Jianzhong Chen

Dendrimeric anticancer prodrugs for targeted delivery of ursolic acid to folate receptor-expressing cancer cells: Synthesis and biological evaluation

The anticancer efficacy of ursolic acid (UA) was limited by poor water solubility, non-specific tumor distribution, and low bioavailability. To overcome this problem, polyamidoamine (PAMAM) conjugated with UA and folic acid (FA) as novel dendrimeric prodrugs were designed and successfully synthesized by a concise one-pot synthetic approach. Both FA and UA were covalently conjugated to the surface of PAMAM through acid-labile ester bonds and the covalently linked UA could be hydrolysed either in acidic (pH 5.4) or in neutral (pH 7.4) PBS solution. The cellular uptake study indicated that the presence of FA enhanced uptake of the dendrimeric prodrugs in folate receptor (FR) over-expressing Hela cells. The enhanced cellular uptake could be due to the electrostatic absorptive endocytosis and FR-mediated endocytosis. In contrast, for HepG2 cells, a FR-negative cell line, FA conjugation on the surface of the dendrimer showed no effect on the cellular uptake. In MTT assay and cell cycle analysis, FA-modified dendrimeric prodrugs showed significantly enhanced toxicity than non-FA-modified ones in Hela cells. These results suggested that FA-modified dendrimeric UA prodrugs have the potential for targeted delivery of UA into cancer cells to improve its anti-tumor efficacy.

Synergism of ursolic acid derivative US597 with 2-deoxy-D-glucose to preferentially induce tumor cell death by dual-targeting of apoptosis and glycolysis

Ursolic acid (UA) is a naturally bioactive product that exhibits potential anticancer effects. The relatively safe and effective molecule intrigued us to explore a way to further improve its anti-cancer activity and tumor-targeting specificity. In the present study, a series of structural modifications of UA was achieved, which resulted in significant increase in growth inhibition on various cancer cell lines with minimal effects on normal cells. The leading molecule US597 (UA-4) caused depolarization of mitochondrial membrane potential, cell arrest in G0/G1 phase and apoptosis/necrosis in a dose-dependent manner. Structural docking suggested that the carbon chains of the modified UA derivatives compete strongly with glucose for binding to glucokinase, the key glycolysis enzyme presumably active in cancer cells. The combination of 2-deoxy-D-glucose (2-DG) and UA-4 induced cell cycle arrest in G2/M phase, promoted caspase-dependent cell death, reduced hexokinase activity, aggravated depletion of intracellular ATP, decreased lactate production and synergistically inhibited cancer cell growth in vitro (HepG2) and in vivo (H22). Collectively, our findings suggest that the structural modification enhances efficacy and selectivity of UA, and the combination of UA-4 with 2-DG produces synergistic inhibition on hepatoma cell proliferation by dual targeting of apoptosis and glycolysis.

The Unique Pharmacological Characteristics of Mifepristone (RU486): From Terminating Pregnancy to Preventing Cancer Metastasis

Mifepristone (RU486) is a born‐for‐woman molecule discovered three decades ago. Unlike those antihypertensive and antipsychotic pharmaceutical blockbusters, this abortifacient offers relatively low profit potential. Current understanding of mechanism of action of mifepristone and its on‐going clinical trials are changing our views on the drug beyond its abortifacient scope. Here we briefly review its metabolism and pharmacokinetic properties including its unique enterohepatic circulation, its mechanisms of actions involving antiprogesterone and antiglucocorticoid, growth inhibition of various cancer cell lines, suppression of invasive and metastatic cancer potential, downregulation of Cdk2, Bcl‐2, and NF‐kappa B, interference of heterotypic cell adhesion to basement membrane, and cell migration. We comprehensively analyze recent results from preclinical and clinical studies using mifepristone as an anticancer drug for breast, meningioma, and gliomas tumors in the central nervous system, prostate cancer, ovarian and endometrial cancer, and gastric adenocarcinoma. Although mifepristone has more benefits for global public health than we originally thought, its effect as a postmetastatic chemotherapeutic agent is limited. Nonetheless, owing to its unique safe, metabolism and other pharmacological properties, metapristone (the primary metabolite of mifepristone) may have potential for cancer metastatic chemoprevention.

