Evaluation of Systemic and Brain Pharmacokinetic Parameters for Repurposing Metformin Using Intravenous Bolus Administration.

Metformin's potential in treating ischemic stroke and neurodegenerative conditions is of growing interest. Yet, the absence of established systemic and brain pharmacokinetic (PK) parameters at relevant pre-clinical doses presents a significant knowledge gap. This study highlights these PK parameters and the importance of using pharmacologically relevant pre-clinical doses to study pharmacodynamics (PD) in stroke and related neurodegenerative diseases. An LC-MS/MS method to measure metformin levels in plasma, brain, and cerebrospinal fluid (CSF) was developed and validated. In vitro assays examined brain tissue binding and metabolic stability. Intravenous (IV) bolus administration of metformin to C57BL6 mice covered low to high dose range maintaining pharmacological relevance. Quantification of metformin in the brain was used to assess brain pharmacokinetic parameters, such as unidirectional blood-to-brain constant (Kin) and unbound brain-to-plasma ratio (Kp, uu, brain). Metformin exhibited no binding in the mouse plasma and brain and remained metabolically stable. It rapidly entered the brain, reaching detectable levels in as little as 5 minutes. A Kin value of 1.87 {plus minus} 0.27 µl/g/min was obtained. As the dose increased, Kp, uu, brain showed decreased value, implying saturation, but this did not affect an increase in absolute brain concentrations. Metformin was quantifiable in the CSF at 30 minutes but decreased over time, with concentrations lower than those in the brain across all doses. Our findings emphasize the importance of metformin dose selection based on pharmacokinetic parameters for pre-clinical pharmacological studies. We anticipate further investigations focusing on pharmacokinetics and pharmacodynamics (PKPD) in disease conditions, such as stroke. Significance Statement The study establishes crucial pharmacokinetic parameters of metformin for treating ischemic stroke and neurodegenerative diseases, addressing a significant knowledge gap. It further emphasizes the importance of selecting pharmacologically relevant pre-clinical doses. The findings highlight metformin's rapid brain entry, minimal binding, and metabolic stability. The necessity of considering pharmacokinetic parameters in pre-clinical studies provides a foundation for future investigations into metformin's efficacy for neurodegenerative disease (s).

Higher Brain Uptake of Gentamicin and Ceftazidime under Isoflurane Anesthesia Compared to Ketamine/Xylazine.

We have recently shown that the volatile anesthetics isoflurane and sevoflurane acutely enhance the brain uptake of the hydrophilic markers sucrose and mannitol about two-fold from an awake condition, while the combined injection of the anesthetic agents ketamine and xylazine has no effect. The present study investigated two small-molecule hydrophilic drugs with potential neurotoxicity, the antibiotic agents ceftazidime and gentamicin. Transport studies using an in vitro blood-brain barrier (BBB) model, a monolayer of induced pluripotent stem cell-derived human brain microvascular endothelial cells seeded on Transwells, and LC-MS/MS analysis demonstrated the low permeability of both drugs in the range of sucrose, with permeability coefficients of 6.62 × 10-7 ± 2.34 × 10-7 cm/s for ceftazidime and 7.38 × 10-7 ± 2.29 × 10-7 cm/s for gentamicin. In vivo brain uptake studies of ceftazidime or gentamicin after IV doses of 25 mg/kg were performed in groups of 5-6 mice anesthetized at typical doses for surgical procedures with either isoflurane (1.5-2% v/v) or ketamine/xylazine (100:10 mg/kg I.P.). The brain uptake clearance, Kin, for ceftazidime increased from 0.033 ± 0.003 µL min-1 g-1 in the ketamine/xylazine group to 0.057 ± 0.006 µL min-1 g-1 in the isoflurane group (p = 0.0001), and from 0.052 ± 0.016 µL min-1 g-1 to 0.101 ± 0.034 µL min-1 g-1 (p = 0.0005) for gentamicin. We did not test the dose dependency of the uptake, because neither ceftazidime nor gentamicin are known substrates of any active uptake or efflux transporters at the BBB. In conclusion, the present study extends our previous findings with permeability markers and suggests that inhalational anesthetic isoflurane increases the BBB permeability of hydrophilic small-molecule endobiotics or xenobiotics when compared to the injection of ketamine/xylazine. This may be of clinical relevance in the case of potential neurotoxic substances.

Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles.

Existing vascular endothelial growth factor-oriented antiangiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel antiangiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters, and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. SIGNIFICANCE STATEMENT: Systemic administration of antiangiogenic therapeutics induces side effects to non-targeted tissues. This study, among others, has shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, this study generated CD31-targeting liposomes with encapsulated Fasudil, a clinically relevant Rho kinase inhibitor, and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations, and angiogenic functions verify the benefits of site-targeted antiangiogenic therapy.

Effects of Volatile Anesthetics versus Ketamine on Blood-Brain Barrier Permeability via Lipid-Mediated Alterations of Endothelial Cell Membranes.

The purpose of this study was to investigate the effects of the volatile anesthetic agents isoflurane and sevoflurane, at clinically relevant concentrations, on the fluidity of lipid membranes and permeability of the blood-brain barrier (BBB). We analyzed the in vitro effects of isoflurane or ketamine using erythrocyte ghosts (sodium fluorescein permeability), monolayers of brain microvascular endothelial cells ([13C]sucrose and fluorescein permeability), or liposomes (fluorescence anisotropy). Additionally, we determined the effects of 30-minute exposure of mice to isoflurane on the brain tight junction proteins. Finally, we investigated in vivo brain uptake of [13C]mannitol and [13C]sucrose after intravenous administration in mice under anesthesia with isoflurane, sevoflurane, or ketamine/xylazine in addition to the awake condition. Isoflurane at 1-mM and 5-mM concentrations increased fluorescein efflux from the erythrocyte ghosts in a concentration-dependent manner. Similarly, in endothelial cell monolayers exposed to 3% (v/v) isoflurane, permeability coefficients rose by about 25% for fluorescein and 40% for [13C]sucrose, whereas transendothelial resistance and cell viability remained unaffected. Although isoflurane caused a significant decrease in liposomes anisotropy values, ketamine/xylazine did not show any effects. Brain uptake clearance (apparent Kin) of the passive permeability markers in vivo in mice approximately doubled under isoflurane or sevoflurane anesthesia compared with either ketamine/xylazine anesthesia or the awake condition. In vivo exposure of mice to isoflurane did not change any of the brain tight junction proteins. Our data support membrane permeabilization rather than loosening of intercellular tight junctions as an underlying mechanism for increased permeability of the endothelial cell monolayers and the BBB in vivo. SIGNIFICANCE STATEMENT: The blood-brain barrier controls the entry of endogenous substances and xenobiotics from the circulation into the central nervous system. Volatile anesthetic agents like isoflurane alter the lipid structure of cell membranes, transiently facilitating the brain uptake of otherwise poorly permeable, hydrophilic small molecules. Clinical implications may arise when potentially neurotoxic drugs gain enhanced access to the central nervous system under inhalational anesthetics.

Systemic and Brain Pharmacokinetics of Milnacipran in Mice: Comparison of Intraperitoneal and Intravenous Administration.

Milnacipran is a dual serotonin and norepinephrine reuptake inhibitor, clinically used for the treatment of major depression or fibromyalgia. Currently, there are no studies reporting the pharmacokinetics (PK) of milnacipran after intraperitoneal (IP) injection, despite this being the primary administration route in numerous experimental studies using the drug. Therefore, the present study was designed to investigate the PK profile of IP-administered milnacipran in mice and compare it to the intravenous (IV) route. First a liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated to accurately quantify milnacipran in biological samples. The method was used to quantify milnacipran in blood and brain samples collected at various time-points post-administration. Non-compartmental and PK analyses were employed to determine key PK parameters. The maximum concentration (Cmax) of the drug in plasma was at 5 min after IP administration, whereas in the brain, it was at 60 min for both routes of administration. Curiously, the majority of PK parameters were similar irrespective of the administration route, and the bioavailability was 92.5% after the IP injection. These findings provide insight into milnacipran's absorption, distribution, and elimination characteristics in mice after IP administration for the first time and should be valuable for future pharmacological studies.

Modeling Blood-Brain Barrier Permeability to Solutes and Drugs In Vivo.

