Poor Brain Uptake Research Articles

Pretargeted Imaging beyond the Blood-Brain Barrier-Utopia or Feasible?

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

Synthesis, radiolabeling, and evaluation of a potent β-site APP cleaving enzyme (BACE1) inhibitor for PET imaging of BACE1 in vivo

The β-site APP-cleaving enzyme 1 (BACE1) plays important roles in the proteolytic processing of amyloid precursor protein, and can be regarded as an important target for the diagnosis and treatment of AD. This study aimed to report the synthesis and evaluation of an 18F-labeled 2-amino-3,4-dihydroquinazoline analog as a potential BACE1 radioligand. A fluoropropyl side chain was introduced to the phenyl of this 3,4-dihydroquinazoline scaffold to generate the radioligand. Our preliminary data indicated that although the 2-amino-3,4-dihydroquinazoline scaffold possessed favorable in-vitro properties as a PET ligand, its poor brain uptake hindered the in-vivo imaging of BACE1. Further investigation would be required to optimize the scaffold for the development of a blood-brain-barrier-permeable BACE1-targeted PET ligand.

Synthesis and evaluation of NLRP3-inhibitory sulfonylurea [11C]MCC950 in healthy animals

The diaryl sulfonylurea MCC950/CRID3 is a potent NLRP3 inhibitor (IC50 = 8 nM) and, in animal models, MCC950 protects against numerous NLRP3-related neurodegenerative disorders. To evaluate the brain uptake and investigate target engagement of MCC950, we synthesised [11C-urea]MCC950 via carrier added [11C]CO2 fixation chemistry (activity yield = 237 MBq; radiochemical purity >99%; molar activity = 7 GBq/µmol; radiochemical yield (decay-corrected from [11C]CO2) = 1.1%; synthesis time from end-of-bombardment = 31 min; radiochemically stable for >1 h). Despite preclinical efficacy in neurodegeneration studies, preclinical positron emission tomography (PET) imaging studies in mouse, rat and rhesus monkey revealed poor brain uptake of low molar activity [11C]MCC950 and rapid washout. In silico prediction tools suggest efflux transporter liabilities for MCC950 at microdoses, and this information should be taken into account when developing next generation NLRP3 inhibitors and/or PET radiotracers.

Development of an Aryloxazole Derivative as a Brain-Permeable Anti-Glioblastoma Agent.

Glioblastoma drug development has been difficult due to the extremely low blood brain barrier (BBB) penetration of conventional anti-cancer agents. P-glycoprotein, an efflux membrane transporter, is responsible for the poor brain uptake of small and hydrophobic drug substances. To develop brain-penetrable anti-tumor agents, we designed colchicine derivatives containing an aryloxazole moiety, which is known to inhibit P-glycoprotein. Among those tested, an aryloxazole derivative named KIST-G1 showed the strongest anti-glioblastoma cell proliferation activity (IC50 = 3.2 ± 0.8 nM). Compared to colchicine, KIST-G1 showed dramatically increased BBB-permeable properties presenting 51.7 ± 0.5 (10−6 cm/s) parallel artificial membrane permeability assay (PAMPA) permeability and 45.0 ± 6.0% of P-gp inhibition. Aid by the BBB-permeable properties, KIST-G1 (5 mg/kg) suppressed glioblastoma cell growth and migration almost completely in the brain of glioblastoma xenograft models by showing 98.2 ± 0.1% reduced tumor area compared with phosphate buffered saline (PBS)-injected control. In comparison, temozolomide, which is the most widely used drug for glioblastoma, showed only moderate effects. Our results demonstrate the effectiveness of an aryloxazole moiety in targeting brain tumors and suggest KIST-G1 as a potent anti-glioblastoma agent.

Development of Positron Emission Tomography Radiotracers for the GABA Transporter 1.

In vivo positron emission tomography (PET) imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[11C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [18F]fluoride was used to prepare the desired candidate radiotracer ( R, E/ Z)-1-(2-((4-fluoro-2-(4-[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid (( R, E/ Z)-[18F]10). PET studies in rats showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of ( R, E/ Z)-[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of ( R, E/ Z)-[18F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 min. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.