Synthesis, Spectral Characterization, and In Vitro Cellular Activities of Metapristone, a Potential Cancer Metastatic Chemopreventive Agent Derived from Mifepristone (RU486)

Mifepristone (RU486) is marketed and used widely by women as an abortifacient, and experimentally for psychotic depression and anticancer treatments. After administration, metapristone is found to be the most predominant metabolite of mifepristone. We hypothesized that adhesion of circulating tumor cells (CTCs) to vascular endothelial bed is a crucial starting point in metastatic cascade, and that metapristone can serve as a cancer metastatic chemopreventive agent that can interrupt adhesion and invasion of CTCs to the intima of microvasculature. In the present study, we modified the synthesis procedure to produce grams of metapristone, fully characterized its spectral properties and in vitro cellular activities, including its cytostatic effects, cell cycle arrest, mitochondrial membrane potential, and apoptosis on human colorectal cancer HT-29 cells. Metapristone concentration dependently interrupted adhesion of HT-29 cells to endothelial cells. Metapristone may potentially be a useful agent to interrupt metastatic initiation.

UP12, a novel ursolic acid derivative with potential for targeting multiple signaling pathways in hepatocellular carcinoma

Targeting cancer cell glucose metabolism is a promising strategy for cancer therapy. In past approaches to cancer drug discovery, ursolic acid (UA) has been chemically modified to improve its antitumor activities and bioavailability. Here, a novel ursolic acid (UA) derivative UP12 was developed via computer-aided drug design to explore potent anti-cancer agents and to examine possible mechanisms. The structural docking analyses suggested that UP12 could bind to the active sites of glucokinase (GK), glucose transporter 1 (GLUT1) and ATPase, which are the main enzymes involved in cancer glucose metabolism. We further investigated the synergistic effect between UP12 and glycolysis inhibitor 2-deoxy-d-glucose (2-DG) in inhibiting glucose metabolism of cancer cells. The pharmacological results showed that the combination enhanced depletion of intracellular ATP and decrease in lactate production, and pushed more cancer cells arrested in the S and G2/M cycle phases. The combination selectively down-regulated the expression of Bcl-2 and HKII proteins, up-regulated the expression of Bax and p53, and collectively resulted in enhanced apoptosis related to caspase-3, -8, and -9 activities, in addition to inhibition on the cell mitochondrial membrane potential. The animal studies further demonstrated that the combination exhibited significant antitumor activity without obvious toxicity. In summary, UP12 can interfere cancer cell metabolism pathway and further enhance the therapeutic effects of 2-DG likely through synergistic suppression of cancer cell glucose metabolism, making UP12 a likely new candidate for anti-cancer drug development.

Drug enterohepatic circulation and disposition: constituents of systems pharmacokinetics

Drug disposition information constitutes a part of systems pharmacokinetics, and becomes imperative when a drug shows significant effects at its disproportionally low blood concentration. The situation could result from outweighing the parent drug in tissues over in blood and/or from its active metabolites. Fractions of certain drugs absorbed from the intestine to the systemic circulation via the portal vein can return to the intestine via the bile duct and the sphincter of Oddi - a complementary nonrenal elimination route termed the enterohepatic circulation (EHC). Here, we critically evaluate the existing methods, techniques and animal models used for determining drug distribution, elimination and EHC, and collectively portray characteristics of 43 drugs that undergo EHC. EHC could represent an unexplored way to excrete unwanted substrates out of the body. The interdisciplinary analysis galvanizes our efforts to overcome technical gaps in drug discovery and development.

Ultrasound/microwave-assisted extraction and comparative analysis of bioactive/toxic indole alkaloids in different medicinal parts of Gelsemium elegans Benth by ultra-high performance liquid chromatography with MS/MS.

Indole alkaloids are the main bioactive/toxic components in Gelsemium elegans Benth. To determine the distribution and contents of indole alkaloids in its different medicinal parts, a novel and rapid method using ultra-high performance LC (UPLC) with MS/MS has been established and validated with an optimized ultrasound/microwave-assisted extraction method. Four constituents, namely, humantenidine, humantenmine, gelsemine, and koumine, were simultaneously determined in 6 min. Chromatographic separation was achieved on an ultra-high performance LC BEH C18 column with a gradient mobile phase consisting of methanol and water (containing 0.1% formic acid both in methanol and water) at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole electrospray MS/MS by positive ion multiple-reaction monitoring mode. All the analytes showed good linearity (r ≥ 0.9934) within a concentration range from 0.1-25 μg/mL with a LOQ of 25-50 ng/mL. The overall intra- and intervariations of four components were <4.7% with an accuracy of 97.3-101.3%. The analysis results showed that there were remarkable differences in the distribution and contents of four chemical markers in the roots, stems, and leaves of G. elegans Benth. The findings can provide necessary and meaningful information for the rational utilization of its resources.

