Human-relevant near-organ neuromodulation of the immune system via the splenic nerve.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve-induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors.

Bromodomain-containing proteins bind acetylated lysine residues on histone tails and are involved in the recruitment of additional factors that mediate histone modifications and enable transcription. A compound, I-BET-762, that inhibits binding of an acetylated histone peptide to proteins of the bromodomain and extra-terminal domain (BET) family, was previously shown to suppress the production of proinflammatory proteins by macrophages and block acute inflammation in mice. Here, we investigated the effect of short-term treatment with I-BET-762 on T-cell function. Treatment of naïve CD4(+) T cells with I-BET-762 during the first 2 d of differentiation had long-lasting effects on subsequent gene expression and cytokine production. Gene expression analysis revealed up-regulated expression of several antiinflammatory gene products, including IL-10, Lag3, and Egr2, and down-regulated expression of several proinflammatory cytokines including GM-CSF and IL-17. The short 2-d treatment with I-BET-762 inhibited the ability of antigen-specific T cells, differentiated under Th1 but not Th17 conditions in vitro, to induce pathogenesis in an adoptive transfer model of experimental autoimmune encephalomyelitis. The suppressive effects of I-BET-762 on T-cell mediated inflammation in vivo were accompanied by decreased recruitment of macrophages, consistent with decreased GM-CSF production by CNS-infiltrating T cells. These effects were mimicked by an inhibitor of c-myc function, implicating reduced expression of c-myc and GM-CSF as one avenue by which I-BET-762 suppresses the inflammatory functions of T cells. Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells.

Identification of clinical candidates from the benzazepine class of histamine H3 receptor antagonists.

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.

The discovery of the benzazepine class of histamine H3 receptor antagonists.

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.

Development of an acute ovine model of polycystic ovaries to assess the effect of ovarian denervation.

Introduction: Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype. Methods: Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary. Results: Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle. Discussion: Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival.

Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A).

A novel series of quinoline isoxazole BET family bromodomain inhibitors are discussed. Crystallography is used to illustrate binding modes and rationalize their SAR. One member, I-BET151 (GSK1210151A), shows good oral bioavailability in both the rat and minipig as well as demonstrating efficient suppression of bacterial induced inflammation and sepsis in a murine in vivo endotoxaemia model.

The utility of pharmacokinetic-pharmacodynamic modeling in the discovery and optimization of selective S1P(1) agonists.

Sphingosine-1-phosphate (S1P(1)) receptor agonists such as Fingolimod (FTY-720) are a novel class of immunomodulators that have clinical utility in the treatment of remitting relapsing multiples sclerosis. This class of compound act by inducing peripheral lymphopenia. Using an integrated pharmacokinetic/pharmacodynamic (PK-PD) approach based on an in vivo rat model, novel S1P(1) agonists were identified with a predicted more rapid rate of reversibility of lymphocyte reduction in human compared to Fingolimod. The in vivo potency of 15 compounds based on PK-PD modelling of the rat lymphocyte reduction model was correlated with in vitro measures of potency at the S1P(1) receptor using ß arrestin recruitment and G-protein signalling. A structurally novel S1P(1) agonist was identified and predictions of human pharmacokinetics and clinical dose are presented.

Pyrazolopyridazine alpha-2-delta-1 ligands for the treatment of neuropathic pain.

Optimization of the novel alpha-2-delta-1 ligand 4 provided compounds 37 and 38 which have improved DMPK profiles, good in vivo analgesic activity and in vitro selectivity over alpha-2-delta-2. An in-house P-gp prediction programme and the MetaSite software package were used to help solve the specific problems of high P-gp efflux and high in vivo clearance.

Quantification of clinically applicable stimulation parameters for precision near-organ neuromodulation of human splenic nerves.

Neuromodulation is a new therapeutic pathway to treat inflammatory conditions by modulating the electrical signalling pattern of the autonomic connections to the spleen. However, targeting this sub-division of the nervous system presents specific challenges in translating nerve stimulation parameters. Firstly, autonomic nerves are typically embedded non-uniformly among visceral and connective tissues with complex interfacing requirements. Secondly, these nerves contain axons with populations of varying phenotypes leading to complexities for axon engagement and activation. Thirdly, clinical translational of methodologies attained using preclinical animal models are limited due to heterogeneity of the intra- and inter-species comparative anatomy and physiology. Here we demonstrate how this can be accomplished by the use of in silico modelling of target anatomy, and validation of these estimations through ex vivo human tissue electrophysiology studies. Neuroelectrical models are developed to address the challenges in translation of parameters, which provides strong input criteria for device design and dose selection prior to a first-in-human trial.