Our understanding of the pharmacokinetic principles governing the uptake of endogenous substances, xenobiotics, and biologicals across the blood-brain barrier (BBB) has advanced significantly over the past few decades. There is now a spectrum of experimental techniques available in experimental animals and humans which, together with pharmacokinetic models of low to high complexity, can be applied to describe the transport processes at the BBB of low molecular weight agents and macromolecules. This review provides an overview of the models in current use, from initial rate uptake studies over compartmental models to physiologically based models and points out the advantages and shortcomings associated with the different methods. A comprehensive pharmacokinetic profile of a compound with respect to brain exposure requires the knowledge of BBB uptake clearance, intra-brain distribution, and extent of equilibration across the BBB. The application of proper pharmacokinetic analysis and suitable models is a requirement not only in the drug development process, but in all of the studies where the brain uptake of drugs or markers is used to make statements about the function or integrity of the BBB.

In-Vivo and Ex-Vivo Brain Uptake Studies of Peptidomimetic Neurolysin Activators in Healthy and Stroke Animals.

PURPOSE: Neurolysin (Nln) is a peptidase that functions to preserve the brain following ischemic stroke by hydrolyzing various neuropeptides. Nln activation has emerged as an attractive drug discovery target for treatment of ischemic stroke. Among first-in-class peptidomimetic Nln activators, we selected three lead compounds (9d, 10c, 11a) for quantitative pharmacokinetic analysis to provide valuable information for subsequent preclinical development. METHODS: Pharmacokinetic profile of these compounds was studied in healthy and ischemic stroke-induced mice after bolus intravenous administration. Brain concentration and brain uptake clearance (Kin) was calculated from single time point analysis. The inter-relationship between LogP with in-vitro and in-vivo permeability was studied to determine CNS penetration. Brain slice uptake method was used to study tissue binding, whereas P-gp-mediated transport was evaluated to understand the potential brain efflux of these compounds. RESULTS: According to calculated parameters, all three compounds showed a detectable amount in the brain after intravenous administration at 4 mg/kg; however, 11a had the highest brain concentration and brain uptake clearance. A strong correlation was documented between in-vitro and in-vivo permeability data. The efflux ratio of 10c was ~6-fold higher compared to 11a and correlated well with its lower Kin value. In experimental stroke animals, the Kin of 11a was significantly higher in ischemic vs. contralateral and intact hemispheres, though it remained below its A50 value required to activate Nln. CONCLUSIONS: Collectively, these preclinical pharmacokinetic studies reveal promising BBB permeability of 11a and indicate that it can serve as an excellent lead for developing improved drug-like Nln activators.

A Semi-Physiological Three-Compartment Model Describes Brain Uptake Clearance and Efflux of Sucrose and Mannitol after IV Injection in Awake Mice.

PURPOSE: To evaluate a three-compartmental semi-physiological model for analysis of uptake clearance and efflux from brain tissue of the hydrophilic markers sucrose and mannitol, compared to non-compartmental techniques presuming unidirectional uptake. METHODS: Stable isotope-labeled [13C]sucrose and [13C]mannitol (10 mg/kg each) were injected as IV bolus into the tail vein of awake young adult mice. Blood and brain samples were taken after different time intervals up to 8 h. Plasma and brain concentrations were quantified by UPLC-MS/MS. Brain uptake clearance (Kin) was analyzed using either the single-time point analysis, the multiple time point graphical method, or by fitting the parameters of a three-compartmental model that allows for symmetrical exchange across the blood-brain barrier and an additional brain efflux clearance. RESULTS: The three-compartment model was able to describe the experimental data well, yielding estimates for Kin of sucrose and mannitol of 0.068 ± 0.005 and 0.146 ± 0.020 µl.min-1.g-1, respectively, which were significantly different (p < 0.01). The separate brain efflux clearance had values of 0.693 ± 0.106 (sucrose) and 0.881 ± 0.20 (mannitol) µl.min-1.g-1, which were not statistically different. Kin values obtained by single time point and multiple time point analyses were dependent on the terminal sampling time and showed declining values for later time points. CONCLUSIONS: Using the three-compartment model allows determination of Kin for small molecule hydrophilic markers with low blood-brain barrier permeability. It also provides, for the first time, an estimate of brain efflux after systemic administration of a marker, which likely represents bulk flow clearance from brain tissue.

