Targeting orphan G protein-coupled receptors for the treatment of diabetes and its complications: C-peptide and GPR146.

G protein-coupled receptors (GPCRs) are the most abundant receptor family encoded by the human genome and are the targets of a high percentage of drugs currently in use or in clinical trials for the treatment of diseases such as diabetes and its associated complications. Thus, orphan GPCRs, for which the ligand is unknown, represent an important untapped source of therapeutic potential for the treatment of many diseases. We have identified the previously orphan GPCR, GPR146, as the putative receptor of proinsulin C-peptide, which may prove to be an effective treatment for diabetes-associated complications. For example, we have found a potential role of C-peptide and GPR146 in regulating the function of the retinal pigment epithelium, a monolayer of cells in the retina that serves as part of the blood-retinal barrier and is disrupted in diabetic macular oedema. However, C-peptide signalling in this cell type appears to depend at least in part on extracellular glucose concentration and its interaction with insulin. In this review, we discuss the therapeutic potential of orphan GPCRs with a special focus on C-peptide and GPR146, including past and current strategies used to 'deorphanize' this diverse family of receptors, past successes and the inherent difficulties of this process.

Prevalence of colonic motor or evacuation disorders in patients presenting with chronic nausea and vomiting evaluated by a single gastroenterologist in a tertiary referral practice.

BACKGROUND: Nausea and vomiting are thought to result from upper gastrointestinal dysfunctions. Our clinical observations led to the hypothesis that colonic motor dysfunction is associated with nausea and vomiting. METHODS: We reviewed electronic medical records (EMR) of 149 patients presenting with complaints of nausea and/or vomiting in a tertiary gastroenterology practice to investigate the association with disorders of colonic motor or evacuation disorders. We extracted demographics, gastric emptying (GE in 149) and colonic transit (CT in 138) of solids, ascending colon emptying half-time (AC t1/2 ), rectal evacuation by anorectal manometry (ARM) in 91 and balloon expulsion test (BE) in 55 patients. We estimated the proportions with delayed GE or CT, based on the 5th percentile of GE (in 319) and CT in 220 healthy volunteers using same method. KEY RESULTS: Among 11 patients with nausea and/or vomiting with only GE measured, five had delayed and six normal GE. Among the 149 patients, 77 (52%) patients had evacuation disorders, confirmed by objective tests in 68 patients, and clinical examination in nine patients. In the 138 patients with both GE and CT measured, 106 (76%) had both normal GE and CT, 11 (8%) only delayed GE, 16 (11%) normal GE with delayed CT, and five (3%) delayed GE and CT. Among 21 patients (15%) with delayed CT, nine had slow AC t1/2 and 12 evacuation disorder. CONCLUSIONS & INFERENCES: In patients with chronic nausea and/or vomiting in gastroenterology practice, evaluation of colonic motility and rectal evacuation should be considered, since about half the patients have abnormal functions that conceivably contribute to the presenting nausea and/or vomiting.

Systematic review with meta-analysis: highly selective 5-HT4 agonists (prucalopride, velusetrag or naronapride) in chronic constipation.

