Novel nalidixic acid derivatives targeting topoisomerase II enzyme; Design, synthesis, anticancer activity and effect on cell cycle profile.

AIM: Design and synthesis of novel nalidixic acid derivatives of potent anticancer and topoisomerase II inhibitory activities were our major aim. MATERIALS & METHODS: All the newly synthesized nalidixic acid derivatives were submitted to the National Cancer Institute (NCI), Bethesda, USA and were accepted for single dose screening. Further investigation via IC50 determination of the most potent compound 6a against K-562 and SR leukemia cell lines. Finally, the topoisomerase II inhibitory activity, the cell cycle analysis and molecular docking of 6a were performed in order to identify the possible mechanism of the anticancer activity. RESULTS: Compound 6a showed interesting selectivity against leukemia especially K-562 and SR subpanels with IC50 35.29 µM and 13.85 µM respectively. Moreover, compound 6a revealed potent topoisomerase IIα and topoisomerase IIß inhibitory activity compared with known topoisomerase inhibitors such as doxorubicin and topotecan with IC50 1.30 µM and 0.017 µM respectively. Cell cycle analysis indicated that compound 6a induced cell cycle arrest at G2-M phase leading to inhibition of cell proliferation and apoptosis. Molecular modeling demonstrated that the potent topoisomerase inhibitory activity of 6a was due to the interaction with the topoisomerase II enzyme through coordinate bonding with the magnesium ion Mg2+, hydrogen bonding with Asp 545 and arene cation interaction with His 759.

Nalidixic acid surrogate test for susceptibility to ciprofloxacin in Salmonella. Revisiting the question.

We investigated the reliability of nalidixic acid (NA) susceptibility as a marker of ciprofloxacin susceptibility in Salmonella, analysing 302 stool isolates. NC53 of the MicroScan system was used for NA susceptibility tests and the E-test was used for ciprofloxacin susceptibility tests. Among the isolates, 178 (58.9 %) were serogroup B, 74 (24.5 %) were serogroup D, 27 (8.9 %) were serogroup C and 23 (7.6 %) were from other minor serogroups. Globally, susceptibility to NA correctly predicted the susceptibility of Salmonella to ciprofloxacin, with a sensitivity of 81.5 %, a specificity of 97.6 %, and positive and negative predictive values of 88 and 96 %, respectively. However, there were differences among the serogroups in terms of sensitivity (P<0.001) and positive predictive values (P=0.013). NA is a reliable marker for serogroup D, but not for serogroups B or C. According to these findings, NA susceptibility measured with the MicroScan system can be used as a marker of ciprofloxacin resistance in some serogroups in our setting.

The effects of dopaminergic/serotonergic reuptake inhibition on maternal behavior, maternal aggression, and oxytocin in the rat.

Studies using dopaminergic and serotonergic agonists or antagonists implicate involvement of these systems in various aspects of early maternal behavior and postpartum aggression towards an intruder in rats, both of which are associated with the presence of oxytocin in specific brain regions. It is unclear however, if or how long-term uptake inhibition of either neurotransmitter system alone or in combination, affects oxytocin system dynamics or maternal behavior/aggression. Pregnant women frequently take drugs (antidepressants, cocaine) that induce long-term reuptake inhibition of dopamine and/or serotonin, thus it is important to understand these effects on behavior and biochemistry. Rat dams were treated throughout gestation with amfonelic acid, fluoxetine, or a combination of both, to investigate effects of reuptake inhibition of dopamine and serotonin systems respectively, on maternal behavior, aggression and oxytocin. The more appetitive aspects of maternal behavior (nesting, licking, touching) and activity were increased by the low dose of amfonelic acid, high dose of fluoxetine, or the high dose combination more than other treatments. Aggression was decreased by amfonelic acid and somewhat increased by fluoxetine. Dopamine uptake inhibition appears to have a strong effect on hippocampal oxytocin levels, while receptor dynamics may be more strongly affected by serotonin uptake inhibition.

Gestational treatment with cocaine and fluoxetine alters oxytocin receptor number and binding affinity in lactating rat dams.

