Pharmacokinetic Analysis of Prognostic Factors in Patients With Advanced-Stage Intrahepatic Cholangiocarcinoma Following the Administration of Capsule Formulation of the Standardized Extract of Atractylodes lancea (Thunb) DC.

BACKGROUND: A statistical model is essential in determining the appropriate predictive indicators for therapies in many types of cancers. Predictors have been compared favorably to the traditional systems for many cancers. Thus, this study has been proposed as a new standard approach. A recent study on the clinical efficacy of Atractylodes lancea (Thunb) DC. (AL) revealed the higher clinical benefits in patients with advanced-stage intrahepatic cholangiocarcinoma (ICC) treated with AL compared with standard supportive care. We investigated the relationships between clinical efficacy and pharmacokinetic parameters of serum bioactivity of AL and its active constituent atractylodin and determined therapeutic ranges. METHODS: Group 1 of advanced-stage ICC patients received daily doses of 1000 mg of standardized extract of the capsule formulation of AL (CMC-AL) for 90 days. Group 2 received daily doses of 1000 mg of CMC-AL for 14 days, followed by 1500 mg for 14 days, and 2000 mg for 62 days. Group 3 (control group) received palliative care. Cox proportional hazard model and Receiver Operating Characteristic (ROC) were applied to determine the cut-off values of AUC0-inf, Cmax, and Cavg associated with therapeutic outcomes. Number needed to treat (NNT) and relative risk (RR) were also applied to determine potential predictors. RESULTS: The AUC0-inf of total AL bioactivity of >96.71 µg hour/ml was identified as a promising predictor of disease prognosis, that is, progression-free survival (PFS) and disease control rate (DCR). Cmax of total AL bioactivity of >21.42 was identified as a predictor of the prognosis of survival. The therapeutic range of total AL bioactivity for PFS and DCR is 14.48 to 65.8 µg/ml, and for overall survival is 10.97 to 65.8 µg/ml. Conclusions: The predictors of ICC disease prognosis were established based on the pharmacokinetics of total AL bioactivity. The information could be exploited to improve the clinical efficacy of AL in patients with advanced-stage ICC. These predictors will be validated in a phase 2B clinical study. TRIAL REGISTRATION: TCTR20210129007 (TCTR: www.clinicaltrials.in.th).

Antiproliferative and Anti-Inflammatory Activities of Deprungsith Formulation and Its Bioactive Compounds Against Mild Psoriasis and Potential of Metabolic Herb-Drug Interactions.

Psoriasis is an incurable, chronic and auto-immune skin disorder with a global prevalence rate of approximately 2-3%. The study investigated the antipsoriasis activities of Deprungsith formulation and its bioactive components and their potential for inhibitory activities on human cytochrome P450 (CYP450). HaCaT and peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 9) and psoriasis patients (n = 10) were exposed to Deprungsith formulation (Thai traditional medicine for psoriasis consisting of 16 plants), ethyl p-methoxycinnamate (EPMC), ligustilide and cyclosporin for 24 and 48 h. The antiproliferative, cell apoptosis and cell cycle arrest activities were evaluated using MTT assay and flow cytometry, respectively. The pro-inflammatory cytokine mRNA expression levels were measured using real-time polymerase chain reaction (RT-PCR). The CYP450 inhibitory effect was investigated using a bioluminescent-based CYP450 assay. Deprungsith formulation and the bioactive compounds inhibited HaCaT cells and PBMCs with weak to moderate potencies. EPMC and ligustilide combination produced an additive effect. Most substances arrested cell transition at sub-G1 and S phases, leading to early and late apoptosis induction. With prolonged exposure (48 h), all test substances decreased PBMCs necrosis. The mRNA expression of all pro-inflammatory cytokines was downregulated. Deprungsith formulation, EPMC, ligustilide and ferulic acid inhibited CYP1A2, CYP2C9, CYP2D6 and CYP3A4 activities with weak to moderate potencies. Deprungsith formulation and bioactive components induced cell apoptosis by inhibiting cell transition at specific cell cycle phases, which was correlated with the mRNA downregulation of interleukin (IL-6, IL-12p19, IL-23) and tumor necrosis factor (TNF-α). There is a low risk of potential adverse drug reactions and toxicity due to CYP450 interaction when Deprungsith formulation is concurrently administered with modern medicines.

