Blood-brain Distribution Research Articles

Development of fentanyl-specific monoclonal antibody (mAb) to antagonize the pharmacological effects of fentanyl

Fentanyl, a critical component of opioid analgesics, poses a severe threat to public health, exacerbating the drug problem due to its potential fatality. Herein, we present two novel haptens designed with different attachment sites conjugated to keyhole limpet hemocyanin (KLH), aiming to develop an efficacious vaccine against fentanyl. KLH-Fent-1 demonstrated superior performance over KLH-Fent-2 in antibody titer, blood-brain distribution, and antinociceptive tests. Consequently, we immunized mice with KLH-Fent-1 to generate fentanyl-specific monoclonal antibodies (mAbs) using the hybridoma technique to compensate for the defects of active immunization in the treatment of opioid overdose and addiction. The mAb produced by hybridoma 9D5 exhibited the ability to recognize fentanyl and its analogs with a binding affinity of 10−10 M. Subsequently, we developed a human IgG1 chimeric mAb to improve the degree of humanization. Pre-treatment with murine and chimeric mAb significantly reduced the analgesic effect of fentanyl and altered its blood-brain biodistribution in vivo. Furthermore, in a mouse model of fentanyl-induced respiratory depression, the chimeric mAb effectively reversed respiratory depression promptly and maintained a certain level during the week. The development of high-affinity chimeric mAb gives support to combat the challenges of fentanyl misuse and its detrimental consequences. In conclusion, mAb passive immunization represents a viable strategy for addressing fentanyl addiction and overdose.

Nose to brain delivery of naringin loaded transniosomes for epilepsy: formulation, characterisation, blood-brain distribution and invivo pharmacodynamic evaluation

The current work limns the preparation of naringin-loaded transnioosomes (NRN-TN) to enhance NRN solubility, permeation and bioavailability via nasal mucosa for intranasal delivery. NRN-TN was created by the thin-film hydration technique, and with the BBD (Box-Behnken design), optimisation was carried out. NRN-TNopt was characterised for the vesicle size, PDI (Polydispersity index), zeta potential, entrapment efficiency (EE) and in vitro NRN release. For further assessment, nasal permeation study, study of Blood-brain distribution, TEM (Transmission Electron Microscopy), and CLSM (Confocal Scanning Laser Microscopy) were conducted withal. The NRN-TNopt exhibited spherical as well as sealed vesicles with a considerable small size of 151.3 nm, an EE of 75.23 percent, a PDI of 0.1257, and an in vitro release of 83.32 percent. CLSM investigation revealed that the new formulation allows for higher NRN permeation across nasal mucosa than the NRN solution. The blood-brain distribution investigation revealed that intranasally administered NRN-TN had a greater Cmax and AUC0-24 h than orally administered NRN-TN. Seizure activity and neuromuscular coordination as measured by the rotarod test, biochemical estimate of oxidative stress indicators, and histological investigations demonstrated that the NRN-TN has superior anti-epileptic potential in comparison to the standard diazepam. In addition, nasal toxicity studies demonstrate that the NRN-TN formulation is safer for intranasal administration. This study confirmed that the created TN vesicle formulation is a valuable carrier for the intranasal administration of NRN for the treatment of epilepsy.

Spanlastics as a Potential Approach for Enhancing the Nose-To-Brain Delivery of Piperine: In Vitro Prospect and In Vivo Therapeutic Efficacy for the Management of Epilepsy