A Quasi-Physiological Microfluidic Blood-Brain Barrier Model for Brain Permeability Studies.

Microfluidics-based organ-on-a-chip technology allows for developing a new class of in-vitro blood-brain barrier (BBB) models that recapitulate many hemodynamic and architectural features of the brain microvasculature not attainable with conventional two-dimensional platforms. Herein, we describe and validate a novel microfluidic BBB model that closely mimics the one in situ. Induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) were juxtaposed with primary human pericytes and astrocytes in a co-culture to enable BBB-specific characteristics, such as low paracellular permeability, efflux activity, and osmotic responses. The permeability coefficients of [13C12] sucrose and [13C6] mannitol were assessed using a highly sensitive LC-MS/MS procedure. The resulting BBB displayed continuous tight-junction patterns, low permeability to mannitol and sucrose, and quasi-physiological responses to hyperosmolar opening and p-glycoprotein inhibitor treatment, as demonstrated by decreased BBB integrity and increased permeability of rhodamine 123, respectively. Astrocytes and pericytes on the abluminal side of the vascular channel provided the environmental cues necessary to form a tight barrier and extend the model's long-term viability for time-course studies. In conclusion, our novel multi-culture microfluidic platform showcased the ability to replicate a quasi-physiological brain microvascular, thus enabling the development of a highly predictive and translationally relevant BBB model.

Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers.

BACKGROUND: The blood-brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group's previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. METHODS: We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC's impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC's effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. RESULTS: Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. CONCLUSION: In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

Enrichment of the erythrocyte miR-451a in brain extracellular vesicles following impairment of the blood-brain barrier.

Extracellular RNAs (exRNAs) are present in all biofluids and incorporate many types of RNAs including miRNA. To enhance their stability outside of the cell, exRNAs are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (EVs). The blood-brain barrier (BBB) is a dynamic interface between the systemic circulation and the CNS and is responsible for maintaining a stable extracellular environment for CNS cells. The intent of this study was to determine if EVs and their contents are transferred from the peripheral circulation to the CNS under conditions of an impaired BBB. The BBB of mice was disrupted by unilateral intracarotid artery infusion with hyperosmolar mannitol solution. To validate barrier opening, the uptake clearance of [13C12]-sucrose in the left forebrain (i.e. the ipsilateral, mannitol injected hemisphere) was quantified and revealed a 14-fold increase in the mannitol perfused hemisphere compared to sham treated mice. EVs were isolated from the extracellular spaces of the left forebrain following gentle tissue lysis and differential ultracentrifugation. EVs were confirmed using nanotracking analysis, electron microscopy and western blotting. qRT-PCR showed that the erythrocyte-enriched miR-451a in brain tissue EVs increased with mannitol treatment by 24-fold. Small RNA sequencing performed on the EVs isolated from the sham and mannitol treated mice showed that miR-9-5p was the most abundant miRNA contained within the brain EVs. qRT-PCR analysis of plasma EVs did not produce a statistically significant difference in the expression of the CNS-enriched miR-9-5p or miR-9-3p, suggesting that transfer of CNS EVs to the peripheral circulation did not occur under the conditions of our experiment. We demonstrate that EVs containing miR-451a, a highly abundant miRNA present within erythrocytes and erythrocyte EVs, are enhanced in the CNS upon BBB disruption.

LC-MS/MS-based in vitro and in vivo investigation of blood-brain barrier integrity by simultaneous quantitation of mannitol and sucrose.

