in HEK293 cells transfected with the influx transporter (OATP)2B1. KEY RESULTS Curcumin was a potent hBCRP inhibitor in vitro (K(i) 0.70 ± 0.41 µM). Curcumin increased the area under the curve (AUC)(0-8) of plasma sulphasalazine eightfold in wild-type mice at 300 and 400 mg·kg(-1), but not in Bcrp(-/-) mice. Curcumin increased AUC(0-24) of plasma sulphasalazine 2.0-fold at microdoses and 3.2-fold at therapeutic doses in humans. Non-linearity of the dose-exposure relationship was observed between microdoses and therapeutic doses of sulphasalazine. Sulphasalazine was a substrate for OATP2B1 (K(m) 1.7 ± 0.3 µM). Its linear index (dose/K(m)) at the therapeutic dose was high and may saturate OATP2B1. CONCLUSIONS AND IMPLICATIONS Curcumin can be used to investigate effects of BCRP on oral bioavailability of drugs in humans. Besides the limited dissolution, OATP2B1 saturation is a possible mechanism underlying non-linearity in the dose-exposure relationship of sulphasalazine.

Rice is the staple food for more than half of the world's population and, hence, the main source of a vital micronutrient, zinc (Zn). Unfortunately, the bioavailability of Zn from rice is very low not only due to low content but also due to the presence of some antinutrients such as phytic acid. We investigated the effect of germination and Zn fortification treatment on Zn bioavailability of brown rice from three widely grown cultivars using the Caco-2 cell model to find a suitable fortification level for producing germinated brown rice. The results of this study showed that Zn content in brown rice increased significantly (p0.05) as the external Zn concentrations increased from 25 to 250 mg/L. In contrast, no significant influence (p0.05) on germination percentage of rice was observed when the Zn supply was lower than 150 mg/L. Zn fortification during the germination process has a significant impact on the Zn content and finally Zn bioavailability. These findings may result from the lower molar ratio of phytic acid to Zn and higher Zn content in Zn fortified germinated brown rice, leading to more bioavailable Zn. Likewise, a significant difference (p0.05) was found among cultivars with respect to the capacity for Zn accumulation and Zn bioavailability; these results might be attributed to the difference in the molar ratio of phytic acid to Zn and the concentration of Zn among the cultivars evaluated. Based on global intake of Zn among the world population, we recommend germinated brown rice fortified with 100 mg/L ZnSO(4) as a suitable concentration to use in the germination process, which contains high Zn concentration and Zn bioavailability. In the current study, the cultivar Bing91185 fortified with Zn through the germination process contained a high amount as well as bioavailable Zn, which was identified as the most promising cultivar for further evaluation to determine its efficiency as an improved source of Zn for target populations.

Zinc (Zn) is an essential micronutrient for humans, but Zn deficiency has become serious as equally as iron (Fe) and vitamin A deficiencies nowadays. Selection and breeding of high Zn-density crops is a suitable, cost-effective, and sustainable way to improve human health. However, the mechanism of high Zn density in rice grain is not fully understood, especially how Zn transports from soil to grains. Hydroponics experiments were carried out to compare Zn uptake and distribution in two different Zn-density rice genotypes using stable isotope technique. At seedling stage, IR68144 showed higher (68)Zn uptake and transport rate to the shoot for the short-term, but no significant difference was observed in both genotypes for the long-term. Zn in xylem sap of IR68144 was consistently higher, and IR68144 exhibited higher Zn absorption ratio than IR64 at sufficient (2.0 µmol/L) or surplus (8.0 µmol/L) Zn supply level. IR64 and IR68144 showed similar patterns of (68)Zn accumulation in new leaves at seedling stage and in developing grains at ripening stage, whereas (68)Zn in new leaves and grains of IR68144 was consistently higher. These results suggested that a rapid root-to-shoot translocation and enhanced xylem loading capacity may be the crucial processes for high Zn density in rice grains.

