Impairment of locomotor activity induced by the novel N-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice.

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg, ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg, ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

Impairment of locomotor activity induced by the novel N-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg, ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg, ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

LASSBio-881: an N-acylhydrazone transient receptor potential vanilloid subfamily type 1 antagonist orally effective against the hypernociception induced by capsaicin or partial sciatic ligation.

BACKGROUND AND PURPOSE: Compound LASSBio-881 is an orally effective antinociceptive that binds to cannabinoid receptors and is active mainly on the neurogenic component of pain models. We investigated whether transient receptor potential vanilloid subfamily type 1 (TRPV1) channels are involved in the effects of LASSBio-881. EXPERIMENTAL APPROACH: Modulation of capsaicin (CAP)- and low pH-induced currents was evaluated in TRPV1-expressing Xenopus oocytes. In vivo effects were evaluated in CAP-induced acute and inflammatory changes in nociception, as well as in partial sciatic ligation-induced thermal hypernociception. KEY RESULTS: LASSBio-881 inhibited TRPV1 currents elicited by CAP with an IC(50) of 14 microM, and inhibited proton-gated currents by 70% at 20 microM. Functional interaction with CAP was surmountable. Locally applied LASSBio-881 decreased time spent in CAP-elicited nocifensive behaviour by 30%, and given orally it reduced measures of CAP- or carrageenan-evoked thermal hypernociception by 60 and 40% respectively. In addition, LASSBio-881 decreased the paw withdrawal responses to thermal stimuli of animals with sciatic neuropathy 7-11 days after nerve ligation, at a dose of 300 micromol*kg(-1)*day(-1) p.o. At this dose, hyperthermia was not observed within 4 h following oral administration. CONCLUSIONS AND IMPLICATIONS: LASSBio-881 is a TRPV1 antagonist that apparently competes with CAP. Accordingly, LASSBio- 881 inhibited nociception in models of acute, inflammatory and neuropathic pain presumed to involve TRPV1 signalling. These in vivo actions were not hindered by hyperthermia, a common side effect of other TRPV1 antagonists. We propose that the antinociceptive properties of LASSBio-881 are due to TRPV1 antagonism, although other molecular interactions may contribute to the effects of this multi-target drug candidate.

In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi.

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Development and validation of HPLC and UV spectrophotometric methods for the determination of lumiracoxib in tablets

In this study, two methods, based on high-performance liquid chromatography (HPLC) and UV spectrophotometry, were developed and validated for the quantitative determination of lumiracoxib in tablets. The HPLC was carried out on a column of propylsulfonic acid bonded to silica gel (250 x 4.6 mm; 5 mium), with a mobile phase of phosphate buffer (pH 7.4; 10 mM)-water-acetonitrile (10:40:50, v/v/v) fl owing at 1.0 mL/min and detection of the drug at 278 nm. The UV method was based on absorbance at 275 nm, with ethanol as solvent. Both assays were linear over the concentration range of 2–30 miug/mL (R approximate 0.999), as wellas accurate and precise, with recoveries between 98 and 100% and relative standard deviation (%RSD) smaller that 2.0%. The proposed methods are highly sensitive, precise and accurate and were successfully applied to the quantitation of lumiracoxib in the commercial formulation. The spectrophotometric method is a simple, cheap and less time-consuming method. However, the chromatographic method is selective for the determination of the degradation products of lumiracoxib.

Development and validation of a LC-MS/MS method with electrospray ionization for determination of LASSBio-579 in rat plasma.

A simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of LASSBio-579 in plasma rat, using fluconazole as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water (80:20, v/v), containing 0.4mM ammonium hydroxide and 0.2 mM acetic acid at a flow rate of 1.0 ml/min (split ratio 1:5). A Micromass triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the positive mode. Plasma samples were deproteinized with acetonitrile (1:2) and 50 microl of the supernatant were injected into the system. The retention times of LASSBio-579 and IS were approximately 4.7 and 2.4 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 30-2000 ng/ml with determination coefficient >0.98. The lower limit of quantification was 30 ng/ml. The accuracy of method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 13.5% and 6.4%, respectively. The applicability of the LC-MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after intraperitoneal administration of LASSBio-579 to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of LASSBio-579 in pre-clinical pharmacokinetic studies.