Cocaine administered chronically throughout gestation has been correlated with deficits in maternal behavior, increased maternal aggressive behavior and decreased oxytocin levels in rats. In addition to its effects on oxytocin levels, cocaine is a potent serotonergic, dopaminergic and noradrenergic reuptake inhibitor. Alterations in the dopaminergic and serotonergic systems have been suggested as possibly having a role in cocaine-induced maternal aggression. This study was in part, an attempt to understand some of the mechanisms by which cocaine increases postpartum aggression, particularly as they relate to changes in the oxytocin system. Oxytocin receptor number and binding affinity in the medial preoptic area of the hypothalamus, ventral tegmental area, hippocampus and amygdala were determined for lactating rat dams on postpartum day 6 (PPD 6) that were gestationally treated with cocaine, fluoxetine, saline or an amfonelic acid/fluoxetine drug combination. Cocaine and fluoxetine treatment both resulted in a significant up-regulation of oxytocin receptor number and lower receptor affinity in the amygdala of lactating rat dams compared to saline controls and the amfonelic acid/fluoxetine combination treatment group. Cocaine treatment also resulted in a significant down-regulation of oxytocin receptors in the medial preoptic area and both cocaine and fluoxetine treated dams had the highest affinity for oxytocin receptors in this brain region. Results of the present study support previous data indicating that alterations in oxytocinergic and perhaps serotonergic system dynamics in the amygdala may play a role in cocaine-induced postpartum aggression.

A prodrug approach toward the development of water soluble fluoroquinolones and structure--activity relationships of quinoline-3-carboxylic acids.

A fluoroquinolone prodrug, PA2808, was prepared and shown to convert to the highly active parent drug PA2789. In vitro and in vivo activation of PA2808 by alkaline phosphatase was demonstrated using disk diffusion and rat lung infection models. The water solubility of PA2808 showed a marked increase compared to PA2789 over a pH range suitable for aerosol drug delivery. A total of 48 analogues based on PA2789 were prepared and screened against a panel of Gram-positive and Gram-negative pathogens. Incorporating a cyclopropane-fused pyrrolidine (amine) at C-7 resulted in some of the most active analogues.

Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice.

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by approximately 95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre-synaptic regulation of both impulse-dependent (exocytotic) and carrier-mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild-type mice using fast cyclic voltammetry. Impulse-dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild-type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 microm) increased extracellular DA via DAT reversal in both wild-type (by 459 nm) and VMAT2 mutant (by 168 nm, p < 0.01 vs. wild-type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 microm). Simultaneously, amphetamine decreased impulse-dependent DA release, albeit less in homozygous (by 55%) than in wild-type (by 78%) mice. In wild-types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (-)sulpiride (1 microm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (-)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse-dependent DA release with a lower EC50 value in homozygous mice (12 nm) compared with wild-types (34 nm), indicating the compensatory presence of functionally supersensitive release-regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse-dependent or carrier-mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.

A comparison of the patterns of striatal Fos-like immunoreactivity induced by various dopamine agonists in rats.

In contrast to the highly patchy patterns of Fos-like immunoreactivity seen in the rostral striatum after administration of a number of dopamine agonists, the monoamine uptake blocker cocaine has been reported to produce a relatively homogeneous pattern of gene expression. In the current study we extended these observations by using a quantitative technique to demonstrate that while amphetamine and apomorphine produce patchy striatal Fos expression, the selective dopamine uptake inhibitors amfonelic acid, nomifensine and GBR-12909 all, like cocaine, produce near random patterns of gene expression. These findings suggest that the production of relatively 'non-patchy' patterns of immediate early gene expression may be a general property of dopamine transport inhibitors unrelated to any unique pharmacological properties of cocaine.

[Effects of methamphetamine on responsiveness to conditioned fear stress].

The present study examined the effect of repeated methamphetamine (MA) pretreatment on conditioned fear stress in male Wistar-King rats. Rats received MA or the vehicle according to the repeated escalating dose schedule (1.25, 2.5, 3.75, 5 mg/kg s.c x2/every other day for a week). After a 5 day drug abstinent period, the rats were exposed to conditioned fear stress (CFS; exposure to an environment paired previously with footshock). Repeated but not single MA pretreatment significantly increased conditioned freezing behavior, suggesting that rats previously exposed to chronic MA are hypersensitive to subsequent stress than control rats. Repeated MA treatment did not decrease basal dopamine concentrations in the brain. Furthermore, repeated co-administration of MK-801 (non-competitive NMDA antagonist), amfonelic acid (dopamine reuptake inhibitor) or fluoxetine (serotonin reuptake inhibitor) with MA did not alter the enhanced freezing behavior. Taken together, it seems that MA-induced hypersensitivity to stress is not due to the neurotoxic effect of MA. While co-administration of SCH23390 (D1/5 antagonist) or raclopride (D2/3 antagonist) had no effect on the MA-induced increase in freezing, co-administration of nemonapride (D2/3/4 antagonist) prevented this increase. These results suggest that MA-induced enhancement of anxiety might be mediated by D4 receptors. The homovanilic acid (HVA) levels in the striatum were elevated by footshock in MA-treated rats but not in saline-treated rats. Furthermore, MA-treated rats showed increased metabolism of dopamine (DA) in the medial prefrontal cortex (mPFC), even when placed in the shock chamber without shocks. The HVA levels in the striatum in MA-treated rats were more elevated by CFS than these in saline-treated rats. These results suggest that the striatum DA system, as well as the mPFC DA system suggested previously, may be associated with emotional hypersensitivity to stress following repeated MA treatment.