Oroxylin A shows reversal activity on multidrug resistance and has an attractive safety profile. A highly sensitive and specific method based on liquid chromatography coupled with tandem mass spectroscopy was developed and validated for simultaneous determination of oroxylin A and its major metabolite, oroxylin A-7-O-glucuronide in rat plasma. The assay procedure involved precipitation of plasma samples with acetonitrile after adding luteolin as an internal standard. Chromatographic separation was achieved on a C18 column using methanol and 0.5% formic acid as the mobile phase, eluting in a gradient system. Detection was achieved with selected reaction monitoring using the electrospray ionization technique. The method was validated by evaluation of selectivity, linearity, precision, accuracy and limit of quantification. Its applicability was demonstrated through the quantification of oroxylin A and oroxylin A-7-O-glucuronide in plasma after intravenous administration of oroxylin A at a dose of 20 mg kg−1 to rats. The developed quantification method can now be used for pharmacokinetic studies after intravenous infusion of oroxylin A injection in rats.

The tissue distribution of wogonin was investigated for study of its anticancer activity. A robust, reliable, and sensitive LC method was developed for simultaneous analysis of wogonin and its major metabolite, wogonoside, in tissues of tumor-bearing nude mice. To reach the required level of detection in different tissues, sample pretreatment was by solid-phase extraction. Good LC separation was achieved on a C18 column with methanol–0.5% formic acid 68:32 (v/v) as mobile phase. UV detection was performed at 275 nm and calibration plots for both analytes were linear over the range 0.16–80.0 μg g−1 for all the biological samples studied with correlation coefficients >0.9980. Inter-day and intra-day precision were no more than 15% at the limit of quantification and 10% at other concentrations; recovery from all tissues was 92.3–101.4% for wogonin and 66.7–74.3% for wogonoside. The method was ideal for this tissue distribution study and results obtained reflect the potential of wogonin for further pharmaceutical development as an anticancer agent.

Zinc (Zn) is an essential micronutrient for humans, and increasing Zn density in rice ( Oryza sativa L.) grains is important for improving human nutrition. The characteristics of Zn translocation and remobilization were investigated in high Zn density genotype IR68144, in comparison with the low Zn density genotype IR64. Stable isotope tracer (68)Zn was supplied at various growth stages, either to the roots in nutrient solution or to the flag leaves to investigate the contribution of (68)Zn absorbed at different growth stages to grain accumulation and the remobilization ability of (68)Zn within plants. Significant differences in (68)Zn allocation were observed between the two rice genotypes. Much higher (68)Zn concentrations were found in grains, stems, and leaves of IR68144 than in IR64, but higher (68)Zn was found in roots of IR64. More than half of the Zn accumulated in the grains was remobilized before anthesis, accounting for 63 and 52% of the total Zn uptake for IR68144 and IR64, respectively. Without supply of external Zn, at vegetative or reproductive stages, more (68)Zn was retranslocated from "old tissues" to "new tissues" in IR68144 than in IR64. Retranslocation of (68)Zn from flag leaves to grains was twice as high in the former when (68)Zn was applied to the flag leaves during booting or anthesis. These results indicate that Zn density in rice grains is closely associated with the ability to translocate Zn from old tissues to new tissues at both early and late growth stages and with phloem remobilization of Zn from leaves and stems to grains.

1. Henagliflozin is a novel sodium-glucose transporter 2 inhibitor and presents a complementary therapy to metformin for patients with T2DM due to its insulin-independent mechanism of action. This study evaluated the potential pharmacokinetic drug-drug interaction between henagliflozin and metformin in healthy Chinese male subjects. 2. In open-label, single-center, single-arm, two-period, three-treatment self-control study, 12 subjects received 25 mg henagliflozin, 1000 mg metformin or the combination. Lack of PK interaction was defined as the ratio of geometric means and 90% confidence interval (CI) for combination: monotherapy being within the range of 0.80-1.25. 3. Co-administration of henagliflozin with metformin had no effect on henagliflozin area under the plasma concentration-time curve (AUC0-24) (GRM: 1.08; CI: 1.05, 1.10) and peak plasma concentration (Cmax) (GRM: 0.99; CI: 0.92, 1.07). Reciprocally, co-administration of metformin with henagliflozin had no clinically significant on metformin AUC0-24 (GRM: 1.09, CI: 1.02, 1.16) although there was an 11% increase in metformin Cmax (GRM 1.12; CI 1.02, 1.23). All monotherapies and combination therapy were well tolerated. 4. Henagliflozin can be co-administered with metformin without dose adjustment of either drug.

