Development and in-vitro evaluation of co-loaded berberine chloride and evodiamine ethosomes for treatment of melanoma.

Berberine chloride (BBR) and evodiamine (EVO) are two main active ingredients of "ZuoJinWan", a classical Chinese herbal medicine, and these compounds are known to have a synergistic inhibitory effect on various cancer cell lines. Several recent studies have reported anti-melanoma effects for both BBR and EVO. However, topical delivery of the two compounds has been challenging, due to their poor aqueous solubility and their low skin penetration. In the current study, we have combined BBR and EVO into an ethosomes delivery system with the future aim to design a novel topical anti-melanoma formulation. The ethosomes formulations were characterized using particle size, entrapment efficiency and an in vitro skin drug deposition study. The ethosome formulation displaying maximum drug deposition in the epidermis was selected for further study. This formulation contained ethosomes with mean size of 171 nm and 90% or above entrapment efficiency for both BBR and EVO. Cell viability tests proved the optimized ethosomes increased the inhibitory effect on B16 melanoma cells. These results corroborate that ethosomes containing a combination of BBR and EVO are a promising delivery system for potential use in melanoma therapy.

In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3.

Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an α2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks α2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.

Discovery of 4-Methylquinazoline Based PI3K Inhibitors for the Potential Treatment of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease, and its molecular pathogenesis remains poorly understood. Recently, emerging evidence demonstrates that the PI3K signaling transduction pathway is linked to the pathology of IPF. In this work, we rationally designed a new series of 4-methylquinazoline derivatives as highly potent PI3K inhibitors that significantly suppress the phosphorylation of the main PI3K downstream effectors and displays marked antiproliferative activity in mouse MLg2908 lung fibroblasts. In a bleomycin-induced mouse pulmonary fibrosis model, 5d from the series improved mouse lung function and slowed the progression of pulmonary fibrosis. Overall, this work promises a therapeutic potential for PI3K inhibitors to treat IPF.

In vivo and in vitro metabolism and pharmacokinetics of cholinesterase inhibitor deoxyvasicine from aerial parts of Peganum harmala Linn in rats via UPLC-ESI-QTOF-MS and UPLC-ESI-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Peganum harmala Linn are a Uighur traditional medicinal herb in China used to treat amnesia, bronchial asthma, and cough. Deoxyvasicine (DVAS), a potent cholinesterase inhibitor exhibiting anti-senile dementia activity, is one of the chief active ingredients in aerial parts of P. harmala and plays a key role in mediating the pharmacological effects of P. harmala. However, the metabolic profiling and in vivo pharmacokinetic characteristics of DVAS still remain unknown. AIM OF THE STUDY: The aim of this present study was to investigate the metabolism and pharmacokinetic properties of DVAS in rats by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method. MATERIALS AND METHODS: The metabolic profiling of DVAS was evaluated in vitro and in vivo by rat liver microsomes (RLMs) incubation and by rat bio-specimens, such as urine, feces, plasma, and bile, after the oral administration of 45 mg/kg DVAS. An efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its major four metabolites, namely, vasicine, deoxyvasicinone, vasicinone, and 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-ß-D-glucuronide in rat plasma. For pharmacokinetic studies, 32 Sprague-Dawley rats were randomly divided into four groups, namely, intravenous dosage group (2 mg/kg DVAS) and three oral dosage groups (5, 15, and 45 mg/kg DVAS). In addition, the activity of the components in plasma after intravenous administration of DVAS was evaluated by in vitro anti-butyrylcholinesterase (BChE) assays. RESULTS: A total of 23 metabolites were found in RLMs, plasma, urine, feces, and bile by UPLC-ESI-QTOF-MS. The metabolic pathway of DVAS in vivo and in vitro mainly involved hydroxylation, dehydrogenation, acetylation, methylation, glucuronidation, and O-sulphate conjugation, and the C-3 and C-9 sites were the main metabolic soft spots. All 23 metabolites were detected in the urine sample, and 13, 8, 22, and 6 metabolites were identified from rat feces, plasma, bile, and RLMs, respectively. The standard curves of DVAS and four metabolites in rat plasma showed good linearity in the concentration range of 0.82-524.00 ng/mL with acceptable selectivity, precision, accuracy, recovery, and stability. DVAS exhibited linear dose-proportional pharmacokinetics at doses of 5, 15, and 45 mg/kg after oral administration, and the average oral absolute bioavailability of DVAS was 47.46%. The in vitro anti-BChE assays implied that the inhibitive activities were mainly due to the different concentrations of prototype DVAS. CONCLUSIONS: DVAS can be rapidly absorbed and excreted by blood, and it is also extensively metabolized in vivo, and the anti-BChE activity in blood is mainly attributed to DVAS. These findings can lay a foundation for new drug development for DVAS.