Preclinical studies of toxicity and anti-cholangiocarcinoma activity of the standardized capsule formulation of Atractylodes lancea (Thunb.) DC.

BACKGROUND: Cholangiocarcinoma (CCA), the adenocarcinoma of the biliary duct, is commonly reported in Asia, with the highest incidence in northeastern Thailand. Chemotherapy of CCA has been limited by the lack of effective chemotherapeutic drugs. A series of previous in vitro and in vivo studies support further research and development of Atractylodes lancea (Thunb.) DC. (AL) as a potential candidate for treating CCA as a crude ethanolic extract. In the present study, we evaluated the toxicity and anti-CCA activity of the CMC (Chemistry, Manufacturing, and Control) capsule formulation of the ethanolic rhizome extract of AL (CMC-AL) in animals. METHODS: Major steps included acute, subchronic and chronic toxicity testing in Wistar rats and anti-CCA activity in a CCA-xenografted nude mouse model. The safety of CMC-AL was determined based on the maximum tolerated dose (MTD) and no-observed-adverse-effect level (NOAEL) according to the OECD guideline. The anti-CCA activity of CMC-AL in nude mice was evaluated after transplantation of CL-6 cells to evaluate inhibitory effects on tumor size progression and metastasis and survival time prolongation. Safety assessments included hematology, biochemistry parameters and histopathological examination. Lung metastasis was investigated using VEGF ELISA kit. RESULTS: All evaluations confirmed satisfactory pharmaceutical properties of oral formulation and safety profile of the CMC-AL with no overt toxicity up to the MTD and NOAEL of 5,000 and 3,000 mg/kg body weight, respectively. CMC-AL exhibited potent anti-CCA efficacy with regard to inhibitory activity on tumor progression and lung metastasis. CONCLUSIONS: CMC-AL is safe and should be further investigated in a clinical trial as a potential therapy for CCA patients.

The Role of Herbal Medicine in Cholangiocarcinoma Control: A Systematic Review.

The growing incidence of cholangiocarcinoma (bile duct cancer) and limited treatment options stimulate a pressing demand for research and the development of new chemotherapeutics against cholangiocarcinoma. This study aimed to systematically review herbs and herb-derived compounds or herbal formulations that have been investigated for their anti-cholangiocarcinoma potential. Systematic literature searches were conducted in three electronic databases: PubMed, ScienceDirect, and Scopus. One hundred and twenty-three research articles fulfilled the eligibility critera and were included in the analysis (68 herbs, isolated compounds and/or synthetic analogs, 9 herbal formulations, and 119 compounds that are commonly found in several plant species). The most investigated herbs were Atractylodes lancea (Thunb.) DC. (Compositae) and Curcuma longa L. (Zingiberaceae). Only A. lancea (Thunb.) DC. (Compositae) has undergone the full process of nonclinical and clinical development to deliver the final product for clinical use. The extracts of A. lancea (Thunb.) DC. (Compositae), Garcinia hanburyi Hook.f. (Clusiaceae), and Piper nigrum L. (Piperaceae) exhibit antiproliferative activities against human cholangiocarcinoma cells (IC50 < 15 µg/mL). Cucurbitacin B and triptolide are herbal isolated compounds that exhibit the most promising activities (IC50 < 1 µM). A series of experimental studies (in vitro, in vivo, and humans) confirmed the anti-cholangiocarcinoma potential and safety profile of A. lancea (Thunb.) DC. (Compositae) and its active compounds atractylodin and ß-eudesmol, including the capsule pharmaceutical of the standardized A. lancea (Thunb.) DC. (Compositae) extract. Future research should be focused on the full development of the candidate herbs to deliver products that are safe and effective for cholangiocarcinoma control.

Effects of ß-eudesmol and atractylodin on target genes and hormone related to cardiotoxicity, hepatotoxicity, and endocrine disruption in developing zebrafish embryos.

