TC-G 1008 facilitates epileptogenesis by acting selectively at the GPR39 receptor but non-selectively activates CREB in the hippocampus of pentylenetetrazole-kindled mice.

The pharmacological activation of the GPR39 receptor has been proposed as a novel strategy for treating seizures; however, this hypothesis has not been verified experimentally. TC-G 1008 is a small molecule agonist increasingly used to study GPR39 receptor function but has not been validated using gene knockout. Our aim was to assess whether TC-G 1008 produces anti-seizure/anti-epileptogenic effects in vivo and whether the effects are mediated by GPR39. To obtain this goal we utilized various animal models of seizures/epileptogenesis and GPR39 knockout mice model. Generally, TC-G 1008 exacerbated behavioral seizures. Furthermore, it increased the mean duration of local field potential recordings in response to pentylenetetrazole (PTZ) in zebrafish larvae. It facilitated the development of epileptogenesis in the PTZ-induced kindling model of epilepsy in mice. We demonstrated that TC-G 1008 aggravated PTZ-epileptogenesis by selectively acting at GPR39. However, a concomitant analysis of the downstream effects on the cyclic-AMP-response element binding protein in the hippocampus of GPR39 knockout mice suggested that the molecule also acts via other targets. Our data argue against GPR39 activation being a viable therapeutic strategy for treating epilepsy and suggest investigating whether TC-G 1008 is a selective agonist of the GPR39 receptor.

Anticonvulsant Profile of Selected Medium-Chain Fatty Acids (MCFAs) Co-Administered with Metformin in Mice in Acute and Chronic Treatment.

In contrast to the other components of the medium-chain triglycerides ketogenic diet (MCT KD), i.e., caprylic acid (CA8), a comprehensive evaluation of caproic (CA6) and lauric acids' (CA12) properties in standard chemical and electrical seizure tests in mice has not yet been performed. We investigated their effects in maximal electroshock seizure threshold (MEST), 6 Hz seizure threshold and intravenous (i.v.) pentylenetetrazole (PTZ) seizure tests. Since ketone body production can be regulated by the activation of 5'AMP-activated protein kinase (AMPK), we hypothesized that metformin (an AMPK activator) enhance ketogenesis and would act synergistically with the fatty acids to inhibit convulsions. We assessed the effects of acute and chronic co-treatment with metformin and CA6/CA8 on seizures. CA6 and CA12 (p.o.) increased seizure threshold in the 6 Hz seizure test. CA6 at the highest tested dose (30 mmol/kg) developed toxicity in several mice, impaired motor performance and induced ketoacidosis. Acute and chronic co-treatment with metformin and CA6/CA8 did not affect seizure thresholds. Moreover, we observed the pro-convulsive effect of the acute co-administration of CA8 (5 mmol/kg) and metformin (100 mg/kg). Since this co-treatment was pro-convulsive, the safety profile and risk/benefit ratio of MCT KD and metformin concomitant therapy in epileptic patients should be further evaluated.