BACKGROUND: Highly selective 5-HT4 agonists have been suggested for the treatment of chronic constipation (CC). AIM: To assess the effects of highly selective 5-HT4 agonists (prucalopride, velusetrag or naronapride) on patient-important clinical efficacy outcomes and safety in adults with CC. METHODS: We searched the medical literature in January 2013 using MEDLINE/Pubmed, Embase, Cochrane Library, and Web of Science/Scopus for randomised, controlled trials of highly selective 5-HT4 agonists in adults with CC, with no minimum duration of therapy (maximum 12 weeks) or date limitations. Data were extracted from intention-to-treat analyses, pooled using a random-effects model, and reported as relative risk (RR), mean differences, or standardised mean differences with 95% confidence intervals (CI). RESULTS: Main outcomes included stool frequency, Patient-Assessment of Constipation Quality of Life (PAC-QOL), PAC of symptoms (PAC-SYM) and adverse events. Thirteen eligible trials were identified: 11 prucalopride, 1 velusetrag, 1 naronapride. Relative to control, treatment with highly selective 5-HT4 agonists was superior for all outcomes: mean ≥3 spontaneous complete bowel movements (SCBM)/week (RR = 1.85; 95% CI 1.23-2.79); mean ≥1 SCBM over baseline (RR = 1.57; 95% CI 1.19, 2.06); ≥1 point improvement in PAC-QOL and PAC-SYM scores. The only active comparator trial of prucalopride and PEG3350 suggested PEG3350 is more efficacious for some end points. Adverse events were more common with highly selective 5-HT4 agonists, but were generally minor; headache was the most frequent. Most trials studied prucalopride. CONCLUSION: Demonstration of efficacy on patient-important outcomes and a favourable safety profile support the continued use and development of highly selective 5-HT4 agonists in the treatment of chronic constipation.

Diversity of the Ig repertoire is maintained with age in spite of reduced germinal centre cells in human tonsil lymphoid tissue.

Humans and almost all species studied to date exhibit a decreased responsiveness to immunization and increased autoimmunity with age. While this has been observed clinically for decades, only recently has an understanding of the molecular basis for these changes begun to be appreciated. Studies of the B-cell aspects of these changes in ageing mice and the very few reports in ageing humans have not been conclusive. Here we examine the nucleotide sequence of over 1250 VH transcripts from the tonsils of individuals of various ages for changes to the VH4 immunoglobulin repertoire. An exhaustive examination of VH, DH and JH gene segment utilization revealed a remarkable similarity of the repertoires. The extent of somatic hypermutation was fully maintained or even increased by some measures into the eighth decade of life. However, we found by middle age that the representation of naïve and germinal centre B-cell subpopulations changed relative to total B lymphocytes in the tonsil. While the percentage of naïve and germinal centre B-cell subpopulations changes during the second half of life, these findings suggest that even with advancing age, humans remain capable of generating an extremely diverse Ig repertoire while maintaining a similar spectrum of Ig rearrangements once the germinal centre reaction begins.

Immunoglobulin heavy-chain receptor editing is observed in the NOD/SCID model of human B-cell development.

Receptor editing and receptor revision are the two mechanisms of antibody diversity that result in either complete V-gene replacement or the formation of hybrid V genes. We do not yet understand how this process unfolds, because they are rare and difficult to study in vivo. In this study, we describe a family of VH4-34:VH4-61 hybrids isolated from a human B-cell chimeric non-obese diabetic/severe combined immunodeficient mouse. The observation of hybrid immunoglobulin sequences in human B cells that developed in this model system makes it useful for the study of this mechanism of diversification and tolerance.

The NOD/SCID chimeric mouse model of human B cell development: studies on the VH4 family immunoglobulin repertoire and implications for SLE.

The use of the NOD/SCID mouse as a transplant recipient for human cord blood B cell progenitors as a tool for investigations into the development of human B cells has become an exciting reality. The characteristics of the immunoglobulin repertoire in such a model is important to investigate, as it is possible that normal or skewed representations could be produced. Here we review our current work in which we describe a normal VH4 repertoire produced in this chimeric mouse model and describe the differences in combinatorial diversity between the human cells that were isolated from the bone marrow and spleen. The implications of this model for studies of systemic lupus erythematosus are also discussed.

Relatively normal human lymphopoiesis but rapid turnover of newly formed B cells in transplanted nonobese diabetic/SCID mice.

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2'-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of V(H) genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.

Acute oral toxicity in rats of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane compared with 3,7-bis-(3-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane and N,N'-oxydimethylenebis(2-trifluoromethylaniline).