The present study delineates the preparation of piperine-loaded spanlastics (PIP-SPL) to improve piperine (PIP) solubility, bioavailability, and permeation through nasal mucosa for intranasal delivery. PIP-SPL was formulated using the thin-film hydration method and optimization was performed using Box-Behnken design (BBD). PIP-SPL optimized formulation (PIP-SPLopt) was characterized for polydispersity index (PDI), vesicle size, entrapment efficiency, zeta potential, and in vitro PIP release. For further evaluation, blood-brain distribution study, transmission electron microscopy (TEM), nasal permeation study, and confocal scanning laser microscopy (CLSM) were performed withal. The PIP-SPLopt presented spherical and sealed shape vesicles with a small vesicle size of 152.4 nm, entrapment efficiency of 72.93%, PDI of 0.1118, and in vitro release of 82.32%. The CLSM study unveiled that the developed formulation has greater permeation of PIP across the nasal mucosa in comparison with the PIP suspension. The blood-brain distribution study demonstrated higher Cmax and AUC0-24h of PIP-SPL via the intranasal route in comparison to PIP-SPL via oral administration. The in vivo study revealed that the PIP-SPL has good antiepileptic potential in comparison with the standard diazepam, which was evinced by seizure activity, neuromuscular coordination by rotarod test, biochemical estimation of oxidative stress markers, and histopathological studies. Furthermore, nasal toxicity study confirm that the developed PIP-SPL formulation is safer for intranasal application. The current investigation corroborated that the prepared spanlastic vesicle formulation is a treasured carrier for the PIP intranasal delivery for the management of epilepsy.

Quantitative Retention (Structure)-Activity Relationships in Predicting the Pharmaceutical and Toxic Properties of Potential Pesticides.

The micellar liquid chromatography technique and quantitative retention (structure)–activity relationships method were used to predict properties of carbamic and phenoxyacetic acids derivatives, newly synthesized in our laboratory and considered as potential pesticides. Important properties of the test substances characterizing their potential significance as pesticides as well as threats to humans were considered: the volume of distribution, the unbonded fractions, the blood–brain distribution, the rate of skin and cell permeation, the dermal absorption, the binding to human serum albumin, partitioning between water and plants’ cuticles, and the lethal dose. Pharmacokinetic and toxicity parameters were predicted as functions of the solutes’ lipophilicities and the number of hydrogen bond donors, the number of hydrogen bond acceptors, and the number of rotatable bonds. The equations that were derived were evaluated statistically and cross-validated. Important features of the molecular structure influencing the properties of the tested substances were indicated. The QSAR models that were developed had high predictive ability and high reliability in modeling the properties of the molecules that were tested. The investigations highlighted the applicability of combined chromatographic technique and QS(R)ARs in modeling the important properties of potential pesticides and reducing unethical animal testing.

Predicting Pharmacokinetic Properties of Potential Anticancer Agents via Their Chromatographic Behavior on Different Reversed Phase Materials.

The Quantitative Structure-Activity Relationship (QSAR) methodology was used to predict biological properties, i.e., the blood–brain distribution (log BB), fraction unbounded in the brain (fu,brain), water-skin permeation (log Kp), binding to human plasma proteins (log Ka,HSA), and intestinal permeability (Caco-2), for three classes of fused azaisocytosine-containing congeners that were considered and tested as promising drug candidates. The compounds were characterized by lipophilic, structural, and electronic descriptors, i.e., chromatographic retention, topological polar surface area, polarizability, and molecular weight. Different reversed-phase liquid chromatography techniques were used to determine the chromatographic lipophilicity of the compounds that were tested, i.e., micellar liquid chromatography (MLC) with the ODS-2 column and polyoxyethylene lauryl ether (Brij 35) as the effluent component, an immobilized artificial membrane (IAM) chromatography with phosphatidylcholine column (IAM.PC.DD2) and chromatography with end-capped octadecylsilyl (ODS) column using aqueous solutions of acetonitrile as the mobile phases. Using multiple linear regression, we derived the statistically significant quantitative structure-activity relationships. All these QSAR equations were validated and were found to be very good. The investigations highlight the significance and possibilities of liquid chromatographic techniques with three different reversed-phase materials and QSARs methods in predicting the pharmacokinetic properties of our important organic compounds and reducing unethical animal testing.