Effects of colistin on amino acid neurotransmitters and blood-brain barrier in the mouse brain

Neurotoxicity is one of the major potential side effects of colistin therapy. However, the mechanistic aspects of colistin-induced neurotoxicity remain largely unknown. The objective of this study was to examine the effects of colistin on the blood-brain barrier (BBB) and amino acid neurotransmitters in the cerebral cortex of mouse. Mice were divided into four groups (n=5) and were administrated intravenously with 15mg/kg/day of colistin sulfate for 1, 3 and 7days successively while the control group was administrated intravenously with saline solution. The permeability and ultrastructure of the BBB were detected using the Evans blue (EB) dye and transmission electron microscopy (TEM), and the expression of Claudin-5 were determined by real-time PCR examination and western blotting. The brain uptake of colistin was measured by high-performance liquid chromatography (HPLC). The effects of colistin on amino acid neurotransmitters and their receptors were also examined by HPLC and real-time PCR. The results of EB extravasation, TEM and expression of Claudin-5 showed that colistin treatment did not affect the BBB integrity. In addition, multiple doses of colistin could induce accumulation of this compound in the brain parenchyma although there was poor brain uptake of colistin. Moreover, colistin exposure significantly increased the contents of glutamate (Glu) and gamma aminobutyric acid (GABA), and enhanced the mRNA expression levels of gamma aminobutyric acid type A receptor (GABAAR), gamma aminobutyric acid type B receptor (GABABR), N-methyl-d-aspartate 1 receptor (NR1), N-methyl-d-aspartate 2A receptor (NR2A) and N-methyl-d-aspartate 2B receptor (NR2B) in the cerebral cortex. Our data demonstrate that colistin is able to accumulate in the mouse brain and elevate the levels of amino acid neurotransmitters. These findings may be associated with colistin-induced neurotoxicity.

Metal chelation, radical scavenging and inhibition of Aβ42 fibrillation by food constituents in relation to Alzheimer’s disease

Various food constituents have been proposed as disease-modifying agents for Alzheimer’s disease (AD), due to epidemiological evidence of their beneficial effects, and for their ability to ameliorate factors linked to AD pathogenesis, namely by: chelating iron, copper and zinc; scavenging reactive oxygen species; and suppressing the fibrillation of amyloid-beta peptide (Aβ). In this study, nine different food constituents (l-ascorbic acid, caffeic acid, caffeine, curcumin, (−)-epigallocatechin gallate (EGCG), gallic acid, propyl gallate, resveratrol, and α-tocopherol) were investigated for their effects on the above factors, using metal chelation assays, antioxidant assays, and assays of Aβ42 fibrillation. An assay method was developed using 5-Br-PAPS to examine the complexation of Zn(II) and Cu(II). EGCG, gallic acid, and curcumin were identified as a multifunctional compounds, however their poor brain uptake might limit their therapeutic effects. The antioxidants l-ascorbic acid and α-tocopherol, with better brain uptake, deserve further investigation for specifically addressing oxidative stress within the AD brain.

Formulation and Development of In Situ Nasal Gelling Systems for Quetiapine Fumarate-Loaded Mucoadhesive Microemulsion

Purpose Quetiapine fumarate (QF), a novel antipsychotic agent, has got poor brain uptake because of its poor oral bioavailability (9 %) due to extensive first-pass metabolism. The aim of this study was to develop QF microemulsion formulation to enhance the bioavailability and to accomplish the intranasal delivery of the drug to the brain. Mucoadhesive in situ gelling systems of QF were formulated to increase the residence time of dosage form and provide sustained release of the drug.

2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, (99m)Tc) complexes as potential imaging probes for β-amyloid plaques.

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates , , and were demonstrated to have moderate to high affinities for Aβ1-42 aggregates with Ki values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ plaques. Moreover, in in vitro autoradiography, (99m)Tc-labeled complexes clearly detected β-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aβ plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g(-1)), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these (99m)Tc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aβ plaques.

Synthesis and evaluation of [(11)C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3β (GSK-3β).