The acute oral toxicity of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane (4-TFMPD) was compared with its 3-substituted isomer, 3,7-bis-(3-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane (3-TFMPD) and with N,N'-oxydimethylenebis(2-trifluoromethylaniline) (N,N'-oxy-DM-bis (2-TFMA)). 4-TFMPD, 3-TFMPD, N,N'-oxy-DM-bis (2-TFMA) and their precursors (4-trifluoromethylaniline (4-TFMA), 3-trifluoromethylaniline (3-TFMA) and 2-trifluoromethylaniline (2-TFMA), respectively) were administered intragastrically to male Wistar rats at a dose of 0.12 mmol/kg body weight/day for three consecutive days and the resulting effects on haematological variables were determined. 4-TFMPD induced the highest methaemoglobinemia as compared with 3-TFMPD and N,N'-oxy-DM-bis (2-TFMA). Haemolytic anaemia with Heinz bodies, neutrophilia, lymphocytosis, enlargement of the spleen and enhanced production of granulocytes/macrophages from multipotential bone marrow cells (as determined by CFU-C test) were observed in animals treated with 4-TFMPD and 4-TFMA, whereas no such effects were observed in the other treatment groups. In conclusion, 4-substituted aniline derivatives exert special haematotoxicity on the red blood cells and induce leucocytosis, which differs from the effects of their 2- and 3-substituted congeners.

Effects of trifluoromethylaniline isomers on enzyme activities in lymphatic organs and hematology of the rat.

Three isomers of trifluoromethylaniline (TFMA) were investigated for their possible different toxic effects on the hematopoietic system in male Wistar rats. The effects of isomeric 2-, 3- and 4-TFMA were compared with those of aniline, the prototypic drug. Strong leukocytosis manifested by considerable increase in the number of all respective white blood elements was observed in the peripheral blood 1 day after the administration of 4-TFMA. In contrast, erythropoiesis, as ascertained by erythrocyte count and hemoglobin concentration, was inhibited by 4-TFMA. The determination of the ED50 revealed lymphocytes to be the most responsive elements towards 4-TFMA administration. Besides hyperemic and proliferative splenomegaly the histological changes in maturation of immunocompetent cells following the 4-TFMA administration were found also in thymus. In accord with an enhanced incorporation of [3H]thymidine, the specific activity of thymidine kinase (TdK) in spleen was increased after a single dose of 4-TFMA. Activities of the catabolic enzymes adenosine deaminase (ADA) and inosine phosphorylase (IP) decreased in both organs with the exception of IP activity in thymus. The effects evoked by the 3-TFMA isomer were regularly less pronounced, and 2-TFMA was nearly inactive.

Toxic side effects of local anaesthetics on the human cornea.

The cytotoxic effects of 0.5% amethocaine (tetracaine) on the human cornea were investigated by scanning electron microscopy. The ultrastructural examination of epithelial cells showed damage of the cell membrane, rare-faction and loss of microvilli, deposits of amethocaine on the corneal surface and accelerated desquamation of superficial epithelial cells.

Trifluoromethylanilines--their effect on DNA synthesis and proliferative activity in parenchymal organs of rats.

Reactive isomeric 3- and 4-trifluoromethylanilines (3-,4-TFMA), and control aniline itself, induced the following effects on biosynthesis of DNA in the liver, kidney, thymus and spleen of rats: (a) The administration of 4-TFMA initially suppressed the utilization of labeled thymidine for splenic DNA synthesis during the early prereplicative stage. However, with progressing time the incorporation of the labeled marker began to increase and in 30 h its level exceeded the controls by more than 200%. As expected, aniline administration resulted in mild depression of incorporation during the whole period studied. (b) 4-TFMA caused a significant increase of incorporation of labeled thymidine into DNA thymine also in the thymus. After administration of aniline the utilization of labeled thymidine for the synthesis of DNA thymine in thymus was suppressed during the first 16 h. (c) The dose-response curve showed a linear increase of incorporation in the spleen within the dose range between 0.125 and 0.500 mmol/kg of 4-TFMA. (d) It appears that enhanced incorporation of labeled thymidine into splenic and thymic DNA is a phenomenon specific for compounds bearing the CF3 group on the 4-position of the phenyl ring, such as 4-TFMA and 4-TFMPD. On the contrary, the analogous 3-CF3 substituted derivatives had no effect. Increased incorporation of labeled thymidine into spleen and thymus DNA apparently represents an increased DNA synthesis and cellular proliferation in lymphatic organs. The proliferative response was possibly evoked by the preceding hemolysis or by other toxic effects caused by the drug.

Effect of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane on pyrimidine and DNA synthesis in rat organs.

The administration of the lipophilic 3,7-bis-(4-trifluoromethylphenyl)- 1,5,3,7-dioxadiazocane (TFMPD) to rats induced the following effects on the biosynthesis of DNA in the liver, kidney, thymus and spleen: (a) The utilization of [3H]thymidine for the synthesis of liver DNA thymine was decreased after the administration of a single dose of the drug. The depression of the specific activities of DNA pyrimidines of liver DNA in experimental groups was observed also after an injection of [14C]orotic acid. (b) A decreased incorporation of labeled thymidine had occurred also in the spleen during the prereplicative period. Thereafter the specific activity of DNA thymine was higher than in the control group. (c) The observed mitogenic response in the spleen showed a protracted effect; after the administration of a single dose of the drug the specific activity of DNA thymine as well as the thymidine kinase activity of spleen cytosol have been rising up to the ninth day. The same holds true for DNA thymine of the thymus; the activity of thymidine kinase was not affected. (d) Both the single and repeated doses of TFMPD had no marked effect on the levels of microsomal cytochromes P-450 and b5 in the liver and kidney.

Synthesis and cytotoxic effects of hydroxymethyl-3-pyridyl- and 2-chloro-5-pyridyltriazene derivatives.

3-Hydroxymethyl-3-methyl-1-(3-pyridyl)-triazene (III) and 3-hydroxymethyl-3-methyl-1-(2-chloro-5-pyridyl)-triazene (VI) were synthesized and their cytotoxic effects towards S180 cells compared with those of the corresponding dimethyltriazene and monomethyltriazene derivatives. The hydroxy-methyltriazenes were one to two orders of magnitude more inhibitory than the corresponding dimethyl analogs, as assessed by cell growth, colony forming and macromolecular synthesis assays. Comparable effects were observed with the related monomethyl triazenes indicating that the activity of (III) and (VI) could result, at least in part, from release of monomethyl derivatives. The corresponding dimethyltriazenes were much less toxic to S180 cells and exerted only an unspecific cytotoxic activity at the maximal attainable dose (5 X 10(-3) M). The cytotoxic activities of the tested triazenes were correlated with their chemical half-lives under near physiological conditions (pH 7.5).

Transnitrosating activity of N-nitroso-N-methyl-p-toluenesulfonamide in rats and human gastric juice.

N-nitroso-N-methyl-p-toluenesulfonamide (NMTS, diazald, CAS 80-11-5) is a widely used compound for laboratory production of diazomethane. The present results showed that the noncarcinogenic NMTS reacts as a transnitrosating agent with amino nitrogen of secondary amines and amide both in vitro (human gastric juice) and in vivo (rats) to yield N-nitroso compounds. Since all compounds formed (NMOR, NDMA, NPIP, NPZ, NMU) are known animal carcinogens, caution should be taken by users handling NMTS.

Reactions of substituted arenediazonium chlorides with methylamine-formaldehyde premix revisited: reactivity and transformations of methylolamine intermediates and their biological significance.

Modulation of the N-azo coupling between ring-substituted arenediazonium chlorides and premixed methylamine-formaldehyde leads not only to 1-aryl-3-hydroxymethyl-3-methyltriazenes and their dimers, but also to unexpected cyclic and complex products. The syntheses comprise reactions with arenediazonium chlorides bearing both -M and +M substituents at para and ortho/para positions of the phenyl ring. One of the major constituents isolated from a mixture of products is the O-acetate of 3-hydroxymethyl-3-methyl-1-(2,4,6-trichlorophenyl)triazene. This product was obtained from the reaction of 2,4,6-trichlorobenzenediazonium chloride and methylamine-formaldehyde mixture which was then stabilized by acetylation. The structures of the isolated products could be derived from reactive methylolamines and electrophilic intermediates that possible occur in vivo and thereby offer a plausible mechanistic explanation for the carcinogenic and tumour-inhibitory activity associated with the open-chain triazene compounds in the living cell.

In vivo metabolism and reaction with DNA of the cytostatic agent, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC).

The cytostatic drug dacarbazine [DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide] is strongly carcinogenic in rats. Bioactivation of DTIC yields a methylating intermediate but the extent of interaction with cellular macromolecules has not previously been reported. Following a single i.p. injection of [14C-methyl]DTIC, exhalation of 14CO2 occurred with a t1/2 max of approximately 2 hr (0.95 mg/kg) and 2.5 hr (95 mg/kg). Of the total radioactivity administered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predominantly as unchanged DTIC. In liver, kidney and lung, formation of 7-[14C]methylguanine in DNA and RNA was directly proportional with dose. DNA methylation by a single dose of DTIC (9.8 mg/kg; 5 hr survival time) was highest in liver (35 mumoles 7-methylguanine/mole guanine), followed by kidney (25 mumoles) and lung (20 mumoles). The remainder tissues showed 7-methylguanine concentrations approximately 50% of those in liver DNA, with the exception of the brain which had a very low extent of DNA modification (approximately 1 mumole/mole guanine). At the specific radioactivity used (48 mCi/mmole), the promutagenic base O6-methylguanine was only detectable in liver, kidney, lung, and stomach DNA (0.6-0.8 mumoles/mole guanine). Autoradiographic studies revealed a diffuse distribution of reaction products in rat liver. In contrast, N-nitrosodimethylamine and related carcinogens known to be bioactivated by the hepatic cytochrome P-450 system show a predominantly centrilobular distribution. This difference may be due to the greater stability of proximate carcinogens generated by alpha-C hydroxylation at one of the methyl groups of DTIC.

Carcinogenicity of cytostatic triazenes.

Introduction of the anticancer drug dacarbazine is the result of an attempt to design antagonists of purine biosynthesis. The mechanism of action of dacarbazine depends mainly on enzymatic transformation into as yet unknown reactive (electrophilic) intermediates. Recent studies have led to the identification and synthesis of 5-(3-hydroxymethyl-3-methyl)imidazole-4-carboxamide (HMTIC), a carbinolamine metabolite with methylating capacity. Although dacarbazine and related cytostatic triazenes are effective in the treatment of malignant melanoma and other human malignancies, dacarbazine has been demonstrated to be a carcinogen in laboratory rodents. Chronic administration of dacarbazine to rats of each sex induced predominantly thymic lymphosarcomas and mammary adenocarcinomas that were transplantable. Intraperitoneally injected 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC), a metabolite of dacarbazine, induced a high incidence of mammary adenofibromas and a low incidence of uterine leiomyosarcomas. Animals treated with 5-diazoimidazole-4-carboxamide developed a low incidence of thymic, stomach, bladder or mammary tumours. Animals receiving 5-aminoimidazole-4-carboxamide developed a variety of tumours. No secondary malignancy has been reported in humans after treatment with dacarbazine alone. Despite their adverse effects, dacarbazine and related cytostatic triazene derivatives are useful clinically since their haematological toxicity is relatively moderate. As a rule, they are not cross-resistant with nitrogen mustard alkylating agents. It is hoped that research and development of second-generation N-(1-hydroxyalkyl)triazene compounds will lead to improvements in their clinical efficacy.