Blood microsampling in cynomolgus monkey and evaluation of plasma PK parameters in comparison to conventional sampling.

Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (Cmax, Tmax and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples. The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution.

Slow dissociation of partial agonists from the D2 receptor is linked to reduced prolactin release.

In this study we investigated the correlation between affinity, efficacy, peripheral receptor occupancy, and kinetic properties of D2 dopamine receptor ligands with time-course evaluations of prolactin release in rat blood. We profiled typical and atypical antipsychotic antagonists at D2 receptors, the partial agonist aripiprazole, and four novel partial agonist compounds with different properties. Clozapine and quetiapine revealed lower prolactin release and fast dissociation kinetics, linking fast dissociation and prolactin-sparing properties. Surprisingly, haloperidol, a highly prolactin-releasing antagonist, shared intermediate dissociation properties. Factors other than kinetic properties may thus contribute to prolactin-releasing properties of antagonists. Partial agonists sharing similar efficacies and receptor occupancies differed markedly in their ability to induce hyperprolactinaemia. Aripiprazole moderately released prolactin even at high receptor occupancies, with slow dissociation from D2 receptors. Other compounds displaying low affinities and fast dissociations released prolactin substantially, although less than haloperidol. The effect augmented after repeated administrations. Compounds with high affinities and slow dissociation rates stimulated moderate prolactin release at high receptor occupancies, reaching a ceiling effect at 50-60% occupancy. Moreover, the effect developed tolerance. In conclusion, we investigated the affinity and kinetic properties of D2 partial agonists associated with their ability to induce prolactin release in blood. We propose that for D2 partial agonists, at comparable intrinsic activities and peripheral occupancies, the prolactin-releasing properties are linked to their kinetic rate properties. Differently from D2 antagonists, partial agonists display slow dissociation and high affinity associated with a low prolactin release profile.

Differential effects of antipsychotic and glutamatergic agents on the phMRI response to phencyclidine.

Acute administration of NMDA receptor (NMDAR) antagonists such as phencyclidine (PCP) or ketamine induces symptoms that closely resemble those of schizophrenia in humans, a finding that has led to the hypothesis that a decreased NMDAR function may be a predisposing or even causative factor in schizophrenia. However, the precise neuropharmacological mechanisms underlying these effects remain to be fully elucidated. Here, we applied pharmacological MRI (phMRI) to examine the brain circuitry underlying the psychotomimetic action of PCP in the anesthetized rat, and investigated how these functional changes are modulated by drugs that possess distinct pharmacological mechanisms. Acute administration of PCP (0.5 mg/kg i.v.) produced robust and sustained positive relative cerebral blood volume (rCBV) changes in discrete cortico-limbo-thalamic regions. Pretreatment with the selective D2 dopamine antagonist raclopride (0.3 mg/kg i.p.) did not significantly affect the rCBV response to PCP, while the atypical antipsychotic clozapine (5 mg/kg i.p.) produced region-dependent effects, with complete suppression of the rCBV response in the thalamus, and weaker attenuation of the response in cortical and hippocampal structures. The response to PCP was strongly suppressed in all regions by pretreatment with two drugs that can inhibit aberrant glutamatergic activity: the anticonvulsant lamotrigine (10 mg/kg i.p.) and the mGluR2/3 agonist LY354740 (10 mg/kg i.p.). Taken together, our findings corroborate the pivotal role of dysfunctional glutamatergic neurotransmission in the functional response elicited by PCP, while the lack of effect of raclopride argues against a primary role of dopamine D2 receptor activation in this process. Finally, the thalamic effect of clozapine could be key to elucidating the functional basis of its pharmacological action.

Pharmacological stimulation of NMDA receptors via co-agonist site suppresses fMRI response to phencyclidine in the rat.

