In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

Neoadjuvant Therapy with Drug Arglabin for Breast Cancer with Expression of H-Ras Oncoproteins.

BACKGROUNDS: In breast cancer, blocking of Ras signaling and inhibition of H-Ras is quite promising. H-Ras may become a target for farnesyl transferase inhibitors, and in combination with other immunohistochemical factors it will contribute to the progression of a breast tumor. PURPOSE: The aim of this study was to evaluate the effectiveness of neoadjuvant therapy for breast cancer with the inclusion of farnesyl transferase inhibitor, arglabin interfering with the expression and concentration of H-Ras oncoproteins. METHODS: Depending on the presence of H-Ras oncoproteins after Western-blot hybridization, the patients were divided a negative and positive expression of H-Ras groups. RESULTS: Correlation analysis of methods used for determining the expression ability and concentration of H-Ras oncoproteins (immunohistochemistry and Western-blot analysis) demonstrated substantial statistical relationship Rs=0.71, p=0.03. The H-Ras oncoproteins were absent in patients receiving either "Arglabin" or standard AC regimen. However, in the AC + Arglabin group, there was a varying degrees of positive concentration of H-Ras oncoproteins (Kruskal-Wallis=6.92; p=0.03). CONCLUSION: These results indicate that Arglabin attenuates H-Ras oncoproteins expression which is a promising therapeutic target for breast cancer.

Long-term every-other-day administration of DMAMCL has little effect on aging and age-associated physiological decline in mice.

The activation of transcription factor NF-κB is currently identified as one of the driving forces to the aging process. Genetic impairment of NF-κB signaling pathway or pharmacological inhibition of NF-κB activity has been shown to extend healthspan and lifespan in animal models, and delay or reduce many age-related symptoms. However, the aging intervention strategies based on NF-κB inhibition by the suitable small molecular compound is currently still lacking. The water-soluble dimethylaminomicheliolide (DMAMCL), can inhibit NF-κB activity and is currently undergoing clinical trials. In this study, we showed that 15 months of DMAMCL administration started in 1-year old male mice was well-tolerated and safe, and improved or had little effect on some age-associated symptoms, such as neurobehavioral phenotypes, physical performance, cardiac function, hematological parameters, immune aging phenotypes, clinical chemistry parameters, and glucose homeostasis. At the molecular level, DMAMCL administration mitigated serum levels of several age-associated inflammatory cytokines, including IL-6, IL-1α, IL-1ß, TNF-α, IFN-γ, and CXCL2, and inhibited NF-κB activity in several aged tissues. Collectively, our results indicate that current strategy of DMAMCL administration may has little effect on aging process in mice, and provide basic clues to further exploit the possibility of DMAMCL-based aging intervention to promote healthy aging.

The anti-tumor growth effect of a novel agent DMAMCL in rhabdomyosarcoma in vitro and in vivo.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with poor survival. New treatment approaches are urgently needed to improve treatment efficacy in RMS patients. DMAMCL is a novel agent from Asteraceae family that has been tested in phase I clinical trials in adult glioma in Australia. METHODS: Five RMS cell lines (RD, RH18, RH28, RH30 and RH41) were used. The in vitro anti-tumor effect of DMAMCL, alone or in combination with VCR or Epirubicin, was studied using MTS assay or IncuCyte-Zoom cell confluency assay, and further validated by xenograft-mouse model in vivo. Changes in caspase-3/7 activity, cell-cycle progression and generation of ROS after DMAMCL treatment were investigated. Bim mRNA expression was measured by RT-qPCR, and protein expressions of Bim and phosphorylated-NF-κB(p65) by Western blotting. Small interfering RNAs (siRNA) of Bim were used to study the role of Bim in DMAMCL-induced cell death. RESULTS: In vitro, DMAMCL treatment induced a dose-dependent increase in cell death that could be blocked by pan-caspase-inhibitor-Z-VAD-fmk in five RMS cell lines. The percent of cells in SubG1 phase and activities of caspase-3/7 increased after DMAMCL treatment; The combination of DMAMCL with VCR or Epirubicin significantly increased cell death compared to each reagent alone. In vivo, DMAMCL(75 mg/kg or 100 mg/kg) inhibited tumor growth and prolonged survival of mice bearing xenograft RMS tumors (RD, RH18, RH30, RH41). Compared to treatment with DMAMCL or VCR, a combination of two reagents caused significant inhibition of tumor growth (RD, RH41), even after treatment termination. The expression of Bim increased at protein level after DMAMCL treatment both in vitro and in vivo. The expression of p-NF-κB(p65) had a transient increase and the generation of ROS increased after DMAMCL treatment in vitro. Transfection of Bim siRNA into RMS cells blocked the DMAMCL-induced increase of Bim and partially attenuated the DMAMCL-induced cell death. CONCLUSION: DMAMCL had an anti-tumor growth effect in vitro and in vivo that potentially mediated by Bim, NF-κB pathway and ROS. A combination of DMAMCL with chemotherapeutic drugs significantly increased the treatment efficacy. Our study supports further clinical evaluation of DMAMCL in combination with conventional chemotherapy.