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists.

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC50 value of 0.073µM, indicating 350-fold potency compared to the hit compound 3.

Effect of elicitors on the production of pyrroloquinazoline alkaloids by stimulating anthranilate synthase activity in Adhatoda vasica Nees cell cultures.

MAIN CONCLUSION: Pyrroloquinazoline alkaloids are medicinally important compounds, determined by HPLC from cell cultures of Adhatoda vasica . The maximum production of vasicinone (12-fold) and vasicine (8.3-fold) was enhanced by stimulating the anthranilate synthase activity via feeding of tryptophan and sorbitol. The decoction of Adhatoda vasica leaves is used for the treatment of throat irritations, inflammations and recommended as expectorant. The plant species contains pyrroloquinazoline alkaloids and has been reported to demonstrate various biological activities. To investigate the effect of elicitors to increase the production of alkaloids, five groups (auxins and cytokinins, biotic elicitors, polysaccharides, amino acids and salts) of elicitors were evaluated. Maximum production of vasicinone (72.74 ± 0.74 mg/g DW; 12-fold) and vasicine (99.44 ± 0.28 mg/g DW; 8.3-fold) was enhanced by feeding of tryptophan and sorbitol at 50 mM concentration in cell cultures. Fourteen free amino acids were estimated from the elicited cells. Sorbitol stimulated up to a maximum accumulation of serine (8.2-fold). The maximal anthranilate synthase (AS) activity (7.5 ± 0.47 pkat/mg protein; 2.9-fold) was induced by salicylic acid and sorbitol. Anthranilate synthase functions as rate-limiting factor for the biosynthesis of pyrroloquinazoline alkaloids. Our results support the widespread use of tryptophan and sorbitol as elicitors to raise the production of vasicinone, vasicine, 2-acetyl benzyl amine and other pyrroloquinazoline alkaloids in cell cultures of A. vasica.

Evodiamine Prevents Glioma Growth, Induces Glioblastoma Cell Apoptosis and Cell Cycle Arrest through JNK Activation.

Evodiamine (EVO) is an active medicinal compound derived from the traditional herbal medicine Evodia rutaecarpa. It has been reported that evodiamine has several beneficial biological properties, including anticancer and anti-inflammatory activities. However, the in vitro and in vivo anticancer activities of EVO against the growth of glioblastoma cells remain undefined. EVO induced significant decreases in the viability of U87 and C6 glioma cells, but not of primary astrocytes, according with the occurrence of apoptotic characteristics including DNA ladders, caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage, and hypodiploid cells. The disruption of the mitochondrial membrane potential (MMP) was detected, and it was found that the peptidyl caspase-9 inhibitor, Z-LEHD-FMK, significantly prevented glioma cells from EVO-induced apoptosis. Increased c-Jun N-terminal kinase (JNK) protein phosphorylation by EVO was observed, and the addition of JNK inhibitors, SP600125 and JNKI inhibited the EVO-induced apoptosis was inhibited. Additionally, EVO treatment induced G2/M arrest with increased polymerized tubulin protein expression in U87 and C6 cells. Elevated expressions of the cyclin B1, p53, and phosphorylated (p)-p53 proteins were detected in EVO-treated glioma cells, and these were inhibited by JNK inhibitors. An in vivo study showed that EVO significantly reduced the growth of gliomas elicited by the subcutaneous injection of U87 cells with increases in cyclin B1, p53, and p-p53 protein expressions in tumors. An analysis of eight EVO-related chemicals showed that alkyl groups at position 14 in EVO are important for its anti-glioma effects which involve both apoptosis and G2/M arrest. Evidence is provided that supports EVO induction of apoptosis and G2/M arrest via the activation of JNK-mediated gene expression and disruption of MMP in glioblastoma cells. EVO was shown to penetrate the blood-brain barrier; EVO is therefore predicted to be a promising compound for the chemotherapy of glioblastomas and deserves further investigations.

