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).

The Acute Impact of Propofol on Blood-Brain Barrier Integrity in Mice.

PURPOSE: We investigated whether short term infusion of propofol, a highly lipophilic agonist at GABAA receptors, which is in widespread clinical use as anesthetic and sedative, affects passive blood-brain barrier (BBB) permeability in vivo. METHODS: Mice were anesthetized with an intraperitoneal injection of ketamine/xylazine followed by a continuous IV infusion of propofol in lipid emulsion through a tail vein catheter. Control groups received ketamine/xylazine anesthesia and an infusion of Intralipid, or ketamine/xylazine anesthesia only. [13C12]sucrose as a permeability marker was injected as IV bolus 15 min after start of the infusions. Brain uptake clearance, Kin, of sucrose was calculated from the brain concentrations at 30 min and the area under the plasma-concentration time curve. We also measured the plasma and brain concentration of propofol at the terminal time point. RESULTS: The Kin value for propofol-infused mice was significantly higher, by a factor of 1.55 and 1.87, compared to the Intralipid infusion and the ketamine/xylazine groups, respectively, while the control groups were not significantly different. No difference was seen in the expression levels of tight junction proteins in brain across all groups. The propofol plasma concentration at the end of infusion (10.7 µM) matched the clinically relevant range of blood concentrations reported in humans, while concentration in brain was 2.5-fold higher than plasma. CONCLUSIONS: Propofol at clinical plasma concentrations acutely increases BBB permeability, extending our previous results with volatile anesthetics to a lipophilic injectable agent. This prompts further exploration, potentially refining clinical practices and ensuring safety, especially during extended propofol infusion schemes.

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.

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.

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.

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.

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.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Subcutaneous Semaglutide during Breastfeeding: Infant Safety Regarding Drug Transfer into Human Milk.

Postpartum mothers and their healthcare providers often face the challenge of limited data regarding the safety of drug therapies during lactation. Pregnancy can lead to sustained weight gain, and obesity can negatively impact both physical and psychological well-being. The introduction of GLP-1 agonists to augment weight loss has become a topic of interest for many postpartum mothers. Our study aims to investigate the transmission of semaglutide into human milk in the first steps to ensure the safety and health of both lactating mothers and their breastfed infants. Semaglutide quantification was performed using high-resolution liquid chromatography-mass spectrometry. InfantRisk Center Human Milk biorepository released milk samples from eight women collected at 0, 12 and 24 h post-semaglutide administration. Semaglutide was extracted using protein precipitation in methanol, followed by chromatographic separation. Linear calibration curves for the method ranged between 2.5-30 ng/mL, with a limit of detection of 1.7 ng/mL and a limit of quantification of 5.7 ng/mL (LLOQ). Semaglutide was not detected in any of the collected human milk samples. A worst-case scenario of the relative infant dose (RID) was calculated using the LLOQ as the drug concentration in milk when considering semaglutide's bioavailability and long-acting dose profile. The maximum RID projected was 1.26%, far below the standard 10% safety threshold. While questions about long-term infant outcomes, the safety of maternal nutrient intake, and the nutrient content of breast milk remain, our findings suggest that semaglutide concentrations in human milk are unlikely to pose clinical concerns for breastfed infants. These results support healthcare providers in making informed decisions regarding postpartum therapeutic interventions.

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.

Tumor-induced endothelial RhoA activation mediates tumor cell transendothelial migration and metastasis.

The endothelial barrier plays an active role in transendothelial tumor cell migration during metastasis, however, the endothelial regulatory elements of this step remain obscure. Here we show that endothelial RhoA activation is a determining factor during this process. Breast tumor cell-induced endothelial RhoA activation is the combined outcome of paracrine IL-8-dependent and cell-to-cell contact ß 1 integrin-mediated mechanisms, with elements of this pathway correlating with clinical data. Endothelial-specific RhoA blockade or in vivo deficiency inhibited the transendothelial migration and metastatic potential of human breast tumor and three murine syngeneic tumor cell lines, similar to the pharmacological blockade of the downstream RhoA pathway. These findings highlight endothelial RhoA as a potent, universal target in the tumor microenvironment for anti-metastatic treatment of solid tumors.

TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms.

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.

Transferrin receptor mediated brain uptake during ischemia and reperfusion.

PURPOSE: Drug delivery by transferrin receptor-mediated transport at the blood-brain barrier has shown beneficial effects in animal models of stroke, but it is unclear whether receptor mediated uptake remains functional in the ischemic tissue. The present study addressed that question in a mouse model of brain focal ischemia, permanent or transient middle cerebral artery occlusion (MCAO). METHODS: Brain accumulation of 125I-labeled 8D3, a mouse-specific transferrin receptor antibody, or of the isotype control UPC-10 used as vascular marker, was measured autoradiographically by phosphorimaging in the core ischemic region on cryostat brain sections up to 24h after ischemia or reperfusion. Cerebral blood flow was quantitatively determined in the same animals after administration of 99mTc-ECD (Neurolite). RESULTS: Apparent volume of distribution obtained with UPC-10 indicated no significant nonspecific leakage of the blood-brain barrier at any time point. Although brain uptake of 8D3 gradually declined compared to healthy tissue under MCAO, VD remained significantly higher than VD of UPC-10 up to 5h. In transient MCAO the brain uptake recovered to levels as in healthy tissue immediately after reperfusion. CONCLUSION: Transferrin receptor-mediated brain uptake, which is an energy dependent vesicular transport process, is sensitive to reduction in blood supply but remains partially functional for several hours after onset of ischemia. The uptake shows complete recovery after reperfusion. These results support the use of transferrin receptor-mediated brain drug delivery in the early phase of ischemia and in the phase when blood flow is restored. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

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.

Liposome-encapsulated polyethylenimine/oligonucleotide polyplexes prepared by reverse-phase evaporation technique.

Liposome-encapsulated polyplex system represents a promising delivery system for oligonucleotide-based therapeutics such as siRNA and asODN. Here, we report a novel method to prepare liposome-encapsulated cationic polymer/oligonucleotide polyplexes based on the reverse-phase evaporation following organic extraction of the polyplexes. The polyplexes of polyethylenimine and oligonucleotide were first formed in aqueous buffer at an N/P ratio of 6. The overall positively charged polyplexes were then mixed with the anionic phospholipids in overall organic media. The overall organic environment and electrostatic interaction between anionic phospholipids and positively charged polyplexes resulted in inverted micelle-like particles with the polyplexes in the core. After phase separation, the hydrophobic particles were recovered in organic phase. Reverse-phase evaporation of the organic solvent in the presence of hydrophilic polymer-grafted lipids resulted in a stable aqueous dispersion of hydrophilic lipid-coated particles with the polyplex in the core. Transmission electron microscopy visualization revealed spherical structures with heavily stained polyplex cores surrounded by lightly stained lipid coats. The lipid-coated polyplex particles showed colloidal stability, complete protection of the loaded oligonucleotide molecules from enzymatic degradation, and high loading efficiency of more than 80%. Thus, this technique represents an alternative method to prepare lipid-coated polyplex particles as a delivery system of oligonucleotide therapeutics.

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.

Acute depression of energy metabolism after microdialysis probe implantation is distinct from ischemia-induced changes in mouse brain.

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13-37% and 25-60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.

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.

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.