Higenamine alleviates allergic rhinitis by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling.

BACKGROUND: Allergic rhinitis (AR) is an inflammatory, immunoglobulin E (IgE)-mediated disease characterized by the typical symptoms of sneezing, rhinorrhea, nasal itching, and congestion. Higenamine (HG) is a plant-based alkaloid, possesses a wide range of activities, including vascular and tracheal relaxation, antioxidative, antiapoptotic, anti-inflammatory, and immunomodulatory activities. So far, the effect and the underlying mechanism of HG on AR have not been studied. HYPOTHESIS/PURPOSE: The purpose of this study was to evaluate the effects of HG on AR and investigate its underlying mechanism. METHODS: The effects of HG on AR were evaluated in an ovalbumin-induced AR mouse model. Network pharmacology-based methods such as target prediction, protein-protein interaction (PPI) network analysis, pathway analysis, and molecular docking were used to identify the likely HG targets. Finally, we validated the mechanism of action of HG through its effects on these targets in human nasal epithelial cells (HNEpCs). RESULTS: Oral administration of 30, 60, and 120 mg/kg HG significantly alleviated rubbing and sneezing in AR mice and attenuated histopathological changes in the lung and nasal tissues. Additionally, HG reduced the levels of IgE, histamine, and IL-4 in the serum of AR mice, and regulated imbalance in Th1/Th2 cells. Using network pharmacology-based methods, we identified 29 HG targets related to AR. These targets are mainly involved in the PD-L1, relaxin, estrogen, HIF-1, Th1 and Th2 cell differentiation, T cell receptor, and the Th17 cell differentiation signaling pathways. Molecular docking showed that HG may well be suited to the receptor binding pockets of key target AKT1, EGFR, c-Jun, NOS2, and JAK2. In HNEpCs, HG inhibited the histamine-induced mRNA expression and secretion of interleukin (IL)-6, and IL-8, as well as the expression of MUC5AC and the phosphorylation of NF-κB. Moreover, HG affected the changes of AKT1, EGFR, c-Jun, iNOS, and JAK2 induced by histamine. CONCLUSION: Overall, our results suggest that HG may alleviate AR by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling. HG, therefore, has great potential as a therapeutic agent for the treatment of AR.

Dauricine inhibits proliferation and promotes death of melanoma cells via inhibition of Src/STAT3 signaling.

Melanoma is the most common type of skin cancer. Signal transducer and activator of transcription 3 (STAT3) signaling has been demonstrated to be a therapeutic target for melanoma. Dauricine (Dau), an alkaloid compound isolated from the root of Menispermum dauricum DC., has shown tumor-suppressing effects in multiple human cancers, but its potential in melanoma remains unexplored. In this study, we demonstrated that Dau significantly inhibited the viability and proliferation of A375 and A2058 melanoma cells. Death of melanoma cells was also markedly promoted by Dau. Moreover, Dau inhibited phosphorylation-mediated activation of STAT3 and Src in a dose-dependent manner. Notably, constitutive activation of Src partially abolished the antiproliferative and cytotoxic activities of Dau on melanoma cells. Molecular docking showed that Dau could dock on the kinase domain of Src with a binding energy of -10.42 kcal/mol. Molecular dynamics simulations showed that Src-Dau binding was stable. Surface plasmon resonance imaging analysis also showed that Dau has a strong binding affinity to Src. In addition, Dau suppressed the growth of melanoma cells and downregulated the activation of Src/STAT3 in a xenograft model in vivo. These data demonstrated that Dau inhibits proliferation and promotes cell death in melanoma cells by inhibiting the Src/STAT3 pathways.

Synthesis, binding, and functional properties of tetrahydroisoquinolino-2-alkyl phenones as selective σ2R/TMEM97 ligands.

