In vitro modeling of hepatocellular carcinoma molecular subtypes for anti-cancer drug assessment.

Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and S2, determined by our previous transcriptome meta-analysis of multiple clinical hepatocellular carcinoma cohorts, are presented in a panel of hepatoma cell lines widely used by the research community. Interestingly, cell line that resembles gene expression pattern of S3 subtype, representing less aggressive tumors, was not identified in the panel. MYC pathway-activated S2-like cell lines showed higher sensitivity to a small molecule BET bromodomain inhibitor, (+)-JQ1, which has anti-MYC activity. These results support the use of hepatoma cell lines as models to evaluate molecular subtype-specific drug response, which is expected to lead to development of tailored, precision care of the patients with hepatocellular carcinoma.

Preclinical assessment of the abuse potential of the orexin receptor antagonist, suvorexant.

Suvorexant (Belsomra®) is a dual orexin receptor antagonist approved for the treatment of insomnia. Because of its pharmacology within the central nervous system, intended therapeutic indication, and first-in-class status, an assessment of suvorexant abuse liability potential was required prior to marketing approval. The nonclinical abuse liability potential studies for suvorexant included: 1) rat drug-dependence model to assess physical dependence following abrupt cessation; 2) rat drug-discrimination model to examine the potential similarity of the interoceptive or subjective effects of suvorexant to those elicited by zolpidem and morphine; 3) self-administration model to assess the relative reinforcing efficacy of suvorexant in rhesus monkeys conditioned to self-administer methohexital. No significant signs of spontaneous drug withdrawal or 'discontinuation syndrome' were observed in rats following abrupt discontinuation of suvorexant. Suvorexant did not elicit complete cross-generalization to either a zolpidem or morphine training/reference stimuli in rats, and suvorexant was devoid of behavioral evidence of positive reinforcing efficacy in monkeys. These nonclinical findings suggested that suvorexant will have low abuse potential in humans. In the final regulatory risk assessment, suvorexant was placed into Schedule IV, likely due to its first-in-class status, its sedative properties, and the outcome of the clinical abuse potential assessment.

Assessment of the Abuse Potential of the Orexin Receptor Antagonist, Suvorexant, Compared With Zolpidem in a Randomized Crossover Study.

Suvorexant is a dual orexin receptor antagonist approved in the United States and Japan for the treatment of insomnia at a maximum dose of 20 mg. This randomized double-blind crossover study evaluated the abuse potential of suvorexant in 36 healthy recreational polydrug users with a history of sedative and psychedelic drug use. Single doses of suvorexant (40, 80, and 150 mg: 2-7.5 × maximum dose), zolpidem (15 and 30 mg: 1.5-3 × maximum dose), and placebo were administered, with a 10-day washout between treatments. Subjective and objective measures, including visual analog scales (VASs), Addiction Research Center Inventory, and cognitive/psychomotor tests, were evaluated for 24-hour postdose. Suvorexant had significantly greater peak effects on "drug liking" VAS (primary endpoint) than placebo. Although effects of suvorexant on abuse potential measures were generally similar to zolpidem, they remained constant across doses, whereas zolpidem often had greater effects at higher doses. Suvorexant (all doses) had significantly fewer effects than zolpidem 30 mg on secondary measures, such as "high" VAS, Bowdle VAS, and Addiction Research Center Inventory morphine-benzedrine group. The overall incidence of abuse-related adverse events, such as euphoric mood and hallucination, was numerically lower with suvorexant than zolpidem. In agreement with its classification as a schedule IV drug, suvorexant demonstrated abuse potential, compared with placebo. The abuse potential was similar to zolpidem using certain measures, but with a reduced incidence of abuse-related adverse events. Although this suggests that the overall abuse liability of suvorexant may be lower than zolpidem, the actual abuse rates will be assessed with the postmarketing experience.

Open Access Could Transform Drug Discovery: A Case Study of JQ1.

INTRODUCTION: The cost to develop a new drug from target discovery to market is a staggering $1.8 billion, largely due to the very high attrition rate of drug candidates and the lengthy transition times during development. Open access is an emerging model of open innovation that places no restriction on the use of information and has the potential to accelerate the development of new drugs. AREAS COVERED: To date, no quantitative assessment has yet taken place to determine the effects and viability of open access on the process of drug translation. This need is addressed within this study. The literature and intellectual property landscapes of the drug candidate JQ1, which was made available on an open access basis when discovered, and conventionally developed equivalents that were not are compared using the Web of Science and Thomson Innovation software, respectively. EXPERT OPINION: Results demonstrate that openly sharing the JQ1 molecule led to a greater uptake by a wider and more multi-disciplinary research community. A comparative analysis of the patent landscapes for each candidate also found that the broader scientific diaspora of the publically released JQ1 data enhanced innovation, evidenced by a greater number of downstream patents filed in relation to JQ1. The authors' findings counter the notion that open access drug discovery would leak commercial intellectual property. On the contrary, JQ1 serves as a test case to evidence that open access drug discovery can be an economic model that potentially improves efficiency and cost of drug discovery and its subsequent commercialization.

