DIFFERENT POTENCIES OF [ASP1, ILE5]- AND [ASN1 VAL5]-ANGIOTENSIN II IN STIMULATING ALDOSTERONE PRODUCTION FROM RAT ADRENAL ZONA GLOMERULOSA CELLS IN VITRO

1. Rat adrenal zona glomerulosa cells were incubated in Krebs Ringer bicarbonate medium containing bovine serum albumin. Aldosterone production was measured by radioimmunoassay. 2. Both [Asp¹, Ile⁵]-angiotensin II ('free acid') and (Asn¹, Val⁵]-angiotensin II ('amide'), (10^?5 mol/1), stimulated aldosterone output but the free acid compound produced a larger increase in steroid output than the amide. 3. This difference in steroidogenic activity could not be explained by differences in purity or initial concentration or by stimulation of the contaminating fasciculata cells. 4. However, serial bioassays of the media revealed that the amide form of angiotensin was degraded more rapidly than the free acid form. 5. The different potency of these two forms of angiotensin II could explain some of the reported discrepancies of the action of angiotensin II on rat adrenal tissue or cells in vitro.     

Nucleus accumbens facilitates nociception

We have previously demonstrated an opioid link in nucleus accumbens (NAc) that mediates antinociception produced by a novel ascending pain modulation pathway. For example, noxious stimulation induces heterosegmental antinociception that is mediated by both mu- and delta-opioid receptors in NAc. However, spinal intrathecal administration of the mu-receptor agonist [d-Ala², N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) also induces heterosegmental antinociception. The aim of the present study in the rat was to identify the intra-NAc opioid receptors that mediate the antinociceptive effects of spinally administered DAMGO and also to determine the effect of NAc efferent activity on nociception. Intra-NAc administration of either the mu-opioid receptor antagonist Cys²,Tyr³, Orn⁵,Pen⁷amide (CTOP) or the delta-opioid receptor antagonist naltrindole blocked the antinociceptive effect of spinally administered DAMGO on the jaw-opening reflex (JOR). Injection of quaternary lidocaine (QX-314) attenuated the JOR, suggesting that the output of NAc is pronociceptive. In support of this, intra-NAc injection of the excitatory amino acid agonist kainate enhanced the JOR. Thus, it is possible to modulate activity in NAc to bidirectionally attenuate or enhance nociception, suggesting a potential role for NAc in setting nociceptive sensitivity.     

Immobilized contrast‐enhanced MRI: Gadolinium‐based long‐term MR contrast enhancement of the vein graft vessel wall

An implantable MR contrast agent that can be covalently immobilized on tissue during surgery has been developed. The rationale is that a durable increase in tissue contrast using an implantable contrast agent can enhance postsurgical tissue differentiation using MRI. For small‐vessel (e.g., vein graft) MRI, the direct benefit of such permanent “labeling” of the vessel wall by modification of its relaxation properties is to achieve more efficient imaging. This efficiency can be realized as either increased contrast leading to more accurate delineation of vessel wall and lesion tissue boundaries, or, faster imaging without penalizing contrast‐to‐noise ratio, or a combination thereof. We demonstrate, for the first time, stable long‐term MRI enhancement using such an exogenous contrast mechanism based on immobilizing a modified diethylenetriaminepentaacetic acid gadolinium³⁺ dihydrogen complex on a human vein using a covalent amide bond. Signal enhancement due to the covalently immobilized contrast agent is demonstrated for excised human vein specimens imaged at 3 T, and its long‐term stability is demonstrated during a 4‐month incubation period. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Amide proton transfer imaging with improved robustness to magnetic field inhomogeneity and magnetization transfer asymmetry using saturation with frequency alternating RF irradiation

Amide proton transfer (APT) imaging has shown promise as an indicator of tissue pH and as a marker for brain tumors. Sources of error in APT measurements include direct water saturation, and magnetization transfer (MT) from membranes and macromolecules. These are typically suppressed by postprocessing asymmetry analysis. However, this approach is strongly dependent on B₀ homogeneity and can introduce additional errors due to intrinsic MT asymmetry, aliphatic proton features opposite the amide peak and radiation damping‐induced asymmetry. Although several methods exist to correct for B₀ inhomogeneity, they tremendously increase scan times and do not address errors induced by asymmetry of the z‐spectrum. In this article, a novel saturation scheme—saturation with frequency alternating RF irradiation (SAFARI)—is proposed in combination with a new magnetization transfer ratio (MTR) parameter designed to generate APT images insensitive to direct water saturation and MT, even in the presence of B₀ inhomogeneity. The feasibility of the SAFARI technique is demonstrated in phantoms and in the human brain. Experimental results show that SAFARI successfully removes direct water saturation and MT contamination from APT images. It is insensitive to B₀ offsets up to 180 Hz without using additional B₀ correction, thereby dramatically reducing scanning time. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

