Identification of optimal reference genes for gene expression studies in a focal cerebral ischaemia model-Spatiotemporal effects.

A proper reference gene (RG) is required to reliably measure mRNA levels in biological samples via quantitative reverse transcription PCR (RT-qPCR). Various experimental paradigms require specific and stable RGs. In studies using rodent models of brain ischaemia, a variety of genes, such as ß-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (Hprt1), peptidyl-propyl isomerase A (Ppia) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh), are used as RGs. However, most of these genes have not been validated in specific experimental settings. The aim of this study was to evaluate the time- and brain region-dependent expression of RG candidates in a rat model of transient middle cerebral artery occlusion (tMCAO). The following genes were selected: Actb, Hprt1, Ppia, Gapdh, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (Ywhaz) and beta-2 microglobulin (B2m). Focal cerebral ischaemia was induced by 90 min of tMCAO in male Sprague-Dawley rats. Expression was investigated at four time points (12 and 24 h; 3 and 7 days) and in three brain areas (the frontal cortex, hippocampus and dorsal striatum) within the ischaemic brain hemisphere. The RT-qPCR results were analysed using variance analysis and the ΔCt, GeNorm, NormFinder and BestKeeper methods. Data from these algorithms were ranked using the geometric mean of ranks of each analysis. Ppia, Hprt1 and Ywhaz were the most stable genes across the analysed brain areas and time points. B2m and Actb exhibited the greatest fluctuations, and the results for Gapdh were ambiguous.

The Slow-Releasing and Mitochondria-Targeted Hydrogen Sulfide (H2S) Delivery Molecule AP39 Induces Brain Tolerance to Ischemia.

Ischemic stroke is the third leading cause of death in the world, which accounts for almost 12% of the total deaths worldwide. Despite decades of research, the available and effective pharmacotherapy is limited. Some evidence underlines the beneficial properties of hydrogen sulfide (H2S) donors, such as NaSH, in an animal model of brain ischemia and in in vitro research; however, these data are ambiguous. This study was undertaken to verify the neuroprotective activity of AP39, a slow-releasing mitochondria-targeted H2S delivery molecule. We administered AP39 for 7 days prior to ischemia onset, and the potential to induce brain tolerance to ischemia was verified. To do this, we used the rat model of 90-min middle cerebral artery occlusion (MCAO) and used LC-MS/MS, RT-PCR, LuminexTM assays, Western blot and immunofluorescent double-staining to determine the absolute H2S levels, inflammatory markers, neurotrophic factor signaling pathways and apoptosis marker in the ipsilateral frontal cortex, hippocampus and in the dorsal striatum 24 h after ischemia onset. AP39 (50 nmol/kg) reduced the infarct volume, neurological deficit and reduced the microglia marker (Iba1) expression. AP39 also exerted prominent anti-inflammatory activity in reducing the release of Il-1ß, Il-6 and TNFα in brain areas particularly affected by ischemia. Furthermore, AP39 enhanced the pro-survival pathways of neurotrophic factors BDNF-TrkB and NGF-TrkA and reduced the proapoptotic proNGF-p75NTR-sortilin pathway activity. These changes corresponded with reduced levels of cleaved caspase 3. Altogether, AP39 treatment induced adaptative changes within the brain and, by that, developed brain tolerance to ischemia.

High-Energy Ball Milling as Green Process To Vitrify Tadalafil and Improve Bioavailability.

In this study, the suitability of high-energy ball milling was investigated with the aim to vitrify tadalafil (TD) and improve its bioavailability. To achieve this goal, pure TD as well as binary mixtures composed of the drug and Soluplus (SL) were coprocessed by high-energy ball milling. Modulated differential scanning calorimetry (MDSC) and X-ray powder diffraction (XRD) demonstrated that after such coprocessing, the crystalline form of TD was transformed into an amorphous form. The presence of a single glass transition (Tg) for all the comilled formulations indicated that TD was dispersed into SL at the molecular level, forming amorphous molecular alloys, regardless of the drug concentration. The high values of Tg determined for amorphous formulations, ranging from 70 to 147 °C, foreshow their high stability during storage at room temperature, which was verified by XRD and MDSC studies. The stabilizing effect of SL on the amorphous form of TD in comilled formulations was confirmed. Dissolution tests showed immediate drug release with sustained supersaturation in either simulated gastric fluid of pH 1.2 or in phosphate buffer of pH 7.2. The beneficial effect of both amorphization and coamorphization on the bioavailability of TD was found. In comparison to aqueous suspension, the relative bioavailability of TD was only 11% for its crystalline form and 53% for the crystalline physical mixture, whereas the bioavailability of milled amorphous TD and the comilled solid dispersion was 128% and 289%, respectively. Thus, the results provide evidence that not only the presence of polymeric surfactant but also the vitrification of TD is necessary to improve bioavailability.

