Moses S.S. Chow

Discovery of Molecular Mechanisms of Traditional Chinese Medicinal Formula Si-Wu-Tang Using Gene Expression Microarray and Connectivity Map

To pursue a systematic approach to discovery of mechanisms of action of traditional Chinese medicine (TCM), we used microarrays, bioinformatics and the “Connectivity Map” (CMAP) to examine TCM-induced changes in gene expression. We demonstrated that this approach can be used to elucidate new molecular targets using a model TCM herbal formula Si-Wu-Tang (SWT) which is widely used for womens health. The human breast cancer MCF-7 cells treated with 0.1 µM estradiol or 2.56 mg/ml of SWT showed dramatic gene expression changes, while no significant change was detected for ferulic acid, a known bioactive compound of SWT. Pathway analysis using differentially expressed genes related to the treatment effect identified that expression of genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) cytoprotective pathway was most significantly affected by SWT, but not by estradiol or ferulic acid. The Nrf2-regulated genes HMOX1, GCLC, GCLM, SLC7A11 and NQO1 were upreguated by SWT in a dose-dependent manner, which was validated by real-time RT-PCR. Consistently, treatment with SWT and its four herbal ingredients resulted in an increased antioxidant response element (ARE)-luciferase reporter activity in MCF-7 and HEK293 cells. Furthermore, the gene expression profile of differentially expressed genes related to SWT treatment was used to compare with those of 1,309 compounds in the CMAP database. The CMAP profiles of estradiol-treated MCF-7 cells showed an excellent match with SWT treatment, consistent with SWTs widely claimed use for womens diseases and indicating a phytoestrogenic effect. The CMAP profiles of chemopreventive agents withaferin A and resveratrol also showed high similarity to the profiles of SWT. This study identified SWT as an Nrf2 activator and phytoestrogen, suggesting its use as a nontoxic chemopreventive agent, and demonstrated the feasibility of combining microarray gene expression profiling with CMAP mining to discover mechanisms of actions and to identify new health benefits of TCMs.

Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography―mass spectrometry

Si-Wu-Tang (SWT), comprising Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular Traditional Chinese Medicine (TCM) formulae for womans health. Data mining from the available Chinese and English literatures indicated that the major bioactive components of SWT consist of paeoniflorin, paeonol, gallic acid, ferulic acid, Z-ligustilide, ligustrazine, butylphthalide, senkyunolide A and catalpol. Since content determination of the marker compounds is generally considered as an initial step for quality control of TCM product, a high performance liquid chromatography-mass spectrometric method employing both positive and negative electrospray ionization was developed for the simultaneous determination of the nine identified compounds in the raw herbs and products of SWT. The LOQ of the developed assay method for the tested components was 10ng/ml for ligustrazine, 200ng/ml for catalpol, and 100ng/ml for the other seven compounds. The intra-day and inter-day variations of the current assay were within 17.5%. Paeoniflorin, ferulic acid, gallic acid, Z-ligustilide and senkyunolide A were found in all SWT products investigated. Variations in the contents of the studied compounds were observed among batches of raw herbs and SWT products. The currently developed method provides a sensitive and rapid quantification approach that can be useful in the quality control of raw herbs and products of SWT.

Effect of sodium caprate on the oral absorptions of danshensu and salvianolic acid B.

The current study aims to investigate the effect of sodium caprate on the intestinal absorption and bioavailabilities of danshensu and salvianolic acid B, the major active components in Salvia miltiorrhiza Bge (Danshen). Biopharmaceutics and pharmacokinetics properties of the two compounds have been characterized by in vitro, in situ models as well as in vivo in rats. Based on the identified biopharmaceutics characteristics of the two compounds, effect of sodium carparate as absorption enhancer on the intestinal absorption and pharmacokinetics of danshensu and salvianolic acid B in pure compound form as well as extract form were investigated both in vitro and in vivo. Both danshensu and salvianolic acid B demonstrated very limited intestinal permeabilities, leading to oral bioavailabilities of only 11.09% and 3.90% in rats, respectively. Results from both in vitro and in vivo studies consistently indicated that sodium caprate could significantly enhance intestinal permeabilities as well as the in vivo bioavailabilities of both danshensu and salvianolic acid B. The current findings not only identified the usefulness of sodium caprate for the improved delivery of Danshen product but also demonstrated the importance of biopharmaceutics characterization in the dosage form development of traditional Chinese medicine.

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.

Nutraceuticals for prostate cancer chemoprevention: from molecular mechanisms to clinical application

Introduction: Nutraceutical is a food, or part of a food, used for the prevention and/or treatment of diseases. A number of nutraceuticals serve as candidates for development of prostate cancer chemopreventive agents because of promising epidemiological, preclinical and pilot clinical findings. Their mechanisms of action may involve an ability to target multiple molecular pathways in carcinogenesis without eliciting toxic side effects. Areas covered: This review provides an overview of several nutraceuticals, including green tea polyphenol, omega-3 fatty acids, vitamin D, lycopene, genistein, quercetin, resveratrol and sulforaphane, for the clinical relevance to chemoprevention of prostate cancer. Their mechanisms of action on regulating key processes of carcinogenesis are also discussed. For each of these agents, a brief summary of completed or currently ongoing clinical trials related to the chemopreventive efficacy on prostate cancer is given. Expert opinion: Even though a few clinical trials have been conducted, review of these results indicate that further studies are required to confirm the clinical efficacy and safety, and to provide a guidance on how to use nutraceuticals for optimal effect. Future cancer prevention clinical trials for the nutraceuticals should recruit men with an increased risk of prostate cancer.

