Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide.
OBJECTIVE
To investigate the potential for delivering large peptides orally by altering their absorptive transport pathways and improving intestinal permeability. The absorptive transport of retro-inverso (R.I.-) K-Tat9 and R.I.-K(biotin)-Tat9, novel peptidic inhibitors of the Tat protein of HIV-1, and their interactions with human SMVT (hSMVT), a high affinity, low capacity transporter, were investigated using Caco-2 and transfected CHO cells.


METHODS
Following synthesis on a PAL resin using Fmoc chemistry, the transport of R.I.-K-Tat9 (0.01-25 microM) and R.I.-K(biotin)-Tat9 (0.1-25 microM) was evaluated across Caco-2 cells. The transport and kinetics of biotin, biocytin and desthiobiotin (positive controls for SMVT) were also determined. Uptake of R.I.-K-Tat9 and R.I.K(biotin)-Tat9 (both 0.1-10 microM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells.


RESULTS
The absorptive transport of R.I.-K-Tat9 was passive, low (Pm approximately 1 x 10(-6) cm/sec) and not concentration dependent. R.I.K(biotin)-Tat9 permeability was 3.2-fold higher than R.I.-K-Tat9 demonstrating active (Ea = 9.1 kcal/mole), concentration dependent and saturable transport (Km = 3.3 microM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 microM) was - 500-fold greater than R.I.-K-Tat9 (at 10 microM). R.I.-K(biotin)-Tat9 transport in Caco-2 and CHO/hSMVT cells was significantly inhibited by known substrates of SMVT including biotin, biocytin, and desthiobiotin. Passive uptake of R.I.-K(biotin)-Tat9 was significantly greater than R.I.-K-Tat9 uptake in CHO/pSPORT cells.


CONCLUSIONS
These results demonstrate that the structural modification of R.I.-K-Tat9 to R.I.-K(biotin)-Tat9 altered its intestinal transport pathway resulting in a significant improvement in its absorptive permeability by enhancing nonspecific passive and carrier-mediated uptake by means of SMVT. The specific interactions between R.I.-K(biotin)-Tat9 and SMVT suggest that targeting approaches utilizing transporters such as SMVT may substantially improve the oral delivery of large peptides.
Ramanathan
S
S
College of Pharmacy, Rutgers, The State University of new Jersey, Piscataway 08854, USA.
Pooyan
S
S
Stein
S
S
Prasad
P D
PD
Wang
J
J
Leibowitz
M J
MJ
Ganapathy
V
V
Sinko
P J
PJ
D000284:Administration, Oral; D000818:Animals; D019380:Anti-HIV Agents / Q000493:pharmacokinetics*; D001693:Biological Transport, Active; D001710:Biotin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D016466:CHO Cells / Q000378:metabolism; D018938:Caco-2 Cells / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D016503:Drug Delivery Systems / Q000379:methods*; D015696:Gene Products, tat / Q000493:pharmacokinetics*; D006801:Humans; D007408:Intestinal Absorption / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D027981:Symporters; D054322:tat Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; D002352:Carrier Proteins; D015696:Gene Products, tat; D008562:Membrane Glycoproteins; D027981:Symporters; C118266:Tat9-K-biotin; C119981:biotin transporter; D054322:tat Gene Products, Human Immunodeficiency Virus; D001710:Biotin