Use of magnoflorine-phospholipid complex to permeate blood-brain barrier and treat depression in the CUMS animal model

To improve the liposolubility and blood-brain barrier permeability of magnoflorine, a new formulation of magnoflorine-phospholipid complex was prepared, characterized, and pharmacologically evaluated in the chronic unpredictable mild stress animal model. In this paper, the magnoflorine-phospholipid complex was synthesized and its characterization was determined. The antidepressant-like and antioxidant activity of magnoflorine-phospholipid complex was investigated by behavioral tests and western blotting analysis. As a result, the magnoflorine-phospholipid complex displayed high encapsulation efficiency and significantly improved the oil-water participate coefficient. In vivo blood-brain distribution study, the magnoflorine-phospholipid complex extended the duration of magnoflorine in blood and help magnoflorine to permeate the blood-brain barrier into brain. In behavioral tests, the magnoflorine-phospholipid complex significantly decreased immobility time compared to model control group in both FST and TST. Furthermore, the magnoflorine-phospholipid complex increased the expression of antioxidative stress-related proteins by the western blotting analysis. These findings strongly suggest that the phospholipid complex could significantly improve liposolubility, drug properties of magnoflorine and help magnoflorine permeate blood-brain barrier and exert the antidepressant effect.

Effects of common anesthetic agents on [18F]flumazenil binding to the GABAA receptor.

BackgroundThe availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [18F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [18F]flumazenil to GABAA receptors in mice.MethodsBrain and whole blood radioactivity concentrations were measured ex vivo by scintillation counting or in vivo by PET in four groups of mice following administration of [18F]flumazenil: awake mice and mice anesthetized with isoflurane, dexmedetomidine, or ketamine/dexmedetomidine. Dynamic PET recordings were obtained for 60 min in mice anesthetized by either isoflurane or ketamine/dexmedetomidine. Static PET recordings were obtained at 25 or 55 min after [18F]flumazenil injection in awake or dexmedetomidine-treated mice acutely anesthetized with isoflurane. The apparent distribution volume (VT*) was calculated for the hippocampus and frontal cortex from either the full dynamic PET scans using an image-derived input function or from a series of ex vivo experiments using whole blood as the input function.ResultsPET images showed persistence of high [18F]flumazenil uptake (up to 20 % ID/g) in the brains of mice scanned under isoflurane or ketamine/dexmedetomidine anesthesia, whereas uptake was almost indiscernible in late samples or static scans from awake or dexmedetomidine-treated animals. The steady-state VT* was twofold higher in hippocampus of isoflurane-treated mice and dexmedetomidine-treated mice than in awake mice.ConclusionsAnesthesia has pronounced effects on the binding and blood-brain distribution of [18F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.

Application of RP-18 thin-layer chromatography and quantitative structure–activity relationship analysis for the prediction of the blood–brain barrier permeation

In this study, a number of computed or chromatographically measured (RP-18 thin-layer chromatography [TLC]) descriptors are presented. The relationships between these descriptors and the observed (BBobs) and calculated (B2) BBB bioavailability were studied by stepwise multiple regression analysis and discriminant function analysis on a group of 34 compounds of diverse structures. Useful models of the blood—brain distribution given by the equations: BBobs = −1.19 + 2.05 B2 + 3.89 RF − 62.31 RF/PSA + 0.290 log D (R2 = 0.85, n = 24) and B2 = 4.06 − 1.61 HA − 1.95 HD + 105.49 RF/PSA (R2 = 0.95, n = 34) were developed and validated. Models for discrimination between CNS+ and CNS− compounds were built on the basis of RF, RF/PSA, HD, and B2 descriptors. The diagnostic power of important parameters was evaluated by cluster analysis. Thirty-four compounds examined throughout this study were successfully assigned to two clusters: CNS+ and CNS−. Analysis of variances for 6 descriptors (HD, HA, RF, RF/PSA, DM, and B2...

A linear free energy analysis of PAMPA models for biological systems

Equation coefficients in the Abraham linear free energy relationship have been obtained for a number of parallel artificial membrane permeability assays, PAMPA, and have been compared to equation coefficients for several biological processes and a large number of water-solvent partitions. From a principal component analysis of the equation coefficients we show that one particular PAMPA (Wohnsland and Faller, 2001) is a good model for human intestinal absorption, but we could find no PAMPA that was a good model for rat brain permeation, mouse brain permeation, blood–brain distribution, skin permeation or water–skin partition, although models that include PAMPA and other descriptors may be useful. The water–octanol system is a poor model for all the biological process we have studied, but water-nonpolar solvent partitions are good models for permeation through egg lecithin membranes.