The dysfunction of glycogen synthase kinase-3β (GSK-3β) has been implicated in a number of diseases, including Alzheimer's disease. The ability to non-invasively quantify GSK-3β activity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3β radiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging. (11)C PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding. 2035±518MBq (55±14mCi) of [(11)C]PyrATP-1 was obtained (1%-2% non-corrected radiochemical yield at end-of-synthesis based upon [(11)C]CO2) with high chemical (>95%) and radiochemical (>99%) purities, and good specific activities (143±52GBq/μmol (3874±1424Ci/mmol)), n=5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3β. (11)C PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3β in neurological and psychiatric disorders.

Design, synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT7 receptor

Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT7 receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-HT1A and adrenergic α1 receptors. The synthesized compounds have chemical features that could enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a) and N-pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were radiolabeled on the methoxy group with carbon-11. Positron emission tomography (PET) analysis revealed that [11C]-23a and [11C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests.

Formulation and Evaluation of Solid Lipid Nanoparticles of Quetiapine Fumarate and Quetiapine Hemifumarate for Brain Delivery in Rat Model

Quatiepine, an effective atypical anti psychotic administered as fumarate or hemifumarate salts by intravenous injection shows poor brain uptake due to low partitioning and/or Pgp efflux. In order to improve their brain availability, solid lipid nanoparticles (SLN) loaded with Quetiapine fumarate or hemifumarate were prepared using glyceryl monostearate (GMS), poloxamer 407 and hydrogenated soya phosphatidylcholine (HSPC) as stabilizers–using a hot melt emulsification high- pressure homogenization technique. They were characterized for physical characteristics like particle size, polydispersity (PDI), shape and entrapment efficiency (EE). Fomulation and process parameters were optimized based on particle size, PDI and EE. SLNs with a mean particle size of 101.1 nm were obtained for quetiapine fumarate and 93.6 nm for quetiapine hemifumarate. In vitro drug release study showed the release followed Higuchi kinetics model for both the formulations. In vivo studies showed a significant increase in the percentage of drug reaching the brain when administered in the form of SLN’s as compared to the respective drug solutions and the increase was greater in case of quetiapine hemifumarate salt.

Radiosynthesis and evaluation of new α1-adrenoceptor antagonists as PET radioligands for brain imaging

Evaluation of the α1-adrenoceptors in relation to brain pathophysiology and drug treatment has been hindered by lack of α1-adrenoceptor specific radioligands with sufficient brain exposure. Our aim was to develop an α1-adrenoceptor specific PET radioligand for brain imaging. Two sertindole analogues 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(1-[(11)C]methylpiperidin-4-yl)-1H-indole [(11)C]3 and 1-(4-fluorophenyl)-3-(1-[(11)C]methylpiperidin-4-yl)-5-(pyrimidin-5-yl)-1H-indole ([(11)C]Lu AA27122) [(11)C]4 were synthesized and evaluated as α1-adrenoceptor PET radioligands in cynomolgus monkey. Compounds 3 and 4 were selected due to their promising in vitro preclinical profile; high affinity and selectivity for the α1-adrenoceptor, favourable blood brain barrier permeability rates in Caco-2 monolayers and promising brain tissue/plasma ratio, assessed by equilibrium dialysis of free fraction in plasma and brain homogenate. Compounds [(11)C]3 and [(11)C]4 were synthesized from their desmethyl piperidine precursors with high specific radioactivity (>370 GBq/μmol) using [(11)C]methyl iodide. The 1,2,4-triazole analogue [(11)C]3 exhibited poor brain uptake, but the corresponding pyrimidyl analogue [(11)C]4 exhibited high brain exposure and binding in α1-adrenoceptor rich brain regions. However, the binding could not be inhibited by pretreatment with prazosin (0.1 mg/kg and 0.3 mg/kg). The results were extended by autoradiography of [(11)C]4 binding in human brain sections and competition with antagonists from different structural families, revealing that only a minor portion of the observed binding of [(11)C]4 in brain was α1-adrenoceptor specific. Though [(11)C]3 and [(11)C]4 proved not suitable as PET radioligands, the study provided further understanding of structural features influencing brain exposure of the chemical class of compounds related to the antipsychotic drug sertindole. It provided valuable insight in the delicacy of blood brain barrier penetration for structurally related compounds and underlines the importance for improved protocols for evaluation of brain penetration of future PET ligands.