Aryl sulphonyl amides as potent agonists of the growth hormone secretagogue (ghrelin) receptor.

As part of an on-going lead optimisation effort, a cross screening exercise identified an aryl sulphonyl amide hit that was optimised to afford a highly potent series of ghrelin receptor agonists.

Effect of Superior Ovarian Nerve and Plexus Nerve Sympathetic Denervation on Ovarian-Derived Infertility Provoked by Estradiol Exposure to Rats.

Sympathetic innervation of the ovary in rodents occurs via two routes: the superior ovarian nerve (SON), which runs along the ovarian ligament, and the plexus nerve (PN), which is mainly associated with the vasculature. SON and ovarian norepinephrine (NE) levels play a major role in regulating ovarian cystic health. Although it was previously described that the polycystic ovarian phenotype (PCO) is causally related to hyperstimulation of the sympathetic nerves of the ovary, much less is known, however, regarding the role of PN in ovarian physiology. We studied the role of SON and PN in relation to the maintenance of the PCO phenotype induced in the rat by exposure to estradiol valerate (EV). EV exposure at 24 days old (juvenile exposure) increases NE in the ovary for up to 90 days after EV injection. SON or PN denervation (SONX and PNX) decreased NE. SONXreversed the acyclic condition from 30 days after surgery (p < 0.05), but PNXdid not. SONX was more effective than PNX to downregulate the increased number of cysts induced by EV, with the presence of the corpora lutea (CL, signifying ovulation) in the SONX group. Seventy percent of SONX rats presented with pregnancy at 60 days post-EV (6 of the 7 sperm-positive rats were pregnant); however, SONX rats had a reduced number (half) of pups compared with vehicle-treated rats and 60% more pups than EV rats. These data suggest that the SON plays a predominant role in follicular development, ovulation and pregnancy during ovarian diseases.

Potent achiral agonists of the growth hormone secretagogue (ghrelin) receptor. Part 2: Lead optimisation.

A series of small molecule orally bioavailable ghrelin receptor agonists have been identified through systematic optimisation of a high throughput screening hit.

BET bromodomain inhibitors show anti-papillomavirus activity in vitro and block CRPV wart growth in vivo.

The DNA papillomaviruses infect squamous epithelium and can cause persistent, benign and sometimes malignant hyperproliferative lesions. Effective antiviral drugs to treat human papillomavirus (HPV) infection are lacking and here we investigate the anti-papillomavirus activity of novel epigenetic targeting drugs, BET bromodomain inhibitors. Bromodomain and Extra-Terminal domain (BET) proteins are host proteins which regulate gene transcription, they bind acetylated lysine residues in histones and non-histone proteins via bromodomains, functioning as scaffold proteins in the formation of transcriptional complexes at gene regulatory regions. The BET protein BRD4 has been shown to be involved in the papillomavirus life cycle, as a co-factor for viral E2 and also mediating viral partitioning in some virus types. We set out to study the activity of small molecule BET bromodomain inhibitors in models of papillomavirus infection. Several BET inhibitors reduced HPV11 E1ˆE4 mRNA expression in vitro and topical therapeutic administration of an exemplar compound I-BET762, abrogated CRPV cutaneous wart growth in rabbits, demonstrating translation of anti-viral effects to efficacy in vivo. Additionally I-BET762 markedly reduced viability of HPV16 infected W12 cells compared to non-infected C33A cells. The molecular mechanism for the cytotoxicity to W12 cells is unknown but may be through blocking viral-dependent cell-survival factors. We conclude that these effects, across multiple papillomavirus types and in vivo, highlight the potential to target BET bromodomains to treat HPV infection.

Therapeutic approaches towards the treatment of gastroinstestinal disorders.