In vitro and in vivo evaluation of paclitaxel-lapatinib-loaded F127 pluronic micelles.

The aim of this study was to evaluate the in vitro and in vivo efficacy of paclitaxel-lapatinib-loaded Pluronic micelles. Lapatinib and pluronic sensitize the cancerous cells to paclitaxel via efflux pump inhibition. In addition, pluronic polymers can trigger intrinsic apoptosis pathways. Furthermore, micellar system can passively target the chemotherapeutic agents by enhanced permeability and retention effect. The paclitaxel-lapatinib-loaded micelles were characterized in means of encapsulation efficacy and size. The in vitro analyses were performed by MTT assay and uptake studies. Real-time imaging and in vivo anti-tumor efficacy studies were also performed. The prepared micelles have acceptable encapsulation ratio and size. Hemolysis assay confirmed that the micelles are hemo-compatible. MTT assay demonstrated that drug-loaded micelles have superior cytotoxicity compared with the naked drugs. The confocal microscopy and flowcytometry analyses showed that micelles are mainly internalized by endocytosis. According to the results of the in vivo imaging, the micelles are accumulated within liver. In vivo anti-tumor efficacy studies confirmed that tumor inhibition of drug-loaded micelles was significant compared to Intaxel®.

Computational Drug Target Screening through Protein Interaction Profiles.

The development of computational methods to discover novel drug-target interactions on a large scale is of great interest. We propose a new method for virtual screening based on protein interaction profile similarity to discover new targets for molecules, including existing drugs. We calculated Target Interaction Profile Fingerprints (TIPFs) based on ChEMBL database to evaluate drug similarity and generated new putative compound-target candidates from the non-intersecting targets in each pair of compounds. A set of drugs was further studied in monoamine oxidase B (MAO-B) and cyclooxygenase-1 (COX-1) enzyme through molecular docking and experimental assays. The drug ethoxzolamide and the natural compound piperlongumine, present in Piper longum L, showed hMAO-B activity with IC50 values of 25 and 65 µM respectively. Five candidates, including lapatinib, SB-202190, RO-316233, GW786460X and indirubin-3'-monoxime were tested against human COX-1. Compounds SB-202190 and RO-316233 showed a IC50 in hCOX-1 of 24 and 25 µM respectively (similar range as potent inhibitors such as diclofenac and indomethacin in the same experimental conditions). Lapatinib and indirubin-3'-monoxime showed moderate hCOX-1 activity (19.5% and 28% of enzyme inhibition at 25 µM respectively). Our modeling constitutes a multi-target predictor for large scale virtual screening with potential in lead discovery, repositioning and drug safety.

Antiproliferative Activity and Cellular Uptake of Evodiamine and Rutaecarpine Based on 3D Tumor Models.

Evodiamine (EVO) and rutaecarpine (RUT) are promising anti-tumor drug candidates. The evaluation of the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids of cancer cells would better recapitulate the native situation and thus better reflect an in vivo response to the treatment. Herein, we employed the 3D culture of MCF-7 and SMMC-7721 cells based on hanging drop method and evaluated the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids, and compared the results with those obtained from 2D monolayers. The drugs' IC50 values were significantly increased from the range of 6.4-44.1 µM in 2D monolayers to 21.8-138.0 µM in 3D multicellular spheroids, which may be due to enhanced mass barrier and reduced drug penetration in 3D models. The fluorescence of EVO and RUT was measured via fluorescence spectroscopy and the cellular uptake of both drugs was characterized in 2D tumor models. The results showed that the cellular uptake concentrations of RUT increased with increasing drug concentrations. However, the EVO concentrations uptaken by the cells showed only a small change with increasing drug concentrations, which may be due to the different solubility of EVO and Rut in solvents. Overall, this study provided a new vision of the anti-tumor activity of EVO and RUT via 3D multicellular spheroids and cellular uptake through the fluorescence of compounds.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

Preclinical pharmacokinetics and disposition of a novel selective VEGFR inhibitor fruquintinib (HMPL-013) and the prediction of its human pharmacokinetics.