BACKGROUND: Understanding the pathophysiology of the blood brain-barrier (BBB) plays a critical role in diagnosis and treatment of disease conditions. Applying a sensitive and specific LC-MS/MS technique for the measurement of BBB integrity with high precision, we have recently introduced non-radioactive [13C12]sucrose as a superior marker substance. Comparison of permeability markers with different molecular weight, but otherwise similar physicochemical properties, can provide insights into the uptake mechanism at the BBB. Mannitol is a small hydrophilic, uncharged molecule that is half the size of sucrose. Previously only radioactive [3H]mannitol or [14C]mannitol has been used to measure BBB integrity. METHODS: We developed a UPLC-MS/MS method for simultaneous analysis of stable isotope-labeled sucrose and mannitol. The in vivo BBB permeability of [13C6]mannitol and [13C12]sucrose was measured in mice, using [13C6]sucrose as a vascular marker to correct for brain intravascular content. Moreover, a Transwell model with induced pluripotent stem cell-derived brain endothelial cells was used to measure the permeability coefficient of sucrose and mannitol in vitro both under control and compromised (in the presence of IL-1ß) conditions. RESULTS: We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99 ± 0.152 × 10-7 and 3.12 ± 0.176 × 10-7 cm/s, respectively) and in vivo (PS products of 0.267 ± 0.021 and 0.126 ± 0.025 µl g-1 min-1, respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1ß. Corrected brain concentrations (Cbr), obtained by washout vs. vascular marker correction, were not significantly different for either mannitol (0.071 ± 0.007 and 0.065 ± 0.009 percent injected dose per g) or sucrose (0.035 ± 0.003 and 0.037 ± 0.005 percent injected dose per g). These data also indicate that Cbr and PS product values of mannitol were about twice the corresponding values of sucrose. CONCLUSIONS: We established a highly sensitive, specific and reproducible approach to simultaneously measure the BBB permeability of two classical low molecular weight, hydrophilic markers in a stable isotope labeled format. This method is now available as a tool to quantify BBB permeability in vitro and in vivo in different disease models, as well as for monitoring treatment outcomes.

Isoflurane increases cell membrane fluidity significantly at clinical concentrations.

There is an on-going debate whether anesthetic drugs, such as isoflurane, can cause meaningful structural changes in cell membranes at clinical concentrations. In this study, the effects of isoflurane on lipid membrane fluidity were investigated using fluorescence anisotropy and spectroscopy. In order to get a complete picture, four very different membrane systems (erythrocyte ghosts, a 5-lipid mixture that mimics brain endothelial cell membrane, POPC/Chol, and pure DPPC) were selected for the study. In all four systems, we found that fluorescence anisotropies of DPH-PC, nile-red, and TMA-DPH decrease significantly at the isoflurane concentrations of 1 mM and 5 mM. Furthermore, the excimer/monomer (E/M) ratio of dipyrene-PC jumps immediately after the addition of isoflurane. We found that isoflurane is quite effective to loosen up highly ordered lipid domains with saturated lipids. Interestingly, 1 mM isoflurane causes a larger decrease of nile-red fluorescence anisotropy in erythrocyte ghosts than 52.2 mM of ethanol, which is three times the legal limit of blood alcohol level. Our results paint a consistent picture that isoflurane at clinical concentrations causes significant and immediate increase of membrane fluidity in a wide range of membrane systems.

Brain Uptake of [13C] and [14C]Sucrose Quantified by Microdialysis and Whole Tissue Analysis in Mice.

Among small, hydrophilic drug-like molecules, [14C]sucrose has long been considered the gold standard for determination of blood-brain barrier permeability. However, we have recently shown in rats that, compared with liquid chromatography-tandem mass spectrometry analysis of stable isotope (13C) of sucrose, [14C]sucrose significantly overestimates the brain tissue concentration and uptake of sucrose by a factor of 6 to 7. This discrepancy is due to the presence of small quantities of lipophilic impurities in [14C]sucrose tracer solutions. Here, we used intracranial microdialysis to measure concentrations of both sucrose variants in brain extracellular fluid (ECF) after intravenous bolus administration to mice. Both markers displayed similar plasma profiles and ECF dialysate concentrations. However, total brain tissue concentrations and apparent brain uptake clearance of [14C]sucrose were 4.1- and 3.6-fold higher, respectively, than those of [13C]sucrose. Therefore, the contaminants of [14C]sucrose with higher permeability were likely sequestered by brain cells, which renders them nondialyzable. It is concluded that although measurement of radioactivity overestimates the concentrations of intact sucrose in the brain tissue, the ECF radioactivity after microdialysis is a relatively accurate reflection of intact sucrose after the systemic administration of the [14C]sucrose marker.