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-ß. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

One-pot microwave assisted synthesis of new 2-alkoxycarbonylmethylene-4-oxo-1,5-benzo-, naphtho-, and pyridodiazepines and assessment of their cytogenetic activity.

1,5-Benzo-, naphtho-, and pyridodiazepines 3 have been synthesized in excellent yields in one-step from the reaction of o-phenylenediamines with acetonedicarboxylates through microwave assisted acid catalysis. In order to ascertain their cytogenetic activity in vitro at doses equivalent to the per os doses of common 1,4-benzodiazepine drugs, Sister Chromatid Exchanges (SCEs) were employed, and for the determination of cytostaticity the Proliferation Rate Index (PRI) on lymphocytes of human whole blood cultures was estimated. It was found that benzodiazepines 3a, 3c, and 3e exhibit significant cytoprotection, but mild cytostatic effect (a statistically significant reduction of SCEs and a confined decrease of PRI values at similar concentrations). The most active compound was found to be 3e.

Assessment of Brd4 inhibition in idiopathic pulmonary fibrosis lung fibroblasts and in vivo models of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of high unmet medical need. Although bromodomain (Brd) and extra terminal domain isoforms have recently been implicated in mediating inflammatory and oncologic indications, their roles in lung fibrosis have not been comprehensively assessed. We investigated the role of Brd on the profibrotic responses of lung fibroblasts (LFs) in patients with rapidly progressing IPF and a mouse bleomycin model of lung fibrosis. The enhanced migration, proliferation, and IL-6 release observed in LFs from patients with rapidly progressing IPF are attenuated by pharmacologic inhibition of Brd4. These changes are accompanied by enhanced histone H4 lysine5 acetylation and association of Brd4 with genes involved in the profibrotic responses in IPF LFs as demonstrated using chromatin immunoprecipitation and quantitative PCR. Oral administration of 200 mg/kg per day Brd4 inhibitor JQ1 in a therapeutic dosing regimen substantially attenuated lung fibrosis induced by bleomycin in C57BL/6 mice. In conclusion, this study shows that the Brd4 inhibitor JQ1, administered in a therapeutic dosage, is capable of inhibiting the profibrotic effects of IPF LFs and attenuates bleomycin-induced lung fibrosis in mice. These results suggest that Brd4 inhibitors may represent a novel therapy for the treatment of rapidly progressing IPF.

A novel transition state analog inhibitor of guanase based on azepinomycin ring structure: Synthesis and biochemical assessment of enzyme inhibition.

Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K(i) of 16.7±0.5µM.

Calcitonin gene-related peptide receptor antagonists for the treatment of migraine: a patent review.

BACKGROUND: Migraine is a debilitating headache disorder which affects approximately 12% of the general population and is the cause of significant loss of productivity (i.e., lost time from work or school) for those afflicted. The current standard of care, the 5-HT(1B/1D) agonists known as triptans, is contraindicated in patients with cardiovascular disease due to their inherent vasoconstrictive activity; thus, there is a need to develop an alternative therapy for the treatment of the disorder. OBJECTIVE: This article reviews patent publications related to the use of small molecule calcitonin gene-related peptide (CGRP) receptor antagonists for the treatment of migraine that have appeared in the literature within the past decade. The commentary is supplemented by information presented in journal articles and focuses on the activity of several major pharmaceutical companies in the field. CONCLUSION: Two small molecule CGRP receptor antagonists, olcegepant and telcagepant, have been shown to be clinically efficacious in the treatment of migraine, and thus provide validation of this novel therapeutic mechanism.

Decision tree modeling predicts effects of inhibiting contractility signaling on cell motility.