Atractylodes lancea, commonly known as Kod-Kamao in Thai, a traditional medicinal herb, is being developed for clinical use in cholangiocarcinoma. ß-eudesmol and atractylodin are the main active components of this herb which possess most of the pharmacological properties. However, the lack of adequate toxicity data would be a significant hindrance to their further development. The present study investigated the toxic effects of selected concentrations of ß-eudesmol and atractylodin in the heart, liver, and endocrine systems of zebrafish embryos. Study endpoints included changes in the expression of genes related to Na/K-ATPase activity in the heart, fatty acid-binding protein 10a and cytochrome P450 family 1 subfamily A member 1 in the liver, and cortisol levels in the endocrine system. Both compounds produced inhibitory effects on the Na/K-ATPase gene expressions in the heart. Both also triggered the biomarkers of liver toxicity. While ß-eudesmol did not alter the expression of the cytochrome P450 family 1 subfamily A member 1 gene, atractylodin at high concentrations upregulated the gene, suggesting its potential enzyme-inducing activity in this gene. ß-eudesmol, but not atractylodin, showed some stress-reducing properties with suppression of cortisol production.

Atractylodes lancea for cholangiocarcinoma: Modulatory effects on CYP1A2 and CYP3A1 and pharmacokinetics in rats and biodistribution in mice.

Atractylodes lancea (Thunb.) DC. (A. lancea: AL) is a promising candidate for the treatment of cholangiocarcinoma (bile duct cancer). The study investigated (i) the propensity of capsule formulation of the standardized extract of AL (formulated AL) to modulate mRNA and protein expression and activities of CYP1A2 and CYP3A1 in rats after long- and short-term exposure, (ii) the pharmacokinetics of atractylodin (ATD: active constituent) after long-term administration of formulated AL, and (iii) the biodistribution of atractylodin-loaded polylactic-co-glycolic acid (ATD-PLGA-NPs) in mice. To investigate CYP1A2 and CYP3A1 modulatory activities following long-term exposure, rats of both genders received oral doses of the formulated AL at 1,000 (low dose), 3,000 (medium dose), and 5,000 (high dose) mg/kg body weight daily for 12 months. For short-term effects, male rats were orally administered the formulated AL at the dose of 5,000 mg/kg body weight daily for 1, 7, 14 and 21 days. The pharmacokinetic study was conducted in male rats after administration of the formulated AL at the dose of 5,000 mg/kg body weight daily for 9 months. The biodistribution study was conducted in a male mouse receiving ATD-PLGA-NPs at the equivalent dose to ATD of 100 mg/kg body weight. The high dose of formulated AL produced an inducing effect on CYP1A2 but an inhibitory effect on CYP3A1 activities in male rats. The low dose, however, did not inhibit or induce the activities of both enzymes in male and female rats. ATD reached maximum plasma concentration (Cmax) of 359.73 ng/mL at 3 h (tmax). Mean residence time (MRT) and terminal phase elimination half-life (t1/2z) were 3.03 and 0.56 h, respectively. The extent of biodistribution of ATD in mouse livers receiving ATD-PLGA-NPs was 5-fold of that receiving free ATD. Clinical use of low-dose AL should be considered to avoid potential herb-drug interactions after long-term use. ATD-PLGA-NPs is a potential drug delivery system for cholangiocarcinoma treatment.

Potential of Alpha-Mangostin-Loaded PLGA Nanoparticles for Cholangiocarcinoma Treatment.

Alpha-mangostin (AM), a significant component isolated from the pericarp of mangosteen (Garcinia mangostana L.), has been demonstrated as a potential compound for the treatment of cholangiocarcinoma (CCA). Due to its hydrophobic nature, however, its clinical uses may be limited by its low aqueous solubility, poor stability, and low bioavailability. The present study aimed to formulate and characterize the AM-loaded PLGA nanoparticles (AM-PLGA-NPs) and further evaluate the antiproliferative and proapoptotic activities, including the inhibitory activities on CCA cell (CL-6 and HuCCT-1) invasion and migration. The AM-PLGA-NPs were prepared using PLGA MW 7000-17,000 and 38,000-54,000 by the solvent displacement method. The methods used to evaluate these activities included a MTT assay, flow-cytometry, QCM ECMatrix cell migration, and cell invasion assays, respectively. The optimized AM-PLGA-NPs were characterized for physical (particle size and morphology, polydispersity index, and zeta potential) and pharmaceutical (encapsulation efficiency, loading efficiency, and drug release profile) parameters. AM-PLGA-NPs showed relatively potent and selective antiproliferative and proapoptotic activities in both CCA cell lines in a concentration- and time-dependent manner. The results revealed that the PLGA nanoparticles could be a suitable nanocarrier to encapsulate AM for its delivery to the CCA cells.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

OBJECTIVES: Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. MATERIALS AND METHODS: Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. RESULTS: The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-ß, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. CONCLUSION: The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-ß signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Phase I clinical trial to evaluate the safety and pharmacokinetics of capsule formulation of the standardized extract of Atractylodes lancea.

