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Custom Evaluation for Drug BBB-Permeability


1. Custom Evaluation Schedule


2. Sample
【assay buffer:DPBS-H 】
10×PBS(+/+)  100 mL
d-glucose   4.5 g
HEPES (1M)  10 mL
DW   890 mL
Total 1,000 mL  (at 37℃)
 
10 x Dulbecco's Phosphate buffered saline (10 x PBS (+/+)
(Sigma: D1283)
d-glucose (Wako Pure Chemical Industories :041-00595)
HEPES buffer (1M) (Sigma: H0887)
 
Your compound will be diluted into the concentration of 1.0 μM with assay buffer, DPBS-H.
Caution 1) You may change the concentration of your compound for cytotoxicity and mesurement sensitibity.
Caution 2) When you need to use organic solvent to dissolve your compound, the final concentraion of the solvent should be under 0.5% (V/V).
Caution 3) Cytotoxicity of your compound can be evaluated by TEER (trans endothelial electrical resistance) after permeability assay.
 


3. Samples for testing
No Sample Solution Concentration Concentration Storage temprature Others
Ex.  PCC-1 DMSO 1mM 20μL -20℃ -
1            
2            
3            
4            


Drug BBB-Permeability Assay
For evaluating the permeability of a molecule through the brain endothelial and pericytes layers of BBB Kit ™ in blood-to-brain direction, the molecule is aplied to the upper compartment of the insert.

   *Depending on types of molecules or sensitivity measurement, the time points are decided.


Schematic Diagram 1
*The value obtained with only Transwell® membrane is the background data. The backgound data is needed in the calculation of apparent permeability.


Schematic Diagram 2(ex.)


Collecting samples
t=0 t=20 t=40 t=60
luminal
(assay buffer)
- -
abluminal
(assay buffer)
cell + membrane - -
(if needs)


Analysis
1.Calculation of apparant permeability
 
2.Evaluation of Papp
We obtained a very good correlation between the BBB Kit™ and in vivo permeabilities of drugs. You can evaluate the BBB-permeability by our BBB Kit™, quantitatively.
When you design molecular modifications of your compound or vectors carrying your compound into the brain, you can easily evaluate the BBB-permeability, quantitatively.
 
Papp(×10-6cm/s) Permeability ex)
Permeabilitis >20 very good Antipyrine
10〜20 good -
2〜10 low Na-F
<2 very low ESA, sucrose
*Transwell® is trademark of Corning, Incorporated, Corning, NY, USA.


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