Pharmacokinetic/Pharmacodynamic Assessment of Selective Phosphodiesterase Inhibitors in a Mouse Model of Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a life-threatening disorder currently treated with nonspecific immunosuppressive drugs. It is postulated that phosphodiesterase (PDE) inhibitors, as agents exerting anti-inflammatory and immunomodulatory activities, may constitute a possible treatment of autoimmune disorders. This study develops a pharmacokinetic/pharmacodynamic (PK/PD) model to assess the effects of PDE-selective inhibitors, namely, cilostazol (PDE3), rolipram (PDE4), and BRL-50481 (PDE7), in a mouse model of AIH. The pharmacokinetics of the PDE inhibitors (PDEi) were assessed in male BALB/c mice after intraperitoneal administration. In pharmacodynamic studies, mice received PDEi and AIH was induced in these animals by intravenous injection of concanavalin A (ConA). Serum drug concentrations, tumor necrosis factor α (TNFα), interleukin 17 (IL-17), and aminotransferase activities were quantified. The PK/PD analysis was performed using ADAPT5 software. The PK/PD model assumes inhibition of cAMP hydrolysis in T cells by PDEi, ConA-triggered formation of TNFα and IL-17, suppression of TNFα and IL-17 production by cAMP, and stimulatory effects of TNFα and IL-17 on the hepatic release of aminotransferases. Selective blockage of PDE4 leads to the highest inhibition of cAMP degradation in T cells and amelioration of disease outcomes. However, inhibition of both PDE3 and PDE7 also contribute to this effect. The proposed PK/PD model may be used to assess and predict the activities of novel PDEi and their combinations in ConA-induced hepatitis. A balanced suppression of different types of PDE appears to be a promising treatment option for AIH; however, this hypothesis warrants testing in humans based on translation of the PK/PD model into clinical settings. SIGNIFICANCE STATEMENT: A novel PK/PD model of PDE inhibitor effects in mice with ConA-induced autoimmune hepatitis was developed involving a mechanistic component describing changes in cAMP concentrations in mouse T cells. According to model predictions, inhibition of PDE4 in T cells causes the highest cAMP elevation in T cells, but suppression of PDE3 and PDE7 also contribute to this effect. A balanced inhibition of PDE3, PDE4, and PDE7 appears to be a promising treatment strategy for AIH.

In silico and in vitro ADME-Tox analysis and in vivo pharmacokinetic study of representative pan-PDE inhibitors from the group of 7,8-disubstituted derivatives of 1,3-dimethyl-7H-purine-2,6-dione.

Phosphodiesterase (PDE) inhibitors represent a wide class of chemically different compounds that have been extensively studied in recent years. Their anti-inflammatory and anti-fibrotic effects are particularly desirable in the treatment of chronic respiratory diseases, including asthma and chronic obstructive pulmonary disease (COPD). Due to diversified expression of individual PDEs within cells and/or tissues as well as PDE signaling compartmentalization, pan-PDE inhibitors (compounds capable of simultaneously blocking various PDE subtypes) are of particular interest. Recently, a large group of 7,8-disubstituted derivatives of 1,3-dimethyl-7H-purine-2,6-dione (theophylline) was designed and synthesized. These compounds were characterized as potent pan-PDE inhibitors and their prominent anti-inflammatory and anti-fibrotic activity in vitro has been proved. Herein, we investigated a general in vitro safety profile and pharmacokinetic characteristics of two leading compounds from this group: a representative compound with N'-benzylidenebutanehydrazide moiety (38) and a representative derivative containing N-phenylbutanamide fragment (145). Both tested pan-PDE inhibitors revealed no cytotoxic, mutagenic, and genotoxic activity in vitro, showed moderate metabolic stability in mouse and human liver microsomes, as well as fell into the low or medium permeation category. Additionally, 38 and 145 revealed a lack of interaction with adenosine receptors, including A1, A2A, and A2B. Pharmacokinetic analysis revealed that both tested 7,8-disubstituted derivatives of 1,3-dimethyl-7H-purine-2,6-dione were effectively absorbed from the peritoneal cavity. Simultaneously, they were extensively distributed to mouse lungs and after intraperitoneal (i.p.) administration were detected in bronchoalveolar lavage fluid. These findings provide evidence that investigated compounds represent a new drug candidates with a favorable in vitro safety profile and satisfactory pharmacokinetic properties after a single i.p. administration. As the next step, further pharmacokinetic studies after multiple i.p. and p.o. doses will be conducted to ensure effective 38 and 145 serum and lung concentrations for a longer period of time. In summary, 7,8-disubstituted derivatives of 1,3-dimethyl-7H-purine-2,6-dione represent a promising compounds worth testing in animal models of chronic respiratory diseases, the etiology of which involves various PDE subtypes.

Effects of new antiseizure drugs on seizure activity and anxiety-like behavior in adult zebrafish.