PURPOSE: This study evaluated the preclinical pharmacokinetics (PK) and disposition of fruquintinib (HMPL-013), a small molecule vascular endothelial growth factor receptors inhibitor. METHODS: In vitro and in vivo PK/ADME assays were conducted. Allometry and PK modeling/simulation were conducted to predict human PK parameters and the time course profiles. RESULTS: HMPL-013 has high permeability without efflux. It shows moderate oral bioavailability of 42-53 % and Tmax < 4 h in mouse, rat, dog and monkey, with exposure-dose linearity proved in rats and dogs. No significant food effect is on dog PK. HMPL-013 has moderately high tissue distribution. It majorly distributes in gastrointestinal tract, liver, kidney, adrenal and adipose. The plasma protein binding fraction is 88-95 % in mouse, rat, dog and human, invariable up to 10 µM. The in vivo clearance of HMPL-013 is low, consistent with the in vitro scaling. Three major oxidative metabolites were identified in liver microsomes of mouse, rat, dog, monkey and human. Dog is mostly similar to human regarding in vitro metabolism. Demethylation, hydroxylation and sequential glucuronidation are the major in vivo metabolic reactions. Direct urinary and biliary excretion of HMPL-013 is negligible. Metabolizing to M1 (demethylation), sequentially glucuronidating, followed by biliary excretion, and to a less extent, by urinary excretion, are important elimination pathways for HMPL-013 in rats. HMPL-013 has low risk of drug-drug interaction. It is predicted to have favorable human PK properties and low efficacious dose. CONCLUSION: HMPL-013 demonstrates good preclinical PK and enables successful human PK and dose projection. It is valuable for further clinical development.

Metabolic activation of the indoloquinazoline alkaloids evodiamine and rutaecarpine by human liver microsomes: dehydrogenation and inactivation of cytochrome P450 3A4.

Evodiamine and rutaecarpine are the main active indoloquinazoline alkaloids of the herbal medicine Evodia rutaecarpa, which is widely used for the treatment of hypertension, abdominal pain, angina pectoris, gastrointestinal disorder, and headache. Immunosuppressive effects and acute toxicity were reported in mice treated with evodiamine and rutaecarpine. Although the mechanism remains unknown, it is proposed that metabolic activation of the indoloquinazoline alkaloids and subsequent covalent binding of reactive metabolites to cellular proteins play a causative role. Liquid chromatography-tandem mass spectrometry analysis of incubations containing evodiamine and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed formation of a major GSH conjugate which was subsequently indentified as a benzylic thioether adduct on the C-8 position of evodiamine by NMR analysis. Several other GSH conjugates were also detected, including conjugates of oxidized and demethylated metabolites of evodiamine. Similar GSH conjugates were formed in incubations with rutaecarpine. These findings are consistent with a bioactivation sequence involving initial cytochrome P450-catalyzed dehydrogenation of the 3-alkylindole moiety in evodiamine and rutaecarpine to an electrophile 3-methyleneindolenine. Formation of the evodiamine and rutaecarpine GSH conjugates was primarily catalyzed by heterologously expressed recombinant CYP3A4 and, to a lesser extent, CYP1A2 and CYP2D6, respectively. It was found that the 3-methyleneindolenine or another reactive intermediate was a mechanism-based inactivator of CYP3A4, with inactivation parameters KI = 29 µM and kinact = 0.029 minute(-1), respectively. In summary, these findings are of significance in understanding the bioactivation mechanisms of indoloquinazoline alkaloids, and dehydrogenation of evodiamine and rutaecarpine may cause toxicities through formation of electrophilic intermediates and lead to drug-drug interactions mainly via CYP3A4 inactivation.

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes. MATERIAL AND METHODS: A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression. RESULTS: The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Clint) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment. CONCLUSION: We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.

Quantitative chemical proteomics profiling differentiates erlotinib from gefitinib in EGFR wild-type non-small cell lung carcinoma cell lines.

Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), α-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATP-binding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors.

Synthesis and biological evaluation of novel 2,4-disubstituted quinazoline analogues as GPR119 agonists.

