| Assay Detail |
The PAMPA was used to measure the capacity of the peptides to cross membranes (such as the BBB) by passive diffusion. The effective permeability (Pe) of the compounds was determined at an initial concentration of 200 μM. The buffer solution was prepared from a concentrated commercial one, supplied by pION. Following the manufacturer's instructions, system solution was adjusted to pH 7.4 using a 0.5 M NaOH solution. The peptides were dissolved in a mixture of buffer solution with 20% of 1-propanol used as co-solvent. The PAMPA sandwich was separated, and the donor well was filled with 195 μL of the compound solution of interest. A magnetic stirrer was placed in each well. The acceptor plate was put into the donor plate, ensuring that the underside of the membrane was in contact with the buffer. Then, 4 μL of a mixture of phospholipids from a porcine polar brain extract (20 mg mL^-1) in dodecane (composition: 12.6% phosphatidylcholine (PC), 33.1% phosphatidylethanolamine (PE), 18.5% phosphatidylserine (PS), 4.1% phosphatidylinositol (PI), 0.8% phosphatidic acid and 30.9% of other compounds) was added to the filter of each well, followed by 200 μL of buffer solution. The plate was then covered and incubated at room temperature in a saturated humidity atmosphere for 4 h under orbital agitation at 25 μm of unstirred water layer (UWL). After this period, 165 μL/well of the solution from the donor and acceptor plates was transferred to the HPLC vials, except for the bicyclic compound, for which all the fractions were pooled. About 100 μLof each linear, stapled and cyclic acceptor samples and 80 μL of the bicyclic acceptor were injected into the HPLC apparatus. Transport was also confirmed by HPLC-MS, which indicated that the integrity of the peptides was preserved. Regarding the donor and initial time samples, the linear compound was injected at 1 μL, the linear stapled peptide at 20 μL, the head-to-tail cyclic peptide at 10 μL, and the bicyclic peptide at 5 μL. The bicyclic acceptor was injected again at 1 and 5 μL owing to the initial large absorbance obtained.
The effective permeability after 4 h was calculated using Equation 1 and the percentage of transport using Equation 2. T(%) = CA(t)/CD(t0)*100 ([Equation 1]) Pe = -218.3/t * log[1-((2*CA(t))/(CD(t0)))]*10^-16 cm/s ([Equation 2]) where t is time (h), CA(t) is the peptide concentration in the acceptor well at time t,and CD(t0) is the peptide concentration in the donor well at 0 h. Membrane retention (%R) was calculated from the difference between the total starting amount of peptide and the amount of peptide in the donor and acceptor wells at the end of the assay after 4 h.
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