Several studies with larvae and adult zebrafish have shown that old and new antiseizure drugs (ASDs) produce discrepant results in seizure tests, locomotor activity or anxiety models. In this study, the pentylenetetrazole seizure test (PTZ) was performed to assess the effectiveness of four new ASDs: lamotrigine (LTG), topiramate (TPM), felbamate (FBM), and levetiracetam (LEV) in the subsequent stages of seizures in adult fish. All ASDs were administered intraperitoneally (i.p.). The time of maximal anticonvulsant effect and the dose-response relationship of the drugs were assessed. The effects of studied ASDs on the locomotor activity and the anxiety-like behavior in the color preference test were also investigated. Furthermore, drug concentrations in zebrafish homogenates were determined. LTG, TPM, and LEV significantly increased the seizure latency at three subsequent stages of seizures (SI-SIII), while FBM was effective only at SI. Locomotor activity decreased after TPM treatment. TPM and FBM exhibited a strong anxiolytic-like effect in the color preference test. LEV at the highest dose tested had a weak anxiolytic-like effect. The HPLC analysis showed average concentrations of the studied ASDs in the fish body during their maximum anticonvulsant activity. The present study shows that FBM cannot inhibit all subsequent PTZ seizure stages in the adult fish. Except for LTG, the studied drugs affected the anxiety-like behavior of treated animals. Furthermore, only TPM significantly changed locomotion parameters. Our findings support the need to accurately characterize the efficacy of new ASDs at different stages of the PTZ-induced seizures in adult zebrafish.

Effects of classic antiseizure drugs on seizure activity and anxiety-like behavior in adult zebrafish.

The zebrafish is extensively used as a model organism for studying several disorders of the central nervous system (CNS), including epilepsy. Some antiseizure drugs (ASDs) have been shown to produce discrepant results in larvae and adults zebrafish, therefore, their anticonvulsant efficacy in subsequent stages of the pentylenetetrazole (PTZ)-induced seizures should be more precisely characterized. The purpose of this study was to investigate behavioral effects of five classic ASDs: valproate (VPA), phenytoin (PHT), carbamazepine (CBZ), diazepam (DZP), and phenobarbital (PB) administered intraperitoneally (i.p.) in the PTZ-induced seizure test in adult zebrafish. We determined the time of maximal effect and the dose-response relationship of the studied ASDs. Furthermore, we assessed changes in the locomotor activity and the anxiety-like behavior in the color preference test. Moreover, drug concentrations in zebrafish homogenates were examined. VPA, DZP, and PB significantly increased the seizure latency at three subsequent stages of seizures (SI-SIII). PHT produced the anticonvulsant-like effect at SI and SII, while CBZ was effective at SII and SIII. Only DZP decreased zebrafish locomotor activity. A strong anxiolytic-like effect was observed after administration of PHT and PB. A weak anxiolytic-like effect occurred after treatment with VPA and DZP. The HPLC analysis showed the average concentrations of the studied ASDs in the fish body during the maximum anticonvulsant activity of each drug. Our results confirm the advantages of using zebrafish with the mature CNS over larval models and its utility to investigate some neuropharmacological properties of the tested drugs.

Synthesis and in vitro evaluation of anti-inflammatory, antioxidant, and anti-fibrotic effects of new 8-aminopurine-2,6-dione-based phosphodiesterase inhibitors as promising anti-asthmatic agents.

Phosphodiesterase (PDE) inhibitors are currently an extensively studied group of compounds that can bring many benefits in the treatment of various inflammatory and fibrotic diseases, including asthma. Herein, we describe a series of novel N'-phenyl- or N'-benzylbutanamide and N'-arylidenebutanehydrazide derivatives of 8-aminopurine-2,6-dione (27-43) and characterized them as prominent pan-PDE inhibitors. Most of the compounds exhibited antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)-induced murine macrophages RAW264.7. The most active compounds (32-35 and 38) were evaluated in human bronchial epithelial cells (HBECs) derived from asthmatics. To better map the bronchial microenvironment in asthma, HBECs after exposure to selected 8-aminopurine-2,6-dione derivatives were incubated in the presence of two proinflammatory and/or profibrotic factors: transforming growth factor type ß (TGF-ß) and interleukin 13 (IL-13). Compounds 32-35 and 38 significantly reduced both IL-13- and TGF-ß-induced expression of proinflammatory and profibrotic mediators, respectively. Detailed analysis of their inhibition preferences for selected PDEs showed high affinity for isoenzymes important in the pathogenesis of asthma, including PDE1, PDE3, PDE4, PDE7, and PDE8. The presented data confirm that structural modifications within the 7 and 8 positions of the purine-2,6-dione core result in obtaining preferable pan-PDE inhibitors which in turn exert an excellent anti-inflammatory and anti-fibrotic effect in the bronchial epithelial cells derived from asthmatic patients. This dual-acting pan-PDE inhibitors constitute interesting and promising lead structures for further anti-asthmatic agent discovery.

