Ramanatha Saralaya

A simple and rapid method to collect the cerebrospinal fluid of rats and its application for the assessment of drug penetration into the central nervous system.

Many central nervous system (CNS) drug discovery programs require the successful collection of cerebrospinal fluid (CSF) for assessing CNS penetration and distribution of new chemical entities. The objective of the present investigation was to simplify the technique for collecting maximum CSF from cisterna magna of the rats. Rat was anesthetized with 5% halothane and positioned in a stereotaxic frame. The rat head was flexed downward at approximately 45 degrees , a depressible surface with the appearance of a rhomb between occipital protuberances and the spine of the atlas becomes visible. The 23 G needle was punctured into the cisterna magna for CSF collection without making any incision at this region. The blunt end of the needle was inserted into a 10 in. length of PE-50 tubing and other end of the tubing was connected to a collection syringe. The non-contaminated sample was drawn into the syringe by simple aspiration. This technique is simple and can be performed by one person. The technique has a greater than 95% success rate of CSF collection and it was free of red blood cell contamination. In addition, it yielded 100-120 microL of CSF per rat. This method is simple, effective, and easy to perform and has been successfully applied in preclinical screening of novel chemical entities in neuropharmacotherapy for CNS use. The present method is demonstrated by studying the CSF concentrations of carbamazepine and raclopride.

Approach to reduce the non-specific binding in microdialysis

Measurement of unbound test compound concentrations at the biophase is routinely carried out in the drug discovery. Microdialysis is an established sampling technique for in vivo measurement of endogenous and exogenous compounds and it is commonly used for monitoring true concentrations. Endogenous compounds like neurotransmitters and neuropeptides in the brain are routinely evaluated as a proof of pharmacological activity of test compounds. Although, microdialysis offers several advantages over the conventional techniques for its use in brain pharmacokinetics, the absolute determination of extracellular concentrations of test compound depends on the predictable non-specific binding to the tubing and probe membrane. In the present investigation, we have demonstrated steps to predict non-specific binding and described approaches to reduce while working with compounds having different degree of adsorption properties. Non-specific binding to the tubing was measured in vitro for seven structurally diverse compounds and based on the binding characteristics, changes were adapted in study conditions. In vitro probe extraction efficiency was evaluated by gain and loss, which was further used as a second layer of measurement for non-specific binding. For selected compounds, in vivo probe extraction efficiencies were carried out and brain pharmacokinetics was evaluated in the prefrontal cortex of male Sprague-Dawley rats. Thus, the present approach demonstrates a systematic approach for evaluating and reducing the non-specific binding of test compounds to the microdialysis tubing and probe membranes. The stepwise approach described will strengthen the applicability of microdialysis in brain pharmacokinetics.

Pharmacokinetic profiling of efavirenz–emtricitabine–tenofovir fixed dose combination in pregnant and non‐pregnant rats

During pregnancy, the disposition of various drugs is altered due to changes in physiological condition, maternal gastrointestinal absorption, gastric secretion and motility. A fixed dose combination of antiretrovirals is commonly prescribed for the treatment of HIV infection. There is a need to understand the pharmacokinetics and placental transfer of efavirenz–emtricitabine–tenofovir in fixed dose combination during pregnancy. The pharmacokinetics and placental transfer of efavirenz–emtricitabine–tenofovir fixed dose combination was evaluated in timed pregnant and non‐pregnant Sprague–Dawley rats at 30, 10, 15 mg/kg p.o., respectively. The plasma, placental tissue, amniotic fluid and fetal tissue concentrations were measured using high performance liquid chromatography combined with tandem mass spectrometric detector (LC‐MS/MS). To summarize, the pharmacokinetic profile of efavirenz remained similar in the pregnant and non‐pregnant rats. However, a considerable difference in the pharmacokinetics of emtricitabine and tenofovir was observed in pregnant and non‐pregnant rats. Efavirenz and emtricitabine showed appreciable placental, amniotic fluid and fetal exposure compared with tenofovir. The present study suggests that a profound impact on antiretroviral pharmacokinetics was observed during pregnancy and there is a need to monitor the exposure levels of each drug when administered as a fixed dose combination during pregnancy. Further studies to explore the pharmacokinetic parameters of fixed dose antiretrovirals during the preclinical stage in a timed‐pregnancy rat model are required. Such studies can help in the development of safe and effective medications with a reduced risk of perinatal transmission of HIV‐1 infection. Copyright

Difference in the norepinephrine levels of experimental and non-experimental rats with age in the object recognition task

In the present study, we investigated the performance of adult and juvenile rats in the Object Recognition Task (ORT). While it is well known that the performance of rat in ORT differs with age, the reason for the difference as well as the underlying neurotransmitter that may have led to these differences were investigated. In the present study, juvenile rats of postnatal day 40-45 (PND 40-45) and adult rats of postnatal day 60+ (PND 60+) were subjected to a two trial ORT. The juvenile rats did not discriminate between the novel object and the familiar object, while the adult rats discriminated the novel from the familiar object. On estimating brain concentrations of norepinephrine (NE), it was observed that the NE level in MTL (medial temporal lobe) of adult experimental rats was significantly higher than the adult non-experimental rats. In juvenile rats, no significant difference was observed in the NE levels of experimental rats in comparison to its non-experimental counterparts. Administration of yohimbine (α(2A) adrenergic receptor antagonist) enhanced the level of NE in juvenile rats and reversed the difference seen with age. From the present study, we conclude that the deficit in memory seen is likely due to the difference in NE levels with task and this can be reversed by yohimbine which enhance NE levels.