BACKGROUND AND AIM: Atractylodes lancea (AL) has been demonstrated in a series of studies to be a potential candidate for the treatment of cholangiocarcinoma. The aim of the current study was to evaluate the safety and pharmacokinetics of the capsule formulation of the standardized AL extract in healthy Thai participants. EXPERIMENTAL PROCEDURE: Forty-eight healthy Thai participants who fulfilled the inclusion and had none of the exclusion criteria were allocated to two study groups. The group 1 participants were randomized to receive a single oral dose of 1,000 mg of AL or placebo (20:4 participants). The group 2 participants were randomized to receive daily oral doses of 1,000 mg AL or placebo daily for 21 days (20:4 participants). Safety and tolerability of the two AL regimens were monitored. Blood samples were collected for measurement of atractylodin concentrations by HPLC and pharmacokinetic analysis was performed using model-dependent and model-independent analysis. RESULTS AND CONCLUSION: The AL extract was well tolerated in both groups. Atractylodin was rapidly absorbed but with low systemic exposure and residence time. There was no difference in the pharmacokinetic parameters of atractylodin following a single or multiple dosing, suggesting the absence of accumulation and dose-dependency in human plasma after continuous dosing for 21 days. The information on human pharmacokinetics of AL, when given as capsule formulation of the standardized extract, would assist in further dose optimization in cholangiocarcinoma patients with the defined pharmacokinetic-pharmacodynamic relationship.

Embryotoxicity evaluation of atractylodin and ß-eudesmol using the zebrafish model.

Atractylodin and ß-eudesmol are the major active ingredients of Atractylodes lancea (Thunb) DC. (AL). Both compounds exhibit various pharmacological activities, including anticancer activity against cholangiocarcinoma. Despite the widespread use of this plant in traditional medicine in China, Japan, Korea, and Thailand, studies of their toxicological profiles are limited. The present study aimed to evaluate the embryotoxicity of atractylodin and ß-eudesmol using the zebrafish model. Zebrafish embryos were exposed to a series of concentrations (6.3, 12.5, 25, 50, and 100 µM) of each compound up to 72 h post-fertilization (hpf). The results showed that atractylodin and ß-eudesmol induced mortality of zebrafish embryos with the 50% lethal concentration (LC50) of 36.8 and 53.0 µM, respectively. Both compounds also caused embryonic deformities, including pericardial edema, malformed head, yolk sac edema, and truncated body. Only ß-eudesmol decreased the hatching rates, while atractylodin reduced the heart rates of the zebrafish embryos. Additionally, both compounds increased reactive oxygen species (ROS) production and altered the transcriptional expression levels of superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2) genes. In conclusion, atractylodin and ß-eudesmol induce mortality, developmental toxicity, and oxidative stress in zebrafish embryos. These findings may imply similar toxicity of both compounds in humans.

In vitro cytotoxic and toxicological activities of ethanolic extract of Kaempferia galanga Linn. and its active component, ethyl-p-methoxycinnamate, against cholangiocarcinoma.

OBJECTIVE: To evaluate the cytotoxic, apoptotic, mutagenic and immunomodulatory activities of Kaempferia galanga Linn. (KG) extract and ethyl-p-methoxycinnamate (EPMC) in vitro. METHODS: The present study investigated the cytotoxic [using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test], apoptotic (using a mitochondrial membrane potential assay), mutagenic (using a micronucleus test) and immunomodulatory (using flow cytometry) activities of the ethanolic extract of KG and its bioactive component, EPMC, against two cholangiocarcinoma (CCA) cell lines, CL-6 and HuCCT1, and one normal human cell line, OUMS-36T-1F. RESULTS: Both KG extract and EPMC exhibited moderate cytotoxic activity against both CCA cells. The cytotoxic activity was supported by their concentration-dependent induction of apoptosis. CL-6 was most sensitive (3-4 fold) and selective to 5-fluorouracil (5-FU), compared with KG extract and EPMC [median half inhibiting concentration (IC50) and selectivity index (SI) were 23.01 µg/mL and 17.32; 78.41 µg/mL and 4.44; 100.76 µg/mL and 2.20, respectively for 5-FU vs. KG extract vs. EPMC]. HuCCT1 was relatively more sensitive and selective to 5-FU and EPMC than KG extract [median IC50 and SI were 66.03 µg/mL and 6.04; 60.90 µg/mL and 3.65; 156.60 µg/mL and 2.23, respectively for 5-FU vs. EPMC vs. KG extract]. EPMC produced relatively potent cytotoxic activity against polymorphonuclear cells (IC50 = 92.20 µg/mL). KG extract and EPMC exhibited concentration-dependent mutagenic activity, as well as inhibition of tumor necrosis factor-α and interleukin-6. CONCLUSION: Considering cytotoxic, apoptotic, immunomodulatory and mutagenic activities, further development of KG as a drug candidate is likely to focus on the oral pharmaceutical formulation of a standardized KG extract rather than isolated compounds.