Identification of New Compounds with Anticonvulsant and Antinociceptive Properties in a Group of 3-substituted (2,5-dioxo-pyrrolidin-1-yl)(phenyl)-Acetamides.

We report herein a series of water-soluble analogues of previously described anticonvulsants and their detailed in vivo and in vitro characterization. The majority of these compounds demonstrated broad-spectrum anticonvulsant properties in animal seizure models, including the maximal electroshock (MES) test, the pentylenetetrazole-induced seizure model (scPTZ), and the psychomotor 6 Hz (32 mA) seizure model in mice. Compound 14 showed the most robust anticonvulsant activity (ED50 MES = 49.6 mg/kg, ED50 6 Hz (32 mA) = 31.3 mg/kg, ED50scPTZ = 67.4 mg/kg). Notably, it was also effective in the 6 Hz (44 mA) model of drug-resistant epilepsy (ED50 = 63.2 mg/kg). Apart from favorable anticonvulsant properties, compound 14 revealed a high efficacy against pain responses in the formalin-induced tonic pain, the capsaicin-induced neurogenic pain, as well as in the oxaliplatin-induced neuropathic pain in mice. Moreover, compound 14 showed distinct anti-inflammatory activity in the model of carrageenan-induced aseptic inflammation. The mechanism of action of compound 14 is likely complex and may result from the inhibition of peripheral and central sodium and calcium currents, as well as the TRPV1 receptor antagonism as observed in the in vitro studies. This lead compound also revealed beneficial in vitro ADME-Tox properties and an in vivo pharmacokinetic profile, making it a potential candidate for future preclinical development. Interestingly, the in vitro studies also showed a favorable induction effect of compound 14 on the viability of neuroblastoma SH-SY5Y cells.

Effect of Ellagic Acid on Seizure Threshold in Two Acute Seizure Tests in Mice.

Ellagic acid (EA) is a natural dietary polyphenol that has many beneficial properties, including anti-inflammatory, antioxidant, antiviral, antibacterial, and neuroprotective effects. Studies have revealed that EA may modulate seizure activity in chemically induced animal models of seizures. Therefore, the aim of the present study was to investigate the effect of EA on the seizure threshold in two acute seizure tests in male mice, i.e., in the intravenous (i.v.) pentylenetetrazole (PTZ) seizure test and in the maximal electroshock seizure threshold (MEST) test. The obtained results showed that EA (100 mg/kg) significantly elevated the threshold for both the first myoclonic twitch and generalized clonic seizure in the i.v. PTZ seizure test. At the highest dose tested (200 mg/kg), EA increased the threshold for tonic hindlimb extension in the MEST test. EA did not produce any significant changes in motor coordination (assessed in the chimney test) or muscular strength (investigated in the grip-strength test). The plasma and total brain concentration-time profiles of EA after intraperitoneal and oral administration were also determined. Although further studies are necessary to confirm the anticonvulsant activity of EA, our findings suggest that it may modulate seizure susceptibility in animal models.

Correction to: Comparative Assessment of the New PDE7 Inhibitor - GRMS-55 and Lisofylline in Animal Models of Immune-Related Disorders: A PK/PD Modeling Approach.

There was a mistake in the unit of clearance (Cl) in Table II. In addition, the descriptions of V1(ROL) and V1(GRMS-55) were imprecise and the reference number in the footnote below this table should be (9). The corrected Table appears below.

Comparative Assessment of the New PDE7 Inhibitor - GRMS-55 and Lisofylline in Animal Models of Immune-Related Disorders: A PK/PD Modeling Approach.

PURPOSE: This study aimed to assess the activity of two phosphodiesterase (PDE) inhibitors, namely GRMS-55 and racemic lisofylline ((±)-LSF)) in vitro and in animal models of immune-mediated disorders. METHODS: Inhibition of human recombinant (hr)PDEs and TNF-alpha release from LPS-stimulated whole rat blood by the studied compounds were assessed in vitro. LPS-induced endotoxemia, concanavalin A (ConA)-induced hepatitis, and collagen-induced arthritis (CIA) animal models were used for in vivo evaluation. The potency of the investigated compounds was evaluated using PK/PD and PK/PD/disease progression modeling. RESULTS: GRMS-55 is a potent hrPDE7A and hrPDE1B inhibitor, while (±)-LSF most strongly inhibits hrPDE3A and hrPDE4B. GRMS-55 decreased TNF-alpha levels in vivo and CIA progression with IC50 of 1.06 and 0.26 mg/L, while (±)-LSF with IC50 of 5.80 and 1.06 mg/L, respectively. Moreover, GRMS-55 significantly ameliorated symptoms of ConA-induced hepatitis. CONCLUSIONS: PDE4B but not PDE4D inhibition appears to be mainly engaged in anti-inflammatory activity of the studied compounds. GRMS-55 and (±)-LSF seem to be promising candidates for future studies on the treatment of immune-related diseases. The developed PK/PD models may be used to assess the anti-inflammatory and anti-arthritic potency of new compounds for the treatment of rheumatoid arthritis and other inflammatory disorders.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Anticonvulsant Activity of Pterostilbene in Zebrafish and Mouse Acute Seizure Tests.

Pterostilbene (PTE), a natural dimethylated analog of resveratrol, possesses numerous health-beneficial properties. The ability of PTE to cross the blood-brain barrier raised the possibility that this compound may modulate central nervous system functions, including seizure activity. The aim of our study was to investigate the activity of PTE in the larval zebrafish pentylenetetrazole (PTZ) seizure assay and three acute seizure tests in mice, i.e., in the maximal electroshock seizure threshold (MEST), 6 Hz-induced psychomotor seizure threshold and intravenous (iv) PTZ tests. Additionally, potential antidepressant activity of PTE was estimated in the forced swim test in mice. The chimney test was used to determine the influence of PTE on motor coordination in mice, while its influence on neuromuscular strength was assessed in the grip strength test in mice. Locomotor activity was determined to verify the results from the forced swim test. PTE revealed an evident anticonvulsant effect both in zebrafish larvae (10 µM; 2 h-incubation) and mice (at doses of 100 and 200 mg/kg, intraperitoneally) but it did not exhibit antidepressant potential in the forced swim test. Furthermore, it did not cause any statistically significant changes in motor coordination, neuromuscular strength and locomotor activity in mice. In conclusion, our present findings demonstrate for the first time the anticonvulsant potential of PTE. The aforementioned results suggest that it might be employed in epilepsy treatment, however, further precise studies are required to verify its activity in other experimental seizure and epilepsy models and its precise mechanism of action should be determined.

Influence of inflammatory disorders on pharmacokinetics of lisofylline in rats: implications for studies in humans.

1. Despite the number of favourable properties of lisofylline (LSF), clinical trials on this compound have not yielded the expected results yet. 2. The aims of this study were to evaluate the pharmacokinetics of LSF enantiomers in rats following intravenous, oral and subcutaneous administration of (±)-LSF and to assess the influence of experimental inflammatory disorders, such as multiple organ dysfunction syndrome and severe sepsis on LSF pharmacokinetics. 3. In addition, based on the results obtained an attempt was made to elucidate the possible reasons for the failure of LSF therapy in clinical trials carried out in patients with severe inflammatory disorders. 4. A subcutaneous route of (±)-LSF administration to rats is more favourable than an oral one due to a high bioavailability and a fast absorption of both LSF enantiomers. Pharmacokinetics of LSF in rats is significantly influenced by inflammatory diseases. Too low LSF serum levels might have been one of the reasons for clinical trial failures. A long-term i.v. infusion of LSF seems to be more effective compared to short-term multiple infusions that were used in clinical trials, as it may provide concentrations above IC50 for inhibition of both TNF-alpha release and cAMP degradation in serum for a longer period of time.