Brain uptake of ketoprofen–lysine prodrug in rats

The blood–brain barrier (BBB) controls the entry of xenobiotics into the brain. Often the development of central nervous system drugs needs to be terminated because of their poor brain uptake. We describe a way to achieve large neutral amino acid transporter (LAT1)-mediated drug transport into the rat brain. We conjugated ketoprofen to an amino acid l-lysine so that the prodrug could access LAT1. The LAT1-mediated brain uptake of the prodrug was demonstrated with in situ rat brain perfusion technique. The ability of the prodrug to deliver ketoprofen into the site of action, the brain intracellular fluid, was determined combining in vivo and in vitro experiments. A rapid brain uptake from blood and cell uptake was seen both in in situ and in vivo experiments. Therefore, our results show that a prodrug approach can achieve uptake of drugs via LAT1 into the brain intracellular fluid. The distribution of the prodrug in the brain parenchyma and the site of parent drug release in the brain were shown with in vivo and in vitro studies. In addition, our results show that although lysine or ketoprofen are not LAT1-substrates themselves, by combining these molecules, the formed prodrug has affinity for LAT1.

Candidate PET radioligands for cannabinoid CB 1 receptors: [ 18F]AM5144 and related pyrazole compounds

Introduction The mammalian brain contains abundant G protein-coupled cannabinoid CB 1 receptors that respond to Δ 9-tetrahydrocannabinol, the active ingredient of cannabis. The availability of a positron emission tomography (PET) radioligand would facilitate studies of the addictive and medicinal properties of compounds that bind to this receptor. Among the known classes of ligands for CB 1 receptors, the pyrazoles are attractive targets for radiopharmaceutical development because they are antagonists and are generally less lipophilic than the other classes. Methods A convenient high-yield synthesis of N-(4-[ 18F]fluorophenyl)-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1 H-pyrazole-3-carboxamide (AM5144) was devised by coupling the appropriate pyrazole-3-carboxyl chloride compound with 4-[ 18F]fluoroaniline. The labeled precursor was synthesized from 1-[ 18F]fluoro-4-nitrobenzene in 60% radiochemical yield for 10 min using an improved procedure involving sodium borohydride reduction with cobalt chloride catalysis. The product was purified by HPLC to give a specific activity >400 mCi/μmol and a radiochemical purity >95%, and a PET study was conducted in a baboon. Results Although the regional uptake of AM5144 in baboon brain was consistent with binding to cannabinoid CB 1 receptors, absolute uptake at <0.003% injected radioactivity per cubic centimeter was lower than the previously reported uptake of the radioiodinated pyrazole AM281. Conclusions The relatively poor brain uptake of AM5144 and other pyrazole CB 1 receptor ligands is not surprising because of their high lipophilicity as compared with most brain PET radiotracers. However, for nine pyrazole compounds for which rodent data are available, brain uptake and calculated log P values are not correlated. Thus, high log P values should not preclude evaluation of radiotracers for targets such as the CB 1 receptor that may require very lipophilic ligands.

Dramatic Effect of the Tridentate Ligand on the Stability of 99mTc "3 + 1" Oxo Complexes Bearing Arylpiperazine Derivatives

Mixed-ligand model complexes of general formula [(99m)Tc(O)(kappa(3)-PNX)(kappa(1)-SPh))] [X = O (1a), S (2a)] were prepared in a one-step procedure from [(99m)TcO(4)(-)] using stannous chloride as reducing agent. Stability studies and challenge experiments with glutathione showed that complex 2a presented promising features for pursuing animal studies. The activity in the brain (% dose injected/organ) at 5 min (0.14% +/- 0.03) and 120 min (0.11% +/- 0.02) pi encouraged the synthesis of several mixed-ligand "3 + 1" oxo complexes of general formula [M(O)(kappa(3)-PNS)(kappa(1)-SL))] (M = (99m)Tc, 3a-6a, Re, 3-6), in which the tridentate ligand is the heterofunctionalized phosphine 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (PNS) and the co-ligands are different arylpiperazine derivatives (HSL1-HSL4). The (99m)Tc complexes have been characterized by comparison of their retention times in the HPLC chromatogram (gamma-detection) with the retention times of the analogous Re complexes (UV detection at 254 nm). The (99m)Tc complexes, obtained with radiochemical purity higher than 95%, after HPLC purification, are stable in saline, 0.01 M PBS (pH 7.4), rat plasma (4 h, 37 degrees C), and glutathione (10 mM solutions, 2h, 37 degrees C). Binding affinity and selectivity for 5-HT(1A) receptors (relative to the 5-HT(2A) receptor) were determined, complex 5 demonstrating the best values (IC(50) for the 5-HT(1A) 2.35 +/- 0.02 nM; competitor 5-HT(2A) 372 +/- 11 nM). Biodistribution and stability studies in mice indicated a preferred hepatobiliary excretion, a high in vivo stability, but a poor brain uptake.