Simultaneous UPLC-MS/MS analysis of two stable isotope labeled versions of sucrose in mouse plasma and brain samples as markers of blood-brain barrier permeability and brain vascular space.

Blood Brain Barrier (BBB) permeability is frequently compromised in the course of diseases affecting the central nervous system (CNS). Sucrose is a low molecular weight, hydrophilic marker with slow permeability at the naive BBB and therefore one of the widely used indicators of barrier integrity. Our laboratory recently developed a highly sensitive UPLC-MS/MS method for stable isotope labeled [13C12]sucrose in biological matrices. Correction of total brain concentration for contribution of intravascular space is required in such experiments in order to accurately measure BBB permeability, and it is often accomplished by vascular perfusion with buffer solutions prior to brain sampling. The purpose of the present study was to develop a UPLC-MS/MS method, which allows simultaneous analysis of two different stable isotope labeled sucrose variants, one of which can be utilized as a vascular marker. The first analyte, [13C12]sucrose, serves to quantify brain uptake clearance as a measure of BBB permeability, while the second analyte, [13C6]sucrose, is administered just before termination of the animal experiment and is considered as the vascular marker. [2H2]sucrose is used as the internal standard for both 13C labeled compounds. Because the majority of recent studies on CNS diseases employ mice, another objective was to validate the new technique in this species. The UPLC-MS/MS method was linear (r2 ≥ 0.99) in the tested concentration ranges, from 10 to 1000 ng/mL for both analytes in plasma, from 2 to 400 ng/g [13C12]sucrose in brain and from 10 to 400 ng/g [13C6]sucrose in brain. It was also validated in terms of acceptable intra and inter run accuracy and precision values (n = 5). The dual analyte technique was applied in a study in mice. One group received intravenous bolus injections of 10 mg/kg [13C12]sucrose at time 0, and 10 mg/kg [13C6]sucrose at 14.5 min, and subsequent terminal blood and brain sampling was performed at 15 min. For comparison, another group received an intravenous bolus dose of 10 mg/kg [13C12]sucrose and was submitted to transcardiac perfusion with buffer after 15 min. We demonstrate that the two alternative techniques to correct for intravascular content deliver equivalent values for brain concentration and brain uptake clearance.

Effects of hepatic ischemia-reperfusion injury on the blood-brain barrier permeability to [14C] and [13C]sucrose.

Hepatic encephalopathy that is associated with severe liver failure may compromise the blood-brain barrier (BBB) integrity. However, the effects of less severe liver diseases, in the absence of overt encephalopathy, on the BBB are not well understood. The goal of the current study was to investigate the effects of hepatic ischemia-reperfusion (IR) injury on the BBB tight junction permeability to small, hydrophilic molecules using the widely used [14C]sucrose and recently-proposed alternative [13C]sucrose as markers. Rats were subjected to 20 min of hepatic ischemia or sham surgery, followed by 8 h of reperfusion before administration of a single bolus dose of [14C] or [13C]sucrose and collection of serial (0-30 min) blood and plasma and terminal brain samples. The concentrations of [14C] and [13C]sucrose in the samples were determined by measurement of total radioactivity (nonspecific) and LC-MS/MS (specific), respectively. IR injury significantly increased the blood, plasma, and brain concentrations of both [14C] and [13C]sucrose. However, when the brain concentrations were corrected for their respective area under the blood concentration-time curve, only [14C]sucrose showed significantly higher (30%) BBB permeability values in the IR animals. Because [13C]sucrose is a more specific BBB permeability marker, these data indicate that our animal model of hepatic IR injury does not affect the BBB tight junction permeability to small, hydrophilic molecules. Methodological differences among studies of the effects of liver diseases on the BBB permeability may confound the conclusions of such studies.

Evaluation of [14C] and [13C]Sucrose as Blood-Brain Barrier Permeability Markers.