Preclinical Toxicology and Anticholangiocarcinoma Activity of Oral Formulation of Standardized Extract of Zingiber Officinale.

Cholangiocarcinoma (CCA) remains a significant public health problem in Thailand. New effective and safe drugs are urgently needed. Zingiber officinale Roscoe (ZO) is a widely used medicinal plant for the treatment of several ailments, and the animal study suggests a potential anti-CCA activity. The present study aimed to develop the oral formulation of standardized extract of ZO and investigate toxicological profiles (acute, repeated dose, and chronic toxicity), including anti-CCA activity of the ZO formulation. The oral pharmaceutical formulation of the standardized ZO extract was successfully developed with an acceptable level of contamination and physicochemical and pharmaceutical properties. Acute, subacute, and chronic toxicity tests were conducted in healthy Sprague Dawley rats according to the OECD guidelines. The results showed no evidence of toxicity and death in the acute and subacute toxicity testing with the maximum tolerated dose (MTD) of 5000 and 2000 mg/kg body weight, respectively. Chronic toxicity revealed MTD and No-Observed-Adverse-Effect level (NOAEL) of 1000 mg/kg body weight. The anti-CCA activity was evaluated in CCA-xenografted mouse model. The formulated ZO powder was fed to animals daily for 30 days. Significant anti-CCA activity on tumor growth inhibition and prolongation of survival time were demonstrated at the high (2000 mg/kg body weight) and moderate (1000 mg/kg body weight) dose levels. Further investigation to elucidate molecular targets of action of ZO against CCA cells is encouraged.

Application of active targeting nanoparticle delivery system for chemotherapeutic drugs and traditional/herbal medicines in cancer therapy: a systematic review.

Patients treated with conventional cancer chemotherapy suffer from side effects of the drugs due to non-selective action of chemotherapeutic drugs to normal cells. Active targeting nanoparticles that are conjugated to targeting ligands on the surface of nanoparticles play an important role in improving drug selectivity to the cancer cell. Several chemotherapeutic drugs and traditional/herbal medicines reported for anticancer activities have been investigated for their selective delivery to cancer cells by active targeting nanoparticles. This systematic review summarizes reports on this application. Literature search was conducted through PubMed database search up to March 2017 using the terms nanoparticle, chemotherapy, traditional medicine, herbal medicine, natural medicine, natural compound, cancer treatment, and active targeting. Out of 695 published articles, 61 articles were included in the analysis based on the predefined inclusion and exclusion criteria. The targeting ligands included proteins/peptides, hyaluronic acid, folic acid, antibodies/antibody fragments, aptamer, and carbohydrates/polysaccharides. In vitro and in vivo studies suggest that active targeting nanoparticles increase selectivity in cellular uptake and/or cytotoxicity over the conventional chemotherapeutic drugs and non-targeted nanoparticle platform, particularly enhancement of drug efficacy and safety. However, clinical studies are required to confirm these findings.

Anticholangiocarcinoma activity and toxicity of the Kaempferia galanga Linn. Rhizome ethanolic extract.