GPR119 agonist has emerged as a promising target for the treatment of type 2 diabetes. A series of novel 2,4-disubstituted quinazoline analogues was prepared and evaluated their agonistic activity against human GPR119. The analogues bearing azabicyclic amine substituents (12a, 12c and 12g) exhibited better EC(50) values than that of OEA though they appeared to be partial agonists.

Investigation of the in vitro metabolism of evodiamine: characterization of metabolites and involved cytochrome p450 isoforms.

Evodiamine is the main active alkaloid of Evodia rutaecarpa (E. rutaecarpa) and has been demonstrated to exhibit many pharmacological activities including vasorelaxation, uterotonic action, anoxia and control of body temperature. The present study focused on the metabolism of evodiamine. Human and phenobarbital-induced rat liver microsomal incubation of evodiamine in the presence of NADPH resulted in the formation of five major metabolites (M-1, M-2, M-3, M-4, M-5). Four metabolites (M-1, M-2, M-3 and M-5) were identified to mono-hydroxylated evodiamine and one metabolite (M-4) was identified to be N-demethylated evodiamine. CYP3A4, CYP2C9 and CYP1A2 were identified to be the main CYP isoforms involved in the metabolism of evodiamine in human liver microsomes. Finding new metabolites can help us decipher novel substance basis of efficiency and toxicity. Elucidation of drug metabolizing enzymes will facilitate explaining the individual difference for response to the same drugs or herbs and the potential drug-drug interaction or herb-drug interaction. Taken together, these results are of significance for better understanding the pharmacokinetic behaviour of evodiamine and helpful for clinical application of evodiamine and E. rutaecarpa.

Transdermal behaviors comparisons among Evodia rutaecarpa extracts with different purity of evodiamine and rutaecarpine and the effect of topical formulation in vivo.

AIM: Evodiamine (EVO) and rutaecapine (RUT), the major active components from Evodia rutaecarpa extract (EE), are recognized as a depended analgesic agent. This study was designed to investigate the effect of purity and chemical enhancers on the transdermal behavior of EVO and RUT, and the pharmacological effect of their topical cream in vivo. MATERIAL AND METHODS: Transdermal delivery across a full thickness pig abdominal skin was detected in vitro by Franz-type diffusion cell, with HPLC for quantification of the permeation of EVO and RUT. The activity of topical cream in vivo was evaluated by a mice pain model induced by formalin and hot plate. RESULTS: Transdermal characters of EVO and RUT showed a low transdermal rate, long lag time and low cumulative amount. The transdermal rate and cumulative amount could be promoted by lipophilic enhancers, whereas lag time was shortened by hydrophilic surfactant, but these permeation parameters were not markedly influenced by purity of EE (p>0.05). The effect in vivo was confirmed by analgesic models in topical cream of EE, which produced a significant (p<0.05) inhibitory effects on pain response in dose-dependent manner. CONCLUSION: The purity of EVO and RUT from EE has no significant effect on their permeation through porcine skin, but oleic acid or nerolidol can markedly elevate the transdermal rate of EVO and RUT. High purity of EE is the best choice for topical preparation to increase the drug loading. The effect of EE in vivo is verified by formalin model and hot plate test.

Sulfur and selenium derivatives of quinazoline and pyrido[2,3-d]pyrimidine: synthesis and study of their potential cytotoxic activity in vitro.

The synthesis, cytotoxic activities and selectivities of 35 derivatives related to quinazoline and pyrido[2,3-d]pyrimidine are described. The synthesized compounds were screened in vitro against four tumoral cell lines - leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54) and breast (MCF-7) - and two cell lines derived from non-malignant cell lines, one mammary (184B5) and one from bronchial epithelium (BEAS-2B). MCF-7 and HTB-54 were the most sensitive cell lines with GI(50) values below 10µM for eleven and ten compounds, respectively. Two compounds (2o and 3a) were identified that evoked a marked cytotoxic effect in all cell lines tested and one compound, 7h, was potent and selective against MCF-7. A preliminary study into the mechanism of the potent derivatives 2o, 3a and 7h indicated that the cytotoxic activities of these compounds might be mediated by inducing cell death without affecting cell cycle phases.