Blood–brain barrier permeability mechanisms in view of quantitative structure–activity relationships (QSAR)

The goal of the present paper was to develop a quantitative structure–activity relationship (QSAR) method using a simple statistical approach, such as multiple linear regression (MLR) for predicting the blood–brain barrier (BBB) permeability of chemical compounds. The “best” MLR models, comprised logP and either molecular mass (M) or isolated atomic energy (Eisol), tested on a structurally diverse set of 66 compounds, is characterized the by correlation coefficients (R) around 0.8. The obtained models were validated using leave-one-out (LOO) cross-validation technique and the correlation coefficient of leave-one-out- RLOO2 (Q2) was at least 0.6. Analysis of a case from legal medicine demonstrated informative value of our QSAR model. To best authors’ knowledge the present study is a first application of the developed QSAR models of BBB permeability to case from the legal medicine. Our data indicate that molecular energy-related descriptors, in combination with the well-known descriptors of lipophilicity may have a supportive value in predicting blood–brain distribution, which is of utmost importance in drug development and toxicological studies.

SDS-Based Biomembrane Mimetic Chromatography for Prediction of Human Drug Transport as an in Vitro Technique

The main oral drug absorption barriers are fluid cell membranes, and generally drugs are absorbed by a passive diffusion mechanism. On the other hand, the blood–brain barrier (BBB) is considered to be the main barrier to drug transport into the central nervous system (CNS). The BBB restricts the passive diffusion of many drugs from blood to brain. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography that uses micellar mobile phases in adequate experimental conditions, can be useful as an in vitro system in mimicking the drug partitioning process into biological systems. In this study, relationships between the BMC retention data of a heterogeneous set of 12 drugs and their pharmacokinetics parameters (human oral drug absorption and BBB penetration ability) are studied and the predictive ability of the models is evaluated. Modeling of log kBMC of these compounds was established by multiple linear regression in two different concentrations (0.07 and 0.09 M) of sodium dodecyl sulfate (SDS). The results showed a fair correlation between human oral drug absorption and BMC retention data in 0.09 M SDS (R2 = 0.864) and a good correlation between the blood–brain distribution coefficient and BMC retention data in 0.07 M of SDS (R2 = 0.887). Application of the developed models to a prediction set demonstrated that the model is also reliable with good predictive accuracy. The external and internal validation results showed that the predicted values are in good agreement with the experimental value.

Association Study of 27 Annotated Genes for Clozapine Pharmacogenetics

Pharmacogenetic studies on clozapine (CLZ) have provided meaningful insights but have shown redundancies owing to wide interindividual variability and insufficient replication. The present study was designed to validate hitherto suggested candidate genes on CLZ pharmacokinetics and pharmacodynamics and explore new markers through an integrative study. Based on a literature review, a total of 127 variations in 27 candidate genes were selected and analyzed. Ninety-six schizophrenic patients of Korean ethnicity with constant CLZ dosing were recruited, and information on body weight and smoking habits was gathered, as well as plasma drug levels and treatment responses. Among the pharmacokinetic-related single nucleotide polymorphisms, rs2069521 and rs2069522 in CYP1A2 for CLZ/(dose/weight) and norclozapine/(dose/weight) and rs1135840 in CYP2D6 for norclozapine/CLZ showed borderline associations that were insignificant after correction for multiple testing. Regarding treatment response, significant associations were exhibited in rs7787082 and rs10248420 of ABCB1 (P = 0.0005 and P = 0.0013, respectively) even after correction, and the rs7787082 G and rs10248420 A alleles in ABCB1 were more frequently observed in nonresponders. We also observed a trend in the associations of rs13064530 in HRH1 and rs4938013 in DRD2/ANKK1 with treatment response. We could not convincingly replicate most of the previous studies, a result that is possibly due to modest association between the suggested genes. Rather, we found a new candidate gene, ABCB1, for treatment response, which may provide a hypothesis on the relationship between the blood-brain distribution of CLZ and its clinical efficacy.

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

BackgroundMolecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined.Materials32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured.ResultsThe gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA.ConclusionThe increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.