BACKGROUND: Computational models of cell signaling networks typically are aimed at capturing dynamics of molecular components to derive quantitative insights from prior experimental data, and to make predictions concerning altered dynamics under different conditions. However, signaling network models have rarely been used to predict how cell phenotypic behaviors result from the integrated operation of these networks. We recently developed a decision tree model for how EGF-induced fibroblast cell motility across two-dimensional fibronectin-coated surfaces depends on the integrated activation status of five key signaling nodes, including a proximal regulator of transcellular contractile force generation, MLC (myosin light chain) [Hautaniemi et al, Bioinformatics 21: 2027 {2005}], but we have not previously attempted predictions of new experimental effects from this model. RESULTS: In this new work, we construct an improved decision tree model for the combined influence of EGF and fibronectin on fibroblast cell migration based on a wider spectrum of experimental protein signaling and cell motility measurements, and directly test a significant and non-intuitive a priori prediction for the outcome of a targeted molecular intervention into the signaling network: that partially reducing activation of MLC would increase cell motility on moderately adhesive surfaces. This prediction was indeed confirmed experimentally: partial inhibition of the activating MLC kinase (MLCK) upstream using the pharmacologic agent ML-7 resulted in increased motility of NR6 fibroblasts. We further extended this exciting finding by showing that partial reduction of MLC activation similarly enhanced the transmigration of the human breast carcinoma cell line MDA-213 through a Matrigel barrier. CONCLUSION: These findings specifically highlight a central regulatory role for transcellular contractility in governing cell motility, while at the same time demonstrating the value of a decision tree approach to a systems "signal-response" model in discerning non-intuitive behavior arising from integrated operation a cell signaling network.

[Actigraphic assessment of the effects of hypnotics on the night before surgery in relation with the severity of surgery].

BACKGROUND: Hypnotics have been used to provide adequate sleep on the night before surgery, but these effects have rarely been investigated. We investigated whether the preoperative hypnotic effects are related with the severity of surgery with quantitative evaluation using actigraph. METHODS: Sleep time on the night before surgery was evaluated after the administration of brotizolam 0.25 mg in 40 patients awaiting surgery; 20 patients for cardiovascular surgeries (Group I) and 20 patients for general surgeries (Group II). Sleep and awake state were identified by wrist activity measured with a motion-logger actigraph. Sleep time was assessed in total period from 22:00 to 6:00 and its 4 subdivided 2-hour periods (22:00-24:00, 24:00-2:00, 2:00-4:00, 4:00-6:00). RESULTS: The total sleep time in Group II (448 +/- 22 min) was significantly longer than that in Group I (409 +/- 44 min). Group I showed a significant reduction in sleep time in the period of 4:00-6:00 compared with other periods, whereas Group II did not show any difference among 4 periods. Group I showed significant shorter sleep time in the period of 4:00-6:00 compared with Group II. CONCLUSIONS: An actigraphic assessment of sleep time has demonstrated the possible influence of the severity of surgery on the sleep time on the night before surgery.

Rapid actin transport during cell protrusion.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.

Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells.

Most drugs have some efficacy so that improved methods to determine the relative intrinsic efficacy of partial agonists should be of benefit to preclinical and clinical investigators. We examined the effects of partial D(1) or partial D(2) dopamine agonists using a partial agonist interaction model. The dependent variable was the modulation of the dopamine-receptor-mediated cAMP response in C6 glioma cells selectively and stably expressing either D(1) or D(2) recombinant dopamine receptors. The dissociation constant (K(B)) and relative intrinsic efficacy (E(r)) for each partial agonist were calculated using a partial agonist interaction null model in which the effects of fixed concentrations of each partial agonist on the dopamine dose-response curve were evaluated. This model is an extension of the competitive antagonist null model to drugs with efficacy and assumes only that the log-dose--response curve is monotonic. Generally, the partial agonist interaction model fit the data, as well as fits of the independent logistic curves. Furthermore, the partial agonist K(B) values could be shared across partial agonist concentrations without worsening the model fit (by increasing the residual variance). K(B) values were also similar to drug affinities reported in the literature. The model was validated in three ways. First, we assumed a common tissue stimulus parameter (beta) and calculated the E(r) values. This provided a qualitative check on the interaction model results. Second, we calculated new relative efficacy values, E(r)(beta), using the beta estimate. Third, we calculated relative efficacy using relative maxima times midpoint shift ratios (J. Theor. Biol. 198 (1999) 347.). All three methods indicated that the present model yielded reasonable estimates of affinity and relative efficacy for the set of compounds studied. Our results provide a quick and convenient method of quantification of partial agonist efficacy. Special applications and limitations of the model are discussed. In addition, the present results are the first report of the relative intrinsic efficacy values for this set of D(2) ligands.