RATIONALE: Increasing experimental evidence suggests that impaired N-methyl-D: -aspartic acid (NMDA) receptor (NMDAr) function could be a key pathophysiological determinant of schizophrenia. Agonists at the allosteric glycine (Gly) binding site of the NMDA complex can promote NMDAr activity, a strategy that could provide therapeutic efficacy for the disorder. NMDAr antagonists like phencyclidine (PCP) can induce psychotic and dissociative symptoms similar to those observed in schizophrenia and are therefore widely used experimentally to impair NMDA neurotransmission in vivo. OBJECTIVES: In the present study, we used pharmacological magnetic resonance imaging (phMRI) to investigate the modulatory effects of endogenous and exogenous agonists at the NMDAr Gly site on the spatiotemporal patterns of brain activation induced by acute PCP challenge in the rat. The drugs investigated were D: -serine, an endogenous agonist of the NMDAr Gly site, and SSR504734, a potent Gly transporter type 1 (GlyT-1) inhibitor that can potentiate NMDAr function by increasing synaptic levels of Gly. RESULTS: Acute administration of PCP induced robust and sustained activation of discrete cortico-limbo-thalamic circuits. Pretreatment with D: -serine (1 g/kg) or SSR504734 (10 mg/kg) completely inhibited PCP-induced functional activation. This effect was accompanied by weak but sustained deactivation particularly in cortical areas. CONCLUSIONS: These findings suggest that agents that stimulate NMDAr via Gly co-agonist site can potentiate NMDAr activity in the living brain and corroborate the potential for this class of drugs to provide selective enhancement of NMDAr neurotransmission in schizophrenia.

A multimodality investigation of cerebral hemodynamics and autoregulation in pharmacological MRI.

Pharmacological MRI (phMRI) methods have been widely applied to assess the central hemodynamic response to pharmacological intervention as a surrogate for changes in the underlying neuronal activity. However, many psychoactive drugs can also affect cardiovascular parameters, including arterial blood pressure (BP). Abrupt changes in BP or the anesthetic agents used in preclinical phMRI may impair cerebral blood flow (CBF) autoregulation mechanisms, potentially introducing confounds in the phMRI response. Moreover, relative cerebral blood volume (rCBV), often measured in small-animal phMRI studies, may be sensitive to BP changes even in the presence of intact autoregulation. We applied laser Doppler flowmetry and MRI to measure changes in CBF and microvascular CBV induced by increasing doses of intravenous norepinephrine (NE) challenge in the halothane-anesthetized rat. NE is a potent vasopressor that does not cross the blood-brain barrier and mimics the rapid BP changes typically observed with acute drug challenges. We found that CBF autoregulation was maintained over a BP range of 60-120 mmHg. Under these conditions, no significant central rCBV responses were observed, suggesting that microvascular rCBV changes in response to abrupt changes in perfusion pressure are negligible within the autoregulatory range. Larger BP responses were accompanied by significant changes in both CBV and CBF that might confound the interpretation of phMRI results.

Rat models of acute inflammation: a randomized controlled study on the effects of homeopathic remedies.

BACKGROUND: One of the cardinal principles of homeopathy is the "law of similarities", according to which patients can be treated by administering substances which, when tested in healthy subjects, cause symptoms that are similar to those presented by the patients themselves. Over the last few years, there has been an increase in the number of pre-clinical (in vitro and animal) studies aimed at evaluating the pharmacological activity or efficacy of some homeopathic remedies under potentially reproducible conditions. However, in addition to some contradictory results, these studies have also highlighted a series of methodological difficulties.The present study was designed to explore the possibility to test in a controlled way the effects of homeopathic remedies on two known experimental models of acute inflammation in the rat. To this aim, the study considered six different remedies indicated by homeopathic practice for this type of symptom in two experimental edema models (carrageenan- and autologous blood-induced edema), using two treatment administration routes (sub-plantar injection and oral administration). METHODS: In a first phase, the different remedies were tested in the four experimental conditions, following a single-blind (measurement) procedure. In a second phase, some of the remedies (in the same and in different dilutions) were tested by oral administration in the carrageenan-induced edema, under double-blind (treatment administration and measurement) and fully randomized conditions. Seven-hundred-twenty male Sprague Dawley rats weighing 170-180 g were used. Six homeopathic remedies (Arnica montana D4, Apis mellifica D4, D30, Atropa belladonna D4, Hamamelis virginiana D4, Lachesis D6, D30, Phosphorus D6, D30), saline and indomethacin were tested. Edema was measured using a water-based plethysmometer, before and at different times after edema induction. Data were analyzed by ANOVA and Student t test. RESULTS: In the first phase of experiments, some statistically significant effects of homeopathic remedies (Apis, Lachesis and Phosporus) were observed (the reduction in paw volume increase ranging from 10% to 28% at different times since edema induction). In the second phase of experiments, the effects of homeopathic remedies were not confirmed. On the contrary, the unblinded standard allopathic drug indomethacin exhibited its anti-inflammatory effect in both experimental phases (the reduction in paw volume increase ranging from 14% to 40% in the first phase, and from 18% to 38% in the second phase of experiments). CONCLUSION: The discrepancies between single-blind and double-blind methods in animal pharmacological research are noteworthy and should be better investigated, also in non-homeopathic research.