Sigma-2 receptor (σ2R/TMEM97) has been implicated to play important roles in multiple cellular dysfunctions, such as cell neoplastic proliferation, neuro-inflammation, neurodegeneration, etc. Selective σ2 ligands are believed to be promising pharmacological tools to regulate or diagnose various disorders. As an ongoing effort of discovery of new and selective σ2 ligands, we have synthesized a series of tetrahydroisoquinolino-2-alkyl phenone analogs and identified that 10 of them have moderate to potent affinity and selectivity for σ2R/TMEM97. Especially, 4 analogs showed Ki values ranging from 0.38 to 5.1 nM for σ2R/TMEM97 with no or low affinity for sigma-1 receptor (σ1R). Functional assays indicated that these 4 most potent analogs had no effects on intracellular calcium concentration and were classified as putative σ2R/TMEM97 antagonists according to current understanding. The σ2R/TMEM97 has been suggested to play important roles in the central nervous system. Based on published pharmacological and clinical results from several regarded σ2R/TMEM97 antagonists, these analogs may potentially be useful for the treatment of various neurodegenerative diseases.

Spirofused tetrahydroisoquinoline-oxindole hybrids as a novel class of fast acting antimalarial agents with multiple modes of action.

Molecular hybridization of privileged scaffolds may generate novel antiplasmodial chemotypes that display superior biological activity and delay drug resistance. In the present study, we describe the in vitro activities and mode of action of 3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones, a novel class of spirofused tetrahydroisoquinoline-oxindole hybrids, as novel antimalarial agents. Whole cell phenotypic screening of these compounds identified (14b), subsequently named (±)-moxiquindole, as the most potent compound in the current series with equipotent antiplasmodial activity against both chloroquine sensitive and multidrug resistant parasite strains with good selectivity. The compound was active against all asexual stages of the parasite including inhibition of merozoite egress. Additionally, (±)-moxiquindole exhibited significant inhibitory effects on hemoglobin degradation, and disrupted vacuolar lipid dynamics. Taken together, our data confirm the antiplasmodial activity of (±)-moxiquindole, and identify 3'4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones as a novel class of antimalarial agents with multiple modes of action.

P-glycoprotein (MDR1/ABCB1) controls brain accumulation and intestinal disposition of the novel TGF-ß signaling pathway inhibitor galunisertib.

Galunisertib (LY2157299), a promising small-molecule inhibitor of the transforming growth factor-beta (TGF-ß) receptor, is currently in mono- and combination therapy trials for various cancers including glioblastoma, hepatocellular carcinoma and breast cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters and the drug-metabolizing CYP3A complex in galunisertib pharmacokinetics. In vitro, galunisertib was vigorously transported by human ABCB1, and moderately by mouse Abcg2. Orally administered galunisertib (20 mg/kg) was very rapidly absorbed. Galunisertib brain-to-plasma ratios were increased by ~24-fold in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice compared to wild-type mice, but not in single Abcg2-/- mice, whereas galunisertib oral availability was not markedly affected. However, recovery of galunisertib in the small intestinal lumen was strongly reduced in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted galunisertib brain accumulation in wild-type mice to equal the levels seen in Abcb1a/1b;Abcg2-/- mice. Oatp1a/1b deficiency did not alter oral galunisertib pharmacokinetics or liver distribution. Cyp3a-/- mice showed a 1.9-fold higher plasma AUC0-1 hr than wild-type mice, but this difference disappeared over 8 hr. Also, transgenic human CYP3A4 overexpression did not significantly alter oral galunisertib pharmacokinetics. Abcb1 thus markedly restricts galunisertib brain penetration and affects its intestinal disposition, possibly through biliary excretion. Elacridar coadministration could fully inhibit both processes, without causing acute toxicity. Moreover, mouse Cyp3a, but not human CYP3A4, may eliminate galunisertib at high plasma concentrations. These insights may help to guide the further clinical development and application of galunisertib.

Behavioral Pharmacology of Novel Kappa Opioid Receptor Antagonists in Rats.