Assessment of correlation between skin target site free drug concentration and the in vivo topical antiviral efficacy in hairless mice for (E)-5-(2-bromovinyl)-2'-deoxyuridine and acyclovir formulations.

Recently, we reported that the in vivo efficacy of acyclovir (ACV) formulations was a single valued function of skin target site free drug concentration (C) irrespective of the formulation compositions. A long-term objective of this research has been to generalize the C concept using model drugs which are similar to as well as different from ACV in their mechanism of actions. (Bromovinyl)deoxyuridine (BVDU) was selected as a model drug based on the reported similarity in its mechanism of action with ACV. The relationship between the C predictions and the in vivo efficacies for some topical formulations containing different concentrations (0.05-10%) of either ACV or BVDU in 95% DMSO as a vehicle with or without 5% Azone as skin permeation enhancer was examined. Hairless mice infected cutaneously with HSV-1 were used to quantitatively estimate the in vivo topical antiviral efficacy. A finite dose of the test antiviral formulation was applied twice a day for 4 days, starting the day after virus inoculation. On the fifth day, the lesions were scored and the efficacy values were calculated. For each formulation, in vitro flux experiments were performed in an in vivo-in vitro experimental design that closely approximated the in vivo study protocol. As was previously shown, with all ACV formulations, a good correlation was found between the C predictions and the in vivo topical efficacy. With the BVDU formulations, on the other hand, this was found not to be the case. BVDU formulations with 5% Azone were generally much more effective than those without Azone at comparable C values. This finding is believed to be the first of its kind showing that skin "permeation enhancers" may enhance efficacy by more than simply increasing skin permeation rates.

Magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice: a nutritional model for discriminatory screening of anticonvulsant drugs and original assessment of neuroprotection properties.

A great many animal models for audiogenic seizures have been described. The extent to which these models may provide insight into neuroscience fields such as abnormal locomotor behavior (wild running), seizures and anticonvulsants, and neuroinsults and neuroprotectors is examined here by our study of magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice. MDDASs were induced in all of the eight tested adult murine strains and are presented as a sequence of four successive components (latency, wild running, convulsion, and recovery phase periods). Compared with several classic seizure tests, the nutritional MDDAS model responded to low doses of prototype antiepileptic drugs (AEDs), including phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), valproic acid (VPA), ethosuximide (ESM), and diazepam (DZP). Modulation by AEDs of the four components of MDDAS indicated that this seizure test was discriminatory, distinguishing between phenytoinergic (PHT, CBZ), GABAergic (PB, VPA, DZP), and ethosuximide (ESM) compounds. Suitability of the MDDAS test for evaluation of neuroprotective compounds was also examined: it showed partial (melatonin) and complete (WEB2170, an anti-PAF agent) reduction of recovery phase by non-anticonvulsant doses of test compounds. These neuroprotective responses were compared with neuroprotective potentials determined in a model of neonatal cerebral injury induced by focal injection of ibotenate (a glutamate analog). WEB2170 and melatonin reduced the size of lesions in white matter, but only WEB2170 protected cortical plate against ibotenate-induced lesions. In addition to the original neuroprotective behavior of WEB2170, studies on the neuroprotectors also supported GABAergic anticonvulsant activity of melatonin in the MDDAS test.

Nocturnal psychometric assessment of the hypnotic activity of low and normal doses of brotizolam.

The hypnotic activity of acute doses of 0.125 mg and 0.250 mg brotizolam (CAS 57801-81-7, Lendormin) was compared in a double-blind randomised placebo-controlled study. Forty-nine healthy female volunteers aged between 23 and 44 years were enrolled. Trial medication was administered sublingually at 9:15 p.m. and 9:30 p.m., respectively. The nocturnal investigations continued until 2:30 a.m. A final examination was performed in the morning after breakfast. Every 30 min mood was measured by visual analogue scales. A computerised psychometric test (CDT) over 8 min was undertaken in order to measure continuous attention under short-term memory load. The Digit Symbol Substitution Test (DSST) was performed every hour. The CDT was not evaluable due to significant baseline differences. A statistically significant sedative effect in the DSST was already found 30 min after administration of 0.250 mg brotizolam, while the effect of 0.125 mg brotizolam just failed to reach the threshold of significance. Both treatments showed equivalent efficacy at 0.5, 1.5 and 2.5 h after administration. After 3.5 and 4.5 h there was no statistically significant difference between placebo and 0.125 mg brotizolam. After 4.5 h 0.250 mg brotizolam still showed significant hypnotic activity compared to placebo and 0.125 mg brotizolam. No treatment effects on mood were apparent according to the visual analogue scales. Furthermore, no hangover effects were detected for any of the parameters measured. The pharmacodynamic results confirmed the duration of action of more than 4.5 of h 0.250 mg brotizolam found in earlier studies and suggest that 0.125 mg is as effective as 0.250 mg with regard to sleep onset disturbances but has a shorter duration of action.