The Society for Medicines Research gathered an international panel of speakers and about 60 delegates for their symposium September 21, 2006, on Therapeutic Approaches Towards the Treatment of Gastroinstestinal Disorders, at the National Heart and Lung Institute, in London, U.K. The focus of the conference was to discuss therapeutic strategies taken towards the treatment of inflammatory bowel disease, acid related disorders and irritable bowel syndrome. Key note lectures addressed the development of tegaserod, a 5-HT(4) receptor agonist, for the treatment of constipation dominant irritable bowel syndrome (cIBS), the use of tumor necrosis factor alpha (TNFalpha) inhibitors in the treatment of chronic inflammatory diseases, including Crohn's disease, the development of effective inhibitors of gastric acid secretion, the role of alpha(4)beta(7) integrin in the development of Crohn's disease and ulcerative colitis, the parts played by the neuropeptides ghrelin and motilin in the control of gastrointestinal motility, and the role of bacteria in functional gastrointestinal disease.

Potent achiral agonists of the ghrelin (growth hormone secretagogue) receptor. Part I: Lead identification.

High throughput screening combined with efficient datamining and parallel synthesis led to the discovery of a novel series of indolines showing potent in vitro ghrelin receptor agonist activity and acceleration of gastric emptying in rats.

Recent disclosures of clinical drug candidates.

On December 8, 2005, the Society for Medicines Research held a one-day meeting in London, United Kingdom, entitled Recent Disclosures of Clinical Drug Candidates. The meeting brought together speakers from Europe representing the pharmaceutical industry and provided an overview of some the latest approaches being taken in a range of therapeutic areas such as oncology, inflammation, CNS disease and reproductive medicine.

Anti-emetic activity of ghrelin in ferrets exposed to the cytotoxic anti-cancer agent cisplatin.

Emesis may be modulated via multiple mechanisms. The actions of ghrelin suggest an ability to couple an induction of hunger with preparation of the stomach for ingestion of food. Such a process might reduce any tendency to vomit, so an anti-emetic activity of ghrelin was investigated in the ferret cisplatin-induced emesis model. In controls, intra-peritoneal cisplatin (10 mg/kg) induced 41.4+/-8.4 episodes of emesis comprising 310.4+/-55.3 retches and 28.8+/-6.9 vomits during the 6h observation; the latency to onset of the first emetic episode was 108.9+/-4.8 min. Intra-peritoneal ghrelin (1mg/kg, split as a 30 min pre- and 30 min-post dose) did not induce a change in behaviour or modify cisplatin-induced emesis (p>0.05). Intracerebroventricular (i.c.v.) administration (third ventricle) was achieved via a pre-implanted cannula. At the first emetic episode following cisplatin, ghrelin or vehicle (20 microl saline) was administered i.c.v. During the 30 min following the initial episode of emesis, control animals exhibited 18.0+/-2.6 emetic episodes comprising 160.3+/-24.1 retches and 13.8+/-2.7 vomits. Ghrelin 10 microg i.c.v. reduced the number of retches by 61.5% (p<0.05) and at a dose of 30 microg i.c.v. ghrelin reduced the number of episodes, individual retches and vomits by 74.4 (p<0.05), 80.4 (p<0.01), and 72.5% (p<0.05), respectively. At subsequent time periods there were no differences between ghrelin- or saline-treated animals (p>0.05). An ability of ghrelin to reduce emesis is consistent with a role in modulating gastro-intestinal functions and identifies a novel approach to the treatment of emesis.

Discovery of Tetrahydropyrazolopyridine as Sphingosine 1-Phosphate Receptor 3 (S1P3)-Sparing S1P1 Agonists Active at Low Oral Doses.

FTY720 is the first oral small molecule approved for the treatment of people suffering from relapsing-remitting multiple sclerosis. It is a potent agonist of the S1P1 receptor, but its lack of selectivity against the S1P3 receptor has been linked to most of the cardiovascular side effects observed in the clinic. These findings have triggered intensive efforts toward the identification of a second generation of S1P3-sparing S1P1 agonists. We have recently disclosed a series of orally active tetrahydroisoquinoline (THIQ) compounds matching these criteria. In this paper we describe how we defined and implemented a strategy aiming at the discovery of selective structurally distinct follow-up agonists. This effort culminated with the identification of a series of orally active tetrahydropyrazolopyridines.

Challenges facing drug discovery in vascular disease.

The Society for Medicines Research symposium Challenges Facing Drug Discovery in Vascular Disease was held September 30, 2005, at Organon Research, Newhouse, Scotland. The conference brought together an international panel of speakers representing academia and the pharmaceutical industry to review approaches to the treatment of diseases affecting the vasculature. The focus of the meeting was on atherosclerosis and one of its clinical manifestations, stroke. The meeting reviewed current and emerging therapeutic approaches and improving technologies to monitor risk and disease progression in patients.