Sensitive and precise HPLC method with back-extraction clean-up step for the determination of sildenafil in rat plasma and its application to a pharmacokinetic study.

A sensitive HPLC method was developed and validated for the determination of sildenafil concentrations in rat plasma (200 µL) using a liquid-liquid extraction procedure and paroxetine as an internal standard. In order to eliminate interferences and improve the peak shape, a back-extraction into an acidic solution was utilized. Chromatographic separation was achieved on a cyanopropyl bonded-phase column with a mobile phase composed of 50 m m potassium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (75:25, v/v), pumped at the flow rate of 1 mL/min. A UV detector was set at 230 nm. A calibration curve was constructed within a concentration range from 10 to 1500 ng/mL. The limit of detection was 5 ng/mL. The inter- and intra-day precisions of the assay were in the ranges 2.91-7.33 and 2.61-6.18%, respectively, and the accuracies for inter- and intra-day runs were within 0.14-3.92 and 0.44-2.96%, respectively. The recovery of sildenafil was 85.22 ± 4.54%. Tests confirmed the stability of sildenafil in plasma during three freeze-thaw cycles and during long-term storage at -20 and -80°C for up to 2 months. The proposed method was successfully applied to a pharmacokinetic study in rats.

Benzophenone-3 Passes Through the Blood-Brain Barrier, Increases the Level of Extracellular Glutamate, and Induces Apoptotic Processes in the Hippocampus and Frontal Cortex of Rats.

Benzophenone-3 is the most commonly used UV filter. It is well absorbed through the skin and gastrointestinal tract. Its best-known side effect is the impact on the function of sex hormones. Little is known about the influence of BP-3 on the brain. The aim of this study was to show whether BP-3 crosses the blood-brain barrier (BBB), to determine whether it induces nerve cell damage in susceptible brain structures, and to identify the mechanism of its action in the central nervous system. BP-3 was administered dermally during the prenatal period and adulthood to rats. BP-3 effect on short-term and spatial memory was determined by novel object and novel location recognition tests. BP-3 concentrations were assayed in the brain and peripheral tissues. In brain structures, selected markers of brain damage were measured. The study showed that BP-3 is absorbed through the rat skin, passes through the BBB. BP-3 raised oxidative stress and induced apoptosis in the brain. BP-3 increased the concentration of extracellular glutamate in examined brain structures and changed the expression of glutamate transporters. BP-3 had no effect on short-term memory but impaired spatial memory. The present study showed that dermal BP-3 exposure may cause damage to neurons what might be associated with the increase in the level of extracellular glutamate, most likely evoked by changes in the expression of GLT-1 and xCT glutamate transporters. Thus, exposure to BP-3 may be one of the causes that increase the risk of developing neurodegenerative diseases.

The effects of benzophenone-3 on apoptosis and the expression of sex hormone receptors in the frontal cortex and hippocampus of rats.

Benzophenone-3 (BP-3) is the most commonly used chemical UV filter. This compound can easily be absorbed through the skin and the gastrointestinal tract and can disturb sex hormone receptor function. BP-3 is lipophilic and should cross the blood-brain barrier and it may reduce the survival of neurons, although so far, its effects on nerve cells have been studied in only in vitro cultures. The aim of the present study was to determine the effects of BP-3 on apoptosis and the expression of oestrogen, androgen and arylhydrocarbon receptors (AhR) in the rat frontal cortex and hippocampus. This compound was administered dermally to female rats during pregnancy and next to their male offspring through 6 and 7 weeks of age. BP-3 in the frontal cortex induced the mitochondrial apoptosis pathway by increasing the active forms of caspase-3 and caspase-9, inducing the pro-apoptotic proteins Bax and Bak and increasing the number of cells with apoptotic DNA fragmentation. In the hippocampus, an increase in the caspase-9 level and a downward trend in the level of anti-apoptotic proteins were observed. In both brain regions, the contents of ERß in the nuclear fraction and GPR30 in the membrane fraction were significantly reduced. BP-3 significantly increased AhR in the cytosol of the frontal cortex but had no effect on the content of this receptor in the hippocampus. This is the first study showing that exposure to BP-3 induces the mitochondrial apoptosis pathway in the rat frontal cortex and this effect may result from a weakening of the neuroprotective effects of oestrogen and/or an intensification of AhR-mediated apoptosis.

The impact of polymers on 3D microstructure and controlled release of sildenafil citrate from hydrophilic matrices.

