Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide

S. Ramanathan, S. Pooyan, S. Stein, P. D. Prasad, J. Wang, M. J. Leibowitz, V. Ganapathy, P. J. Sinko

Research output: Contribution to journalArticle

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Abstract

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 μM) and R.I.-K(biotin)-Tat9 (0.1-25 μM) 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 μM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (Pm ∼ 1 × 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 μM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 μM) was ∼ 500-fold greater than R.I.-K-Tat9 (at 10 μM). 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.

Original languageEnglish (US)
Pages (from-to)950-956
Number of pages7
JournalPharmaceutical Research
Volume18
Issue number7
DOIs
StatePublished - Sep 11 2001

Fingerprint

Sodium
Permeability
CHO Cells
Biotin
Human Immunodeficiency Virus tat Gene Products
tat Gene Products
Tat9-K-biotin
Peptides
Caco-2 Cells
HIV-1
Resins
biotin transporter
Kinetics
Substrates

Keywords

  • Biotin
  • CHO
  • Caco-2
  • Oral absorption
  • Tat peptide
  • Vitamin transporter

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

Cite this

Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide. / Ramanathan, S.; Pooyan, S.; Stein, S.; Prasad, P. D.; Wang, J.; Leibowitz, M. J.; Ganapathy, V.; Sinko, P. J.

In: Pharmaceutical Research, Vol. 18, No. 7, 11.09.2001, p. 950-956.

Research output: Contribution to journalArticle

Ramanathan, S. ; Pooyan, S. ; Stein, S. ; Prasad, P. D. ; Wang, J. ; Leibowitz, M. J. ; Ganapathy, V. ; Sinko, P. J. / Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide. In: Pharmaceutical Research. 2001 ; Vol. 18, No. 7. pp. 950-956.
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abstract = "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 μM) and R.I.-K(biotin)-Tat9 (0.1-25 μM) 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 μM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (Pm ∼ 1 × 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 μM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 μM) was ∼ 500-fold greater than R.I.-K-Tat9 (at 10 μM). 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.",
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T1 - Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide

AU - Ramanathan, S.

AU - Pooyan, S.

AU - Stein, S.

AU - Prasad, P. D.

AU - Wang, J.

AU - Leibowitz, M. J.

AU - Ganapathy, V.

AU - Sinko, P. J.

PY - 2001/9/11

Y1 - 2001/9/11

N2 - 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 μM) and R.I.-K(biotin)-Tat9 (0.1-25 μM) 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 μM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (Pm ∼ 1 × 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 μM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 μM) was ∼ 500-fold greater than R.I.-K-Tat9 (at 10 μM). 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.

AB - 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 μM) and R.I.-K(biotin)-Tat9 (0.1-25 μM) 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 μM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (Pm ∼ 1 × 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 μM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 μM) was ∼ 500-fold greater than R.I.-K-Tat9 (at 10 μM). 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.

KW - Biotin

KW - CHO

KW - Caco-2

KW - Oral absorption

KW - Tat peptide

KW - Vitamin transporter

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