HPLC-MS/MS measurement of lidocaine in rat skin and plasma. Application to study the release from medicated plaster.

A simple, sensitive HPLC-MS/MS method was developed and validated for the determination of lidocaine in skin and plasma of rats. The methods were established and validated assessing lower limit of quantitation (LLOQ), linearity, intra and inter-day precision and accuracy, selectivity, recovery and matrix effect. Chromatography was done on a Gemini column embedded with C18 stationary phase (50 mm × 2.0 mm, 5 µm particle size), using a gradient with mobile phases consisting of 0.1% HCOOH in bidistilled water and 0.1% HCOOH in acetonitrile. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring, using target ions m/z 235.10 for lidocaine and m/z 245.10 for lidocaine-d10, used as internal standard. RESULTS: The linearity of the method was in the ranges of lidocaine concentrations 10.0-200.0 ng/mL for skin homogenate (accuracy 94.1-105.5%; R2 ≥ 0.998) and 0.025-2 ng/mL for plasma (accuracy 96.2-104.8%; R2 ≥ 0.996). The intra- and inter-day precision and accuracy determined on three quality control samples (20, 75 and 170 ng/mL for skin and 0.075, 0.4 and 1.5 ng/mL for plasma) were ≤4.2% and 103.8-108.2% for skin and ≤12.4% and 95.5-101.4% for plasma. The LLOQ was 10 ng/mL in skin homogenate and 0.025 ng/mL in plasma. The applicability of the method was demonstrated by measuring lidocaine in skin and plasma after exposure to medicated patches containing 5% lidocaine.

Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia.

Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, ß, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1ß had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer.

Oleamide-mediated sleep induction does not depend on perturbation of membrane homeoviscosity.

To verify whether the sleep-inducing properties of oleamide were related to its ability to perturb membrane homeoviscosity, affecting 5-HT(2A) receptors, we compared the effects of oleamide and oleic acid, the latter lacking both the sleep-inducing effect and the action on 5-HT(2A) receptors. In binding studies the two compounds did not directly interact with rat brain cortex 5-HT(2A) receptors, nor did they increase the affinity of a 5-HT(2A) agonist, either in vitro or ex vivo. They had similar fluidizing effects, in vitro at high concentrations (>/=10 microM), and ex vivo after a dose of 100 mg/kg, and they reduced locomotor activity with similar potency. There thus appears to be no causal relationship between the fluidizing effects of oleamide and its sleep-inducing properties.

Cocaine-seeking behavior in response to drug-associated stimuli in rats: involvement of D3 and D2 dopamine receptors.

Previous studies employed a second-order schedule paradigm maintained by cocaine reinforcement to show that BP897, a dopamine D(3) partial agonist, selectively modulated drug-seeking behavior. We investigated its effect on drug-seeking behavior induced by presentation of stimuli associated with and predictive of cocaine availability after a period of extinction and in the absence of any further cocaine. Male rats were trained to associate discriminative stimuli (S(D)) with the availability of intravenous (i.v.) 0.25 mg/0.1 ml/infusion cocaine (S(D+)) or no-reward (S(D-)) saline solution. Each infusion of cocaine or saline was followed by a response-cue signaling 20-s time-out (TO). After meeting the self-administration training criterion rats were placed on extinction conditions during which i.v. solutions and S(D)s were withheld. Every other 3 days on which rats met the extinction criterion, reinstatement tests were conducted, presenting the S(D+) or S(D-) noncontingently together with a contingent presentation of cocaine- or saline-cues signaling 20-s TO. Regardless of the order of presentation or the nature of the stimuli (auditory or visual), cocaine-associated but not saline-associated stimuli reinstated responding on the previously active lever. Presentation of cocaine-associated stimuli induced lasting drug-seeking behavior for at least eight test sessions. BP897 (1.0 mg/kg i.p.) significantly attenuated this behavior. Since it has been reported that BP897 can interact with a panel of different receptors with high affinity, we evaluated the effects of 7-OH-DPAT, an agonist to D(3) receptors, raclopride, a preferential antagonist to D(2) receptors, and WAY 100,635, an antagonist at 5-HT(1A) receptors, on drug-seeking behavior. 7-OH-DPAT (0.1-3.0 mg/kg i.p.) had biphasic effects on reinstatement induced by the cocaine-associated cues, low dosages reducing and high dosages increasing the impact of cocaine-associated stimuli on rats' behavior. Raclopride (0.1, 0.3 mg/kg s.c.) completely prevented drug-seeking behavior induced by the reintroduction of cocaine-associated stimuli. WAY 100,635 (0.1-1.0 mg/kg s.c.) had no effect on this behavior. These results, while confirming that the partial agonist at the D(3) receptors, BP897, might be a useful medication, also suggest a role of D(2) receptors in cue-induced cocaine-seeking behavior.