Sildenafil citrate has short biological half-life in humans. Thus, matrix tablets of controlled release were designed and prepared by compaction on the basis of hydrophilic polymers, i.e. HPMC, sodium alginate, carbomer, poloxamer and their mixtures. The impact of these polymers on sildenafil release in vitro and its pharmacokinetics in vivo was evaluated. Since drug release rate from hydrophilic matrices can be govern by the porosity of the matrix, the microstructure of tablets was studied using X-ray microcomputed tomography. 3D network of either open (percolating) or closed (non-percolating) pores was reconstructed. The tortuosity and the diameter of both kinds of pores were determined. Their spatial distribution within the matrix was analyzed in linear and radial direction. Polymer-dependent characteristics of the open pores (Ø > 2 µm) architecture was shown. The release profiles of sildenafil from matrix tablets fitted to Korsmeyer-Peppas model (r2: 0.9331-0.9993) with either Fickian diffusion or anomalous transport involved. Mean dissolution time (MDT) from tablets made of HPMC, carbomer or a mixture of HPMC and sodium alginate (2:1) was ca. 100 min, which was more than twelve times longer as compared to matrices prepared of silicified microcrystalline cellulose (MDT = 8 min). MDT correlated with the number of the open pores (Pearson's r = 0.94). Sustained release of sildenafil from ground carbomer tablets reflected in the slow absorption of the drug (tmax = 5.0 ±â€¯1.2 h) in vivo and the relative bioavailability of 151%. Interestingly, the relative bioavailability of sildenafil from binary matrices composed of HPMC and sodium alginate (2:1) was almost four times higher than that of sildenafil alone.

In vitro and in vivo behavior of ground tadalafil hot-melt extrudates: How the carrier material can effectively assure rapid or controlled drug release.

Different types of ground hot-melt extrudates loaded with 10, 20 or 30 % of the poorly water-soluble drug tadalafil were prepared and characterized in vitro and in vivo (in rats). Soluplus was used as an amorphous carrier material, whereas mannitol and lactitol were studied as crystalline matrix formers. The systems were characterized using X-ray powder diffraction, thermogravimetric analysis coupled with quadruple mass spectrometry, differential scanning calorimetry, X-ray computed microtomography, in vitro drug release measurements and monitoring of drug plasma levels upon oral administration to rats. The pure drug substance and physical mixtures of tadalafil with the carrier materials were used as references. Importantly, the bioavailability of this poorly water-soluble drug could be substantially increased with the proposed formulations, and the in vitro and in vivo release rates could be effectively adjusted by choosing the appropriate type of carrier material: Whereas mannitol-based ground hot-melt extrudates rapidly released the drug and led to an early rise in drug plasma concentrations, Soluplus-based systems released tadalafil more slowly, resulting in delayed plasma peaks. These behaviors could be explained by the rapid disintegration/dissolution of the porous mannitol-based formulations, whereas Soluplus significantly swelled and the dissolved drug had to diffuse through the polymeric network prior to release. Blending these formulations can be expected to allow providing elevated drug concentrations in vivo during prolonged periods of time upon one single administration with a rapid onset of drug action.

Novel, highly potent and in vivo active inhibitor of GABA transporter subtype 1 with anticonvulsant, anxiolytic, antidepressant and antinociceptive properties.

BACKGROUND AND PURPOSE: Since GABAergic dysfunction underlies a variety of neurological and psychiatric disorders, numerous strategies leading to the augmentation of GABAergic neurotransmission have been introduced. One of them is the inhibition of GABA reuptake from the synaptic cleft mediated by four plasma membrane GABA transporters (GAT1-4). GAT1 which is exclusively expressed in the brain is an interesting target for centrally acting drugs. In this research, pharmacological properties of a novel, highly potent and selective inhibitor of GAT1, the guvacine derivative named DDPM-2571, were assessed in vivo. EXPERIMENTAL APPROACH: Pharmacological effects and pharmacokinetics of intraperitoneally administered DDPM-2571 were assessed in CD-1 mice. KEY RESULTS: DDPM-2571 was quickly distributed into the brain and was highly effective in the prevention of chemically-induced seizures (pentylenetetrazole and pilocarpine models) and 6-Hz convulsions. It demonstrated significant anxiolytic-like and antidepressant-like properties. DDPM-2571 had antinociceptive properties, both in the hot plate test and in the second phase of the formalin test. Within the dose range tested, it did not impair animals' motor skills, but it impaired cognition and potentiated scopolamine-induced cognitive deficits in the passive avoidance task. CONCLUSIONS AND IMPLICATIONS: Due to GAT1 inhibition, DDPM-2571 is effective in mouse models of chemically-induced seizures, anxiety, depression, acute and tonic pain. At biologically active doses, it does not impair animals' motor skills, but it might induce memory deficits. Taken together, DDPM-2571 can be regarded as a promising lead structure in the search for new centrally acting drugs and a potent pharmacological tool to study the biological role of GAT1.