BACKGROUND: New treatments for stress-related disorders including depression, anxiety, and substance use disorder are greatly needed. Kappa opioid receptors are expressed in the central nervous system, including areas implicated in analgesia and affective state. Although kappa opioid receptor agonists share the antinociceptive effects of mu opioid receptor agonists, they also tend to produce negative affective states. In contrast, selective kappa opioid receptor antagonists have antidepressant- and anxiolytic-like effects, stimulating interest in their therapeutic potential. The prototypical kappa opioid receptor antagonists (e.g., norBNI, JDTic) have an exceptionally long duration of action that complicates their use in humans, particularly in tests to establish safety. This study was designed to test dose- and time-course effects of novel kappa opioid receptor antagonists with the goal of identifying short-acting lead compounds for future medication development. METHODS: We screened 2 novel, highly selective kappa opioid receptor antagonists (CYM-52220 and CYM-52288) with oral efficacy in the warm water tail flick assay in rats to determine initial dose and time course effects. For comparison, we tested existing kappa opioid receptor antagonists JDTic and LY-2456302 (also known as CERC-501 or JNJ-67953964). RESULTS: In the tail flick assay, the rank order of duration of action for the antagonists was LY-2456302 < CYM-52288 < CYM-52220 << JDTic. Furthermore, LY-2456302 blocked the depressive (anhedonia-producing) effects of the kappa opioid receptor agonist U50,488 in the intracranial self-stimulation paradigm, albeit at a higher dose than that needed for analgesic blockade in the tail flick assay. CONCLUSIONS: These results suggest that structurally diverse kappa opioid receptor antagonists can have short-acting effects and that LY-2456302 reduces anhedonia as measured in the intracranial self-stimulation test.

Novel pharmacological actions of trequinsin hydrochloride improve human sperm cell motility and function.

BACKGROUND AND PURPOSE: Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. EXPERIMENTAL APPROACH: A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. KEY RESULTS: Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. CONCLUSION AND IMPLICATIONS: Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.

Higenamine, a Dual Agonist for ß 1- and ß 2-Adrenergic Receptors Identified by Screening a Traditional Chinese Medicine Library.

Chronic heart failure is the terminal stage of various cardiovascular diseases. Despite the availability of several classes of drugs, there is still an unmet need for effective treatment. Based on bench work during the past two decades, we have proposed that enhancement of ß 2-adrenergic receptor signaling in combination with the presently preferred ß 1-adrenergic receptor blockade would be a promising strategy. Chinese herbal medicines have been shown to be effective in the treatment of heart failure, although the mechanisms largely remain unknown. In the present study, we screened an herbal medicine compound/extract library for ß-adrenergic receptor ligands to determine the target of certain effective botanical remedies and seek a leading compound(s) for chronic heart failure treatment. Using a high-throughput screening assay, we identified higenamine, which has a long history in chronic heart failure treatment in traditional Chinese medicine, to be a potent ß-adrenergic receptor agonist. Further experiments using specific inhibitors showed that higenamine activated both ß 1-adrenergic receptor and ß 2-adrenergic receptor. Inhibition of its action by pertussis toxin (a Gi inhibitor) indicated that it is a ß 2-adrenergic receptor Gs/Gi dual agonist. Contractility experiments demonstrated a positive inotropic effect of higenamine. In conclusion, we found an herbal compound, higenamine, to be a dual agonist for ß 1/ß 2-adrenergic receptors with no preference in stimulating the Gs and Gi pathways in ß 2-adrenergic receptor signaling. Our results elucidated not only the target of higenamine to explain its pharmacological effect in treating chronic heart failure, but also the mechanisms of its cardiac toxicity.

Structure-Aided Identification and Optimization of Tetrahydro-isoquinolines as Novel PDE4 Inhibitors Leading to Discovery of an Effective Antipsoriasis Agent.

Psoriasis is a common, chronic inflammatory disease characterized by abnormal skin plaques, and the effectiveness of phosphodiesterase 4 (PDE4) inhibitor to lessen the symptoms of psoriasis has been proved. Aiming to find a novel PDE4 inhibitor acting as an effective, safe, and convenient therapeutic agent, we constructed a library consisting of berberine analogues, and compound 2 with a tetrahydroisoquinoline scaffold was identified as a novel and potent hit. The structure-aided and cell-based structure-activity relationship studies on a series of tetrahydro-isoquinolines lead to efficient discovery of a qualified lead compound (16) with the high potency and selectivity, well-characterized binding mechanism, high cell permeability, good safety and pharmacokinetic profile, and impressive in vivo efficacy on antipsoriasis, in particular with a topical application. Thus, our study presents a prime example for efficient discovery of novel, potent lead compounds derived from natural products using a combination of medicinal chemistry, biochemical, biophysical, and pharmacological approaches.