Evidence that dopamine mechanisms in the nucleus accumbens are selectively involved in the effect of desipramine in the forced swimming test.

Bilateral injections of 5 or 1 (but not 0.5) micrograms sulpiride into the nucleus accumbens reduced the effect of a 7-day treatment with 10 mg/kg/day desipramine in the forced swimming test. Bilateral injections of 5 or 1 micrograms sulpiride in the caudate-putamen did not modify the anti-immobility effect of desipramine. The data support the hypothesis that dopamine mechanisms in the limbic regions of the brain of the rat are involved in the effect of repeated treatment with desipramine in the forced swimming test.

The role of serotonin and dopamine in brain in the antidepressant-like effect of clonidine in the forced swimming test.

The effect of clonidine (0.1 mg/kg, i.p.), as a three-injection course, on behaviour in the forced swimming test was studied in rats injected intracerebroventricularly (i.c.v.) with 150 micrograms 5,7-dihydroxy-tryptamine (5,7-DHT) to destroy serotonin (5-HT) neurones or treated with 100 mg/kg (i.p.) (+/-)-sulpiride or 0.5 micrograms/0.5 microliter (-)-sulpiride in the nucleus accumbens. Clonidine significantly increased struggling and reduced floating and the effects were antagonized by both treatments with sulpiride but not by 5,7-DHT which markedly depleted 5-HT in brain. The results suggest that the mesolimbic dopaminergic system but not 5-HT neurones, plays a permissive role in the antidepressant-like effect of clonidine in the forced swimming test.

8-Hydroxy-2-(di-n-propylamino) tetralin, a selective serotonin1A receptor agonist, blocks haloperidol-induced catalepsy by an action on raphe nuclei medianus and dorsalis.

The selective serotonin1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was studied for its ability to reverse haloperidol-induced catalepsy in rats. Given subcutaneously 8-OH-DPAT (0.06-0.5 mg/kg), dose-dependently antagonized the catalepsy induced by 1 mg/kg of haloperidol. Intraventricular injection of the serotonin (5-HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which caused marked depletion of 5-HT in brain, did not change haloperidol-induced catalepsy per se, but completely antagonized the anticataleptic effect of subcutaneously administered 8-OH-DPAT. When injected directly into the median or dorsal raphe nucleus, 8-OH-DPAT, in doses ranging from 0.2 to 5 micrograms/0.5 microliter, reduced the catalepsy induced by haloperidol. The results suggest that the activation of 5-HT1A receptors, probably those located presynaptically on 5-HT-containing cell bodies, reduces the catalepsy induced by haloperidol.

Roles of 5-HT(1A) receptors in the dorsal raphe and dorsal hippocampus in anxiety assessed by the behavioral effects of 8-OH-DPAT and S 15535 in a modified Geller-Seifter conflict model.

