The novel phosphodiesterase 4 inhibitor, CI-1044, inhibits LPS-induced TNF-alpha production in whole blood from COPD patients.

Chronic obstructive pulmonary disease (COPD) is a common, progressive respiratory disease that causes great morbidity and mortality despite treatment. Tumor necrosis factor alpha (TNF-alpha) plays a central role as a pro-inflammatory cytokine in COPD. TNF-alpha release is markedly inhibited by phosphodiesterase type 4 (PDE4) inhibitors that have proven efficacious in COPD clinical trials. The aim of this study was to compare the in vitro activities of the novel selective PDE4 inhibitors CI-1044 compared to well-known PDE4 inhibitors, rolipram and cilomilast, and to the glucocorticoid dexamethasone at reducing lipopolysaccharide (LPS)-induced TNF-alpha release in whole blood from COPD patients and healthy subjects. In the whole blood from COPD patients pre-incubation with PDE4 inhibitors or dexamethasone resulted in a dose-dependent inhibition of LPS-induced TNF-alpha release with IC(50) values of 1.3+/-0.7, 2.8+/-0.9 microM, higher to 10 microM and lesser than 0.03 microM for CI-1044, rolipram, cilomilast and dexamethasone, respectively. We observed a similar inhibition in the whole blood from healthy volunteers with, however, higher IC(50) values. These results indicate that CI-1044 inhibits in vitro LPS-induced TNF-alpha release in whole blood from COPD patients better than rolipram and cilomilast and suggested that it could be a useful anti-inflammatory therapy in COPD.

Synthesis, structure-activity relationships, and pharmacological profile of 9-amino-4-oxo-1-phenyl-3,4,6,7-tetrahydro[1,4]diazepino[6, 7,1-hi]indoles: discovery of potent, selective phosphodiesterase type 4 inhibitors.

The synthesis, structure-activity relationships, and biological properties of a novel series of potent and selective phosphodiesterase type 4 (PDE4) inhibitors are described. These new aminodiazepinoindoles displayed in vitro PDE4 activity with submicromolar IC(50) values and PDE4 selectivity vs PDE1, -3, and -5. Specifically, one compound (CI-1044, 10e) provided efficient in vitro inhibition of TNFalpha release from hPBMC and hWB with IC(50) values of 0.34 and 0.84 microM, respectively. This compound was found to exhibit potent in vivo activity in antigen-induced eosinophil recruitment in Brown-Norway rats (ED(50) = 3.2 mg/kg po) and in production of TNFalpha in Wistar rats (ED(50) = 2.8 mg/kg po). No emetic side effects at therapeutic doses were observed in ferrets.

Synthesis and structure-activity relationships of 4-oxo-1-phenyl-3,4,6,7-tetrahydro-[1,4]diazepino[6,7,1-hi]indoles: novel PDE4 inhibitors.

A novel series of benzodiazepine derivatives have been discovered as inhibitors of PDE4 enzymes. We have found that our compounds are selective versus other PDE enzymes, and that the activity can be modulated by specific structural modifications. One compound exhibited a strong eosinophilic infiltration inhibiting action on sensitized Brown-Norway rats (compound 9, 5.1 mg/kg p.o.), moreover this compound is not emetic at 3 mg/kg i.v.

A simple, sensitive method of analyzing bacterial ribosomal DNA polymorphism.

A rapid DNA extraction procedure and random primer labelling of Escherichia coli ribosomal RNA with cloned reverse transcriptase have been used to establish a simple and highly sensitive method for studying ribosomal DNA polymorphism in bacteria. Examples of inter- and intraspecies differentiation of bacteria are given and potential applications in bacterial epidemiology and taxonomy are discussed.

Retrieval of precursors for white-type adipose conversion in brown adipose tissue.

A cellular compartment from brown adipose tissue (BAT) of newborn rats was isolated by Percoll-density-gradient centrifugation and was shown to proliferate and to undergo adipose conversion in vitro in primary culture. The features of the effector requirement for adipose conversion as well as the differentiated morphological and biochemical phenotype are almost identical with that of a compartment designated HCF, from white adipose tissue (WAT). A possible role for these precursors from BAT and WAT in the involution of BAT into WAT, on the one hand, and in the development of brown adipose cells among typical WAT deposits, on the other, is discussed.