Dauricine negatively regulates lipopolysaccharide- or cecal ligation and puncture-induced inflammatory response via NF-κB inactivation.

Acute lung injury (ALI) is a challenging clinical problem worldwide characterized by severe pulmonary inflammation. Dauricine, extracted from the root of traditional Chinese medicine Menispermum dauricum DC, is employed as anti-inflammatory herbs. In this study, we explored the inhibitory effects of dauricine on lipopolysaccharide (LPS)-induced inflammation in macrophages and LPS- or cecal ligation and puncture (CLP)-induced ALI in C57BL/6 mice. Our in vitro study identified that pretreatment of dauricine dose-dependently inhibited pro-inflammatory cytokines including nitric oxide (NO), interleukin-1ß (IL1ß), IL6, tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) in LPS-stimulated macrophages. Moreover, dauricine could suppress LPS-mediated nuclear translocation and transcriptional activity of nuclear factor-kappaB (NF-κB) by suppressing the phosphorylation of NF-κB inhibitors (IκB). In vivo studies, administration of dauricine, especially high-dose dauricine, potently improved the survival rate, reduced the production of pro-inflammatory cytokines in serum, and ameliorated ALI induced by LPS or CLP via blockage of NF-κB activation. Collectively, the present study discovers a new biological effect of dauricine in prevention of inflammation, indicating that dauricine can be served as a potential therapeutic agent to treat inflammatory diseases.

Identification of higenamine as a novel α1 -adrenergic receptor antagonist.

The α1 -adrenoceptor (α1 -AR) antagonists are potential candidates for the treatment of blood pressure. Higenamine (HG) is a novel α1 -AR antagonist. In this study, we investigated the effects of HG in HEK293A cells transfected with α1A -, α1B -, and α1D -AR in vitro, rat mesenteric artery ex vivo, Wistar-Kyoto rats and spontaneously hypertensive rats in vivo. The radioligand binding assay showed that HG competitively inhibited the binding of [3 H]-prazosin to α1 -AR in a concentration-dependent manner. The affinities (pKi) of HG for the cloned α1A -, α1B -, and α1D -AR were 6.57, 6.48, and 6.35, respectively, indicating that HG displayed no selectivity for the three α1 -AR subtypes. In in vitro studies, HG was able to blunt inositol monophosphate production. It also displayed an inhibitory effect on the influx and entry of calcium ions and phosphorylation of extracellular signal-regulated kinase 1 and 2 induced by phenylephrine (PE). In ex vivo studies, PE caused a dose-dependent inotropic response curve, and the pA2 value for HG was 6.86 ± 0.29. In addition, the in vivo results showed that HG could decrease the blood pressure in normotension, spontaneous hypertension, and PE-induced hypertension models. These results indicate that HG can directly bind to α1 -AR and it appears to be a novel antagonist for α1 -AR, which may contribute to its hypotensive effect.

Synthesis and Evaluation of Orexin-1 Receptor Antagonists with Improved Solubility and CNS Permeability.

Orexins are hypothalamic neuropeptides playing important roles in many functions including the motivation of addictive behaviors. Blockade of the orexin-1 receptor has been suggested as a potential strategy for the treatment of drug addiction. We have previously reported OX1 receptor antagonists based on the tetrahydroisoquinoline scaffold with excellent OX1 potency and selectivity; however, these compounds had high lipophilicity (clogP > 5) and low to moderate solubility. In an effort to improve their properties, we have designed and synthesized a series of analogues where the 7-position substituents known to favor OX1 potency and selectivity were retained, and groups of different nature were introduced at the 1-position where substitution was generally tolerated as demonstrated in previous studies. Compound 44 with lower lipophilicity (clogP = 3.07) displayed excellent OX1 potency ( Ke = 5.7 nM) and selectivity (>1,760-fold over OX2) in calcium mobilization assays. In preliminary ADME studies, 44 showed excellent kinetic solubility (>200 µM), good CNS permeability ( Papp = 14.7 × 10-6 cm/sec in MDCK assay), and low drug efflux (efflux ratio = 3.3).

Discovery of a tetrahydroisoquinoline-based HDAC inhibitor with improved plasma stability.

