CNS access of selected H3-antagonists: ex vivo binding study in rats

Inflammation Research -     

Synthesis and biological assays of new H3-antagonists with imidazole and imidazoline polar groups

New histamine H3-receptor antagonists were synthesised and tested on rat brain membranes and on electrically stimulated guinea-pig ileum. The new compounds have a central polar group represented by a 2-alkylimidazole or a 2-thioimidazoline nucleus. The effect of the polar group basicity on the optimal length of the alkyl chain, connecting this group to a 4(5)-imidazolyl ring, was investigated. The best affinity values, obtained by displacement of [3H]-RAMHA from rat brain, were obtained for the 2-alkylimidazole derivatives (2a-f) with tetramethylene chain (pKi 8.03-8.97), having an intermediate basicity between that of the previously reported 2-thioimidazoles (1a-i) and that of 2-alkylthioimidazolines (3a-h). In contrast, a general lowering of affinity (pKi 5.90-7.63) was observed for compounds of the last series (3a-h), with a complex dependence on the terminal lipophilic group and chain length.     

Biphenyl-3-yl alkylcarbamates as fatty acid amide hydrolase (FAAH) inhibitors: steric effects of N-alkyl chain on rat plasma and liver stability

Secondary alkylcarbamic acid biphenyl-3-yl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which include the reference compounds URB597 and URB694. Given the intrinsic reactivity of the carbamate group, the in vivo potency of these molecules in rats is strongly affected by their hydrolysis in plasma or hepatic metabolism. In the present study, in vitro chemical and metabolic stability assays (rat plasma and rat liver S(9) fraction) were used to investigate the structure-property relationships (SPRs) for a focused series of title compounds, where lipophilicity and steric hindrance of the carbamate N-substituent had been modulated. The resulting degradation rates indicate that a secondary or tertiary alkyl group at the carbamate nitrogen atom increases hydrolytic stability towards rat plasma esterases. The calculated solvent accessible surface area (SASA) of the carbamate fragment was employed to describe the differences observed in rate constants of hydrolysis in rat plasma (log k(plasma)), suggesting that stability in plasma increases if the substituent exerts a shielding effect on the carbamate carbonyl. Stability in rat liver S(9) fraction is increased when a tertiary carbon is bound to the carbamate nitrogen atom, while other steric effects showed complex relationships with degradation rates. The SPRs here described may be applied at the pharmacokinetic optimization of other classes of carbamate FAAH inhibitors.     

Development and validation of a LC-MS method with electrospray ionization for the determination of the imidazole H3 antagonist ROS203 in rat plasma

A rapid, simple and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the determination of the imidazole H(3) antagonist ROS203 in rat plasma, using the superior homologue ROS287 as internal standard. Analyses were performed on an Agilent 1100 Series HPLC system employing a Supelco Ascentis C(18) column and isocratic elution with acetonitrile-10mM ammonium acetate buffer pH 4.0 (30:70, v/v) at a flow rate of 0.25 mL/min. An Applied Biosystems/MDS Sciex 150-EX single quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. Plasma samples were deproteinized with acetonitrile (1:2), evaporated under nitrogen stream, reconstituted in the mobile phase and 5 microL were injected into the system. The retention times of ROS203 and IS were 2.20 and 2.90 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 2610-2.61 ng/mL with determination coefficients >0.99. The lower limit of quantification (LLOQ) was 2.61 ng/mL. The accuracy of the method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 9.50% or 7.19%, respectively. The applicability of the LC-MS method was tested employing plasma samples obtained after i.p. administration of ROS203 to female Wistar rats to support a behavioral in vivo study. The specificity of the method was confirmed by the absence of interferences from endogenous substances. The reported method can provide the necessary sensitivity, linearity, precision, accuracy and specificity to allow the determination of ROS203 in rat plasma samples to support further pharmacokinetic assays.     

MRI patterns of invasive lobular cancer: T1 and T2 features

Purpose

The aim of this study was to describe magnetic resonance imaging (MRI) patterns in 21 patients with histologically proven invasive lobular cancer (ILC) of the breast.

Materials and methods

We retrospectively reviewed MR images of 21 out of 24 women with ILC of the breast. Three women were excluded from the study because they underwent neoadjuvant chemotherapy after MRI. Thirteen of the 24 women had positive clinical findings. All 24 patients underwent mammography, sonography and MRI. MRI was performed to evaluate disease extent and multifocality/multicentricity before modified radical mastectomy (n=5) or quadrantectomy (n=16). Two experienced radiologists reviewed the MRI scans and described the tumour patterns.

Results

We identified five morphological patterns of ILC: a solitary mass with irregular margins (n=8); a mass with smooth margins (n=5); multiple small enhancing foci with interconnecting enhancing strands (n=4); dominant lesion surrounded by small foci (n=3); one MR examination was negative.

Conclusions

Architectural and dynamic features are important in the interpretation of breast MRI findings. ILC may be detected on MRI as solitary or multiple lesions that correspond to tumour morphology on pathologic examination. False-negative MRI findings do occur in a small percentage of ILC.     

Size assessment of breast lesions by means of a computer-aided detection (CAD) system for magnetic resonance mammography

Purpose

The aim of this study was to investigate the efficacy of a dedicated software tool for automated volume measurement of breast lesions in contrast-enhanced (CE) magnetic resonance mammography (MRM).

Material and methods

The size of 52 breast lesions with a known histopathological diagnosis (three benign, 49 malignant) was automatically evaluated using different techniques. The volume of all lesions was measured automatically (AVM) from CE 3D MRM examinations by means of a computer-aided detection (CAD) system and compared with the size estimates based on maximum diameter measurement (MDM) on MRM, ultrasonography (US), mammography and histopathology.

Results

Compared with histopathology as the reference method, AVM understimated lesion size by 4% on average. This result was similar to MDM (3% understimation, not significantly different) but significantly better than US and mammographic lesion measurements (24% and 33% size underestimation, respectively).

Conclusions

AVM is as accurate as MDM but faster. Both methods are more accurate for size assessment of breast lesions compared with US and mammography.     

Structure-property relationships on histamine H3-antagonists: binding of phenyl-substituted alkylthioimidazole derivatives to rat plasma proteins

The binding of a series of H3-antagonists to rat plasma proteins was investigated by dialysis experiments, with RP-HPLC measurement of the free ligand. The series was composed of 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles having, on the phenyl ring, meta- and para-substituents, with different physico-chemical characteristics. As high protein binding had been proposed as being one of the features limiting brain access for the reference H3-antagonist thioperamide, the title series was employed to test the possibility of achieving lower protein binding by modulation of lipophilicity, while maintaining good receptor affinity. The compounds tested showed quotas of bound drug ranging from 60 to 97.5%, while for thioperamide a 78% bound drug quota was observed at high total concentrations, with a steep increase in bound percentage at lower concentrations. Two of the tested compounds, having a carboxamide substituent, showed lower protein binding compared to thioperamide over a wide range of total concentration, without a significant loss in affinity with respect to the parent compound. A strict dependence of protein binding on lipophilicity was observed, and a QSPR model was derived which could also account for the protein binding observed for thioperamide, while receptor affinity had been reported to be quite insensitive to phenyl ring substitution. It is therefore possible to modulate protein binding of these H3-antagonists, through lipophilicity adjustment, without losing receptor affinity; this finding could help in the design of new compounds with improved brain access.     

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of α–β-heteromeric glycine receptors. Glycine receptors were also activated by taurine and β-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the β subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (∼13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor β subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.