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography.

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15-100 micrograms ml-1, with a correlation coefficient greater than 0.999 and detection limits in the 0.2-0.7 ng ml-1 range. Intra- and inter-day precision values of about 0.8-1.2% RSD (n = 11) and 1.3-2.0% RSD (n = 30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml-1.

Effects of dopamine agonists on appetitive and consummatory male sexual behavior in Japanese quail.

The effects of pharmacological manipulations of dopaminergic transmission on appetitive and consummatory aspects of male sexual behavior were investigated in castrated male Japanese quail treated with exogenous testosterone. Appetitive male sexual behavior was assessed by measuring a learned social proximity response and consummatory behavior was assessed by measuring copulatory behavior per se. The nonselective dopamine receptor agonist, apomorphine, inhibited in a dose-dependent manner both components of male sexual behavior. Two indirect dopamine agonists were also tested. Nomifensine, a dopamine re-uptake inhibitor, decreased appetitive sexual behavior but increased the frequency of mount attempts, a measure of consummatory sexual behavior. Amfonelic acid, a compound that enhances dopaminergic tone by a complex mechanism, increased aspects of both appetitive and consummatory behaviors. These data suggest that, in quail, as in rodents, increases in dopaminergic tone facilitate both appetitive and consummatory aspects of male sexual behavior. Apomorphine may be inhibitory in quail because it acts primarily on D2-like receptors, unlike in rats, where it stimulates sexual behavior and acts primarily on D1-like receptors at low doses but interacts with D2-like receptors at higher doses. This is supported by the observation that stereotyped pecking, a behavior stimulated selectively in quail by D2 agonists, was increased by apomorphine but not by the two indirect agonists. The observed partial dissociation between the effects of these dopaminergic agonists on appetitive and consummatory sexual behaviors suggests that these two components of male sexual behavior may be controlled by the action of dopamine through different neuronal systems.

Amphetamine induces hydroxyl radical formation in the striatum of rats.

Amphetamine-induced hydroxyl radical formation in the striatum of rats was investigated in this study. With the utilization of the microdialysis and HPLC-ECD, the striatal dopamine (DA) release and the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA), derived from the reaction of hydroxyl radicals (.OH) and salicylate in perfusion, were monitored and detected during desipramine and/or amphetamine (AMPH) administration. Our data revealed that after desipramine treatment AMPH injections not only amplified striatal DA release and 2,3-DHBA formation, but also intensified the stereotyped behaviors induced by AMPH. Furthermore, we discovered that alpha-methyl-para-tyrosine (alpha-MT) pretreatment prevented the onset of the above responses. In desipramine-treated rats, the tissue homogenization study demonstrated that a single dose of AMPH produced long-term depletion of striatal DA; this was not seen in saline-treated rats. Moreover, striatal DA depletion could be lessened by pretreatment with mannitol, a .OH scavenger. These results indicate that AMPH-induced striatal .OH formation might be DA-related in desipramine-treated rats, and suggest that .OH formation might be correlated with AMPH-induced neurodegeneration.

A rat model of distractibility: effects of drugs modifying dopaminergic, noradrenergic and GABAergic neurotransmission.