QSAR Analysis of Blood–Brain Distribution: The Influence of Plasma and Brain Tissue Binding

The extent of brain delivery expressed as steady-state brain/blood distribution ratio (log BB) is the most frequently used parameter for characterizing central nervous system exposure of drugs and drug candidates. The aim of the current study was to propose a physicochemical QSAR model for log BB prediction. Model development involved the following steps: (i) A data set consisting of 470 experimental log BB values determined in rodents was compiled and verified to ensure that selected data represented drug disposition governed by passive diffusion across blood-brain barrier. (ii) Available log BB values were corrected for unbound fraction in plasma to separate the influence of drug binding to brain and plasma constituents. (iii) The resulting ratios of total brain to unbound plasma concentrations reflecting brain tissue binding were described by a nonlinear ionization-specific model in terms of octanol/water log P and pK(a). The results of internal and external validation demonstrated good predictive power of the obtained model as both log BB and brain tissue binding strength were predicted with residual mean square error of 0.4 log units. The statistical parameters were similar among training and validation sets, indicating that the model is not likely to be overfitted.

Prediction of Blood-Brain Distribution: Effect of Ionization

Two simple multiple linear regression models were proposed to calculate the logarithm of the blood to brain concentration ratio (log BB) of drugs or drug-like compounds. The drugs were classified into two groups according to their ionization state in blood, and the significant parameters were selected using the train sets for each group. For un-ionizable compounds, the logarithm of distribution coefficient in octanol-water in pH 7.4 (log D(7.4)) and molecular weight are the significant parameters, whereas for ionizable compounds, log D(7.4) and number of hydrogen bond acceptor are significant parameters. The developed models were validated and their prediction capabilities checked using an external dataset of 25 compounds. In addition to the acceptable prediction errors, comparison of the external data analysis results with previously proposed models confirmed superior prediction capability of newly developed models.

Water–Solvent Partition Coefficients and Δ Log P Values as Predictors for Blood–Brain Distribution; Application of the Akaike Information Criterion

It is shown that log P values for water–alkane or water–cyclohexane partitions, and the corresponding Δ log P values when used as descriptors for blood–brain distribution, as log BB, yield equations with very poor correlation coefficients but very good standard deviations, S from 0.25 to 0.33 log units. Using quite large data sets, we have verified that similar S‐values apply to predictions of log BB. A suggested model, based on log P for water–dodecane and water–hexadecane partition coefficients, has 109 data points and a fitted S = 0.254 log units. It is essential to include in the model an indicator variable for volatile compounds, and an indicator variable for drugs that contain the carboxylic group. A similar equation based on water–chloroform partition coefficients has 83 data points and a fitted S = 0.287 log units. We can find no causal connection between these log P values and log BB in terms of correlation or in terms of chemical similarity, but conclude that the log P descriptor will yield excellent predictions of log BB provided that predictions are within the chemical space of the compounds used to set up the model. We also show that model based on log P(octanol) and an Abraham descriptor provides a simple and easy method of predicting log BB with an error of no more than 0.31 log units. We have used the Akaike information criterion to investigate the most economic models for log BB. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2492–2501, 2010

MRI measurement of change in vascular parameters in the 9L rat cerebral tumor after dexamethasone administration

To demonstrate in the rat 9L cerebral tumor model that repeated MRI measurements can quantitate acute changes in the blood-brain distribution of Gadomer after dexamethasone administration. A total of 16 Fischer 344 rats were studied at 7T, 15 days after cerebral implantation of a 9L tumor. MRI procedures employed a T-One by Multiple Read Out Pulses (TOMROP) sequence to estimate R(1) (R(1) = 1/T(1)) at 145-second intervals before and after administration of Gadomer (Bayer), a macromolecular contrast agent (CA). Two baseline studies preceded Gadomer administration and 10 subsequent R(1) maps tracked CA concentration in blood and brain for 25 minutes. Thereafter, either dexamethasone (N = 10) or normal saline (N = 6) was administered intravenously. A total of 90 minutes later a second series of 12 TOMROP measurements of Gadomer distribution was performed. The influx constant, K(1), plasma distribution volume, v(D), backflux constant, k(b), and interstitial space, v(e), were determined, and the test-retest differences of each of four vascular parameters were calculated. Dexamethasone decreased K(1) approximately 60% (P = 0.02), lowered k(b) and v(D) (P = 0.03 and P < 0.01, respectively), and marginally but insignificantly decreased v(e). This noninvasive MRI technique can detect drug effects on blood-brain transfer constants of CAs within two hours of administration.