Increased seizure susceptibility and other toxicity symptoms following acute sulforaphane treatment in mice.

Activation of Nrf2 with sulforaphane has recently gained attention as a new therapeutic approach in the treatment of many diseases, including epilepsy. As a plant-derived compound, sulforaphane is considered to be safe and well-tolerated. It is widely consumed, also by patients suffering from seizure and taking antiepileptic drugs, but no toxicity profile of sulforaphane exists. Since many natural remedies and dietary supplements may increase seizure risk and potentially interact with antiepileptic drugs, the aim of our study was to investigate the acute effects of sulforaphane on seizure thresholds and activity of some first- and second-generation antiepileptic drugs in mice. In addition, some preliminary toxicity profile of sulforaphane in mice after intraperitoneal injection was evaluated. The LD50 value of sulforaphane in mice was estimated at 212.67mg/kg, while the TD50 value - at 191.58mg/kg. In seizure tests, sulforaphane at the highest dose tested (200mg/kg) significantly decreased the thresholds for the onset of the first myoclonic twitch and generalized clonic seizure in the iv PTZ test as well as the threshold for the 6Hz-induced psychomotor seizure. At doses of 10-200mg/kg, sulforaphane did not affect the threshold for the iv PTZ-induced forelimb tonus or the threshold for maximal electroshock-induced hindlimb tonus. Interestingly, sulforaphane (at 100mg/kg) potentiated the anticonvulsant efficacy of carbamazepine in the maximal electroshock seizure test. This interaction could have been pharmacokinetic in nature, as sulforaphane increased concentrations of carbamazepine in both serum and brain tissue. The toxicity study showed that high doses of sulforaphane produced marked sedation (at 150-300mg/kg), hypothermia (at 150-300mg/kg), impairment of motor coordination (at 200-300mg/kg), decrease in skeletal muscle strength (at 250-300mg/kg), and deaths (at 200-300mg/kg). Moreover, blood analysis showed leucopenia in mice injected with sulforaphane at 200mg/kg. In conclusion, since sulforaphane was proconvulsant at a toxic dose, the safety profile and the risk-to-benefit ratio of sulforaphane usage in epileptic patients should be further evaluated.

Chronic treatment with caffeine and its withdrawal modify the antidepressant-like activity of selective serotonin reuptake inhibitors in the forced swim and tail suspension tests in mice. Effects on Comt, Slc6a15 and Adora1 gene expression.

Recent preclinical and clinical data suggest that low dose of caffeine enhances the effects of common antidepressants. Here we investigated the effects of chronic administration of caffeine (5mg/kg, twice daily for 14days) and its withdrawal on day 15th on the activity of per se ineffective doses of fluoxetine (5mg/kg) and escitalopram (2mg/kg) given on day 15th. We found decreased immobility time in the forced swim and tail suspension tests in mice in which caffeine was administered simultaneously with antidepressants on day 15th following a 14-day caffeine treatment and no alterations in the spontaneous locomotor activity. A decrease in the level of escitalopram and an increase in the level of caffeine in serum were observed after concomitant administration of these compounds, while the joint administration of caffeine and fluoxetine was not associated with changes in their levels in serum or brain. Caffeine withdrawal caused a decrease in Adora1 mRNA level in the cerebral cortex (Cx). Administration of escitalopram or fluoxetine followed by caffeine withdrawal caused an increase in this gene expression, whereas administration of escitalopram, but not fluoxetine, on day 15th together with caffeine caused a decrease in Adora1 mRNA level in the Cx. Furthermore, antidepressant-like activity observed after joint administration of the tested drugs with caffeine was associated with decreased Slc6a15 mRNA level in the Cx. The results show that withdrawal of caffeine after its chronic intake may change activity of antidepressants with concomitant alterations within monoamine, adenosine and glutamate systems.

