Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [12-5-labeled Tyr"]Jsomatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (ICs0, 0.17, 0.1, and 21 nM, respectively) and low affinity for SSTR1 and -4 (ICso, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10%o (vol/vol) fetal calf serum, 1 ,uM insulin, or 0.1 ,uM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses ofRC-160 (ECsD, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbacholstimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.Somatostatin is a tetradecapeptide with diverse biological effects on cellular function, including inhibition of secretory and proliferative processes (1, 2). These effects are mediated by specific receptors that belong to the guanine nucleotide binding protein (G-protein)-linked receptor family (3-7).Five somatostatin receptor subtypes SSTR1 to -5 and one splice variant have been cloned from human (h), murine (m), and rat (r) sources (3-7). After expression of SSTR gene clones in mammalian cell lines, distinct profiles for binding soma-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.tostatin analogues and functional coupling to adenylate cyclase v...

BackgroundCharcot-Marie-Tooth type 1A disease (CMT1A) is a rare orphan inherited neuropathy caused by an autosomal dominant duplication of a gene encoding for the structural myelin protein PMP22, which induces abnormal Schwann cell differentiation and dysmyelination, eventually leading to axonal suffering then loss and muscle wasting. We favour the idea that diseases can be more efficiently treated when targeting multiple disease-relevant pathways. In CMT1A patients, we therefore tested the potential of PXT3003, a low-dose combination of three already approved compounds (baclofen, naltrexone and sorbitol). Our study conceptually builds on preclinical experiments highlighting a pleiotropic mechanism of action that includes downregulation of PMP22. The primary objective was to assess safety and tolerability of PXT3003. The secondary objective aimed at an exploratory analysis of efficacy of PXT3003 in CMT1A, to be used for designing next clinical development stages (Phase 2b/3).Methods80 adult patients with mild-to-moderate CMT1A received in double-blind for 1 year Placebo or one of the three increasing doses of PXT3003 tested, in four equal groups. Safety and tolerability were assessed with the incidence of related adverse events. Efficacy was assessed using the Charcot-Marie-Tooth Neuropathy Score (CMTNS) and the Overall Neuropathy Limitations Scale (ONLS) as main endpoints, as well as various clinical and electrophysiological outcomes.ResultsThis trial confirmed the safety and tolerability of PXT3003. The highest dose (HD) showed consistent evidence of improvement beyond stabilization. CMTNS and ONLS, with a significant improvement of respectively of 8% (0.4% - 16.2%) and 12.1% (2% - 23.2%) in the HD group versus the pool of all other groups, appear to be the most sensitive clinical endpoints to treatment despite their quasi-stability over one year under Placebo. Patients who did not deteriorate over one year were significantly more frequent in the HD group.ConclusionsThese results confirm that PXT3003 deserves further investigation in adults and could greatly benefit CMT1A-diagnosed children, usually less affected than adults.Trial registrationEudraCT Number: 2010-023097-40. ClinicalTrials.gov Identifier: NCT01401257. The Committee for Orphan Medicinal Products issued in February 2014 a positive opinion on the application for orphan designation for PXT3003 (EMA/OD/193/13).Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0199-0) contains supplementary material, which is available to authorized users.

This study used a novel computational pipeline to exploit draft bacterial genome sequences in order to predict, automatically and rapidly, PCR primer sets for Dickeya spp. that were unbiased in terms of diagnostic gene choice. This pipeline was applied to 16 draft and four complete Dickeya genome sequences to generate >700 primer sets predicted to discriminate between Dickeya at the species level. Predicted diagnostic primer sets for both D. dianthicola (DIA-A and DIA-B) and 'D. solani' (SOL-C and SOL-D) were validated against a panel of 70 Dickeya reference strains, representative of the known diversity of this genus, to confirm primer specificity. The classification of the four previously sequenced strains was re-examined and evidence of possible misclassification of three of these strains is presented.

1 The toxic e ects of nonsteroidal anti-in¯ammatory drugs for the lower gastrointestinal tract share certain features with in¯ammatory processes, suggesting that the release of in¯ammation cytokines such as TNF-a may damage the intestine. 2 Rats received a s.c. injection of indomethacin. Then, jejunum-ileum was taken up for the quanti®cation of ulcerations, production of TNF-a, nitrites and PGE2 ex vivo and activity of calciumindependent NO synthase and myeloperoxydase. Activation of NO metabolism and myeloperoxydase were measured as potential e ectors of TNF-a. 3 Jejunum-ileum from rats having received indomethacin (10 mg kg 71 ) produced TNF-a ex vivo. Cytokine production was associated with the onset of macroscopic ulcerations of the small intestine, and preceded nitrite production and tissue activity of myeloperoxidase. 4 Similar intestinal ulcerations and upregulation of TNF-a were obtained with¯urbiprofen (30 mg kg 71 ), chemically unrelated to indomethacin. 5 TNF-a production was proportional to the indomethacin dose (from 3 ± 20 mg kg 71 ) and correlated with the surface area of ulcerations and nitrite production, 24 h after indomethacin administration. 6 Pretreatment of rats with RO 20-1724, a type-IV phosphodiesterase inhibitor which inhibits TNF-a synthesis, substantially reduced jejunum-ileum ulcerations, TNF-a and nitrite production and tissue enzyme activities. 7 These ®ndings provide evidence that TNF-a is increased in indomethacin-induced intestinal ulcerations and support suggestions that TNF-a is involved at an early stage of nonsteroidal antiin¯ammatory drug toxicity for the small intestine.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model. Based on these observations in preclinical models, a clinical trial of PTX3003 in CMT1A, a neglected orphan disease, is warranted. If the efficacy of PTX3003 is confirmed, rational polytherapy based on novel combinations of existing non-toxic drugs with pleiotropic effects may represent a promising approach for rapid drug development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0201-x) contains supplementary material, which is available to authorized users.