Transcriptional profiling of Chinese medicinal formula Si-Wu-Tang on breast cancer cells reveals phytoestrogenic activity

BackgroundSi-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of β-estradiol (E2) on MCF-7 cells in the Connectivity Map database.MethodsFurther data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells.ResultsMany estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT’s estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells.ConclusionsThese results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases and possibly for cancer prevention. This study also demonstrated the feasibility of using microarray transcriptional profiling to discover phytoestrogenic components that are present in natural products.

Bioavailability enhancement of glucosamine hydrochloride by chitosan

Glucosamine, as a dietary supplement for management of osteoarthritis, has a low and erratic oral bioavailability due to its transport-mediated absorption and presystemic loss in liver and GI tract. The present study described an effective approach to improve glucosamine intestinal absorption and hence its bioavailability using chitosan. Effects of chitosan on intestinal permeability and pharmacokinetics of glucosamine were evaluated in Caco-2 cell monolayer and rats, respectively. In addition, randomized crossover pharmacokinetic studies in beagle dogs were performed to evaluate the oral bioavailabilities of the developed glucosamine oral formulations containing chitosan (QD-Glu solution and QD-Glu tablet) in comparison to its commercial products. Caco-2 permeability studies demonstrated that chitosan could enhance the absorptive transport of glucosamine by 1.9-4.0-fold via the reversible opening of the cell tight junction. After oral administration of glucosamine solutions containing chitosan in rats, it was found that 0.5% (w/v) chitosan exhibited the highest enhancement in Cmax (2.8-fold) and AUC0-∞ (2.5-fold) of glucosamine. Further pharmacokinetic studies in beagle dogs demonstrated that QD-Glu solution and QD-Glu tablet showed much higher relative bioavailabilities of 313% and 186%, when comparing with Wellesse™ solution and Voltaflex™ tablet, respectively. In conclusion, chitosan could serve as a promising oral absorption enhancer for glucosamine.

HO-1-u-1 model for screening sublingual drug delivery--influence of pH, osmolarity and permeation enhancer.

HO-1-u-1 (Ueda-1) is a human tumor cell line established from human sublingual squamous cell carcinoma. In previous study, HO-1-u-1 cell line was grown on cell culture inserts and utilized as in vitro model for screening sublingual drug delivery. The aim of current study was to further investigate the effects of pH, osmolarity and permeation enhancer, sodium glycodeoxycholate (GDC) on the permeability of three beta-blockers with different lipophilicities. The cytotoxicity was evaluated by MTS/PES assay. The permeability studies were carried out using the cell culture model and compared with that obtained from fresh porcine sublingual mucosa. The results showed the enhancement effects caused by pH, osmolarity and GDC were highly lipophilicity-dependent and in the order atenolol>metoprolol>propranolol. The apparent permeability coefficients (P(app)) of all the three beta-blockers were significantly increased by increasing pH. However, less enhancing effects were observed by non-physiological osmolarity or the presence of GDC in permeability study using both cell culture and porcine sublingual mucosa. The present results suggested that the HO-1-u-1 cell culture model maybe a useful and effective in vitro model for evaluating the enhancement effects and mechanism in sublingual drug delivery.

A bio-activity guided in vitro pharmacokinetic method to improve the quality control of Chinese medicines, application to Si Wu Tang.

The purpose of this study was to demonstrate the feasibility of using a bio-activity guided in vitro pharmacokinetic (BAPK) method in identifying relevant (absorbable and bioactive) markers for quality control (QC) of Chinese medicines (CM), using Si Wu Tang (SWT), a popular CM for womens health, as an example. A stepwise BAPK approach was utilized for relevant marker determination and evaluating of six SWT products: (1) data mining to identify active components of SWT, (2) quantification of the identified active components in each SWT product, (3) determination of in vitro dissolution and metabolism of the components under simulated gastrointestinal conditions, (4) identification of absorbable components or marker(s) via in vitro Caco-2 cell model, (5) stability testing of the permeable marker(s). Our results showed considerable variations in the amount of active components in different SWT products. Of the nine active components identified from data mining, three (ferulic acid, ligustilide, senkyunolide A) were found to be well permeated and stable over three months. Paeoniflorin, the marker designated by Chinese Pharmacopoeia, was poorly permeable and thus could not be considered a relevant marker for SWT. Our preliminary evaluation of the BAPK method appears to be feasible and may offer as a useful approach for identifying relevant markers of other TCM products in the future.

Improving sublingual delivery of weak base compounds using pHmax concept: Application to propranolol

The purpose of the present work was to provide theoretical and experimental support in generating an optimal pH (pH(max)) for a representative weak base compound (propranolol), that can lead to enhanced sublingual absorption. Initially equations for pH-solubility and pH-permeability profiles were derived and compared to the profiles obtained experimentally. Excellent correlation (R(2)=0.999) of solubility profiles was obtained using non-linear regression, and the permeability profiles further predicted that at certain pH (pH(max)), optimal mucosal permeation could be achieved. Subsequently, in a pharmacokinetics study, a buffered sublingual propranolol tablet, designed to achieve its pH(max) (when dissolved in saliva), were compared to that from a marketed product (Inderal) which could not achieve pH(max)) in 8 healthy subjects. Each subject received the products sublingually for 15 min followed by swallowing the remaining drug-saliva. The plasma propranolol concentrations of AUC during first 30 min from the buffered tablet were significantly higher than that from the Inderal tablet (p<0.05), and no significant differences in the remaining AUC were observed. These in vitro and in vivo results on propranolol provided experimental confirmation of the pH(max) concept as well as its utility in sublingual drug delivery. Such an approach may be applicable to other similar compounds to improve sublingual drug delivery.