Histone deacetylase inhibitors with desirable pharmacokinetic profiles which can be delivered to solid tumor tissues in large amount might be promising to treat solid tumor effectively. Herein, structural modification of a previously reported tetrahydroisoquinoline-based HDAC inhibitor 1 was carried out to improve its plasma stability for more feasible drug delivery. Among three newly synthesized analogs, the in vitro rat plasma stability of compound 2 (t1/2=630min) was over 5-fold improved than its parent 1 (t1/2=103min). In vitro activity evaluation showed that compound 2 and 1 exhibited similar HDACs inhibitory activity, which was validated by western blot analysis and antiproliferative assay. Moreover, compared with 1, compound 2 exhibited comparable, if not higher, in vivo antitumor activity in a human breast carcinoma (MDA-MB-231) xenograft model.

A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases.

Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

NS1209/SPD 502, A Novel Selective AMPA Antagonist for Stroke, Neuropathic Pain or Epilepsy? Drug Development Lessons Learned.

Preclinical Research The selective AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist, NS1209 (also known as SPD 502) has been explored in several research and development campaigns since its selection as a lead drug candidate in the early 1990s by the Danish biotechnology company, NeuroSearch. The compound was successively tested in animal models of stroke, neuropathic pain and epilepsy. The preclinical data to support development for the treatment of stroke were incomplete, as the compound was administered after the stroke episode, and did not protect subcortical areas of the brain. Preclinical data for neuropathic pain and epilepsy appeared more promising, but the design of the Phase IIa studies in both indications was suboptimal, and an exploratory study in neuropathic pain, like one in refractory epilepsy gave inconclusive results. Preclinical data in pain models were much less convincing than reported by the authors in abstract and discussion sections. Due to a long preclinical sequential testing phase and insufficiently powered clinical trials, NS 1209 disappeared from the CNS development pipeline, while its patent protection exclusivity was markedly reduced due to the unfortunate slow speed in development-a phenomenon far from unusual in CNS drug discovery. NeuroSearch ceased operations as an R&D entity in 2012 and its R & D portfolio was transferred to Teva Pharmaceuticals and to a spin off, Saniona A/S. Based on an in-depth case analysis of the development of NS 1209, a number of recommendations are given to reduce chances of failure during clinical development of neuropathic pain compounds and, more generally, of CNS compounds. Drug Dev Res 78 : 75-80, 2017. © 2017 Wiley Periodicals, Inc.

Applications of Higenamine in pharmacology and medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum has been used as local and traditional medicines in many asian regions for the treatment of various diseases such as collapse, syncope, painful joints, oedema, bronchial asthma et al. Higenamine, a plant-based alkaloid, was initially isolated from Aconitum and identified as the active cardiotonic component of Aconitum. It has been tested as a candidate of pharmacologic stress agent in the detection of coronary artery diseases (CADs) and now researchers have just accomplished the phase III clinical studies successfully in China. Besides, a large number of studies have revealed the various pharmacological properties and potentially multi-spectral medical applications of higenamine. However, to date, no comprehensive review on higenamine has been published. AIM OF THE REVIEW: This present paper aims to compile a comprehensive update regarding the biochemistry, pharmacokinetic features, pharmacological activities, clinical and potential clinical uses and toxicities on higenamine with the ultimate objective of providing a guide for future research on this drug. MATERIALS AND METHODS: The selection of relevant data was made through a search using the keyword "higenamine" in "Web of science", "Pubmed", and "China Knowledge Resource Integrated (CNKI)". Information was also acquired from local classic herbal literature, government reports and conference papers. RESULTS: In addition to Aconitum, higenamine also exists in many other plants including Tinospora crispa, Nandina domestica THUNBERG, Gnetum Parvifolium C.Y. Cheng, sarum Heterotropoides,Nelumbo nucifera,N.nucifera. The pharmacokinetic studies conducted in animals and humans showed that higenamine conformed to a two-compartment pharmacokinetic model. Studies over the last four decades on higenamine have revealed its various pharmacological properties such as positive inotropic and chronotropic effect, activating slow channel effect, vascular and tracheal relaxation effect, anti-thrombotic, anti-apoptotic and anti-oxidative effect, anti-inflammatory and immunomodulatory effect. This phytochemical constituent has shown its potential therapeutic effects for diseases like heart failure, disseminated intravascular coagulation (DIC), shock, arthritis, asthma, ischemia/reperfusion (I/R) injuries and erectile dysfunction. CONCLUSIONS: Extensive basic and clinical studies on higenamine showed valuable therapeutic effects on different disorders. However, the underlying mechanisms of higenamine have not been established. Therefore, the safety, tolerability and efficacy of higenamine are as yet, not fully understood. Additionally, some of the studies were small sample-sized and unreliable. To sum up, there is a need for deeper investigation in the mechanisms of higenamine action, as well as well-designed preclinical and clinical trials studies to test the safety and clinical value of the drug.

