PURPOSE: 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.
PURPOSE: 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 humanSMVT (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.
Authors: P J Sinko; Y H Lee; V Makhey; G D Leesman; J P Sutyak; H Yu; B Perry; C L Smith; P Hu; E J Wagner; L M Falzone; L T McWhorter; J P Gilligan; W Stern Journal: Pharm Res Date: 1999-04 Impact factor: 4.200
Authors: B S Weeks; K Desai; P M Loewenstein; M E Klotman; P E Klotman; M Green; H K Kleinman Journal: J Biol Chem Date: 1993-03-05 Impact factor: 5.157
Authors: B E Vogel; S J Lee; A Hildebrand; W Craig; M D Pierschbacher; F Wong-Staal; E Ruoslahti Journal: J Cell Biol Date: 1993-04 Impact factor: 10.539
Authors: Shahriar Pooyan; Bo Qiu; Marion M Chan; Dunne Fong; Patrick J Sinko; Michael J Leibowitz; Stanley Stein Journal: Bioconjug Chem Date: 2002 Mar-Apr Impact factor: 4.774
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