In vivo receptor occupancy assay of histamine H3 receptor antagonist in rats using non-radiolabeled tracer

INTRODUCTION Rapid and reliable preclinical receptor occupancy measurement at the target organ in relevant species is critical in accelerating the drug hunting process. The aim of this study was to develop in vivo receptor occupancy assay for histamine H₃ receptors (H₃R) using the non-radiolabeled GSK189254 as a tracer and to correlate the occupancy-exposure relationship for H₃R antagonists in the rats. METHODS In vivo tracer characterization studies like brain regional distribution, dose and time dependent uptake were carried out for GSK189254 in the male Wistar rats after intravenous administration. The tracer specificity was validated by pretreatment with H₃ antagonists like ciproxifan, thioperamide, and GSK334429. The brain regional tracer levels and H₃R antagonist concentrations in plasma and brain were quantified using liquid chromatography tandem mass spectrometry. Receptor occupancy was calculated using the ratio of total binding (striatum or frontal cortex) to the nonspecific binding (cerebellum) of the tracer in animals pretreated with H₃R antagonist. RESULTS High degree of selective distribution of GSK189254 was found in striatum, frontal cortex, and low level in the cerebellum. Regional distribution of GSK189254 in the rat brain was consistent to that of H₃R distribution mapped using ³H or ¹¹C-GSK189254 in human, porcine, and rat. The calculated occupancy ED₅₀ values in the frontal cortex were 0.14, 1.58, and 0.14 mg/kg for ciproxifan, thioperamide, and GSK334429, respectively. The plasma EC₅₀ values (ng/mL) were found to be 2.33, 292.2, and 3.54 for ciproxifan, thioperamide and GSK334429, respectively. DISCUSSION Results from mass spectroscopy based approach to determine H₃R occupancy in rat brain is comparable with reported radiolabeled method by scintillation spectroscopy. In conclusion, non-radiolabeled GSK189254 was successfully employed as a tracer for assessing the H₃R occupancy in rats and it can be used as a preclinical tool for evaluation of novel H₃R ligands in the drug discovery.

In-vivo rat striatal 5-HT4 receptor occupancy using non-radiolabelled SB207145

The objective of the current investigation was to develop a simple, rapid method for determining in‐vivo 5‐hydroxytryptamine type 4 receptor (5‐HT4R) occupancy in rat brain using non‐radiolabelled SB207145 as a tracer for accelerating the drug discovery process.

Rat thalamic α4β2 neuronal nicotinic acetylcholine receptor occupancy assay using LC–MS/MS

INTRODUCTION In vivo brain receptor occupancy has been the key assay in driving preclinical drug discovery program and there is a need to hasten this screening step. Radiolabeled methods, which are time consuming and expensive, are most widely employed to measure receptor occupancy. Thus we sought to develop and validate an alternative novel approach for measuring rat brain α₄β₂ neuronal nicotinic acetylcholine receptor occupancy using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS). METHODS Tracer optimization studies like in vivo dose and time dependent brain regional distribution; saturation binding and blocking study with nicotine and atropine were carried out for ZW-104 in rats. Assay validity was tested by pretreatment with potent α₄β₂ ligands; TC-1734, cytisine, ABT-089, ABT-594 and A-366833. Receptor occupancy along with plasma and brain exposure levels of α₄β₂ ligand was measured in the same set of animals. RESULTS The regional distribution of ZW-104 in rat was found to be, thalamus>frontal cortex>striatum>hippocampus>cerebellum, and is in accordance with the distribution and regional densities of α₄β₂ nAChRs measured using [¹⁸F]ZW-104 in mice and baboons. Pretreatment with nicotine and α₄β₂ ligands dose dependently reduced the binding of ZW-104 in the thalamus. Non-nicotinic antagonist atropine did not alter the binding of ZW-104 in the thalamus, indicating the tracer specificity. The ED₅₀ values calculated for occupancy were found to be 3.01, 0.83, 14.81, 0.001 and 0.11 mg/kg for TC-1734, cytisine, ABT-089, ABT-594, and A-366833, respectively. DISCUSSION These findings demonstrate that non-radiolabeled ZW-104 is suitable for determining the α₄β₂ receptor occupancy in rat brain. The LC-MS/MS based receptor occupancy assay is a rapid method and allows the generation of occupancy data along with the brain and plasma concentration in the same group of animals.

Methyllycaconitine: A non-radiolabeled ligand for mapping α7 neuronal nicotinic acetylcholine receptors — In vivo target localization and biodistribution in rat brain

INTRODUCTION Reduction of cerebral cortical and hippocampal α7 neuronal nicotinic acetylcholine receptor (nAChR) density was observed in the Alzheimers disease (AD) and other neurodegenerative diseases. Mapping the subtypes of nAChRs with selective ligand by viable, quick and consistent method in preclinical drug discovery may lead to rapid development of more effective therapeutic agents. The objective of this study was to evaluate the use of methyllycaconitine (MLA) in non-radiolabeled form for mapping α7 nAChRs in rat brain. METHODS MLA pharmacokinetic and brain penetration properties were assessed in male Wistar rats. The tracer properties of MLA were evaluated in rat brain by dose and time dependent differential regional distribution studies. Target specificity was validated after blocking with potent α7 nAChR agonists ABBF, PNU282987 and nicotine. High performance liquid chromatography combined with triple quad mass spectral detector (LC-MS/MS) was used to measure the plasma and brain tissue concentrations of MLA. RESULTS MLA has shown rapid brain uptake followed by a 3-5 fold higher specific binding in regions containing the α7 nAChRs (hypothalamus - 1.60 ng/g), when compared to non-specific regions (striatum - 0.53 ng/g, hippocampus - 0.46 ng/g, midbrain - 0.37 ng/g, frontal cortex - 0.35 ng/g and cerebellum - 0.30 ng/g). Pretreatment with potent α7 nAChR agonists significantly blocked the MLA uptake in hypothalamus. The non-radiolabeled MLA binding to brain region was comparable with the α7 mRNA localization and receptor distribution reported for [(3)H] MLA in rat brain. DISCUSSION The rat pharmacokinetic, brain penetration and differential brain regional distribution features favor that MLA is suitable to use in preclinical stage for mapping α7 nAChRs. Hence, this approach can be employed as an essential tool for quicker development of novel selective ligand to map variation in the α7 receptor densities, as well as to evaluate potential new chemical entities targeting neurodegenerative diseases.

Aripiprazole in an Animal Model of Chronic Alcohol Consumption and Dopamine D2 Receptor Occupancy in Rats

Background: Epidemiologic studies and clinical assessment of schizophrenic population have revealed a high incidence of overlap between schizophrenia and addictive disorders. Objective: The aim of the present investigation was to study the effect of aripiprazole in a preclinical animal model of chronic alcohol self-administration (CASA) and also to evaluate the influence of CASA on plasma pharmacokinetics and dopamine D2 receptor (D2R) occupancy in rats. Methods: The effect of oral administration of aripiprazole (1, 3, and 10 mg/kg) on 4% alcohol intake in CASA was studied for a period of 45 min after a post-dosing interval of 60 min. Brain penetration, pharmacokinetics, and D2R occupancy of aripiprazole were evaluated in normal and CASA rats. Results: Aripiprazole reduced alcohol consumption in CASA rats by 13, 28, and 86% at 1, 3, and 10 mg/kg, respectively, and the effect reached statistical significance at 10 mg/kg (p < .01). At this behavioral effective dose, a decrease (75%) in total plasma apparent clearance and an increase in oral area under the concentration–time curve (3.98-fold) and bioavailability (3.50-fold) of aripiprazole was observed in CASA rats. Striatal D2R occupancy and brain exposure of aripiprazole were significantly higher (∼twofold) in CASA rats when compared to normal rats (p < .01). Conclusion: Chronic alcohol intake results in a significant increase in exposure of aripiprazole in plasma and brain and striatal D2R occupancy. Scientific significance: Chronic alcohol intake would increase aripiprazole exposure, thus aripiprazole dose might have to be decreased (assuming this same phenomenon occurs in humans).

SUVN-G1031, a novel selective H3 receptor antagonist in animal models of cognitive deficits

26 of them completed the study. Electrocardiogram (ECG) records werereceived at the baseline and at the each dose of rivastigmine patch therapy (5cm2, and 10 cm2 respectively), and also blood pressure was measured baseline. Results: We studied 15 male and 11 female elderly subjects (mean age 78.2 6 7.6) with newlydiagnosed LOAD. Mean 6 SD valuesof heart rates are 72 6 15, 666 10, and 67 6 10,PR intervals are 171 6 224, 1786 21,and 1726 28, QRS durations are 886 17, 926 14 and 896 15, QT intervals are 393 6 41,411 6 31 and 408 6 33, and QTc intervals are 421 6 17, 426 6 12 and 426 6 21 (at baseline,5 cm, and 10 cm, respectively). Over two four-week each dose of rivastigmine treatment periods, there were no significant change in all ECG parameters when compared with baseline ECG parameters (Figure, table). Conclusions: According to these results, it is clear that the usual dosage of rivastigmine patch seems to be safe for elderly patients. Nevertheless,it is recommended that these patients are closely monitored for electrocardiographic changes during therapy.