Benchmarking of QSAR Models for Blood-Brain Barrier Permeation

Using the largest available database of 328 blood-brain distribution (logBB) values, a quantitative benchmark was proposed to allow for a consistent comparison of the predictive accuracy of current and future logBB/quantitative structure-activity relationship (-QSAR) models. The usefulness of the benchmark was illustrated by comparing the global and k-nearest neighbors (kNN) multiple-linear regression (MLR) models based on the linear free-energy relationship (LFER) descriptors, and one non-LFER-based MLR model. The leave-one-out (LOO) and leave-group-out Monte Carlo (MC) cross-validation results (q(2) = 0.766, qms = 0.290, and qms(mc) = 0.311) indicated that the LFER-based kNN-MLR model was currently one of the most accurate predictive logBB-QSAR models. The LOO, MC, and kNN-MLR methods have been implemented in the QSAR-BENCH program, which is freely available from www.dmitrykonovalov.org for academic use.

Relative distribution of plasma flow markers and red blood cells across BBB openings in acute cerebral ischemia

Acute blood–brain barrier (BBB) opening in cerebral ischemia is an often observed but seldom studied phenomenon. Increased permeability has been implicated with several consequences including exacerbating ischemic injury, leading to hemorrhagic transformation (HT) and also predictive of chronic damage and a way of delivering therapeutics to the diseased parts of brain. Very few studies have investigated the 'size' of such acute openings. Herein the blood–brain distribution of fluorescent isothiocyanate (FITC)- labeled red blood cells (RBCs; ∼5 μm in diameter) and two different sized plasma flow markers in cerebral microvessels was studied by laser scanning confocal microscopy (LSCM) 6 and 24 hours after the onset of a 3 hour period of focal ischemia. At hour 6, Evans blue-tagged albumin [EB-Alb; molecular weight (MW)= 68 kDa, Stokes-Einstein radius= 37 Å], a marker of both plasma flow and BBB opening, was seen both inside and around microvessels whereas the RBCs were only intravascular. FITC-labeled dextran (FITC-dextran; MW=2000 kDa, Stokes-Einstein radius= ∼150 Å), another plasma flow tracer, had not leaked across the BBB into the tissue at this time. At hour 24, both RBCs and FITC-dextran were found extravascularly along with EB-Alb. We postulate that smaller sized openings in BBB at hour 6 limited the leaking of the two large tracers (RBCs and FITC-dextran) and that such size-dependency was lost by 24 hours with the progression of the ischemic injury.

Blood–brain distribution of morphine‐6‐glucuronide in sheep

At present there are few data regarding the rate and extent of brain-blood partitioning of the opioid active metabolite of morphine, morphine-6-glucuronide (M6G). In this study the cerebral kinetics of M6G were determined, after a short-term intravenous infusion, in chronically instrumented conscious sheep. Five sheep received an intravenous infusion of M6G 2.2 mg kg(-1) over a four-minute period. Non-linear mixed-effects analysis, with hybrid physiologically based kinetic models, was used to estimate cerebral kinetics from the arterio-sagittal sinus concentration gradients and cerebral blood flow measurements. A membrane limited model was selected as the final model. The blood-brain equilibration of M6G was relatively slow (time to reach 50% equilibration of the deep compartment 5.8 min), with low membrane permeability (PS, population mean, 2.5 ml min(-1)) from the initial compartment (V1, 13.7 ml) to a small deep distribution volume (V2) of 18.4 ml. There was some between-animal variability (%CV) in the initial distribution volume (29%), but this was not identified for PS or V2. Pharmacokinetic modelling of M6G showed a delayed equilibration between brain and blood of a nature that is primarily limited by permeability across the blood-brain-barrier, in accordance with its physico-chemical properties.