Prolyl hydroxylase inhibition corrects functional iron deficiency and inflammation-induced anaemia in rats.

Background And Purpose: Small-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated.

Experimental Approach: JNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model].

Effects of meal size on the release of GLP-1 and PYY after Roux-en-Y gastric bypass surgery in obese subjects with or without type 2 diabetes.

Changes in gastrointestinal peptide release may play an important role in improving glucose control and reducing body weight following Roux-en-Y gastric bypass (RYGB), but the impact of low caloric intake on gut peptide release post-surgery has not been well characterized. The purpose of this study was to assess the relationships between low caloric intake and gut peptide release and how they were altered by RYGB. Obese females including ten normoglycemic (ON) and ten with type 2 diabetes mellitus (T2DM) (OD) were studied before, 1 week, and 3 months after RYGB.

Roux-en-Y gastric bypass corrects hyperinsulinemia implications for the remission of type 2 diabetes.

Context: Roux-en-Y gastric bypass (RYGB) has been shown to induce rapid and durable reversal of type 2 diabetes.

Objective: The aim of the study was to investigate a possible mechanism for the remission of type 2 diabetes after RYGB.

Design: A cross-sectional, nonrandomized, controlled study was conducted.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide]: a novel, potent, and selective cholecystokinin 2 receptor antagonist with good oral bioavailability.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide] is a representative of a new chemical class of competitive antagonists of cholecystokinin 2 (CCK2) receptors. In this study, the primary in vitro pharmacology of JNJ-26070109 was evaluated along with the pharmacokinetic and pharmacodynamic properties of this compound in rat and canine models of gastric acid secretion. JNJ-26070109 expressed high affinity for human (pK(I) = 8.

Pharmacological characterization of 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), a potent and selective hypoxia-inducible factor prolyl hydroxylase inhibitor.

The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structure-based drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and whole-animal systems and was compared with other compounds described in the literature as PHD inhibitors.

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

One hundred years of histamine research.

In this introductory chapter, we revisit some of the landmarks in the history of histamine research. Since histamine was first synthesized (1907) and isolated as a bacterial contaminant of an extract of ergot (1910), the elucidation of its role in health and disease and its molecular mechanism of action have been continuous, reflecting the application of advances in scientific knowledge, technology and therapeutics over the last 100 years. It appears that the research will continue indefinitely as the nature of the problem is inherently fractal.

Functional Characterization of the Canine Heme-Regulated eIF2alpha Kinase: Regulation of Protein Synthesis.

The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2alpha (eIF2alpha) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase.

Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase.

A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: tuning of receptor selectivity and in vivo efficacy.

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists I: discovery of CCKR1 selectivity in a previously CCKR2-selective lead series.

A series of CCK2R-selective anthranilic amides is shown to derive CCK1R affinity via selective substitution of the amide side chain. Thus, extending the length of the original benzamide side chain by a single methylene unit imparts CCK1R affinity to the series, and further fine tuning of the affinity results in CCK1R selectivity of greater than 100-fold.

Characterization of a robust enzymatic assay for inhibitors of 2-oxoglutarate-dependent hydroxylases.

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1alpha for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2(181-417)), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1alpha, with succinic acid produced as an end product, radiolabeled [5-(14)C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2(181-417) enzymatic activity.

Discovery of potent cholecystokinin-2 receptor antagonists: elucidation of key pharmacophore elements by X-ray crystallographic and NMR conformational analysis.

A novel series of cholecystokinin-2 receptor (CCK-2R) antagonists has been identified, as exemplified by anthranilic sulfonamide 1 (pK(i)=7.6). Pharmacokinetic and stability studies indicated that this series of compounds suffered from metabolic degradation, and that both the benzothiadiazole and piperidine rings were rapidly oxidized by liver enzymes.

Synthesis and solid-phase purification of anthranilic sulfonamides as CCK-2 ligands.

A novel strategy for the synthesis of cholecystokinin-2 receptor ligands was developed. The route employs a solution-phase synthesis of a series of anthranilic sulfonamides followed by a resin capture purification strategy to produce multi-milligram quantities of compounds for bioassay. The synthesis was used to produce >100 compounds containing various functional groups, highlighting the general applicability of this strategy and to address specific metabolism issues in our CCK-2 program.

SAR studies of 1,5-diarylpyrazole-based CCK1 receptor antagonists.

A high throughput screening campaign revealed compound 1 as a potent antagonist of the human CCK(1) receptor. Here, we report the syntheses and SAR studies of 1,5-diarylpyrazole analogs with various structural modifications of the alkane side chain of the molecule. The difference in affinity between the two enantiomers for the CCK(1) receptor and the flexible nature of the linker led to the design of constrained analogs with increased potency.

Pharmacology and antitussive efficacy of 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), a transient receptor potential vanilloid 1 antagonist in guinea pigs.