Synthesis and Structure-Activity Relationships of a Series of Aporphine Derivatives with Antiarrhythmic Activities and Acute Toxicity.

Some aporphine alkaloids, such as crebanine, were found to present arrhythmic activity and also higher toxicity. A series of derivatives were synthesized by using three kinds of aporphine alkaloids (crebanine, isocorydine, and stephanine) as lead compounds. Chemical methods, including ring-opening reaction, bromination, methylation, acetylation, quaternization, and dehydrogenation, were adopted. Nineteen target derivatives were evaluated for their antiarrhythmic potential in the mouse model of ventricular fibrillation (VF), induced by CHCl3, and five of the derivatives were investigated further in the rat model of arrhythmia, induced by BaCl2. Meanwhile, preliminary structure-activity/toxicity relationship analyses were carried out. Significantly, N-acetamidesecocrebanine (1d), three bromo-substituted products of crebanine (2a, 2b, 2c), N-methylcrebanine (2d), and dehydrostephanine (4a) displayed antiarrhythmic effects in the CHCl3-induced model. Among them, 7.5 mg/kg of 2b was able to significantly reduce the incidence of VF induced by CHCl3 (p < 0.05), increase the number of rats that resumed sinus rhythm from arrhythmia, induced by BaCl2 (p < 0.01), and the number of rats that maintained sinus rhythm for more than 20 min (p < 0.01). Therefore, 2b showed remarkably higher antiarrhythmic activity and a lower toxicity (LD50 = 59.62 mg/kg, mice), simultaneously, indicating that 2b could be considered as a promising candidate in the treatment of arrhythmia. Structural-activity analysis suggested that variationsin antiarrhythmic efficacy and toxicity of aporphines were related to the C-1,C-2-methylenedioxy group on ring A, restricted ring B structural conformation, N-quaternization of ring B, levoduction of 6a in ring C, and the 8-, 9-, 10-methoxy groups on ring D on the skeleton.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

In vitro anti-proliferative activity of Argemone gracilenta and identification of some active components.

BACKGROUND: Cancer is one of the leading causes of death worldwide. Natural products have been regarded as important sources of potential chemotherapeutic agents. In this study, we evaluated the anti-proliferative activity of Argemone gracilenta's methanol extract and its fractions. We identified those compounds of the most active fractions that displayed anti-proliferative activity. METHODS: The anti-proliferative activity on different cancerous cell lines (M12.C3F6, RAW 264.7, HeLa) was evaluated in vitro using the MTT colorimetric method. Identification of the active compounds present in the fractions with the highest activity was achieved by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) analyses. RESULTS: Both argemonine and berberine alkaloids, isolated from the ethyl acetate fraction, displayed high anti-proliferative activity with IC50 values of 2.8, 2.5, 12.1, and 2.7, 2.4, 79.5 µg/mL on M12.C3F6, RAW 264.7, and HeLa cancerous cell lines, respectively. No activity was shown on the normal L-929 cell line. From the hexane fraction, a mixture of fatty acids and fatty acid esters of 16 or more carbon atoms with anti-proliferative activity was identified, showing a range of IC50 values of 16.8-24.9, 34.1-35.4, and 67.6-91.8 µg/mL on M12.C3F6, RAW 264.7, and HeLa cancerous cell lines, respectively. On the normal L-929 cell line, this mixture showed a range of IC50 values of 85.1 to 100 µg/mL. CONCLUSION: This is the first study that relates argemonine, berberine, and a mixture of fatty acids and fatty acid esters with the anti-proliferative activity displayed by Argemone gracilenta.