Region-specific effects of nicotine on brain activity: a pharmacological MRI study in the drug-naïve rat.

We have applied pharmacological magnetic resonance imaging (phMRI) methods to map the functional response to nicotine in drug-naïve rats. Nicotine (0.35 mg/kg intravenous (i.v.)) increased relative cerebral blood volume (rCBV) in cortical (including medial prefrontal, cingulate orbitofrontal, insular) and subcortical (including amygdala and dorsomedial hippocampus) structures. The pharmacological specificity of the effect was demonstrated by acute pretreatment with the nicotinic acetylcholine receptor (nAChR) ion-channel-blocking agent mecamylamine, which suppressed the rCBV response to nicotine. Control experiments with norepinephrine, a potent non-brain-penetrant vasopressor, at a dose that mimics the cardiovascular response induced by nicotine were performed to assess the potential confounding effects of peripheral blood pressure changes induced by nicotine. In an attempt to highlight the relative contribution of different nAChR subtypes to the observed activation pattern of nicotine, we also investigated the central phMRI response to an acute challenge with (R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (cpdA, at 5, 10, 20, and 30 mg/kg i.v.) and 5-iodo-A-85380 (5IA, 5 mg/kg i.v.). CpdA is a selective agonist at homomeric alpha7 nAChRs, while 5IA features high in vivo affinity for the alpha4beta2* and other less-abundant beta2-containing nicotinic receptors. CpdA did not produce significant rCBV changes at any of the doses tested, whereas 5IA induced a pattern of activation very similar to that induced by nicotine. The lack of phMRI response to cpdA together with the high spatial overlap between the activation profile of nicotine and 5IA, suggest that the acute functional response to nicotine in drug-naïve rats is mediated by beta2-containing nAChR isoforms, presumably belonging to the alpha4beta2* subtype.

Functional magnetic resonance mapping of intracerebroventricular infusion of a neuroactive peptide in the anaesthetised rat.

Pharmacological magnetic resonance imaging (phMRI) methods map the cerebral haemodynamic response to challenge with psychotropic agents as a surrogate for drug-induced changes in brain activity. However, many neuroactive compounds present low blood-brain barrier penetration and thus systemic administration may result in insufficient brain concentration. Intracerebroventricular (ICV) administration has been long used as an effective way of bypassing the blood-brain barrier in studies with non-brain-penetrant compounds, such as neuropeptides. In order to extend the range of pharmacological substances accessible to phMRI, we have developed methods to map relative cerebral blood volume (rCBV) changes induced by in situ ICV administration of neuroactive agents in the anaesthetised rat. We have applied this method to study for the first time the phMRI response to central administration of a neuropeptide, the metabolically stable and potent NK1 receptor agonist GR-73632. ICV administration of 4.2 pmol of GR-73632 produced a rapid onset and sustained rCBV increase in several brain structures, such as the amygdala, the caudate putamen and the cortex. These results demonstrate the feasibility of phMRI as a tool to study the functional correlates of brain activity induced by central administration of neuroactive agents.

A robust experimental protocol for pharmacological fMRI in rats and mice.

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity of specific brain circuitries and neurotransmitter systems. However, the application of phMRI to animal models is constrained by a number of experimental factors. Firstly, in order to prevent motion artefacts and reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia. Moreover, several psychoactive drugs produce blood pressure changes and alterations in respiratory frequency that may perturb central haemodynamic readouts of brain function. Hence, the quality and outcome of phMRI studies is critically dependent on the ability to monitor and control peripheral physiological parameters (i.e. blood pressure, arterial blood gases) that could alter phMRI readouts. Here we provide a thorough methodological description of a robust protocol to measure drug-induced cerebral blood volume changes in anaesthetised rats and mice. We show that the protocol ensures stable physiological parameters and robust phMRI response to the psychostimulant drug d-amphetamine in three different rat strains. We also document the successful application of the protocol to map the central effects produced by d-amphetamine in C57Bl/6J mice, a strain commonly used as background for the generation of transgenic lines, thus paving the way to the implementation of phMRI in genetically engineered animals.

Effect of the p38 MAPK inhibitor SB-239063 on Lipopolysaccharide-induced psychomotor retardation and peripheral biomarker alterations in rats.

