Background
- The blood brain barrier (BBB) behaves as a highly selective semipermeable membrane separating circulating blood from the central nervous system while allowing the passage of glucose, water, and amino acids into the cerebrospinal fluid [1].
- The BBB protects brain nervous tissue from the fluctuation of plasma composition, from pathogenic agents, and maintains homeostasis of the brain parenchyma by restricting non-specific flux of ions, peptides, proteins and cells into and out the brain.
- The BBB is permeable to hydrophobic molecules and utilizes active transport to move ions, glucose, and other critical molecules across the membrane. When functioning properly, the blood brain barrier is a complex hurdle that must be overcome when delivering drugs to the brain.
- Assessment of BBB permeability of drug compounds is critical for drugs targeting regions of the brain; many drugs developed to treat Central Nervous System (CNS) disorders are unable to reach the brain parenchyma in therapeutically relevant concentrations.
- Poor penetration of the BBB is the cause for attrition for 95% of drugs developed for neurological disorders. As such, it is of great interest to explore potential molecules that can modulate BBB permeability [2].
- Disruption of the BBB is observed in many neurological disorders, including multiple sclerosis, stroke, Alzheimer’s disease, epilepsy, and traumatic brain injury, and is frequently induced by neuroinflammation [1].
- The BBB is a complex system, which is difficult to mimic in vitro, which typically had to be addressed with costly, low-throughput animal studies.
- Utilizing a novel BBB in vitro model, we offer an in vitro assay capable of assessing the penetration kinetics of molecules passing across the BBB.
- Additionally, we offer a complementary assay to assess modulation of BBB permeability due to drug treatment.
- This assay service allows for both compound transport across the barrier to be studied as well as the effect of compounds on the structure and function of the BBB.
Protocol
| Instrument | ThermoFisher Varioskan Lux Agilent UPLC/DAD |
| Analysis Method | Plate Reader UPLC Analysis |
| Markers | 70 kDa fluorescent dextran conjugate (for assessment of alterations to BBB permeability) |
| Cell model | Neuromics Human Blood Brain Barrier Model |
| Time points | 30 min, 1 hr, 4 hr, 24 hr (custom time points available) |
| Test Article Concentration | Single point assay (1 µM) (custom concentrations available) |
| Number of Replicates | 3 replicates per time point |
| Quality Controls | 0.5% DMSO (vehicle control) Propanolol (positive control) |
| Test Article Requirements | 50 uL of 20 mM solution or equivalent amount of solid |
| Data Delivery | Apparent Permeability of test compounds in either the apical or basal direction Change in Apparent Permeability of BBB due to effect of drug compound (optional) |
General Procedure
- 3D Blood Brain Barrier models are thawed and cultured for 4 days to establish viable barrier as measured by transendothelial electrical resistance (> 150 Ω x cm2).
- The test compound and fluorescent probe are added and the cultures are returned to incubate for designated times.
- If assessment of alterations to BBB permeability due to drug treatment is required, fluorescent dextran conjugate is added to wells
- Media is sampled for quantitation of test articles by UPLC, and for quantification of fluorescent probe by plate reader
- Apparent permeability is calculated
Data
Figure 1. Apparent permeability (Papp) of various known drugs measured using in vitro BBB assay. CNS+ drugs show excellent Papp, CNS- drugs show very low Papp
Figure 2. In vitro results measuring apparent permeability of BBB to known drug compounds showed excellent correlation to in vivo results (R2 = 0.8919)
Figure 3. Alterations in apparent permeability of blood brain barrier due to drug treatment. Data presented as mean ± SEM x 10-6 cm/s. * Indicates p<0.05 for Fisher’s LSD t-test comparisons to DMSO controls in a post hoc analysis following two-way ANOVA.
Figure 4. Transendothelial electrical resistance measurement of BBB model by day illustrating intact and functional barrier
References
- Daneman, R. (2012). The blood–brain barrier in health and disease. Annals of neurology, 72(5), 648-672.
- Bourassa, P., Alata, W., Tremblay, C., Paris-Robidas, S., & Calon, F. (2019). Transferrin Receptor-Mediated Uptake at the Blood–Brain Barrier Is Not Impaired by Alzheimer’s Disease Neuropathology. Molecular pharmaceutics, 16(2), 583-594.