Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2 days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals.

Benzotriazole: An overview on its versatile biological behavior.

Discovered in late 1960, azoles are heterocyclic compounds class which constitute the largest group of available antifungal drugs. Particularly, the imidazole ring is the chemical component that confers activity to azoles. Triazoles are obtained by a slight modification of this ring and similar or improved activities as well as less adverse effects are reported for triazole derivatives. Consequently, it is not surprising that benzimidazole/benzotriazole derivatives have been found to be biologically active. Since benzimidazole has been widely investigated, this review is focused on defining the place of benzotriazole derivatives in biomedical research, highlighting their versatile biological properties, the mode of action and Structure Activity Relationship (SAR) studies for a variety of antimicrobial, antiparasitic, and even antitumor, choleretic, cholesterol-lowering agents.

Encapsulation and modified-release of thymol from oral microparticles as adjuvant or substitute to current medications.

The aim of this study was to encapsulate, thymol, in natural polymers in order to obtain (i) taste masking effect and, then, enhancing its palatability and (ii) two formulations for systemic and local delivery of herbal drug as adjuvants or substitutes to current medications to prevent and treat several human and animal diseases. Microspheres based on methylcellulose or hydroxypropyl methylcellulose phthalate (HPMCP) were prepared by spray drying technique. Microparticles were in vitro characterized in terms of yield of production, drug content and encapsulation efficiency, particle size, morphology and drug release. Both formulations were in vivo orally administered and pharmacokinetic analysis was carried out. The polymers used affect the release and, then, the pharmacokinetic profile of thymol. Encapsulation into methylcellulose microspheres leads to short half/life but bioavailability remarkably increases compared to the free thymol. In contrast, enteric formulation based on HPMCP shows very limited systemic absorption. These formulations could be proposed as alternative or adjuvants for controlling pathogen infections in human or animal. In particular, methylcellulose microspheres can be used for thymol systemic administration at low doses and HPMCP particles for local treatment of intestinal infections.

Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE) line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30 mg/kg) or vehicle for 15 days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal  DA by indicating an enhancement of selective attention and working memory in a deficit model.

Effect of free thymol on differential gene expression in gastric mucosa of the young pig.

Thymol is the most common molecule in thyme and has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers. The knowledge of the in vivo physiological effects of thymol on tissues is limited, particularly its impact on the gastric mucosa, where it is primarily absorbed when it is orally supplied. In this study, thymol (TH, 50 mg/ kg BW) or a placebo (CO) was introduced directly into the stomach of 8 weaned pigs that were slaughtered 12 h later and sampled for gastric oxyntic and pyloric mucosa. The analysis of whole transcript expression was performed using Affymetrix© Porcine Gene 1.1 ST array strips. Affymetrix Transcripts IDs were associated with 13 406 human gene names based on Sus scrofa Ensemble. Gene Set Enrichment Analysis was performed, comparing TH and CO pigs. For each gene set, the normalized enrichment score (NES) was defined as significant when the false discovery rate % was <25 and the P-value of NES was <0.05. In response to TH, 72 and 19 gene sets were significantly enriched in the oxyntic and pyloric mucosa, respectively. Several gene sets involved in mitosis and its regulation ranked near the top, primarily in the oxyntic mucosa; the gene set DIGESTION ranked first and ninth in the pyloric and oxyntic mucosa, respectively. Within this group, somatostatin (SST), SST receptors, peptide transporter 1 (SLC15A1) and calpain 9 (gastrointestinal tract-specific calpain) were the most strongly upregulated genes. Thymol reduced the enrichment of 120 and 59 gene sets in the oxyntic and pyloric mucosa, respectively. Several gene sets related to ion transport and channeling and aqueous pores across membranes, including short transient receptor potential (TRP) channel 4, potassium voltage-gated channel members 1 and 2, and ryanodine receptors 2 and 3, were less enriched. The downregulation of these genes sensitive to thymol in vitro could depend on the thymol dose and contact with the gastric tissues that causes an adaptive response with their reduced activation. Conversely, the activation of the TRPA1 gene (ranked 1072 and 128 among all the genes in the oxyntic and pyloric mucosa, respectively) indicates the involvement of another TRP-regulating cellular calcium storage. In conclusion, the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs. These properties should be considered in addition to thymol's antimicrobial properties when supplementation of this molecule in feed is evaluated.

Galactosylated dopamine enters into the brain, blocks the mesocorticolimbic system and modulates activity and scanning time in Naples high excitability rats.