Nonspecific quantitation of [14C]sucrose in blood and brain has been routinely used as a quantitative measure of the in vivo blood-brain barrier (BBB) integrity. However, the reported apparent brain uptake clearance (Kin) of the marker varies widely (∼100-fold). We investigated the accuracy of the use of the marker in comparison with a stable isotope of sucrose ([13C]sucrose) measured by a specific liquid chromatography-tandem mass spectrometry method. Rats received single doses of each marker, and the Kin values were determined. Surprisingly, the Kin value of [13C]sucrose was 6- to 7-fold lower than that of [14C]sucrose. Chromatographic fractionation after in vivo administration of [14C]sucrose indicated that the majority of the brain content of radioactivity belonged to compounds other than the intact [14C]sucrose. However, mechanistic studies failed to reveal any substantial metabolism of the marker. The octanol:water partition coefficient of [14C]sucrose was >2-fold higher than that of [13C]sucrose, indicating the presence of lipid-soluble impurities in the [14C]sucrose solution. Our data indicate that [14C]sucrose overestimates the true BBB permeability to sucrose. We suggest that specific quantitation of the stable isotope (13C) of sucrose is a more accurate alternative to the current widespread use of the radioactive sucrose as a BBB marker.

Preparation and preliminary characterization of recombinant neurolysin for in vivo studies.

The goal of this study was to produce milligram quantities of pure, catalytically active, endotoxin-free recombinant neurolysin (rNln) in standard laboratory conditions for use as a research tool. To this end, we transformed E. coli cells with a plasmid construct for polyhistidine-tagged rNln, selected a high-expressing clone and determined the optimal time-point for translation of rNln. rNln was purified to homogeneity from the soluble pool of the cell lysate using Ni-NTA affinity and size-exclusion chromatography, followed by removal of endotoxins. Using this protocol ∼3mg pure, catalytically active and nearly endotoxin-free (≈0.003EU/µg protein) rNln was reproducibly obtained from 1l of culture. Lack of cytotoxicity of rNln preparation was documented in cultured mouse cells, whereas stability in whole mouse blood. Intraperitonealy administered rNln in mice reached the systemic circulation in intact and enzymatically active form with Tmax of 1h and T1/2 of ∼30min. Administration of rNln (2 and 10mg/kg) did not alter arterial blood pressure, heart rate, body temperature and blood glucose levels in mice. These studies demonstrate that the rNln preparation is suitable for cell culture and in vivo studies and can serve as a research tool to investigate the (patho)physiological function of this peptidase.

Development and validation of a sensitive UPLC-MS/MS method for the quantitation of [(13)C]sucrose in rat plasma, blood, and brain: Its application to the measurement of blood-brain barrier permeability.

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [(14)C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC-MS/MS method for the analysis of stable isotope-modified [(13)C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [(13)C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90-106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥0.99) in the concentration ranges tested for the diluted blood and plasma (10-1000 ng/mL) and brain homogenate (1-200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [(13)C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [(13)C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.

Effects of Pringle maneuver and partial hepatectomy on the pharmacokinetics and blood-brain barrier permeability of sodium fluorescein in rats.

Liver diseases are known to affect the function of remote organs. The aim of the present study was to investigate the effects of Pringle maneuver, which results in hepatic ischemia-reperfusion (IR) injury, and partial hepatectomy (Hx) on the pharmacokinetics and brain distribution of sodium fluorescein (FL), which is a widely used marker of blood-brain barrier (BBB) permeability. Rats were subjected to Pringle maneuver (total hepatic ischemia) for 20 min with (HxIR) or without (IR) 70% hepatectomy. Sham-operated animals underwent laparotomy only. After 15 min or 8h of reperfusion, a single 25-mg/kg dose of FL was injected intravenously and serial (0-30 min) blood and bile and terminal brain samples were collected. Total and free (ultrafiltration) plasma, total brain homogenate, and bile concentrations of FL and/or its glucuronidated metabolite (FL-Glu) were determined by HPLC. Both IR and HxIR caused significant reductions in the biliary excretions of FL and FL-Glu, resulting in significant increases in the plasma AUC of the marker. Additionally, the free fraction of FL in plasma was significantly increased by HxIR. Although the brain concentrations of FL were increased by almost twofold in both IR and HxIR animals, the brain concentrations corrected by the free FL AUC (and not the total AUC) were similar in both groups at either time points. It is concluded that Pringle maneuver and/or partial hepatectomy substantially alters the hepatobiliary disposition, plasma AUC, plasma free fraction, and brain accumulation of FL without altering the BBB permeability to the marker.