A procedure for analyzing effects of drugs on distractibility is proposed. Rats are trained to traverse a straight runway with a sucrose solution as reinforcement. Once the response has been acquired, an additional runway ending in an empty box is connected. The time spent investigating this additional runway is the measure of distractibility. Amphetamine, 1 mg/kg i.p., increased distractibility. In rats that were never reinforced, amphetamine at a dose of 1 mg/kg reduced the time spent in the additional runway. This shows that the effects of amphetamine in the reinforced animals cannot be interpreted as enhanced exploration. Furthermore, the benzodiazepines diazepam (2 and 4 mg/kg, i.p.) and chlordiazepoxide (2.5, 5 and 10 mg/kg, i.p.), known to enhance exploration of novel environments, did not affect the time spent in the additional runway in sucrose-reinforced animals. It was concluded that the procedure indeed is a model of distractibility. The dopamine antagonist cis(Z)-flupenthixol, at a dose of 0.25 mg/kg, i.p., blocked the effects of amphetamine, 1 mg/kg. Flupenthixol itself, in doses of 0.25 and 0.5 mg/kg, did not affect the time spent in the additional runway. This suggests that enhanced dopaminergic activity indeed is responsible for the effects. This proposal is further supported by experiments showing that the noradrenaline precursor dihydroxyphenylserine (10 mg/kg + carbidopa, 50 mg/kg, both i.p.) and the noradrenergic neurotoxin DSP4 (50 mg/kg, i.p.) had no effect on distractibility. Moreover, amfonelic acid, a dopamine releaser with slight or no effect on noradrenergic neurotransmission, had effects very similar to those of amphetamine when given in doses of 0.25 and 0.5 mg/kg, i.p. A lower dose, 0.125 mg/ kg, was ineffective. Taken together, these data suggest that enhanced dopaminergic neurotransmission increases distractibility in the rat. However, both amphetamine and amfonelic acid may stimulate serotonin release. Until serotonergic drugs have been tested, a contribution of this transmitter cannot be ruled out. The distraction procedure may constitute an animal model of some kinds of disordered information processing.

Central nervous stimulants facilitate sexual behavior in male rats with medial prefrontal cortex lesions.

Male rats with lesions of the cerebral cortex near the midline in the frontal region destroying most of the cingulate cortex and producing some damage to adjacent frontal areas have very long mount and intromission latencies. Otherwise their sexual behavior is essentially normal. The dopamine releasers amfonelic acid, 0.5 mg/kg, and amphetamine, 1 mg/kg, reduced the mount and intromission latencies in males with such lesions. Caffeine, 30 mg/kg, had similar effects. None of the drugs modified sexual behavior in intact males. It has been suggested that medial prefrontal lesions reduce the animal's reactivity to environmental stimuli, and hence renders the activation of sexual behavior difficult. Present results show that stimulant drugs are capable of compensating for this reduced reactivity. The possible mechanisms behind this effect are discussed. The lesion had also a small but consistent effect on the intromission ratio, suggesting some motor impairment. The effect on intromission ratio was not reduced by the drugs, suggesting that the lesion's motor consequences are mediated by mechanisms different from those controlling behavioral reactivity. The noradrenaline precursor dl-threo-dihydroxyphenylserine, 10 mg/kg, in combination with carbidopa, 50 mg/kg, increased mount and intromission latencies in both intact and lesioned males. Thus, activation of noradrenergic neurotransmission had effects opposite to those found after activation of dopaminergic transmission. Noradrenergic stimulation cannot, therefore, be important for the effects of amphetamine or amfonelic acid.

Physico-chemical properties and biological activities of erythromycin nalidixate.

Erythromycin nalidixate was prepared by combining nalidixic acid with erythromycin base. Thin-layer chromatographic studies and infrared absorption spectrum confirmed homogeneity of the new salt. The salt is very soluble in nonpolar solvent and freely soluble in polar solvent. The salt is quite stable at room temperature (30 +/- 1 degrees C). The antimicrobially active dose of the salt was found to be 820 micrograms/mg. Serum protein binding amounted to 85% and was reversible. LD50 in mice was found to be 371.5 mg/kg when the intraperitoneal route was used.

The effects of amfonelic acid, a dopamine uptake inhibitor, on methamphetamine-induced dopaminergic terminal degeneration and astrocytic response in rat striatum.

