Development and validation of the stability indicating RP-UHPLC method for the determination of the chemical purity and assay of bimatoprost.

A simple, rapid and accurate ultra-high performance liquid chromatographic (UHPLC) method with a UV detection for the determination of the chemical purity and assay of bimatoprost (BT-1) was developed. The chromatographic separation was achieved with the use of an Acquity BEH C8, 150 × 2.1 mm, 1.

Use of the hyphenated LC-MS/MS technique and NMR/IR spectroscopy for the identification of exemestane stress degradation products during the drug development.

Exemestane (6-Methyleneandrosta-1,4-diene-3,17-dione) active pharmaceutical ingredient (EE-3) was subjected to thermal, photolytic, oxidative, acidic and base stress conditions prescribed by the ICH (International Conference on Harmonization) guideline Q1A(R2). EE-3 was found to degrade in base, acidic and oxidative conditions. Eleven new degradation products of EE-3 were characterized by the LC-MS/MS technique.

A novel method for the determination of chemical purity and assay of menaquinone-7. Comparison with the methods from the official USP monograph.

An HPLC method with UV detection and separation with the use of a C30 reversed phase analytical column for the determination of chemical purity and assay of menaquinone-7 (MK7) in one chromatographic run was developed. The method is superior to the methods published in the USP Monograph in terms of selectivity, sensitivity and accuracy, as well as time, solvent and sample consumption. The developed methodology was applied to MK7 samples of active pharmaceutical ingredient (API) purity, MK7 samples of lower quality and crude MK7 samples before purification.

Development and Validation of a UHPLC UV Method for the In-Process Control of Bosentan Monohydrate Synthesis.

Bosentan monohydrate (4--butyl--[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]benzene-1-sulfonamide monohydrate) is a dual endothelin receptor antagonist (ERA) applied in the treatment of pulmonary arterial hypertension. To achieve effective process control of the bosentan monohydrate synthesis, it was necessary to develop a selective and not highly time-consuming method for ultra-high performance liquid chromatography (UHPLC). The method is characterized by adequate sensitivity, reproducibility and selectivity for the determination of bosentan monohydrate and related compounds from all synthetic stages.

A Validated TLC-Densitometric Method for the Determination of Mesterolone in Bulk Material and in Tablets.

Mesterolone is a synthetic androgenic steroid indicating a weak anabolic activity. A new, simple in use, and economical TLC-densitometric method in normal phase system (NP-TLC) has been developed and validated for the identification and quantitative determination of mesterolone in bulk drug and in tablet formulation. NP-TLC analysis was performed on aluminium plates precoated with silica gel 60F254 as the stationary phase using chloroform-acetone (40 : 10, v/v) as mobile phase.

Determination of organic volatile impurities in nepafenac by GC method.

The methods for controlling volatile impurities, including reagent and starting material, in Nepafenac active pharmaceutical ingredient, are reported. The proposed methods were developed using gas chromatography (GC) and gas chromatography with headspace injection (GC-HS) and validated according to the requirements of the ICH (International Conference of Harmonization) validation guidelines Q2R1 and the guideline for residual solvents Q3C.

Searching for new derivatives of neocryptolepine: synthesis, antiproliferative, antimicrobial and antifungal activities.

A series of novel amino acid and dipeptide derivatives of neocryptolepine were synthesized and tested for their antimicrobial, antifungal and antiproliferative activity in vitro against cancer cell lines (KB, A549, MCF-7, LoVo) and normal mice fibroblast cells (BALB/3T3). Biological evaluation revealed that almost all of the new compounds displayed high antiproliferative activity against the tested cells and moderate to potent antibacterial activities. Interestingly, these compounds were active against Candida albicans biofilms at doses significantly lower than those required against free-floating planktonic fungal cells.

Development and validation of high performance liquid chromatography with a spectrophotometric detection method for the chemical purity and assay of nepafenac.

The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyze the chemical purity and assay of nepafenac, an active pharmaceutical ingredient (API). During literature search only a few publications were found about nepafenac. HPLC UV methods were mainly presented in patent documents about nepafenac synthesis and chemical purity.

Stability of sample solution as a crucial point during HPLC determination of chemical purity of temozolomide drug substance.

An HPLC method for determination of related substances in temozolomide drug substance was developed. Particular attention was paid to the stability studies due to the fact that temozolomide is unstable in a solution and quickly decomposes to its main degradation product 5-amino-4-imidazolecarboxamide (AIC). A mixture of diluted acetic acid and acetonitrile (4:1, v/v) as a diluent guaranteed lowering the decomposition of temozolomide in the solution.

Synthesis and biological evaluation of new amino acid and dipeptide derivatives of neocryptolepine as anticancer agents.

The syntheses of neocryptolepine derivatives containing an amino acid or a dipeptide at the C-9 position and their evaluation for antitumor activity in vitro and in vivo are reported. To establish the influence of an amino acid or a peptide on the physicochemical properties of 5H-indolo[2,3-b]quinoline (DiMIQ), lipophilic and hemolytic properties were investigated. Most of the compounds displayed a high antiproliferative activity in vitro and strongly inhibited growth of tumor in mice compared to cyclophosphamide.

Age increases anxiety and reactivity of the fear/anxiety circuit in Lewis rats.

A growing body of data indicates that changes in emotional behavior occur with age. Young Lewis rats are known to display hypofunction of the HPA axis. With age the reactivity of this axis is thought to increase with a concomitant rise in anxiety.

Diverse sensitivity of RHA/Verh and RLA/Verh rats to emotional and spatial aspects of a novel environment as a result of a distinct pattern of neuronal activation in the fear/anxiety circuit.