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma.

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C18 column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25-8.0 microg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 microg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 microg/ml, 7.44 to 13.81% at 1.5 microg/ml and 6.10 to 13.94% at 6.0 microg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 microg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.

Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives

Dopamine constitutes about 80 percent of the content of central catecholamines and has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to report the structural design of two N-phenylpiperazine derivatives, compound 4: 1-[1-(4-chlorophenyl)-1H-4-pyrazolylmethyl]-4-phenylhexahydropyrazine and compound 5: 1-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-ylmethyl]-4-phenylhexahydropyrazine, planned to be dopamine ligands, and their dopaminergic action profile. The two compounds were assayed (dose range of 15-40 mg/kg) in three experimental models: 1) blockade of amphetamine (30 mg/kg, ip)-induced stereotypy in rats; 2) the catalepsy test in mice, and 3) apomorphine (1 mg/kg, ip)-induced hypothermia in mice. Both derivatives induced cataleptic behavior (40 mg/kg, ip) and a hypothermic response (30 mg/kg, ip) which was not prevented by haloperidol (0.5 mg/kg, ip). Compound 5 (30 mg/kg, ip) also presented a synergistic hypothermic effect with apomorphine (1 mg/kg, ip). Only compound 4 (30 mg/kg, ip) significantly blocked the amphetamine-induced stereotypy in rats. The N-phenylpiperazine derivatives 4 and 5 seem to have a peculiar profile of action on dopaminergic functions. On the basis of the results of catalepsy and amphetamine-induced stereotypy, the compounds demonstrated an inhibitory effect on dopaminergic behaviors. However, their hypothermic effect is compatible with the stimulation of dopaminergic function which seems not to be mediated by D2/D3 receptors

Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives.

Dopamine constitutes about 80% of the content of central catecholamines and has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to report the structural design of two N-phenylpiperazine derivatives, compound 4: 1-[1-(4-chlorophenyl)-1H-4-pyrazolylmethyl]-4-phenylhexahydropyrazine and compound 5: 1-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-ylmethyl]-4-phenylhexahydropyrazine, planned to be dopamine ligands, and their dopaminergic action profile. The two compounds were assayed (dose range of 15-40 mg/kg) in three experimental models: 1) blockade of amphetamine (30 mg/kg, ip)-induced stereotypy in rats; 2) the catalepsy test in mice, and 3) apomorphine (1 mg/kg, ip)-induced hypothermia in mice. Both derivatives induced cataleptic behavior (40 mg/kg, ip) and a hypothermic response (30 mg/kg, ip) which was not prevented by haloperidol (0.5 mg/kg, ip). Compound 5 (30 mg/kg, ip) also presented a synergistic hypothermic effect with apomorphine (1 mg/kg, ip). Only compound 4 (30 mg/kg, ip) significantly blocked the amphetamine-induced stereotypy in rats. The N-phenylpiperazine derivatives 4 and 5 seem to have a peculiar profile of action on dopaminergic functions. On the basis of the results of catalepsy and amphetamine-induced stereotypy, the compounds demonstrated an inhibitory effect on dopaminergic behaviors. However, their hypothermic effect is compatible with the stimulation of dopaminergic function which seems not to be mediated by D2/D3 receptors.

Selective PGHS-2 inhibitors: a rational approach for treatment of the inflammation.

Prostaglandin-H synthase exists in two isoforms, PGHS-1 and PGHS-2. PGHS-1 is present and is constitutively expressed in most cells and tissues, whereas PGHS-2 is mainly thought to mediate inflammation. Selective prostaglandin-H synthase-2 (or cyclooxygenase-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). This review addresses the main classes of the selective PGHS-2 inhibitors whose selectivity is documented by supporting PGHS-1 and PGHS-2 enzyme data. In addition, we also describe our experience in design, synthesis and pharmacological in vivo evaluation of new 1,2-benzodioxole derivatives as candidate of the selective PGHS-2 inhibitors, with special attention to molecular dynamics simulations of these derivatives attached to the active site of PGHS-2.