Ionic liquid salt bridge based on N-alkyl-N-methylpyrrolidinium bis(pentafluoroethanesulfonyl)amide for low ionic strength aqueous solutions

The phase-boundary potential between the moderately hydrophobic ionic liquid and a low ionic strength aqueous solution is demonstrated to be stable and constant with the standard deviation of 0.4 mV down to 20 μmol kg⁻¹ HBr, LiBr, and KBr solutions, for three ionic liquids that consist of either N-methyl-N-octylpyrrolidinium, N-heptyl-N-methylpyrrolidinium, or N-hexyl-N-methylpyrrolidinium and a common anion species, bis(pentafluoroethanesulfonyl)amide. This stability is promising for accurate measurements of pH of low ionic strength samples and reliable estimates of single ion activities in general. The phase-boundary potential deviates from the value determined by the partition of the ionic liquid in further dilute aqueous solutions. The magnitude of the deviation ranges from 3 to 11 mV at 5 μmol kg⁻¹ MBr (M is H⁺, Li⁺, or K⁺). The solubility of these ionic liquids in water is 0.2 mmol dm⁻³ at most at 25 °C, which is another advantage of ionic liquid salt bridge in electroanalytical chemistry.     

Morphology of Novel PEAs Containing Two Consecutive Amide Bonds Randomly Distributed Along the Polyester Backbone

The morphology of a series of novel segmented PEAs with varying amide content was studied by a combination of DSC, SFM and proton solid state NMR. Semicrystalline polymers organised in a lamellar morphology were obtained for all amide contents. The results of different NMR experiments revealed that a three phase system composed of a rigid phase, an interface and a mobile amorphous phase is the most appropriate model to describe the morphology of these materials. The amount, the chain dynamics and the domain size of each phase were estimated and correlated with the amount of the amide fraction. Such morphological investigations help to understand the macroscopic properties of segmented PEAs for their applications as biomaterials.

     

Genetic Deletion of Fatty Acid Amide Hydrolase Alters Emotional Behavior and Serotonergic Transmission in the Dorsal Raphe, Prefrontal Cortex, and Hippocampus

Pharmacological blockade of the anandamide-degrading enzyme, fatty acid amide hydrolase (FAAH), produces CB₁ receptor (CB₁R)-mediated analgesic, anxiolytic-like and antidepressant-like effects in murids. Using behavioral and electrophysiological approaches, we have characterized the emotional phenotype and serotonergic (5-HT) activity of mice lacking the FAAH gene in comparison to their wild type counterparts, and their response to a challenge of the CB₁R antagonist, rimonabant. FAAH null-mutant (FAAH^−/−) mice exhibited reduced immobility in the forced swim and tail suspension tests, predictive of antidepressant activity, which was attenuated by rimonabant. FAAH^−/− mice showed an increase in the duration of open arm visits in the elevated plus maze, and a decrease in thigmotaxis and an increase in exploratory rearing displayed in the open field, indicating anxiolytic-like effects that were reversed by rimonabant. Rimonabant also prolonged the initiation of feeding in the novelty-suppressed feeding test. Electrophysiological recordings revealed a marked 34.68% increase in dorsal raphe 5-HT neural firing that was reversed by rimonabant in a subset of neurons exhibiting high firing rates (33.15% mean decrease). The response of the prefrontocortical pyramidal cells to the 5-HT_2A/2C agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ((±)-DOI) revealed desensitized 5-HT_2A/2C receptors, likely linked to the observed anxiolytic-like behaviors. The hippocampal pyramidal response to the 5-HT_1A antagonist, WAY-100635, indicates enhanced tonus on the hippocampal 5-HT_1A heteroreceptors, a hallmark of antidepressant-like action. Together, these results suggest that FAAH genetic deletion enhances anxiolytic-like and antidepressant-like effects, paralleled by altered 5-HT transmission and postsynaptic 5-HT_1A and 5-HT_2A/2C receptor function.     