A simple strategy to amplify specifically the HLA-DQ beta gene region with genomic DNA as template.

The nature of codon 57 in the HLA-DQ beta gene was recently reported as a potential marker of genetic susceptibility to insulin-dependent diabetes mellitus. When exploring the relevance of this marker by using genomic DNA amplification, we encountered difficulties resulting from the coamplification of the homologous DX beta region. A simple strategy is proposed to amplify the DQ beta region exclusively. It involves the preliminary digestion of genomic DNA with a restriction enzyme which cleaves DX beta specifically, leaving intact the DQ beta sequence. The amplified material is suitable for dot blot analysis and restriction enzyme digestion. This strategy is of general interest when homologous sequences impair the specificity of enzymatic DNA amplification.

Response of the exocrine pancreas to quantitative and qualitative variations in dietary lipids.

In the rat, pancreatic amylase and, to a lesser extent, lipase adapt quantitatively to the amount of their respective substrates in the diet by an increase in specific activity and total contents (range of variation, fivefold for amylase and twofold for lipase). Colipase responded to protein intake (r = 0.85, P less than 0.01) and not to lipids provided protein intake was below 3.5 g or above 6.0 g. With this latter amount of protein, a maximal level was obtained, even with 2% lipid in the diet. Between 3.5 and 6.0 g, lipid intake was found to modulate colipase in parallel with lipase. When different types of fat were compared, the degree of saturation was found to have no impact on lipase, colipase, and amylase. Diets containing medium-chain triglycerides (C8-C10) did not maximally increase specific activity and total content of lipase and colipase, whereas they did not repress amylase as much longer chain triglycerides did. With coconut oil (45% C12), lipase was maximally stimulated but amylase was not maximally repressed, showing that the regulation of the hydrolases may be partly reciprocal and partly independent.

Comparative effects of some carbohydrates on serum sugars, triglycerides and digestive hydrolases.

For 3 weeks, rats were fed diets containing 60 p. 100 carbohydrate in the form of starch (wheat flour), purified sucrose, commercial sugar or a commercial sweetener containing a mixture of glucose and fructose. Glycemia was lower during the day than at night, and it was lowest in the starch-fed group. Fructosemia, high in all groups during the day, suggested endogenous production; it was low at night, showing efficient clearance of exogenous fructose. Triglyceridemia was highest in the rats fed purified sucrose and exhibited no light/dark variation in that group. It was higher in all the other groups during the day. Regarding pancreatic hydrolases, starch, rather than sugars, raised pancreatic amylase, while lipase did not correlate with endogenous hyperglyceridemia and was similar in all groups. Commercial preparations significantly lowered chymotrypsinogen contents. These results confirm that sucrose and equimolar mixtures of glucose and fructose are not equivalent (disaccharide effect). The data evidence an endogenous fructose production during the day and suggest that commercial sugar, often used in the preparation of diets, may have different effects than purified sucrose.

Differential regulation of lipase and colipase in the rat pancreas by dietary fat and proteins.

The adaptative response of the exocrine pancreas to diets rich in lipids and the influence of dietary proteins are reconsidered taking into account colipase. This was particularly necessary in the rat since colipase is often limiting with respect to lipase in this species. In a first experiment, adult male rats received diets containing 10, 15, 18, 40 or 86% casein together with 2, 8 or 40% lard. Under these conditions, lipase was found to be sensitive to variations in the lipid contents of the diet and was two-fold higher with 40% than with 8% lipid, in diets containing 40% casein. Proteins exerted a permissive effect since no response was recorded with diets containing less than 18% casein. Colipase responded to protein intake, even in lipid-poor diets (2% lard), and was increased by a factor 3 when casein was raised from 18 to 40%. In a second experiment, proteins were fed as a separate meal, and the remainder of the diet (provided ad libitum) contained either 8 or 40% lard. In a diurnal study, lipase and colipase were followed every 3 hours over 24 hours. Both lipase and colipase were found to accumulate after the protein meal. Colipase was found to accumulate much faster than lipase in all cases and reach 3 times basal levels 9 hours after the protein meal. This resulted in important diurnal variations in the ratio of colipase to lipase which modulates lipolytic activity. It is concluded that colipase is particularly sensitive to protein intake, perhaps more than to lipid intake and may become and limiting factor of lipid digestion.