Psychogenetically selected Roman high (RHA/Verh) and Roman low (RLA/Verh) avoidance rats constitute a well-recognized model of diverse emotional reactivity. The two Swiss lines display marked behavioral and endocrine differences in reaction to a novel environment. In our study we found that these differences are accompanied by a distinct, line-specific pattern of neuronal activation within the fear/anxiety circuit.

The effect of age on the dynamics and the level of c-Fos activation in response to acute restraint in Lewis rats.

Recent studies have reported an age-related increase of anxiety in rodents with a concomitant decrease in neuronal activity in some of the key structures of the fear/anxiety circuit. In the present study we present evidence that distinct parts of this circuit are differentially affected by age in Lewis rats. The effect of ageing is observed both at the actual level of neuronal activation and its time-course.

Expression of c-Fos in response to stressogenic stimuli in the amygdala of old vs. young rats--a preliminary study.

During aging many of the brain functions became deteriorated or altered. One of the most important age related changes is an increase of anxiety level, reported both in humans and in animals. Our study was intended to compare c-fos gene expression in amygdala, the key structure in anxiety/fear regulation, in old (24 months) and young (4 months) rats exposed to various behavioral stimulations.

22-kHz ultrasonic vocalization in rats as an index of anxiety but not fear: behavioral and pharmacological modulation of affective state.

Ultrasonic vocalization (USV) was found useful for differentiating fear and anxiety in rats. These affective states were established through a Pavlovian conditioning procedure. Danger stimulus, preceding unavoidable tail shock, elicited acute fear.

Emotional changes related to age in rats--a behavioral analysis.

The present study investigated age-related differences in the emotional behavior of rats using factor analysis to identify motivational factors influencing spontaneous behavior in open field with illuminated center (OF), plus maze (EPM) and social interactions test. Animals of the same strain, bred under the same conditions, formed two experimental groups: young adults (YA, N=20) tested at the age of 4 months and old rats (OA, N=16) tested at the age of 24 months. The computer video based tracking system EthoVision was used for automated acquisition and analysis of data.

Heart rate changes in partially restrained rats during behaviorally and pharmacologically evoked emotional states.

The effect of fear and relief from fear on heart rate (HR) was studied in partially restrained adult male rats. The emotional state was influenced behaviorally with the use of Pavlovian aversive conditioning procedure, and pharmacologically by injections of the selected anxiolytics and anxiogenics. A signal of danger (DS)--light or tone, preceded tail-shock (excitatory trials), while a signal of safety (SS), respectively tone or light, overlapped last 3s of 5s DS and predicted an omission of this expected aversive event (inhibitory trials).

Bicuculline administration into ventromedial hypothalamus: effects on fear and regional brain monoamines and GABA concentrations in rats.

The effects of bicuculline methiodide administration into ventromedial hypothalamus (15 ng per site, bilaterally) on fear behavior and monoamines (NA, DA, 5-HT) and GABA in structures of the brain defensive system (hypothalamus, midbrain gray matter, amygdala, hippocampus and frontal cortex) were studied. Fear behavior was examined in the modified version of light-dark transition test. The time out from the illuminated compartment of chamber, the time spent there and number of returns to the illuminated compartment was measured.

Contrasting expressions of aggressive behavior released by lesions of the central nucleus of the amygdala during wakefulness and rapid eye movement sleep without atonia in cats.

Whether damage to the central nucleus of the amygdala (Ace) contributes to the predatorylike attack sometimes observed in rapid eye movement sleep without atonia (REM-A), created in cats by bilateral pontine lesions, was examined. Such lesions eliminate REM sleep skeletal muscle atonia and release elaborate behavior. Unilateral damage to the Ace alone increased affective defensive aggressive behavior toward humans and conspecifics without altering predatory behavior in wakefulness.

Electrophysiological correlates of the limbic-motor interactions in various behavioral states in rats.

Depth electroencephalographic (EEG) activity was recorded from basolateral amygdala (BLA), ventral subiculum (VSB), n. accumbens (ACC) and subpallidal area (SPL) in freely moving rats, during locomotor tasks with various types of reinforcement in order to compare the strength of limbic-motor interactions in selected behavioral situations. For all EEG signals multichannel coherences (ordinary, multiple and partial) were calculated using autoregression model.

Social interactions in rats: behavioral and neurochemical alterations in DSP-4-treated rats.

Noradrenergic neurotoxin DSP-4, preceded by zimelidine to prevent serotonin depletion, was administered IP to rats behaving in a defensive-submissive manner in a resident-intruder paradigm. Computer-based ethological analysis revealed the decrease of frequency and duration of defensive episodes and marked increase of offensive aggression. This might suggest an increase of aggressiveness and therefore support the notion of an inhibitory role of the noradrenergic system in aggressive behavior independently of the model used.

DSP-4 lesion of locus coeruleus does not affect spontaneous predatory behaviour in cats.

The effect of the destruction of locus coeruleus noradrenergic (LC NA) projection on spontaneous predatory attack, predatory competition and food intake was studied in cats. Selective noradrenergic neurotoxin DSP-4 injected into LC caused 71% decrease of noradrenaline content in amygdala and 41% decrease in hypothalamus. Predatory behaviour, predatory competition as well as food intake remained unchanged.

Scopolamine-induced alterations in predatory behaviour pattern in cats.

Predatory behaviour in its full pattern (i.e., following the mouse, killing it and consuming the carcass) was tested in semi-natural conditions in cats.

Changes in the aggressive behavior of cats treated with amphetamine.

Male, adult cats were tested in three different experimental conditions which might lead to aggressive display: (1) Cat-mouse interaction, (2) Predatory competition between two mice killers, (3) Conspecific interactions. DL-amphetamine in a dose of 1.5 mg/kg was administered every day for three weeks to one group of cats, whereas the other group was treated with saline.