In vitro blood–brain barrier permeability and cerebral endothelial cell uptake of the neuroprotective nitrone compound NXY-059 in normoxic, hypoxic and ischemic conditions

The free radical trapping nitrone compounds α-phenyl- N- tert-butylnitrone (PBN), 2-sulfophenyl- N- tert-butylnitrone (S-PBN) and disodium 2,4-disulfophenyl- N- tert-butyl nitrone (NXY-059) are effective neuroprotective agents in experimental models of both transient and permanent focal ischemia. A recent in vivo study suggested that NXY-059 had poor brain uptake in a transient ischemia model. We have now examined its blood–brain barrier permeability and cerebral endothelial uptake during hypoxic and ischemic conditions using an in vitro model of the blood–brain barrier. The in vitro blood–brain barrier permeability and cerebral endothelial uptake of NXY-059 and S-PBN were low during normoxic conditions. In contrast, PBN had very high blood–brain barrier penetration in vitro which confirmed earlier in vivo results. The permeability of [ 14C]NXY-059 increased 3.5 times after 9 h of hypoxia or 3 h of ischemia. There was, respectively, a 5-fold and more than 10-fold increase, after 6 and 9 h of ischemia. The control molecule [ 3H]inulin ( M r≈5000) showed a similar increase in permeability under the same experimental conditions indicating a major change in the transport properties of the endothelium. There was a 60% reduction in the ATP levels of astrocytes after 3 h of ischemia and a 90% reduction after 9 h. The reduction in ATP levels in endothelial cells was somewhat lower. The uptake of NXY-059 in cerebral endothelial cells under normoxic, hypoxic or 9 h of ischemic conditions was negligible. NXY-059, S-PBN and PBN showed no effects on vesicular transport or the integrity of the blood–brain barrier in normoxic or ischemic conditions, nor did the compounds induce any change in the ATP levels of the cells. In conclusion, it is possible that the increase in blood–brain barrier permeability of [ 14C]NXY-059 which occurs during prolonged ischemia in vitro reflects a change which may be of importance to the neuroprotective effects of this nitrone free radical trapping agent.

Brain perfusion systems for studies of drug uptake and metabolism in the central nervous system.

Recent advances in biotechnology and the pharmaceutical sciences have greatly expanded the number of drugs that are being developed for the treatment of central nervous system (CNS) diseases. These drugs include a wide array of antibody, protein, peptide, and recombinant DNA agents as well as novel small agents (e. g., amino acids, nucleic acid precursors, and receptor agonists or blockers) for the treatment of brain tumors, strokes, infections, and neurodegenerative diseases. Though such agents often show excellent activity in vitro, many exhibit only restricted activity in vivo because of poor brain uptake and penetration. This problem has stimulated new work on factors that control drug uptake into brain and on methods to enhance CNS drug delivery.

Use of a biophysical-kinetic model to understand the roles of protein binding and membrane partitioning on passive diffusion of highly lipophilic molecules across cellular barriers.

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

Some current and future applications and goals for nuclear imaging at Eli Lilly and Company

Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT7 receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-HT1A and adrenergic α1 receptors. The synthesized compounds have chemical features that could enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a) and N-pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were radiolabeled on the methoxy group with carbon-11. Positron emission tomography (PET) analysis revealed that [11C]-23a and [11C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests.