Brain Penetration Studies Research Articles

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 β-cells by pentamidine may be secondary to pentamidine-mediated activation of β-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

Characterization of Ubrogepant: A Potent and Selective Antagonist of the Human Calcitonin Gene‒Related Peptide Receptor.

A growing body of evidence has implicated the calcitonin gene-related peptide (CGRP) receptors in migraine pathophysiology. With the recent approval of monoclonal antibodies targeting CGRP or the CGRP receptor, the inhibition of CGRP-mediated signaling has emerged as a promising approach for preventive treatments of migraine in adults. However, there are no small-molecule anti-CGRP treatments available for treating migraine. The current studies aimed to characterize the pharmacologic properties of ubrogepant, an orally bioavailable, CGRP receptor antagonist for the acute treatment of migraine. In a series of ligand binding assays, ubrogepant exhibited a high binding affinity for native (K i=0.067 nM) and cloned human (K i=0.070 nM) and rhesus CGRP receptors (K i=0.079 nM), with relatively lower affinities for CGRP receptors from rat, mouse, rabbit and dog. In functional assays, ubrogepant potently blocked human α-CGRP stimulated cAMP response (IC50 of 0.08 nM) and exhibited highly selective antagonist activity for the CGRP receptor compared with other members of the human calcitonin receptor family. Furthermore, the in vivo CGRP receptor antagonist activity of ubrogepant was evaluated in a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans. Results demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV with a mean EC50 of 3.2 and 2.6 nM in rhesus monkeys and humans, respectively. Brain penetration studies with ubrogepant in monkeys showed a CSF/plasma ratio of 0.03 and low CGRP receptor occupancy. In summary, ubrogepant is a competitive antagonist with high affinity, potency, and selectivity for the human CGRP receptor. SIGNIFICANCE STATEMENT: Ubrogepant is a potent, selective, orally delivered, small-molecule competitive antagonist of the human calcitonin generelated peptide receptor. In vivo studies using a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV, indicating a predictable pharmacokinetic-pharmacodynamic relationship.

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.

Pharmacokinetics and brain penetration study of chlorogenic acid in rats

1. The present study is designed to investigate the brain distribution and plasma pharmacokinetics profiles of chlorogenic acid (CGA) after intranasal administration in Charles–Foster rats to evaluate whether the CGA molecules are transported directly via the nose-to-brain path.2. The CGA is administered intravenously (IV) and intranasally (IN) at the dose of 10 mg/kg. Further, its concentration in the plasma, cerebrospinal fluid (CSF) and the whole brain is analyzed by HPLC-UV method.3. The study observes that CGA is rapidly absorbed in plasma with tmax of 1 min similar to IV route after IN administration. The peak plasma concentration and AUC0–24 are higher by 3.5 and 4.0 times respectively in IV administration, compared to IN delivery that represents the significant less systemic exposure of CGA in IN route.4. However, the concentration of CGA in the brain is 4, 6.5, 5.3, 5.2 and 4.5 times higher at 30, 60, 120, 240 and 360 min, respectively in IN administration compared to IV administration. The exposure of CGA in the brain after IN administration (AUCbrain, IN) was significantly greater (4 times) as compared to the exposure of CGA in the brain (AUCbrain, IV) after IV administration reflecting significant brain uptake of CGA through nasal route. Therefore, IN delivery of CGA can be a promising approach for the treatment of stroke and neurodegenerative disorders.

Correlation of pharmacokinetics and brain penetration data of adult zebrafish with higher mammals including humans

IntroductionAdult zebrafish pharmacology is evolving rapidly for creating efficacy and safety models for drug discovery. However, there is very limited research in understanding pharmacokinetics (PK) in adult zebrafish. Methods for understanding PK will help in conducting pharmacokinetic – pharmacodynamic (PK-PD) correlations and improving the quality and applicability of data obtained using zebrafish. MethodsWe conducted adult zebrafish PK and brain penetration studies on two known compounds (irinotecan and lorcaserin) with distinct PK and brain penetration properties using validated LCMS/MS method. Irinotecan was studied at a dose of 100mg/kg i.p. and levels of the parent drug and active metabolite SN-38 were measured. Loracserin was studies at a dose of 10mg/kg by two routes i.p. and p.o. ResultsZebrafish PK and brain penetration profiles for both compounds were very similar to that of higher mammals including humans. Irinotecan was metabolised to SN-38 in ratios similar to ratios seen in other species and the compound had long half life with very low brain penetration in our studies. Loracasin was highly permeable in brain as compared to the exposure in blood, with long half life and high relative bioavailability, similar to other mammalian species including humans. DiscussionAdult zebrafish PK studies are relatively an unexplored area of zebrafish research. The zebrafish data for key parameters of irinotecan and loracserin shows a high correlation to the data from higher species, including human. This report explores and discusses the use of adult zebrafish as a predictive PK tool for higher animal studies.

Pharmacokinetic Study of Piracetam in Focal Cerebral Ischemic Rats.

Cerebral ischemia affects hepatic enzymes and brain permeability extensively. Piracetam was investigated up to phase III of clinical trials and there is lack of data on brain penetration in cerebral ischemic condition. Thus, knowledge of the pharmacokinetics and brain penetration of piracetam during ischemic condition would aid to improve pharmacotherapeutics in ischemic stroke. Focal cerebral ischemia was induced by middle cerebral artery occlusion for 2h in male Wistar rats followed by reperfusion. After 24h of middle cerebral artery occlusion or 22h of reperfusion, piracetam was administered for pharmacokinetic, brain penetration, and pharmacological experiments. In pharmacokinetic study, blood samples were collected at different time points after 200-mg/kg (oral) and 75-mg/kg (intravenous) administration of piracetam through right external jugular vein cannulation. In brain penetration study, the cerebrospinal fluid, systemic blood, portal blood, and brain samples were collected at pre-designated time points after 200-mg/kg oral administration of piracetam. In a separate experiment, the pharmacological effect of the single oral dose of piracetam in middle cerebral artery occlusion was assessed at a dose of 200mg/kg. All the pharmacokinetic parameters of piracetam including area under curve (AUC0-24), maximum plasma concentration (C max), time to reach the maximum plasma concentration (t max), elimination half-life (t 1/2), volume of distribution (V z), total body clearance, mean residence time, and bioavailability were found to be similar in ischemic stroke condition except for brain penetration. Piracetam exposure (AUC0-2) in brain and CSF were found to be 2.4- and 3.1-fold higher, respectively, in ischemic stroke compared to control rats. Piracetam significantly reduced infarct volume by 35.77% caused by middle cerebral artery occlusion. There was no change in the pharmacokinetic parameters of piracetam in the ischemic stroke model except for brain penetration. This indicates that variables influencing brain penetration may not be limiting factors for use of piracetam in ischemic stroke.

Safety and pharmacological characterization of the molecular tweezer CLR01 - a broad-spectrum inhibitor of amyloid proteins' toxicity.

BackgroundThe “molecular tweezer” CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer’s disease and other amyloidoses, here we studied its safety and pharmacokinetics.MethodsToxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer’s disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration.ResultsExamination of CLR01’s effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound’s putative “process-specific” mechanism of action.A single-injection of 100 mg/kg CLR01 in mice – 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h.ConclusionEstimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

LC‐MS/MS method for the determination of pitolisant: application to rat pharmacokinetic and brain penetration studies

A simple and sensitive LC-MS/MS method was developed and validated for the quantitation of pitolisant, an H3 receptor antagonist/inverse agonist. Acetonitrile protein precipitation technique was used to prepare rat blood and brain tissue homogenate samples by using aripiprazole as internal standard (IS). Chromatographic separation was performed by using Xbridge column (2.1 × 50 mm, 3.5 µm) with a gradient elution program. The mobile phase consists of ammonium formate (10 mm) with 0.2% formic acid and acetonitrile. Multiple reaction monitoring mode was used in positive polarity with a transition of m/z 296.3 → 98.2 for the pitolisant and m/z 448.2 → 285.3 for the IS. The calibration curves were linear in the range of 0.1-100 ng/mL in both the blood and brain homogenate samples. This method was applied to quantify samples obtained from the pharmacokinetic and brain penetration studies in male wistar rats. Mean maximum concentration, area under the curve from zero to infinity and half-life of the pitolisant were found to be 3.4 ± 1.7 ng/mL, 5 ± 4 ng h/mL and 1.9 ± 0.3 h, respectively, after a 3 mg/kg oral dose. The mean calculated concentrations in the brain were found to be 38, 60 and 52 ng/g at 0.5, 1 and 2 h, respectively.

Quantification of methyllycaconitine, selective α7 nicotinic receptor antagonist, in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry – application to neuropharmacokinetics of methyllycaconitine in rats

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 683-216 for MLA and m/z 260-116 for the internal standard. The assay exhibited a linear dynamic range of 0.5-250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study.

A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents

Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects. Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated. AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice. Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications.

TAK-285, a Novel HER2/EGFR Inhibitor, Penetrates the CNS in Rats with an Intact Blood Brain Barrier (BBB).

Abstract Background: Approximately one third of patients with HER2-overexpressing (HER2+) metastatic breast cancer develop brain metastases. Treatment options for patients whose brain metastases have progressed after radiotherapy are urgently needed. The currently approved HER2-targeted therapies, trastuzumab and lapatinib, have generally shown limited activity against brain metastases, likely due to the limited brain penetration of these drugs. Brain penetration is mediated by multiple factors including drug permeability, tissue binding, and the expression of efflux transporters (P-gp and BCRP) and their interactions with the drug. TAK-285 is a novel, orally active, dual HER2/EGFR inhibitor currently under clinical development. In this nonclinical work, we evaluated the permeability, interactions with efflux transporters, and brain penetration of TAK-285, along with lapatinib and neratinib, using in vitro and animal models.Material and Methods: The first study evaluated the permeability of TAK-285, lapatinib, and neratinib and their interactions with efflux transporters using the Caco-2 cell monolayer system in the absence or presence of efflux pump inhibitors. The second study evaluated brain penetration of TAK-285, lapatinib and neratinib after a single oral administration in rats with an intact BBB.Results: In the Caco-2 cell model, TAK-285 showed high permeability with an efflux ratio of 1.7, indicating it is not a substrate for efflux pumps. The efflux pump inhibitors LY335979 (an inhibitor of P-gp), Ko143 (an inhibitor of BCRP), and GF120918 (an inhibitor of BCRP and P-gp) had no marked effect on the efflux ratio of TAK-285, further confirming that TAK-285 is not a substrate for P-gp and BCRP. Lapatinib and neratinib showed low to medium permeability and appeared to be substrates for P-gp/BCRP in this testing system. In the rat brain penetration study, the brain-to-plasma AUC ratio for total (free and bound) TAK-285, lapatinib, and neratinib was 0.202, 0.0243, and 0.0263, respectively.Discussion: These nonclinical studies showed that, unlike lapatinib and neratinib, TAK-285 is not a substrate for the efflux transporters, P-gp and BCRP. Based on published information, all of the approved receptor tyrosine kinase inhibitors are substrates for efflux transporters. Additionally, in rats with an intact BBB, the brain-to-plasma AUC ratio for TAK-285 was substantially higher than that of lapatinib and neratinib. The AUC ratios observed in this study for lapatinib and neratinib are similar to the proportion of blood volume in the rat brain (ie, outside the CNS) relative to the total rat brain volume. This suggests the actual brain-to-plasma AUC ratio for TAK-285 in rats may be much greater than 8-fold of that of lapatinib and neratinib calculated based on these measured AUC ratios. Consequently, TAK-285 may have a unique role in the treatment of brain lesions in patients with HER2- or EGFR-dependent tumors. Furthermore, TAK-285 may not be subject to drug resistance conferred by efflux transporters. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5098.

EFFECT OF P-GLYCOPROTEIN ON INTESTINAL ABSORPTION AND BRAIN PENETRATION OF ANTIALLERGIC AGENT BEPOTASTINE BESILATE

The antiallergic agent bepotastine besilate is a nonsedating, second-generation H1-antagonist with high oral absorption and negligible distribution into brain. To clarify the role of P-glycoprotein (P-gp) in the pharmacokinetics of bepotastine, intestinal absorption and brain penetration studies were performed. [(14)C]Bepotastine transport in P-gp-overexpressed LLC-PK1 cells indicated that bepotastine was a substrate of P-gp. The affinity of bepotastine to P-gp estimated by ATPase activity assay was low, with a K(m) value of 1.25 mM. After i.v. administration, the brain/plasma free concentration ratio in mdr1-knockout mice was 3 times higher than that in wild-type mice. The in situ intestinal absorption studies of [(14)C]bepotastine in rats showed a clear regional difference, showing highest permeability at the upper part of small intestine with a decreasing permeability in the descending part of small intestine. The apparent absorption rate constant (ka) of [(14)C]bepotastine in the small intestine was greatly increased by cyclosporin A and verapamil, especially in the distal portion, and the site-specific absorption of [(14)C]bepotastine disappeared. The concentration dependence of ka of [(14)C]bepotastine was observed with a higher ka at higher concentration (20 mM) compared with that at lower concentration (1 microM). In conclusion, bepotastine is a substrate for P-gp, and P-gp clearly limited the brain distribution of bepotastine, whereas the effect of P-gp on intestinal absorption of bepotastine was minimal, presumably because of high membrane permeability at the upper region of small intestine where P-gp is less expressed. Such intestinal absorption property of bepotastine is distinctly different from the low membrane-permeable P-gp substrate fexofenadine.

Preformulation studies and estimation of brain penetration for two alpidem analogues having anticonvulsant activity

The dual aim of this study was to gain insight into the potential development of two alpidem analogues 2 and 3 containing a GABA or glycine moiety and to explore the brain penetration properties accounting for their anticonvulsant effects. For these purposes, solubility, stability and brain penetration studies were performed. In contrast to that found in acidic medium, in phosphate buffer pH 7.4 compounds 2 and 3 were poorly soluble. In solution buffered at pH 7.4 compounds 2 and 3 showed half-lives of 140 and 64 h, but they were found to undergo faster cleavage in dilute rat serum at 37°C. To gain insights into the brain penetration properties of compounds 2 and 3 after intraperitoneal administration, capillary electrophoresis, semi-microbore liquid chromatography and electron spray ionization-mass spectrometry techniques were used to analyze microdialysis samples. The detection of intact compounds 2 and 3, along with the absence of acid solutes in the dialysate, lend support to a mechanism whereby the alpidem analogues 2 and 3 act as intact molecules rather than in a prodrug-type mechanism, implying that GABA and Gly, respectively, are delivered to the brain.

P-glycoprotein plays a role in the oral absorption of BMS-387032, a potent cyclin-dependent kinase 2 inhibitor, in rats.

BMS-387032, a novel cyclin-dependent kinase 2 inhibitor, is currently in phase I clinical trials for anticancer therapy. The oral bioavailability of BMS-387032 has been found to be about 31% in rats. Absorption and first-pass metabolism were evaluated as possible reasons for the incomplete oral bioavailability in rats. Male Sprague-Dawley rats were given single doses of BMS-387032 intraarterially (9.1 mg/kg), orally (9.1 mg/kg), or intraportally (10 mg/kg). The routes of excretion of BMS-387032 after intravenous dosing were investigated in bile-duct-cannulated rats. The rate of metabolism of BMS-387032 was investigated in liver microsomes. The permeability of BMS-387032 was evaluated using Caco-2 cells, an in vitro model of the intestinal epithelium. To determine if BMS-387032 was a P-glycoprotein substrate, brain uptake studies were conducted in P-glycoprotein knockout versus wildtype mice. The exposure in rats after an intraportal dose was similar to that after an intraarterial dose, indicating that absorption may play a greater role than liver first-pass metabolism in the low oral bioavailability seen in rats. After an intravenous dose, the percent of dose excreted unchanged in the urine and bile over a 9-h period was 28% and 11%, respectively. In vitro studies in rat liver microsomes showed low rates of metabolism of BMS-387032. The Caco-2 cell permeability of BMS-387032 was <15 nm/s in the apical to basolateral direction, and 161 nm/s in the basolateral to apical direction, indicating that it may be a substrate for an intestinal efflux transporter. A P-glycoprotein binding assay showed that BMS-387032 might be a P-glycoprotein modulator. Brain penetration studies in mice showed brain levels of BMS-387032 about 3.5-fold higher in P-glycoprotein knockout mice than in wildtype mice, providing evidence of BMS-387032 being a P-glycoprotein substrate. Poor absorption may be playing a greater role than extensive first-pass metabolism in the incomplete oral bioavailability of BMS-387032 seen in rats. The efflux transporter, P-glycoprotein, may be responsible for limiting absorption, as BMS-387032 appears to be a substrate of P-glycoprotein.