BACKGROUND: Cholangiocarcinoma (CCA) is an important public health problem in several tropical and subtropical parts of the world particularly Thailand. Chemotherapy of CCA is largely ineffective and discovery and development of effective alternative drugs is urgently needed. The objective of the study was to confirm the anti-CCA potential as well as toxicity of the crude extract of Kaempferia galangal Linn. (rhizome) both in vitro and in animal models. METHODS: The ethanolic extract of K. galanga Linn. rhizome, ethyl-p-methoxycinnamate (EPMC) and 5-fluorouracil (5-FU) were evaluated for their cytotoxic activities against CCA cell line (CL-6) using MTT cell proliferation assay. Acute and subacute toxicity of the extract were evaluated in ICR (Imprinting Control Region) mice according to the OECD (International Organization for Economic Co-operation and Development) Guideline. Anti-CCA activity was evaluated in CCA- xenografted nude mice. RESULTS: Results of cytotoxicity test showed moderate activity of the extract and EPMC with median (95% confidence interval: 95% CI) 50% inhibitory concentration (IC50) of 64.2 (57.76-72.11) and 49.19 (48.16-52.29) µg/ml, respectively. The IC50 of 5-FU was 107.1 (103.53-109.64) µg/ml. The selectivity index (SI) values for the extract, EPMC and 5-FU against human normal cell line (OUMS) and cancer cell line (CL-6) were 2.2, 2.09 and 1.31, respectively. Toxicity testing revealed no overt toxic effect up to the maximum single oral dose of 5000 mg/kg body weight and up to daily dose of 1000 mg/kg body weight for 30 days. The extract at the maximum tolerated dose level of 1000 mg/kg body weight for 30 days exhibited promising anti-CCA activity in CL6-xenografted nude mice as determined by inhibitory activity on tumor growth (58.41%) and lung metastasis (33.3%), as well as prolongation of survival time (62 days). CONCLUSION: The K. galangal Linn. rhizome extract and its bioactive compound EPMC exhibited moderate cytotoxic activity against human CCA tumor (CL-6) cell line. Results of toxicity testing suggest that the extract was well tolerated up to the maximum single oral dose of 5000 mg/kg body weight and daily dose of 1000 mg/kg body weight for 30 days. The extract exhibited promising anti-CCA activity in CL6-xenografed nude mice as determined by significant inhibitory activity on tumor growth and lung metastasis, as well as prolongation of survival time.

Research and Development of Atractylodes lancea (Thunb) DC. as a Promising Candidate for Cholangiocarcinoma Chemotherapeutics.

Treatment and control of cholangiocarcinoma (CCA): the bile duct cancer is limited by the lack of effective chemotherapeutic drugs and alternative drugs are needed, particularly those from natural sources. This article reviews steps of research and development of Atractylodes lancea (Thunb) DC. (AL) as potential candidate for CCA chemotherapy, with adoption of the reverse pharmacology approach. Major steps include (1) reviewing of existing information on its phytochemistry and pharmacological properties, (2) screening of its activities against CCA, (3) standardization of AL, (4) nonclinical studies to evaluate anti-CCA activities, (5) phytochemistry and standardization of AL extract, (6) development of oral pharmaceutical formulation of standardized AL extract, and (7) toxicity testing of oral pharmaceutical formulation of standardized AL extract. Results from a series of our study confirm anti-CCA potential and safety profiles of both the crude extract and the finished product (oral pharmaceutical formulation of the standardized AL extract). Phases I and II clinical trials of the product to confirm tolerability and efficacy in healthy subjects and patients with advanced stage CCA will be carried out soon.

Anticancer Activity of Atractylodes lancea (Thunb.) DC in a Hamster Model and Application of PET-CT for Early Detection and Monitoring Progression of Cholangiocarcinoma.

Opisthorchis viverrini (OV)-induced cholangiocarcinoma (CCA) is an important cancer in the Great Mekong region, particularly in Thailand. Limitations of treatment options and the lack of an effective diagnostic tool for early detection of CCA are major concerns for the control of this type of cancer. The aim of the study was to investigate anti-CCA activity of the ethanolic extract of Atractylodes lancea (Thunb.) DC., and the applicability of positron emission tomography-computed tomography (PET-CT) as a tool for detection and monitoring the progression of CCA in Opisthorchis viverrini (OV)/dimethylnitrosamine (DMN)-induced CCA hamsters. Male Syrian hamsters were used for toxicity tests and anti-CCA activity evaluation. Development of CCA was induced by initial feeding of 50 metacercariae of OV, followed by drinking water containing 12.5 ppm of DMN in hamsters. The ethanolic extract of A. lancea (Thunb.) DC. was administered orally for 30 days. PET-CT was performed every 4 weeks after initiation of CCA using 18F-fluorodeoxyglucose (18F-FDG). Results from the present study suggest that the ethanolic extract of A. lancea (Thunb.) DC. rhizome exhibited promising anti-CCA activity and safety profile in the OV/DMN-induced hamster model. To successfully apply PET-CT as a tool for early detection of tumor development and progression, modification of radiolabeling approach is required to improve its specificity for CCA cells.