Influence of the selective antagonist of the NR2B subunit of the NMDA receptor, traxoprodil, on the antidepressant-like activity of desipramine, paroxetine, milnacipran, and bupropion in mice.

Pre-clinical and clinical studies indicated that a blockade of the NMDA receptor complex creates new opportunities for the treatment of affective disorders, including depression. The aim of the present study was to assess the influence of traxoprodil (10 mg/kg) on the activity of desipramine (10 mg/kg), paroxetine (0.5 mg/kg), milnacipran (1.25 mg/kg), and bupropion (10 mg/kg), each at sub-therapeutic doses. Moreover, brain levels of traxoprodil and tested agents were determined using HPLC. The obtained results were used to ascertain the nature of occurring interaction between traxoprodil and studied antidepressants. The experiment was carried out on naïve adult male Albino Swiss mice. Traxoprodil and other tested drugs were administered intraperitoneally. The influence of traxoprodil on the activity of selected antidepressants was evaluated in forced swim test (FST). Locomotor activity was estimated to exclude false positive/negative data. To assess the influence of traxoprodil on the concentration of used antidepressants, their levels were determined in murine brains using HPLC. Results indicated that traxoprodil potentiated activity of all antidepressants examined in FST and the observed effects were not due to the increase in locomotor activity. Only in the case of co-administration of traxoprodil and bupropion, increased bupropion concentrations in brain tissue were observed. All tested agents increased the traxoprodil levels in the brain. Administration of a sub-active dose of traxoprodil with antidepressants from different chemical groups, which act via enhancing monoaminergic transduction, caused the antidepressant-like effect in FST in mice. The interactions of traxoprodil with desipramine, paroxetine, milnacipran, and bupropion occur, at least partially, in the pharmacokinetic phase.

Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high-performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study.

A direct fluorometric high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100 µl) and tissues (brain, liver, kidneys) using liquid-liquid extraction and 4-tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ-H chiral column based on cellulose tris(4-methylbenzoate) coated on 5 µm silica-gel, 250 x 4.6 mm at 22 °C with a mobile phase composed of n-hexane, 2-propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1 ml min-1 . A fluorometric detector was set at: λexcit . = 220 nm and λemis. = 290 nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within-run and between-run precision and accuracy. The LLOQ for the two enantiomers was 0.125 µg ml-1 in plasma, 0.09 µg g-1 in brain, and 0.25 µg g-1 in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35 µg ml-1 for plasma, 0.09-1.44 µg g-1 for brain, and 0.25-20 µg g-1 for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10 mg kg-1 of racemate.

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.

Antidepressant-like activity of sildenafil following acute and subchronic treatment in the forced swim test in mice: effects of restraint stress and monoamine depletion.

Sildenafil is a highly effective oral agent for the treatment of erectile dysfunction of multiple etiologies. Although in clinical practice sildenafil is often used in depressed patients, its influence on the pathophysiology of depression remains unclear. The aim of the present study was to evaluate the antidepressant-like activity following acute and subchronic treatment with sildenafil in naïve mice as well as in mice with reserpine- and restraint stress-induced depressive-like behavior. Since corticosterone is released in response to acute stress, we also aimed to assess the influence of sildenafil on serum corticosterone level in non-stressed and stressed animals. The antidepressant activity of sildenafil was assessed in the forced swim test. Corticosterone serum level was determined by using ELISA method, while brain and serum sildenafil level via HPLC method. Sildenafil administered acutely exerted an antidepressant-like effect. Subchronic (14 days) administration of sildenafil resulted only in a weak antidepressant-like effect when evaluated 24 h after the last dose. Acute but not subchronic sildenafil administration reversed the reserpine- and stress-induced immobility in the forced swim test. The lack of effects of sildenafil after subchronic treatment could have been related to its complete elimination from the brain within 24 h from the last injection. Interestingly, acute administration of sildenafil produced a marked increase in serum corticosterone level in both non-stressed and stressed animals. Sildenafil exerts differential effects in the forced swim test after acute and subchronic administration. Further studies on the antidepressant activity of sildenafil are required.