Matrine is the major bioactive alkaloid found in certain Sophora plants and has been used for the treatment of liver diseases and protection of liver function. The aim of this study was to investigate the human liver uptake mechanism of matrine by using HepG2 cells as the in vitro model. Matrine was transported into HepG2 cells in a time- and temperature-dependent manner. The cellular uptake was saturable and was significantly reduced by the metabolic inhibitors, such as sodium azide and rotenone. Furthermore, the uptake of matrine was found to be regulated by a protonophore (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) and pH, indicating that this influx transporter may be a proton-coupled antiporter. The uptake of matrine was sensitive to inhibition by the cationic drugs including pyrilamine, quinidine, verapamil, amantadine, diphenhydramine, and cimetidine but insensitive to other typical substrates or inhibitors of well-known organic cation transport systems. The present study reveals that, for the first time, in HepG2 cells, the existence of a proton-coupled organic cation antiporter that contributes substantially to the hepatic uptake of matrine.

Diacylglycerol kinases ζ (DGKζ) is a critical lipid kinase which is involved in phosphatidic acid (PA) generation via diacylglycerol (DAG) phosphorylation. DGKζ is highly expressed in central nervous system and essential for brain development. Studies have indicated that DGKζ is associated with colon cancer invasion and metastasis. However, the involvement of DGKζ in human glioma development remains elusive. Here, we explored the impact and possible mechanisms of DGKζ knockdown on the proliferation and survival of glioma cells. The relationship between DGKζ expression status and human glioma stages was explored in 111 specimens of human gliomas via immunohistochemistry technology. Then the impact of DGKζ on cell proliferation, cell cycle, survival, and colony formation ability was determined in U-87 MG glioma cell lines via lentiviral-mediated small interfering (shRNA) strategy. The influence of DGKζ knockdown on global gene expression in U-87 MGglioma cell lines was further analyzed by microarray platform to reveal the possible molecular mechanisms underlying DGKζ-mediated glioma development and progression. Immunohistochemistry analysis revealed that DGKζ expression is positively correlated with human gliomagrade. Lentiviral-mediated small interfering (shRNA) strategyefficiently reduced DGKζ expression and DGKζ knockdown impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and cell apoptosis in U-87 MG glioma cells. Finally, microarray analysis revealed that multiple cancer-associated pathways and oncogenes were regulated by DGKζ knockdown, which provides insights into underlying mechansims of DGKζ-associated glioma development and progression. Our results established the positive correlation between DGKζ expression and gliomagrade. Furthermore, DGKζ knockdown in human glioma cell lines U-87 MG impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and apoptosis which microarray analysis showed that DGKζ knockdown interrupted multiple oncogenes and cancer-associated pathways. Taken together, we provided confidential evidence for the causal role of DGKζ in glioma development and progression.

A sensitive, specific and rapid high performance liquid chromatography - tandem mass spectrometric (LC/MS/MS) method for the determination of cyclovirobuxine D in human plasma was developed and validated. The triple-quadrupole tandem mass spectrometric detector with an electrospray interface (ESI) was operated under the selected reaction monitoring (SRM) mode. After the addition of citalopram as an internal standard (IS), plasma samples were extracted with ethyl acetate. Chromatographic separation of the analytes was performed on a Kromasil CN column with a mobile phase of methanol/water (88/12, v/v) containing 0.4% formic acid. Linearity was established for the range of concentration 0.2-40ng/ml. Under optimized conditions, the mean recovery was 86.6%. The intra-day precision ranged from 4.56% to 7.81%, while the intra-day accuracy ranged from 2.75% to 11.0%. The inter-day precision was in the range 3.87-10.7%, and the inter-day accuracy was in the range -4.00% to 2.50%. The cyclovirobuxine D was stable in human plasma after three freeze-thaw cycles, under storage at room temperature for 12h, in a freezer at -20 degrees C for 15 days and during processing (in autosampler) at 10 degrees C for 24h. The validated method is suitable for quantitative determination of cyclovirobuxine D in human plasma in pharmacokinetics study and has been successfully applied to the analysis of clinical samples.