Administration of methamphetamine (MA) induces degeneration of dopaminergic nerve terminals and astrogliosis, such as hypertrophy and an increase in apparent number, in the neostriatum. In this experiment adult rats were treated with MA (10 mg/kg, i.p.) 4 times in one day at 2 h intervals. Amfonelic acid (AFA), a dopamine reuptake inhibitor, was administered (20 mg/kg, i.p.) at the same time the last MA dose was given. Three days later, dopaminergic terminals and astrocytes were examined immunohistochemically and the contents of striatal dopamine and its metabolites were analyzed by HPLC. The results showed that MA-induced the typical depletion of dopaminergic terminals, reduction of dopamine content and astrogliosis in the neostriatum. AFA treatment completely prevented the effects of MA on the dopaminergic system, both morphologically and biochemically. However, the reaction of astrocytes remained in the region where the most severe depletion of dopaminergic terminals was seen in MA treated animals (ventral-lateral portion of neostriatum). The results support the concept that the dopamine transporter is involved in MA-induced dopaminergic nerve terminal degeneration. The results also indicate that blocking the dopamine transporter cannot completely prevent the reaction of astrocytes in the neostriatum, which indicates that the astrocytic reaction can be induced by factors other than degeneration of dopaminergic terminals in this region. Based on these and other data, it is hypothesized that MA may cause degeneration of corticostriatal glutamate pathways and this effect may be responsible for the astrogliosis in MA-AFA treated animals.

Prenatal exposure to cocaine: effects on aggression in Sprague-Dawley rats.

Social/aggressive behavior in adult rat offspring (beginning at postnatal Day 180) prenatally exposed to saline, cocaine, or amfonelic acid (AFA) was examined. Pregnant rats received injections of 15 mg/kg of cocaine, or 0.9% saline twice daily, s.c., or on 2 consecutive days at 4-day intervals, or 1.5 mg/kg amfonelic acid daily throughout gestational Days 1-20. Frequency, duration, and latency of 11 social/aggressive behaviors were recorded for two 15-min sessions during which a smaller male intruder replaced an ovariectomized female in the resident's home cage. Subjects received a s.c. saline injection before Session 1 and 2.0 mg/kg of gepirone, a 5HT1a partial agonist, prior to Session 2. Prenatal cocaine treatment resulted in alterations of aggressive behavior. Aggressive behavior was reduced by gepirone in all groups but to a lesser extent in the AFA group.

Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula.

In rats treated systemically with either amphetamine, amfonelic acid or apomorphine, large numbers of cells displaying Fos-like immunoreactivity (FLI) could be seen in the lateral zone of the lateral habenula. The induction of FLI by amphetamine could be blocked either by pretreatment with haloperidol or by 6-hydroxydopamine lesions of ascending dopamine fibers at the level of the lateral hypothalamus. In contrast, a variety of stressors selectively induced FLI in the most medial portion of the lateral habenula. These findings support the concept of a functional differentiation of the medial and lateral regions of the lateral habenula and provide further evidence for involvement of the habenula in the circuitry of the basal ganglia.

Changes in dopamine, serotonin and their metabolites in discrete brain areas of rat offspring after in utero exposure to cocaine or related drugs.

The concentrations of dopamine (DA), serotonin (5HT) and their metabolites were quantified in 5 brain areas of rats exposed to saline, cocaine (15 mg/kg b.i.d.), amitriptyline (10 mg/kg), or amfonelic acid (AFA, 1.5 mg/kg) throughout gestation. Male pups from 3 similarly treated dams were fostered to 2 surrogate dams. The process of breeding and rearing was repeated 4 times with new dams to build the groups to 4-12, since only one pup per litter was used for any one measurement. AFA was used to mimic the dopamine (DA) uptake blockade and stimulant properties of cocaine and amitriptyline was used to mimic the other pharmacological effects of cocaine. At postnatal days (PND) 30, 60, and 180, one pup per litter was removed for HPLC analysis of monoamines. A second pup received 0.3 mg/kg haloperidol, catalepsy assessed after 1 hr, and the brain used for analysis. The cataleptic response to haloperidol was unaffected by any prenatal treatment. The striatum from PND 30 cocaine rats had decreased levels of DA without a decrease in DA metabolites. At PND 60 in cocaine exposed rats, DA and DOPAC concentrations were increased, and 5HT levels were decreased in the striatum. The amitriptyline-exposed group exhibited decreased 5HT and 5-HIAA levels in the striatum. The hypothalamus of the cocaine group had lower levels of 5-HIAA, and other brain areas had a trend for lower levels of 5HT and 5-HIAA. At PND 180, DOPAC was increased in the striatum and prefrontal cortex of the cocaine group. Haloperidol-induced altered monoamine metabolism was unaffected by any prenatal treatment at any age. These data suggest that age-related changes in the DA and 5HT neurotransmission systems occur in rats exposed prenatally to cocaine. However, the ability of the dopaminergic system to respond to a challenge by a DA receptor blocker is unaltered by these in utero treatments.