A novel UPLC/MS/MS method for rapid determination of metapristone in rat plasma, a new cancer metastasis chemopreventive agent derived from mifepristone (RU486)

Mifepristone (RU486) is a chemical abortifacient used by hundreds of millions of women world-wide. It has recently been used in clinical trials for psychotic depression and cancer chemotherapy. Metapristone is the most predominant biological active metabolite of mifepristone, and being developed as a novel cancer metastasis chemopreventive agent based on its unique pharmacological properties. In this study, a novel rapid and sensitive method using UPLC/MS/MS was developed and validated for quantitative analysis of metapristone in plasma, which used less plasma volume and was demonstrated to be more simple and low-cost than the published methods. Metapristone in plasma was recovered by liquid-liquid extraction using 1 mL of ethyl acetate and chromatographic separation was carried on a C₁₈ column at 35 °C, with a gradient mobile phase consisting of methanol and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. The mass spectrometric detection was carried out using a triple-quadrupole system via positive electrospray ionization. Multiple reaction monitoring was used for quantitation of m/z transitions from 416.3 to 119.9 for metapristone and from 313.1 to 109 for levonorgestrel (internal standard). Good linearity (r²> 0.9926) was achieved over a concentration range from 7.1 to 2840 ng/mL with a lower limit of quantification of 7.1 ng/mL for metapristone. The intra- and inter-day variations of the assay were 2.4-10.0% relative standard deviation with an accuracy of -5.6 to 8.6% relative error. This newly developed method was successfully applied to a pharmacokinetic study that revealed, for the first time, that there was a significant difference in pharmacokinetic profile between genders.

Development of a sensitive and rapid UPLC-MS/MS method for the determination of koumine in rat plasma: application to a pharmacokinetic study.

A rapid, selective and sensitive method using UPLC-MS/MS was first developed and validated for quantitative analysis of koumine in rat plasma. A one-step protein precipitation with methanol was employed as a sample preparation technique. Plasma samples were separated on an Acquity UPLC BEH C18 column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of methanol with 0.1% (v/v) formic acid and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. Detection and quantification were performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization. Good linearity (r > 0.9997) was achieved using weighted (1/x(2) ) least squares linear regression over a concentration range of 0.025-15 µg/mL with a lower limit of quantification of 0.025 µg/mL for koumine. The intra- and inter- precisions (relative standard deviation) of the assay at all three quality control samples were 5.6-14.1% with an accuracy (relative error) of 5.0-14.0%, which meets the requirements of the US Food and Drug Administration guidance. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 20 mg/kg koumine.

Pharmacokinetics and metabolism study of isoboldine, a major bioactive component from Radix Linderae in male rats by UPLC–MS/MS

ETHNOPHARMACOLOGICAL RELEVANCE Isoboldine is one of the major bioactive constituents in the total alkaloids from Radix Linderae (TARL) which could effectively alleviate inflammation and joints destruction in mouse collagen-induced arthritis. To better understand its pharmacological activities, we need to determine its pharmacokinetic and metabolic profiles. MATERIALS AND METHODS In this study, a sensitive and simple UPLC-MS/MS method was developed and validated for determination of isoboldine in rat plasma. Isoboldine in plasma was recovered by liquid-liquid extraction using 1 mL of methyl tert-butyl ether. Chromatographic separation was performed on a C18 column at 45°C, with a gradient elution consisting of acetonitrile and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. The detection was performed on an electrospray triple-quadrupole MS/MS by positive ion multiple-reaction monitoring mode. This newly developed method was successfully applied to a pharmacokinetic study after oral and intravenous dosing in rats. For metabolites identification, isoboldine was orally administered to rats and the metabolite in plasma, bile, urine and feces were characterized by the established UPLC-MS/MS method. RESULTS Good linearity (r(2)>0.9956) was achieved in a concentration range of 4.8-2400 ng/mL with a lower limit of quantification of 4.8 ng/mL for isoboldine. The intra- and inter-day precisions of the assay were 1.7-5.1% and 2.2-4.4% relative standard deviation with an accuracy of 91.3-102.3%. A total of five phase II metabolites in rat plasma, bile, urine and feces were characterized by comparing retention time in UPLC, and by molecular mass and fragmentation pattern of the metabolites by mass spectrometry with those of isoboldine. CONCLUSION isoboldine has extremely low oral bioavailability due to the strong first-pass effect by the rats, and glucuronidation and sulfonation were involved in metabolic pathways of isoboldine in rats. These results have paved the way for further clarifying therapeutic ingredients and provided new knowledge regarding pharmacokinetic features of this category of isoquinoline alkaloids.