Blood-Brain Barrier (BBB) & CNS Drug Delivery

• During CNS drug research, considerable drug candidate loss occurs due to hampered brain access by the blood-brain barrier (BBB)
• The goal is to develop BBB models that retain fundamental characteristics while meeting pharmaceutical industry demands
• A review describes the roles of the neurovascular unit elements, their influence on brain penetration, in vitro/vivo models
• Addresses their contribution to enhancing brain drug delivery and transport

BBB and Neurovascular Unit

• Molecular exchanges between the blood and neural tissue are restricted by the blood-CSF barrier, arachnoid barrier, and BBB
• Cerebral endothelial cells (CECs) form the BBB, crucial for blood-CNS exchange and CNS homeostasis regulation
• The neurovascular unit contains CECs, astrocytes, pericytes, microglia, neurons, and extracellular matrix
• This coordinated system regulates cerebral microvascular permeability, impacting drug penetration

Cerebral Endothelial Cells (CECs)

• CECs constitute a selective barrier covering the inner surface of cerebral capillaries, determining BBB permeability
• CECs differ from peripheral endothelial cells by not having fenestrae, exhibiting low pinocytotic activity, and having more mitochondria
• CECs poses interconnections between adjacent cells, namely adherens junctions (AJs) and tight junctions (TJs)
• AJ cadherin proteins mediate CEC adhesion, cell polarity, and regulate paracellular permeability
• TJs occlude the intercellular cleft by forming a multi-protein complex composed by transmembrane proteins and cytoplasmic proteins
• Limited ion movement yields higher trans-endothelial electrical resistance (TEER) in brain endothelium
• Anionicity is another determinant factor of BBB permeation
• Gaseous and lipophilic molecules use passive transcellular diffusion, while nutrients use solute-carrier transporters (SLC)
• Large molecules cross via receptor-mediated transcytosis (RMT)
• Cationized proteins are carried by adsorptive-mediated transcytosis (AMT)
• Efflux carriers like P-glycoprotein (P-gp) mediate lipophilic compound efflux
• Other ABC protein family members include multidrug resistance-associated proteins ABCG2
• Role of ABCG2 on drug efflux remains debated
• CECs form a metabolic barrier via enzymes like alkaline phosphatase (AP) and cytochrome P450 (CYP)

Astrocytes

• Astrocytes closely interact with CECs, ensuring neuronal function and controlling cerebral blood flow
• Astrocytic end-feet cover CECs, maintaining tight junctions,enzyme systems and transporter location
• Properties influencing BBB maturation include glial fibrillary acidic protein, orthogonal arrays of particles (OAPs)
• OAPs contain potassium channel kir4.1 and water channel aquaporin-4 (AQP-4)
• Accumulation of heparin sulfate proteoglycan(agrin) supports BBB integrity
• Matrix adhesion receptors contribute to BBB properties

Pericytes

• Pericytes stabilize small vessels and influence angiogenesis
• Pericyte loss causes abnormal vascular morphology, endothelial hyperplasia, and permeability increase
• Pericytes regulate BBB-gene expression patterns and astrocyte end-feet polarization
• Brain pericytes are directly or indirectly involved in CNS pathologies

Microglia

• Microglia, also known as cerebral perivascular macrophages, control immune responses and regulate paracellular permeability

In Vitro BBB Models

• In vitro models are required to possess low paracellular permeability and express key enzymes with carrier systems
• In vitro models with as many BBB characteristics that remain time-practical and feasible for high-moderate-throughput screening
• No specific in vitro BBB model has been adopted by pharmaceutical industries as the "gold standard"
• Current strategy should use combined approaches with distinct in vitro models should be applied for rationalizing and optimizing with brain penetration during CNS studies
• High throughput capacity, lower cost, direct quantification of compounds in physiological buffer, early identification of cell toxicity, and few animals for testing

Surrogate Models

• Parallel Artificial Membrane Permeability Assay, enables the prediction of gastrointestinal absorption
• Modified PAMPA models can differentiate BBB crossing compounds using lipid solutions
• Porcine brain lipids (PBL) mimic blood-brain barrier
• Artificial lipid membranes do not include influx or efflux to BBB
• PAMPA shows high power for predicting BBB permeability of passive diffusion across BBB

Cell-Based Models

• Mimic the BBB, begins with isolation of brain capillaries, culture cells mimic BBB is in vitro
• Cells of non-cerebral origin or cells from primary are used in monoculture, coculture, and triple cell cultures
• Lack if exposure to physiological conditions may result in differing relevant cell biological transporters ligands etc.
• In vitro, permeability assays are carried out in multi-well plates, that stimulates contact with different media in compartments
• Polycarbonate used for permeability studies of hydrophilic and lipophilic
• The permeability by paracellular diffusion of compounds.between endothelial cells must be limited by TJs in order to make the model discriminative

Techniques

• Transendothelial Electrical Resistance used voltage-measuring electrode is placed in each compartment, the resistance data read in the apparatus
• Need to have a sufficient the high TEER value, and a low apparent permeability for the specific molecules uses for integrity markers to evaluate the cell monolayer to be considered adequate to be used
• Commonly use cell culture medium to control tight junction level

Non-Cerebral Cells

• Mammalian kidneys,Madin-Darby Canine Kidney the most commonly used
• Easy to grow compared to CEC are most frequently transferred to multidrug resistance one gene order for polarity The expression of B-gp and subsequent influence of the P-gp flow through The BBB model.
• Caco-2: not commonly used compared to BBB cells
• It was also considered more discriminatory than other in vitro models including primary Bovine one and human CCC line co cultured with astrocytes
• A red and Mouse as immortalized cell line and other non cereal cells

In Vivo Methods

• Better understand the biology but have low throughput capabilities used in more advanced phases
• Among the several in vivo studies, the key concepts used for estimating the ability of a drug to overpass BBB include the rate of BBB permeation and the extent of equilibration.
• The extent depends passive diffusion

Types

• In Situ brain perfusion, involves carotid perfusion animal decapitated, but cannot circumvent metabolism from microcirculation
• Brain uptake index, involves bolus, inject radio labeled, the test into common catarrhal and or the animal decapacitated. Quick and and therefore potential efflux
• Intravenous injection technique, involved in cannulation of femural/tail- vein plasma collect blood example the assessment
• Intra cerebral microdialysis complex non clean need to calculate any of the samples but reduce number animals for kinetics investigation

Drug Delivery to the CNS

• Treatment of CNS disorders includes delivery of molecules with properties safely across the BB - include diseases and deficiencies associated with development for treatment that makes development a urgent challenge
• Several strategies have temporarily disrupt. - drug transport temporary destruction. - drug chemical. - combination and nanoscale drug all to aid delivery
• Disruption is a transient increase permeability, the brain
• Causes can be modified

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