Lipopolysaccharide (LPS) administration in rats induces a characteristic syndrome termed 'sickness behavior', including profound changes on locomotor activity and circulating stress and inflammatory mediators. The aim of the present investigation was to evaluate whether the behavioral and the peripheral biomarker responses induced by LPS could be modified by acute treatment with the p38 mitogen-activated protein kinase inhibitor SB-239063. Male Sprague-Dawley rats were treated orally either with vehicle or SB-239063 (3, 10 and 30 mg/kg) 1h before an intraperitoneal injection of either saline or LPS 125 µg/kg. Two hours after LPS injection, rats were placed in a novel open field arena for locomotion assessment during both the light and dark periods. Inflammation and stress mediators were evaluated in plasma 2, 3, 5 or 14 h into the dark phase. Pre-treatment with SB-239063 significantly reversed the locomotor deficits induced by LPS injection. Interleukin (IL)-1ß, IL-6, IL-10, Granulocyte-Macrophage-Colony Stimulating Factor, Interferon-γ, and C-reactive-protein levels were increased significantly by LPS, but not when LPS was preceded by SB-239063 treatment. LPS significantly decreased growth-hormone and Prolactin, and this effect was attenuated by SB-239063. Tumor Necrosis Factor-α, Adrenocorticotropic Hormone and Corticosterone levels were significantly higher in LPS-treated rats and were not normalized by SB-239063. Thus, we demonstrate that acute treatment with SB-239063 may have ameliorating effects in early changes of LPS-induced sickness behavior and alteration in the peripheral cytokines/hormones. As such, our procedure may offer an opportunity to test the activity of novel anti-inflammatory compounds on specific symptoms of sickness associated with neuroimmune dysfunctions.

Circadian profile of peripheral hormone levels in Sprague-Dawley rats and in common marmosets (Callithrix jacchus).

AIM: in the present study, we report the circadian profiles of a wide panel of hormones measured in rats and common marmosets (Callithrix jacchus), under physiological conditions, paying special attention to minimising the stress imposed on the animals. MATERIALS AND METHODS: blood collections were performed over a 24-hour period for the analysis of stress and pituitary hormones, metabolic markers and cytokines from male cannulated rats connected to a fully automatic system, and healthy marmosets in which gender differences were also evaluated. RESULTS: in rats, a significant time effect was observed for corticosterone, prolactin (PRL), thyroid stimulating hormone (TSH), growth hormone, follicle-stimulating hormone, brain-derived neurotrophic factor, total ghrelin, insulin, leptin, insulin-like growth factor-1, adiponectin and interleukin-10. In marmosets, a significant time effect for cortisol, adrenocorticotropic hormone (ACTH), PRL and TSH, with gender effect for ACTH and PRL only, was observed. On the contrary, luteinizing hormone in the rat and active ghrelin, peptide YY, pancreatic polypeptide and gastric inhibitory polypeptide in the marmoset did not show any significant circadian variation. CONCLUSION: the present work confirmed that, due to time-of-day dependent modulation of hormones, circadian rhythmicity is relevant in physiological studies and should also be taken into consideration when performing pharmacological studies.

Selective dopamine D(3) receptor antagonist SB-277011-A potentiates phMRI response to acute amphetamine challenge in the rat brain.

Dopamine (DA) receptors are a major target for drugs employed in the treatment of neuropsychiatric disorders such as schizophrenia and drug dependence. The D(3) subtype of the D(2) DA receptor family presents a particularly focal distribution in limbic brain areas known to be associated with cognitive and emotional functions. This study examined the modulation of brain activation induced by acute administration of amphetamine in the rat by the highly selective DA D(3) receptor antagonist SB-277011-A using relative cerebral blood volume (rCBV) pharmacological MRI (phMRI). The acute administration of D-amphetamine (1 mg/kg i.v.) produced a widespread rCBV response that was strongest in cortical regions. SB-277011-A (20 mg/kg i.p.) itself did not produce significant changes in rCBV, but potentiated the phMRI response to 1 mg/kg i.v. D-amphetamine in a regionally specific manner, involving a number of structures outside the focal distribution of the D(3) receptor. Potentiated regions included the accumbens, dorsal caudate putamen, islands of Calleja, thalamus, cingulate cortex, ventral tegmental area, dorsal Raphe nucleus, and ventral subiculum. The increased response following D(3) receptor antagonism is consistent with this receptor mediating an inhibitory action on brain activity following a dopaminergic stimulus.