8-OH-DPAT [8-hydroxy-2-(di-N-propylamino)tetralin], a 5-HT(1A) receptor agonist, and S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine, a partial agonist at 5-HT(1A) receptors, were administered into the dorsal raphe nucleus and dorsal hippocampus and their behavioral effects were assessed in a modified Geller-Seifter conflict model. Injected into the dorsal raphe nucleus 8-OH-DPAT, 1 microg but not 0.04 or 0.2 microg 0.5 microl(-1), and S 15535, 2.5 microg but not 0.1 or 0.5 microg 0.5 microl(-1), significantly increased punished responding with no effect on rates of unpunished or time-out responding. WAY 100635, a selective 5-HT(1A) receptor antagonist, injected subcutaneously at 0. 3 mg kg(-1) 30 min before 1 microg 8-OH-DPAT or 2.5 microg S 15535 in the dorsal raphe, completely antagonized their effects on punished responding. At doses ranging from 1 to 10 microg microl(-1) injected into the CA1 region of the dorsal hippocampus neither 8-OH-DPAT nor S 15535 modified punished responding or the rates of time-out. At the highest doses, 8-OH-DPAT significantly reduced unpunished responding whereas S 15535 had the opposite effect. The results suggest that stimulation of 5-HT(1A) receptors in the dorsal raphe nucleus has anxiolytic-like effects whereas stimulation of postsynaptic receptors in the dorsal hippocampus has no anxiolytic or anxiogenic effects, at least judging from changes in rates of punished responding. These results are compatible with the hypothesis that 5-HT(1A) receptor agonists and partial agonists attenuate anxiety by reducing serotonergic transmission in brain areas innervated by the dorsal raphe nucleus.

Pyrroloquinoxaline derivatives as high-affinity and selective 5-HT(3) receptor agonists: synthesis, further structure-activity relationships, and biological studies.

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a series of novel pyrroloquinoxalines and heteroaromatic-related derivatives are described. The new pyrroloquinoxaline-related ligands were tested in rat cortex, a tissue expressing high density of 5-HT(3) receptors, and on NG108-15 cells and exhibited IC(50) values in the low nanomolar or subnanomolar range, as measured by the inhibition of [(3)H]zacopride binding. The SAR studies detailed herein delineated a number of structural features required for improving affinity. Some of the ligands were employed as "molecular yardsticks" to probe the spatial dimensions of the lipophilic pockets L1, L2, and L3 in the 5-HT(3) receptor cleft, while the 7-OH pyrroloquinoxaline analogue was designed to investigate hydrogen bonding with a putative receptor site H1 possibly interacting with the serotonin hydroxy group. The most active pyrroloquinoxaline derivatives showed subnanomolar affinity for the 5-HT(3) receptor. In functional studies ([(14)C]guanidinium accumulation test in NG108-15 hybrid cells, in vitro) most of the tested compounds showed clear-cut 5-HT(3) agonist properties, while some others were found to be partial agonists. Several heteroaromatic systems, bearing N-substituted piperazine moieties, have been explored with respect to 5-HT(3) affinity, and novel structural leads for the development of potent and selective central 5-HT(3) receptor agonists have been identified. Preliminary pharmacokinetic studies indicate that these compounds easily cross the blood-brain barrier (BBB) after systemic administration with a brain/plasma ratio between 2 and 20, unless they bear a highly hydrophilic group on the piperazine ring. None of the tested compounds showed in vivo anxiolytic-like activity, but potential analgesic-like properties have been possibly disclosed for this new class of 5-HT(3) receptor agonists.

Evidence that 5-hydroxytryptamine in the forebrain is involved in naloxone-precipitated jumping in morphine-dependent rats.

A withdrawal syndrome was precipitated by naloxone in morphine-dependent rats injected with 5,7-dihydroxytryptamine (5,7-DHT) in the ventromedial tegmentum (VMT) at the level of the nucleus interpeduncularis. 5,7-DHT, which markedly depleted 5-hydroxytryptamine (5-HT) in the forebrain but not in the brainstem, significantly reduced jumping in abstinent rats with no significant effect on other withdrawal signs. The effect of morphine 10 mg kg-1 on responses on the hot plate was unchanged in 5,7-DHT-treated rats. The findings suggest that 5-HT in the forebrain is selectively involved in the jumping of morphine-abstinent rats.