Design and synthesis of trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB-277011): A potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and CNS penetration in the rat.

A selective dopamine D(3) receptor antagonist offers the potential for an effective antipsychotic therapy, free of the serious side effects of currently available drugs. Using clearance and brain penetration studies as a screen, a series of 1,2,3, 4-tetrahydroisoquinolines, exemplified by 13, was identified with high D(3) affinity and selectivity against the D(2) receptor. Following examination of molecular models, the flexible butyl linker present in 13 was replaced by a more conformationally constrained cyclohexylethyl linker, leading to compounds with improved oral bioavailability and selectivity over other receptors. Subsequent optimization of this new series to improve the cytochrome P450 inhibitory profile and CNS penetration gave trans-N-[4-[2-(6-cyano-1, 2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarbo xamide (24, SB-277011). This compound is a potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and brain penetration in the rat and represents an excellent new chemical tool for the investigation of the role of the dopamine D(3) receptor in the CNS.

Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry.

A method is described for the evaluation of drug concentrations in plasma and brain from treated rats. The analyte is recovered from plasma or brain homogenate by liquid-liquid extraction and subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A simple experimental protocol renders the procedure valuable for obtaining information rapidly on brain penetration and plasma exposure of specific classes of compounds. This methodology has been applied to evaluate brain penetration with 30 different compounds from the same discovery program. In an attempt to increase throughput in our screening efforts, mixture dosing was evaluated. Results from single compound administration were compared with results following administration of a mixture of four compounds. Preliminary results, with specific classes of compounds, show no major differences (ranking order) in brain or plasma concentrations between mixture dosing and single compound administration, suggesting that mixture dosing could be applicable to brain penetration studies in the drug discovery phase.

Sample pooling to enhance throughput of brain penetration study

The advent of combinatorial synthesis and high throughput screening in pharmaceutical research has inevitably given rise to a large number of interesting prelead compounds that requires rapid analytical throughput for kinetic characterization. The traditional approach of one-compound-at-a-time bioanalysis has not been able to meet the demand for high productivity of pharmacokinetic screening. This report demonstrates the application of sample pooling in expediting the pharmacokinetic screening, including assessment of brain penetration, of six NK 1 receptor antagonists in rats: CAM 6108 (C1), CAM 6122 (C2), CAM 6178 (C3), CAM 5825 (C4), CAM 6182 (C5), and CAM 6121 (C6). The approach was adopted to avoid complications associated with cocktail dosing where multiple compounds are administered to one animal. The present investigation features individualized dosing (one compound per animal), followed by sample pooling of brain and plasma and bioanalysis via a conventional LC-fluorescence method. Rats were dosed intravenously with each of the six NK 1 receptor antagonists and blood and brain samples were harvested at suitable post-dose time intervals. Plasma or brain homogenate samples from the same time points were pooled into two groups (C1–C3 and C4–C6) for assay. Drug compounds in plasma or brain were extracted by protein precipitation and quantitated using a validated gradient HPLC/fluorescence method, which was made feasible for both groups of compounds with a modification in gradient scheme. Plasma assay precision and accuracy for C1–C6 were ≤4.7% and within ±9.8%, respectively. Brain homogenate assay accuracy for C1–C6 was within ±7.0%. Brain penetration of these compounds was evaluated as the AUC of brain and plasma and their respective brain/plasma AUC ratio. The sample pooling approach helped to quickly identify C1 as the NK 1 receptor antagonist with the greatest extent of brain penetration, followed by C2, C6, C4, C5, and C3 in that order. By employing sample pooling approach, pharmacokinetic parameters and brain penetration of all six compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D 3 receptor

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF 3SO 2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D 3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D 2 receptor.