Transient receptor potential vanilloid 1 (TRPV1) plays an integral role in modulating the cough reflex, and it is an attractive antitussive drug target. The purpose of this study was to characterize a TRPV1 antagonist, 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), against the guinea pig TRPV1 receptor in vitro followed by a proof-of-principle study in an acid-induced model of cough. The affinity of JNJ17203212 for the recombinant guinea pig TRPV1 receptor was estimated by radioligand binding, and it was functionally characterized by antagonism of low-pH and capsaicin-induced activation of the ion channel (fluorometric imaging plate reader and electrophysiology).

3-[5-(3,4-Dichloro-phenyl)-1-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-2-m-tolyl-propionate (JNJ-17156516), a novel, potent, and selective cholecystokinin 1 receptor antagonist: in vitro and in vivo pharmacological comparison with dexloxiglumide.

3-[5-(3,4-Dichloro-phenyl)-1-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-2-m-tolyl-propionate (JNJ-17156516) is a novel, potent, and selective cholecystokinin (CCK)1-receptor antagonist. In this study, the pharmacology of JNJ-17156516 was investigated both in vitro and in vivo, and the pharmacokinetic profile was evaluated in rats. JNJ-17156516 expressed high-affinity at the cloned human (pK(I) = 7.

Obestatin reduces food intake and suppresses body weight gain in rodents.

Obestatin was recently described as a bioactive peptide encoded for by the same gene as ghrelin but with opposite actions on food intake. Although some groups have confirmed these findings others find no effect. We investigated the effect of obestatin on feeding in rodents over a wide range of doses.

Identification and optimization of anthranilic sulfonamides as novel, selective cholecystokinin-2 receptor antagonists.

A high throughput screening approach to the identification of selective cholecystokinin-2 receptor (CCK-2R) ligands resulted in the discovery of a novel series of antagonists, represented by 1-[2-[(2,1,3-benzothiadiazol-4-ylsulfonyl)amino]-5-chlorobenzoyl]-piperidine (1; CCK-2R, pK(I) = 6.4). Preliminary exploration of the structure-activity relationships around the anthranilic ring and the amide and sulfonamide moieties led to a nearly 50-fold improvement of receptor affinity and showed a greater than 1000-fold selectivity over the related cholecystokinin-1 receptor.

Neuromedin U stimulates contraction of human long saphenous vein and gastrointestinal smooth muscle in vitro.

The neuropeptide Neuromedin U (NMU) stimulates smooth muscle contraction, and modulates local blood flow and adrenocortical function via two endogenous receptors, NMU1 and NMU2. Although its amino-acid sequence is highly conserved across species, the physiological effects of NMU are variable between species and little is known of its effects on human tissues. We have examined the contractile effects of NMU-25 on human smooth muscles of the gastrointestinal (GI) tract (ascending colon, gallbladder) and long saphenous vein (LSV) using in vitro organ bath bioassays.

Species-dependent smooth muscle contraction to Neuromedin U and determination of the receptor subtypes mediating contraction using NMU1 receptor knockout mice.

The peptide ligand neuromedin U (NMU) has been implicated in an array of biological activities, including contraction of uterine, intestinal and urinary bladder smooth muscle. However, many of these responses appear to be species-specific. This study was undertaken to fully elucidate the range of smooth muscle-stimulating effects of NMU in rats, mice and guinea-pigs, and to examine the extent of the species differences.

Advanced qRT-PCR technology allows detection of the cholecystokinin 1 receptor (CCK1R) expression in human pancreas.

Objectives: To help clarify the controversy over the detection of expression of the cholecystokinin 1 receptor (CCK1R; CCKAR) in human pancreas.

Methods: Applied qRT-PCR to detect CCK1R expression using the SYBR green/Smart Cycler II and the QZyme oligonucleotide/ABI PRISM 7500 systems to detect CCK1R expressed message in highly purified cDNAs from human pancreas and other tissues. Samples of normal pancreas were obtained at operation (pancreaticoduodenectomy; Whipple's procedure) and used to ascertain the expression of CCK1R in human tissue and investigate donor individual variability in expression levels by semi-quantitative RT-PCR and scanning densitometry.

Optimization of the in vitro and in vivo properties of a novel series of 2,4,5-trisubstituted imidazoles as potent cholecystokinin-2 (CCK2) antagonists.

The systematic optimization of the structure of a novel 2,4,5-trisubstituted imidazole-based cholecystokinin-2 (CCK(2)) receptor antagonist afforded analogues with nanomolar receptor affinity. These compounds were now comparable in their potency to the bicyclic heteroaromatic-based compounds 5 (JB93182) and 6 (JB95008), from which the initial examples were designed using a field-point based molecular modeling approach. They were also orally active as judged by their inhibition of pentagastrin stimulated acid secretion in conscious dogs, in contrast to the bicyclic heteroaromatic-based compounds, which were ineffective because of biliary elimination.