Pathological conditions, such as Parkinson's disease and attention deficit hyperactivity disorder, have been linked to alterations of specific dopamine (DA) pathways. However, since exogenous DA does not cross the blood-brain barrier, DA levels can be modulated e.g. by DA precursors or DA reuptake blockers. Hereby histochemical, analytical and behavioral evidence shows that a galactosylated form of DA (GAL-DA) carries DA into the brain, thus modulating activity and nonselective attention in rats. To this aim adult male rats of the Naples high-excitability (NHE) and random bred controls (NRB) lines were given a single i.p. injection of GAL-DA (10 or 100 mg/kg). Three hours later the behavior was videotaped and analyzed for horizontal activity, orienting frequency and scanning duration. The dose of 100 mglkg of GAL-DA reduced by 25% the horizontal activity in NHE rats, mainly in the first part of the testing period. No effect was observed on orienting frequency or on scanning duration. However, GAL-DA 100 mg/kg was associated with longer rearing episodes in the second part of the testing period in NHE rats. In parallel experiments histochemistry with a galactose-specific lectin showed 10% increase in galactose residues into the striatum between 0.5 and 3.0 h. To quantify the level of GAL-DA, its metabolite DA-succinate and DA in the prefrontal cortex, neostriatum, and cerebellum, rats were killed 2.0 h after the injection of prodrug. Mass high performance liquid chromatography (HPLC) was used for analysis of GAL-DA and DA succinate whereas electrochemical HPLC for DA. Both HPLC techniques demonstrate that GAL-DA carries and releases DA into the brain. Specifically 100 mg/kg of GAL-DA increased DA level in the striatum in the NHE rats only. Moreover, DA in the mesencephalon (MES) was correlated positively with striatal and prefrontal cortex DA in NHE rats. In contrast DA in the MES was negatively correlated with striatal DA in NRB. GAL-DA disrupted these correlations in both rat lines. Thus, this new DA prodrug may modify DA neurotransmission and might have a potential clinical application.

Thienocinnolinone alkanoic acid derivatives as aldose reductase inhibitors.

A new series of 8-halogen-4,4a,5,6-tetrahydrothieno[2,3-h]cinnolinone-N2-alkanoic acids was prepared and tested for aldose reductase (ALR2) inhibitory activities. These compounds showed significant inhibitory activity against bovine lens ALR2, with the best compound 2e showing an IC(50) value of 31.4 microM. The presence of the C8-substituents here studied (Cl, Br) on the thienocinnolinone scaffold caused a decrease of the inhibitory potency by a factor of about 4 with respect to the unsubstituted parent compound, while the presence of a C8-methyl group, considered in a previous paper decreased the activity by a factor of about 2. Moreover, the length of the N2 alkanoic chain influences strongly the enzyme inhibitory activity. While most of the carboxylic acids ALR2 inhibitors are acetic acid derivatives, in the case of thienocinnolinone compounds, homologues higher than acetic acids showed to be more active.

Determination of phenol and o-cresol by GC/MS in a fatal poisoning case.

A fatality due to the ingestion of solution containing phenol and o-cresol is described. The pathological findings were typical of acute substantial poisoning. Blood, urine and stomach content were obtained during post mortem examinations. Phenol and o-cresol were identified using GC/MS. The extractions from autopsy materials were obtained as follows: by gel permeation with cyclohexane/dichloromethane from stomach content, by solid phase extraction (SPE) from urine and by deproteinization with acetonitrile from blood. The phenol and o-cresol concentrations in the samples were found, respectively, as follows: 115.0 and 5.0 microg/g in the stomach contents, 58.3 and 1.9 microg/ml in the blood, 3.3 and 20.5 microg/ml in the urine. Distributions of phenol in fatal poisonings have been reported, but, usually, colorimetry was used as the analytical method and it cannot exclude the interference of other phenolic compounds.

Synthesis and aldose reductase inhibitory activities of novel thienocinnolinone derivatives.

A novel series of tetrahydrothieno[2,3-h]cinnolinone derivatives were synthesized and evaluated in vitro for their ability to inhibit aldose reductase (ALR2), an enzyme involved in the appearance of diabetic complications. Compounds 2e and 2j exert a remarkable inhibitory effect, with IC(50) of 7.6 and 18 microM, respectively. These compounds incorporate a valid pharmacophore for aldose reductase inhibitory activity represented by a thienocinnolinone template linked through a pentamethylene spacer to a carboxylic function.

Antidermatophytic action of new 1-naphthylmethyl and benzo[b]thiophen-7-ylmethyl hydrazones related to inhibitors of squalene epoxidase.

Two series of hydrazonic compounds related to main classes of inhibitors of fungal squalene epoxidase (SE) were designed and prepared on the hypothesis of a pharmacophoric model. The antifungal activity of the new compounds was evaluated in vitro against dermatophytes, moulds and yeasts. Antidermatophytic activity resulted for several hydrazones, particularly for those containing a tett-butylacetylenic group, supporting the hypothesis that the introduction of a hydrazonic function in the model could retain the antimycotic activity.

Synthesis, pharmacokinetics and anticonvulsant activity of 7-chlorokynurenic acid prodrugs.

7-Chlorokynurenic acid 1 is a potent glycine-N-methyl-D-aspartate (NMDA) receptor antagonist, but it shows weak activity after systemic administration. In order to overcome the Blood-brain barrier (BBB), we synthetized three new esters 2-4 of 1 obtained by chemical conjugation with essential nutrients such as glucose and galactose, that are actively transported across the BBB. These compounds were assayed to evaluate their in vitro chemical and enzymatic hydrolysis. In addition the prodrugs 2-4 were tested for their ability to protect mice against NMDA-induced seizures after systemic administration. All the prodrugs 2-4 appeared moderately stable in pH 7.4 buffered solution and were susceptible to in vitro enzymatic hydrolysis. Intraperitoneal administration of either esters 2 or 4 was highly protective against seizures induced by NMDA in mice, with the latter prodrug showing the highest anticonvulsive activity. In addition, ester 4 undergoes a time-dependent extracellular hydrolysis into 1 when applied to mixed cultures of mouse cortical cells, a model that reproduces in vitro the cellular milieu encountered by the prodrugs once they penetrate the brain parenchyma.