Pharmacokinetics, tissue distribution and excretion of ACT001 in Sprague-Dawley rats and metabolism of ACT001.

This study investigated pharmacokinetics, tissue distribution and excretion of ACT001 in Sprague-Dawley rats. Stability study and metabolism study of ACT001 are conducted. The absolute bioavailability of ACT001 is 50.82%. ACT001 has no accumulation effect and displayed wide tissue distribution. ACT001 can be rapidly distributed to tissues after oral administration and can diffuse through the blood-brain barrier. The total cumulative excretion of ACT001 in feces, urine and bile were found to be 0.05, 3.42 and 0.012%, respectively. UPLC/ESI-QTOF-MS coupled with MetaboLynx XS software was utilized to detect the metabolites of ACT001 in vitro. Five metabolites (M1, M2, M3, M4 and M5) were detected. M2 wasn't discovered in human liver microsome samples and bile samples. M1 and M2 weren't discovered in rat plasma and human plasma. M3, M4 and M5 weren't discovered in bile samples. M5 is an active metabolite named micheliolide (MCL). There is no significant difference in half-life, type of identified metabolites and the amount of each metabolites between using rat plasma and human plasma. Owing to the species differences of hepatomicrosome enzymes, significant differences were shown in half-life, type of identified metabolites and the amount of each metabolites between using rat liver microsome and human liver microsome.

Zedoary Guaiane-Type Sesquiterpenes-Eluting Stents Accelerate Endothelial Healing Without Neointimal Hyperplasia in a Porcine Coronary Artery Model.

OBJECTIVE: The effects of zedoary guaiane-type sesquiterpenes (ZGS)-based eluting stent (ZES) in accelerating reendothelialization and inhibiting neointimal hyperplasia were examined in a porcine coronary artery model. METHODS: The ZES was prepared by polymer-free 316L stainless metal stents. Sirolimus-eluting stents (SES) and bare metal stents (BMS) with identical platforms were used as controls. Stents with 15 mm in length and 2.0 to 3.5 mm in diameter were implanted in porcine coronary arteries. Scanning electron microscopy (SEM) and histopathology were performed to assess the reendothelialization and neointimal hyperplasia. The 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-2H-tetrazoliumbromide assay and flow cytometry were used to assess the influence of ZGS on human umbilical vascular endothelial cells (HUVECs). RESULTS: At 7 days, SEM showed that percentage of endothelial coverage area was 94.04% ± 5.01% for ZES, 47.59% ± 19.91% for SES ( P < .01 for ZES vs SES), and 59.58% ± 19.61% for BMS ( P < .05 for ZES vs BMS). At 28 days, the percentage of coverage area was 98.51% ± 1.86% for ZES, 86.18% ± 8.16% for SES ( P < .05 for ZES vs SES), and 94.26% ± 5.58% for BMS. Neointimal area and stenosis were significantly lower in ZES (1.07 ± 0.48 mm2, 27.66% ± 12.20%) compared to BMS (1.73 ± 0.69 mm2, 44.08% ± 15.03%, both P < .01, respectively), with no difference in SES (0.94 ± 0.12 mm2, 28.87% ± 6.00%, both P > .05, respectively). The ZGS also promoted HUVECs viability and improved HUVECs proliferation compared to sirolimus. CONCLUSION: The ZES accelerated reendothelialization and suppressed neointimal hyperplasia in a porcine coronary artery model, with beneficial effects on HUVECs.

Synthesis of a stable and orally bioavailable englerin analogue.

Synthesis of analogues of englerin A with a reduced propensity for hydrolysis of the glycolate moiety led to a compound which possessed the renal cancer cell selectivity of the parent and was orally bioavailable in mice.

Therapeutic effects of micheliolide on a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease and collagen-induced arthritis (CIA) is an animal model for RA. Micheliolide (MCL) is a novel compound with strong anti-inflammatory effects. The present study was conducted to evaluate the therapeutic effects of MCL on RA. Mice were randomly divided into four groups and the CIA model mice were treated with methotrexate (MTX), MCL and dimethyl sulfoxide. A score associated with the severity of arthritis was assigned on alternate days from the 22nd day for 60 days. Histopathological changes and the serum levels of cytokines were measured on day 85. The results demonstrated that the MCL treatment group had arthritis scores lower than the CIA group and higher than the MTX group; compared with the CIA group, MCL and MTX significantly reduced the swelling of the paws and suppressed the degeneration of articular cartilage. Expression levels of macrophage colony-stimulating factor (M-CSF), tissue inhibitors of metalloproteinases-1 (TIMP-1) and complement component 5a (C5/C5a) were lower in the mice with arthritis compared with normal mice, however, following treatment with MCL and MTX, all the mice exhibited significant recovery to differing degrees. Unlike the MTX group, the MCL group failed to recover the level of soluble intercellular adhesion molecule-1. In addition, the cytokine of B-lymphocyte chemoattractant (BLC) solely presented in the MCL group. These results suggest that MCL may be considered for use as a novel therapeutic treatment against RA and that changes in the expression of cytokines C5/C5a, TIMP-1, M-CSF and BLC may underlie the mechanism by which MCL effects changes in this disease.