Antimalarial activity of plumbagin in vitro and in animal models.

BACKGROUND: Plumbagin is the major active constituent in several plants including Plumbago indica Linn. (root). This compound has been shown to exhibit a wide spectrum of biological and pharmacological activities. The present study aimed to evaluate the in vitro and in vivo antimalarial activity of plumbagin including its acute and subacute toxicity in mice. METHODS: In vitro antimalarial activity of plumbagin against K1 and 3D7 Plasmodium falciparum clones were assessed using SYBR Green I based assay. In vivo antimalarial activity was investigated in Plasmodium berghei-infected mouse model (a 4-day suppressive test). RESULTS: Plumbagin exhibited promising antimalarial activity with in vitro IC50 (concentration that inhibits parasite growth to 50%) against 3D7 chloroquine-sensitive P. falciparum and K1 chloroquine-resistant P. falciparum clones of 580 (270-640) and 370 (270-490) nM, respectively. Toxicity testing indicated relatively low toxicity at the dose levels up to 100 (single oral dose) and 25 (daily doses for 14 days) mg/kg body weight for acute and subacute toxicity, respectively. Chloroquine exhibited the most potent antimalarial activity in mice infected with P. berghei ANKA strain with respect to its activity on the reduction of parasitaemia on day 4 and the prolongation of survival time. CONCLUSIONS: Plumbagin at the dose of 25 mg/kg body weight given for 4 days was safe and produced weak antimalarial activity. Chemical derivatization of the parent compound or preparation of modified formulation is required to improve its systemic bioavailability.

Cytotoxicity, toxicity, and anticancer activity of Zingiber officinale Roscoe against cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/ DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and 27.86 µg/ml, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.

Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models.

BACKGROUND: Chemotherapy of cholangiocarcinoma (CCA), a devastating cancer with increasing worldwide incidence and mortality rates, is largely ineffective. The discovery and development of effective chemotherapeutics is urgently needed. METHODS/DESIGN: The study aimed at evaluating anticancer activities, toxicity, and pharmacological activities of the curcumin compound (CUR), the crude ethanolic extracts of rhizomes of Zingiber officinale Roscoe (Ginger: ZO) and Atractylodes lancea thung. DC (Khod-Kha-Mao: AL), fruits of Piper chaba Hunt. (De-Plee: PC), and Pra-Sa-Prao-Yhai formulation (a mixture of parts of 18 Thai medicinal plants: PPF) were investigated in animal models. Anti-cholangiocarcinoma (anti-CCA) was assessed using CCA-xenograft nude mouse model. The antihypertensive, analgesic, anti-inflammatory, antipyretic, and anti-ulcer activities and effects on motor coordination were investigated using Rota-rod test, CODA tail-cuff system, writhing and hot plate tests, carrageenan-induced paw edema test, brewer's yeast test, and alcohol-induced gastric ulcer test, respectively. Acute and subacute toxicity tests were performed according to the OECD guideline for testing of chemicals with modification. RESULTS: Promising anticancer activity against CCA in nude mouse xenograft model was shown for the ethanolic extract of AL at all oral dose levels (1000, 3000, and 5000 mg/kg body weight) as well as the extracts of ZO, PPF, and CUR compound at the highest dose level (5000, 4000, and 5000 mg/kg body weight, respectively). PC produced no significant anti-CCA activity. Results from acute and subacute toxicity tests both in mice and rats indicate safety profiles of all the test materials in a broad range of dose levels. No significant toxicity except stomach irritation and general CNS depressant signs were observed. Investigation of pharmacological activities of the test materials revealed promising anti-inflammatory (ZO, PPF, and AL), analgesic (CUR and PPF), antipyretic (CUR and AL), antihypertensive (ZO and AL), and anti-ulcer (CUR, ZO, and AL) activities. CONCLUSION: Plants used in Thai traditional medicine for the treatment of various ailments may provide reservoirs of promising candidate chemotherapeutics for the treatment of CCA.