Quantitative assessment of increased airway microvascular permeability to 125I-labelled plasma fibrinogen induced by platelet activating factor and bradykinin.

1. We have used 125I-labelled fibrinogen (I-FN) in experiments monitoring plasma extravasation from vessels within guinea-pig trachea and peripheral lung tissue in response to platelet activating factor (PAF) and bradykinin (BK). Retained tissue radioactivity derived from I-FN was detected by direct measurement and by autoradiography. 2. Both PAF and BK caused concentration-dependent increases in radioactivity in trachea and peripheral lung, with PAF being approximately 1000 times more potent than BK at both sites. On a wet weight basis, mean tracheal leakage responses to PAF and BK were approximately 6 times and 2 times greater respectively than those in peripheral lung. Furthermore, in trachea, the maximal response to PAF was nearly twice that to BK, although they were approximately equiactive in peripheral lung. The dipeptidyl carboxypeptidase inhibitor, enalapril (1 mg kg-1, i.v.), increased the potency of BK by approximately 40 fold. 3. In trachea, PAF (50 ng kg-1, i.v.)-induced leakage was selectively inhibited by the PAF receptor antagonist, WEB 2086 (5-50 micrograms kg-1), while responses to BK (50 micrograms kg-1, i.v.) were selectively inhibited by the BK2 receptor antagonist NPC 349 (0.5-1 mg kg-1). Neither PAF nor BK-induced leakage were significantly altered by pretreatment with the histamine H1-receptor antagonists mepyramine (10 micrograms kg-1) or ketotifen (50 micrograms kg-1) or the leukotriene receptor antagonist SKF 104353. These data indicate that both agonists caused direct, specific receptor operated increases in tracheal vascular permeability to plasma macromolecules.The alpha/beta1-adrenoceptor agonist adrenaline (100 pgkg-1) caused modest inhibition of leakage induced by BK, but not of the leakage response to PAF.4. Peripheral airway leakage responses to both PAF and BK were also detected by light microscopic autoradiography in paraffin-embedded tissue sections. This was possible since a significant amount of extravasated I-FN was apparently precipitated and fixed in the extravascular space as 125-labelled fibrin. Autoradiograms showed that both agonists caused increases in peripheral bronchial circulation microvascular permeability to I-FN. No evidence for leakage in alveolar wall capillaries or in pulmonary blood vessels was observed. Quantitation of such autoradiographic data will allow a comprehensive evaluation of the effects of putative asthma mediators on microvascular permeability throughout the respiratory tree.

In vivo assessment of rat renal alpha adrenoceptors.

Despite a preponderance of alpha-2 adrenoceptors in homogenates of rat renal cortex, alpha-2 adrenoceptor agonists do not vasoconstrict the isolated buffer-perfused rat kidney. Both alpha-1 and alpha-2 adrenoceptor agonists can constrict the kidneys of dogs, cats and rabbits in vivo. Because alpha-2 adrenoceptor-mediated vasoconstriction is often difficult to demonstrate in vitro, and both subtypes of alpha agonists cause large increases in peripheral resistance in pithed rats, we tested the hypothesis that both alpha-1 and alpha-2 agonists would also constrict the rat kidney in vivo. Cannulation of the suprarenal artery and utilization of a high pressure liquid chromatography valve enabled random and reproducible intrarenal arterial bolus injections of agonists, and renal blood flow was monitored using Doppler flowmetry. Cirazoline, phenylephrine and norepinephrine bitartrate caused large renal vasopressor responses with minimal systemic effects. Although administered in a dosage range 100 to 1000 times that of alpha-1 agonists, the alpha-2 agonists (B-HT 920, UK 14,304 and guanabenz) produced only minimal renal vasoconstriction before systemic pressor effects. The low potency and efficacy of alpha-2 agonists could not be attributed to concomitant vasodilatory effects of these agents. Rat renal resistance vessels were less responsive to alpha-2 agonists than other species that have been examined. These studies are consistent with conclusions from in vitro examinations that only alpha-1 adrenoceptors mediate changes in renal vascular resistance and also autoradiographic studies reporting the localization of alpha-2 binding sites to rat renal tubules.