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.

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.

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.

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.

Inhibition of osteoclastogenesis and inflammatory bone resorption by targeting BET proteins and epigenetic regulation.

Emerging evidence suggests that RANKL-induced changes in chromatin state are important for osteoclastogenesis, but these epigenetic mechanisms are not well understood and have not been therapeutically targeted. In this study, we find that the small molecule I-BET151 that targets bromo and extra-terminal (BET) proteins that 'read' chromatin states by binding to acetylated histones strongly suppresses osteoclastogenesis. I-BET151 suppresses pathologic bone loss in TNF-induced inflammatory osteolysis, inflammatory arthritis and post-ovariectomy models. Transcriptome analysis identifies a MYC-NFAT axis important for osteoclastogenesis. Mechanistically, I-BET151 inhibits expression of the master osteoclast regulator NFATC1 by suppressing expression and recruitment of its newly identified upstream regulator MYC. MYC is elevated in rheumatoid arthritis macrophages and its induction by RANKL is important for osteoclastogenesis and TNF-induced bone resorption. These findings highlight the importance of an I-BET151-inhibited MYC-NFAT axis in osteoclastogenesis, and suggest targeting epigenetic chromatin regulators holds promise for treatment of inflammatory and oestrogen deficiency-mediated pathologic bone resorption.

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.

Discovery of epigenetic regulator I-BET762: lead optimization to afford a clinical candidate inhibitor of the BET bromodomains.

The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure-activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.

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.

Evidence that stimulation of ghrelin receptors in the spinal cord initiates propulsive activity in the colon of the rat.

Previous studies have failed to reveal an effect of the gastrointestinal peptide hormone ghrelin on colonic motility. In the present work, ghrelin was applied into the lumbo-sacral spinal cord in the region of defecation control centres, and a synthetic ghrelin receptor agonist, CP464709, which crosses the blood-brain barrier, was applied intravenously or into the lumbo-sacral cord. Both ghrelin and CP464709 elicited propulsive contractions and emptying of the colon in anaesthetized rats. In conscious rats, subcutaneous CP464709 caused fecal expulsion. The sites of action and nerve pathways involved in the stimulation of the colon by ghrelin receptor activation were investigated in anaesthetized rats. Intrathecal application of CP464709 at L6-S1, but not application at ponto-medullary levels or to the thoracic spinal cord, elicited propulsive contractions. The stimulation evoked by intravenous CP464709 was prevented if the pelvic nerve outflows were severed, but not if the spinal cord was cut rostral to the defecation centre at L6-S3. The response was also blocked by hexamethonium. When ghrelin, applied intrathecally, was used to desensitize its receptors, the effect of intravenous CP464709 was blocked. CP464709 did not affect small intestine motility or the amplitudes of visceromotor reflexes caused by colorectal distension. It is concluded that activation of ghrelin receptors in the lumbo-sacral spinal cord triggers co-ordinated propulsive contractions that empty the colo-rectum. The pathways through which these responses are generated pass out of the spinal cord via the pelvic nerves and cause propulsive contractions through activation of enteric neurons.

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.