Anti-inflammatory and analgesic amides: new developments.

A series of substituted N-cycloalkyl benzamides, cinnamamides, and indole-3-carboxamides were synthesized and evaluated for their analgesic, antiinflammatory activities as well as for their gastrointestinal irritation liability. Indomethacin was used as reference drug in both tests. Compounds 1k, 1b, 1h, 1j, and 1g were the most active in the antiinflammatory paw edema inhibition test, with a sharply dose-dependent effect. In terms of the analgesic activity (acetic acid writhing test), the most active compound was 5a followed by 3a, but many other compounds were found to have a non-negligible potency. Even in this case, the effect was dose dependent.

Synthesis of substituted N-(4-piperidyl)-N-(3-pyridyl)amides with antiarrhythmic activity. Note 1.

The synthesis of analogues of N,2-diphenyl-N-(4-piperidyl)acetamide endowed with antiarrhythmic activity is reported. Benzoyl, cinnamoyl, acetyl and propionyl groups replace the phenacyl group as N-acyl substituent, while pyridine replaces benzene as aromatic ring bound to the amide nitrogen. The title compounds were evaluated for antiarrhythmic activity on experimental arrhythmias induced by aconitine in rats. The presence of a n-propyl chain and an unsubstituted cinnamoyl moiety (1j) gives the highest protection against aconitine induced extrasystoles while the best efficacy against lethal effects is due to the presence of a n-propyl chain and an acetyl moiety (1m).

Synthesis of N-[4-(propyl)cyclohexyl]-amides with anti-inflammatory and analgesic activities.

Seventeen (un)substituted N-[4-(propyl)cyclohexyl]-amides (6a-h, 7a-h and 8) were synthesized and tested as anti-inflammatory and analgesic agents. The substituents on the aromatic ring were chosen in order to study the influence of electron-withdrawing or electron-donating residues, that change the electronic density on the aromatic moiety. The pharmacological results allow drawing some preliminary considerations on structure-activity relationships.

Synthesis of N-[4-(alkyl)cyclohexyl]-substituted benzamides with anti-inflammatory and analgesic activities.

Two series of N-[4-(alkyl)cyclohexyl]-substituted benzamides, i.e. a series of N-[4-(tert-butyl)cyclohexyl]-substituted benzamides and a series of N-[4-(ethyl)cyclohexyl]-substituted benzamides, were synthesised and evaluated for their anti-inflammatory and analgesic potencies, and gastrointestinal irritation liability.

Monitoring of oxytetracycline in ovine milk by high-performance liquid chromatography.

A method for 'in vivo' determination of the oxytetracyclin residues in ovine milk at low levels is described. Two groups of Sardinian breed sheep were treated with a dose of oxytetracycline by intramammary infusion and intramuscular administration, respectively. Oxytetracycline residues in extracts obtained from a preliminary cleanup procedure, were detected by an isocratic high-performance liquid chromatography (HPLC) method. Linear calibration plots were obtained over a large concentration range of 1 mg ml(-1)-10 ng ml(-1), with correlation coefficients higher than 0.996. Recoveries between 85.8 and 98.9% were obtained. Limit of detection (LOD) and limit of determination (LOQ) were 5.2 and 17.5 ng ml(-1), respectively. This method would be useful for routine monitoring of oxytetracycline residues in ovine dairy milk.

Monitoring of benzylpenicillin in ovine milk by HPLC.

A method for determination in vivo of the benzylpenicillin residues in ovine milk at low levels was described. Two groups of Sardinian breed sheep were treated with a dose of penicillin G sodium salt by intramammary infusion and intramuscular administration respectively. The residues were detected by isocratic HPLC method of the extract obtained from a previous cleanup procedure. Linear calibration plots were obtained over a large concentration range of 1 mg ml-1 -10 ng ml-1, with correlation coefficients greater than 0.998. Recoveries between 78.6 and 87.3% were obtained. Limit of detection (LOD) and limit of determination (LOQ) were 2.6 and 8.8 ng ml-1 respectively. This method would be useful for routine monitoring of penicillin G residues in ovine dairy milk.

Synthesis of 1-methyl-4-(N-aroyl)-piperidinamides with anti-inflammatory and analgesic activities.

Two series of 1-methyl-4-(N-aroyl)-piperidinamides were synthesized and evaluated for their anti-inflammatory and analgesic properties, as well as for their gastrointestinal irritation liability. A non-aromatic derivative, 1-methyl-4-(N-cyclohexanoyl)-piperidinamide, was synthesized and evaluated in order to obtain a more exhaustive knowledge of the structure-activity relationship.