Identification of a novel anti-inflammatory compound, α-cubebenoate from Schisandra chinensis.

AIMS OF THE STUDY: Extracts of Schisandra chinensis have been used as an anti-fatigue and tonic agent. Because chronic fatigue syndrome is related to inflammatory and oxidative stress, we assessed whether Schisandra chinensis has anti-inflammatory constituents and studied the effect of a novel α-cubebenoate isolated from Schisandra chinensis. MATERIALS AND METHODS: α-Cubebenoate was isolated from an extract of Schisandra chinensis fruits. The inductions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS) were quantified by RT-PCR and Western blotting in mouse peritoneal macrophages. Nitric oxide (NO) and prostaglandin E2 (PGE2) were also measured in the media by Griess reagent and EIA method. A mouse model of LPS-induced peritonitis was used to test the in vivo efficacy of α-cubebenoate. RESULTS: α-Cubebenoate (5-10µg/ml) inhibited the inductions of iNOS and COX-2 in mouse peritoneal macrophages at the mRNA and protein levels. LPS-induced productions of NO and PGE2 were inhibited by α-cubebenoate (5-10µg/ml). In addition, α-cubebenoate inhibited the LPS-induced activation of JNK, but not those of ERK and p38 MAPK in mouse peritoneal macrophages. Furthermore, in the LPS-induced in vivo peritonitis model, α-cubebenoate (1mg/kg) strongly inhibited the accumulation of polymorph nuclear lymphocytes in the peritoneal cavity. CONCLUSION: α-Cubebenoate inhibited LPS-induced expression of iNOS and COX-2 in a concentration-dependent manner, thereby suppressing productions of NO and PGE2 in vitro in peritoneal macrophages. α-Cubebenoate also inhibited LPS-induced accumulation of polymorph nuclear lymphocytes in LPS-induced peritonitis model in vivo. α-Cubebenoate may act as an anti-fatigue constituent of Schisandra chinensis through anti-inflammation and could be of therapeutic use as a treatment for inflammatory diseases.

Japonicone A suppresses growth of Burkitt lymphoma cells through its effect on NF-κB.

PURPOSE: NF-κB, a transcriptional regulator of diverse genes involved in cell survival, proliferation, adhesion, and apoptosis, has been implicated in various malignancies. We discovered a potent natural NF-κB inhibitor, Japonicone A, from the traditional herb Inula japonica Thunb, evaluated its preclinical pharmacology and therapeutic activity, and investigated the underlying mechanisms of action for its antitumor activity. EXPERIMENTAL DESIGN: Various types of cancer and normal cells were exposed to Japonicone A for cytotoxicity screening, followed by determination of cell apoptosis and cell-cycle arrest. Western blotting, immunostaining, and gene reporter assay were used to analyze NF-κB activity. Two xenograft models were used for therapeutic efficacy evaluation. RESULTS: Japonicone A killed cancer cells but had low cytotoxicity to normal cells. Burkitt lymphoma cells were particularly sensitive. Japonicone A inhibited the growth and proliferation of Raji, BJAB, and NAMALWA lymphoma cells and resulted in G2-M phase arrest and apoptosis. Furthermore, exposure of cells to Japonicone A caused inactivation of the TNF-α-TAK1-IKK-NF-κB axis and inhibition of TNF-α-stimulated NF-κB activity and nuclear translocation, followed by downregulation of NF-κB target genes involved in cell apoptosis (Bcl-2, Bcl-xL, XIAP, TRAF2) and in the cell cycle and growth (cyclin D, c-Myc). Moreover, Japonicone A inhibited local growth and dissemination of cancer cells to multiple organs in vivo. CONCLUSION: Japonicone A exerts significant anticancer effects on Burkitt lymphoma cells in vitro and in vivo through targeting of the NF-κB signaling cascade. These results highlight the potential of Japonicone A as a chemotherapeutic agent and warrant its development as a therapy for lymphomas.

Pseudoguaianolide sesquiterpene lactones with high activities against the human malaria parasite Plasmodium falciparum.

Four sesquiterpene lactones of the pseudo-guaianolide type (helenalin, dihydrohelenalin and their acetates), have shown activities against asexual blood forms of Plasmodium falciparum in vitro. Their IC(50) values were situated in the range of 0.23 to 7.41 micro M. The activity found for helenalin was high, even compared to the activity found for artemisinin.

New guaianolides from leaves and stems of Chrysanthemum boreale.

The 8-acetoxy-2-methoxy-10-hydroxy-3,11(13)-guaiaden-12,6-olide 1 and 8,10-diacetoxy-2-methoxy-3,11(13)-guaiadiene-12,6-olide 2 were isolated from Chrysanthemum boreale Makino. Compound 1 inhibited the etoposide-induced apoptosis in U 937 cells with an IC50 value 8.6 microg/mL.