Neuropeptide FF distribution in the human and rat forebrain: a comparative immunohistochemical study

Neuropeptide FF (NPFF) is an octapeptide implicated in a variety of physiological functions, including nociception, cardiovascular responses, and neuroendocrine regulation. The NPFF gene and its mRNA are highly conserved across species. A comparative study of NPFF distribution in the human and rat forebrain was carried out by using single NPFF and double NPFF + vasopressin (VP) immunohistochemistry. NPFF is extensively localized within neurochemical circuits of human and rat forebrain. Semiquantitative analysis revealed that the densities of NPFF cells and fibers in many forebrain nuclei in the human correlate well with those observed for the same structures in the rat. High numbers of NPFF positive neurons in the dorsomedial hypothalamic nucleus and a dense plexus of NPFF fibers surrounding the fornix within the bed nucleus of the stria terminalis were identified in the human and rat forebrain. Within the hypothalamus of both species, dense NPFF innervation was observed in the perinuclear zone of the supraoptic nucleus (SO) just dorsolateral to the VP-positive neurons. Extensive NPFF innervation of ventricular ependyma and brain microvasculature were common for both species. At the same time, obvious differences in NPFF localization between the two species were also apparent. For example, in contrast to the rat SO, no NPFF- or NPFF- + VP-immunostained cells were observed in the human SO. Knowledge of NPFF neuroanatomical localization in the human brain and the relationship of these observations to those in the rat brain may provide insight into the role of this peptide in central cardiovascular and neuroendocrine regulation.     

Utility of the carboxylesterase inhibitor bis-para-nitrophenylphosphate (BNPP) in the plasma unbound fraction determination for a hydrolytically unstable amide derivative and agonist of the TGR5 receptor

1. The potent, functional agonist of the bile acid Takeda G-protein-coupled receptor 5 (TGR5), (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (3), represents a useful tool to probe in vivo TGR5 pharmacology. Rapid degradation of 3 in both rat and mouse plasma, however, hindered the conduct of in vivo pharmacokinetic/pharmacodynamic investigations (including plasma-free fraction (f_{u plasma}) determination) in rodent models of pharmacology. Studies were therefore initiated to understand the biochemical basis for plasma instability so that appropriate methodology could be implemented in in vivo pharmacology studies to prevent the breakdown of 3.

2. Compound 3 underwent amide bond cleavage in both rat and mouse plasma with half-lives (T_1/2) of 39?±?7 and 9.9?±?0.1?min. bis(p-nitrophenyl) phosphate (BNPP), a specific inhibitor of carboxylesterases, was found to inhibit hydrolytic cleavage in a time- and concentration-dependent manner, which suggested the involvement of carboxylesterases in the metabolism of 3. In contrast with the findings in rodents, 3 was resistant to hydrolytic cleavage in both dog and human plasma.

3. The instability of 3 was also observed in rat and mouse liver microsomes. β-Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent metabolism of 3 occurred more rapidly (T_1/2 approximately 2.22–6.4?min) compared with the metabolic component observed in the absence of the co-factor (T_1/2 approximately 89–130?min). Oxidative metabolism dominated the NADPH-dependent decline of 3, whereas NADPH-independent metabolism of 3 proceeded via simple amide bond hydrolysis.

4. Compound 3 was highly bound (approximately 95%) to both dog and human plasmas. Rat and mouse plasma, pre-treated with BNPP to inhibit carboxylesterases activity, were used to determine the f_{u plasma} of 3. A BNPP concentration of 500 μM was determined to be optimal for these studies. Higher BNPP concentrations (1000 μM) appeared to displace 3 from its plasma protein-binding sites in preclinical species and human. Under the conditions of carboxylesterases-inhibited rat and mouse plasma, the level of protein binding displayed by 3 was similar to those observed in dog and human.

5. In conclusion, a novel system has been devised to measure f_{u plasma} for a plasma-labile compound. The BNPP methodology can be potentially applied to stabilize hydrolytic cleavage of structurally diverse carboxylesterase substrates in the plasma (and other tissue), thereby allowing the characterization of pharmacology studies on plasma-labile compounds if and when they emerge as hits in exploratory drug-discovery programmes.

     

Optimized prodrug approach: A means for achieving enhanced anti-inflammatory potential in experimentally induced colitis

In the present study, prodrug of ketoprofen was synthesized and evaluated in vitro to optimize the prodrug, and in vivo to observe the reduction in gastrointestinal disturbance and enhanced colonic anti- inflammatory potential for the prodrug. The prodrug was synthesized by coupling ketoprofen with l-glycine (KET-GLY). In vitro reversion of KET-GLY to ketoprofen was carried out in different pHs and in pH 6.8 containing optimized rat fecal material. In vivo healing potential of KET-GLY was evaluated in acetic acid-induced experimental colitis model. In vitro reversion studies suggested that KET-GLY remained intact in stomach but released the free drug at pH 6.8 containing fresh rat fecal material, where the colonic microfloral enzymes (amidase) hydrolyzed the KET-GLY amide linkage, releasing the free drug. In vivo evaluation indicated KET-GLY to be less toxic in stomach, with enhanced anti-inflammatory potential in the colonic region. These findings suggested KET-GLY to be better in action compared with the parent drug.