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Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery.

Song KH, Kim SB, Shim CK, Chung SJ, Kim DD, Rhee SK, Choi GJ, Kim CH, Kim K - Drug Des Devel Ther (2015)

Bottom Line: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002.In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration-time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone.Thus, Pep1 increased the stability or possibly reduced the instability of AT1002, which resulted in an increased permeation-enhancing effect of AT1002.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Engineering, Soonchunhyang University, Asan, Republic of Korea.

ABSTRACT

Background: The identification of permeation enhancers has gained interest in the development of drug delivery systems. A six-mer peptide, H-FCIGRL-OH (AT1002), is a tight junction modulator with promising permeation-enhancing activity. AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. However, AT1002 is not stable in neutral pH or after incubation under physiological conditions, which is necessary to fully uncover its permeation-enhancing effect. Thus, we increased the stability or mitigated the instability of AT1002 by modifying its terminal amino acids and evaluated its subsequent biological activity.

Methods: C-terminal-amidated (FCIGRL-NH2, Pep1) and N-terminal-acetylated (Ac-FCIGRL, Pep2) peptides were analyzed by liquid chromatography-mass spectrometry. We further assessed cytotoxicity on cell monolayers, as well as the permeation-enhancing activity following nasal administration of the paracellular marker mannitol.

Results: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002. In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration-time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone. In contrast, no increase in mannitol concentration was shown with mannitol/AT1002 or mannitol/Pep2 compared to the control. Thus, Pep1 increased the stability or possibly reduced the instability of AT1002, which resulted in an increased permeation-enhancing effect of AT1002.

Conclusion: These results suggest the potential usefulness of C-terminal-amidated AT1002 in enhancing nasal drug delivery, which may lead to the development of a practical drug delivery technology for drugs with low bioavailability.

No MeSH data available.


Related in: MedlinePlus

The LDH assay for FCIGRL (AT1002), FCIGRL-NH2 (Pep1), Ac-FCIGRL (Pep2), PBS (negative control), and Triton X-100 (positive control) at 1 hour incubation time in Caco-2 cell monolayers.Notes: The control for PBS viability (%) is LDH activity of PBS at 0 hours incubation time. Each data point represents the mean ± SEM (n=3).Abbreviations: LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; SEM, standard error of the mean.
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f5-dddt-9-1815: The LDH assay for FCIGRL (AT1002), FCIGRL-NH2 (Pep1), Ac-FCIGRL (Pep2), PBS (negative control), and Triton X-100 (positive control) at 1 hour incubation time in Caco-2 cell monolayers.Notes: The control for PBS viability (%) is LDH activity of PBS at 0 hours incubation time. Each data point represents the mean ± SEM (n=3).Abbreviations: LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; SEM, standard error of the mean.

Mentions: An LDH assay was used to examine the toxic effects of terminally modified AT1002 peptides on epithelial cells. An increase in LDH released from the cytosol of cells resulted from an increase in dead or plasma membrane–damaged cells. Caco-2 cell monolayers were incubated with AT1002, Pep1, and Pep2 in PBS for 1 hour. The viability of cells was not significantly different for each peptide compared to the PBS negative control (Figure 5). These results suggest that the terminally modified peptides of AT1002 did not produce a cytotoxic response to Caco-2 cells during the incubation time. AT1002 was also not cytotoxic, which correlates with our previous results.17


Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery.

Song KH, Kim SB, Shim CK, Chung SJ, Kim DD, Rhee SK, Choi GJ, Kim CH, Kim K - Drug Des Devel Ther (2015)

The LDH assay for FCIGRL (AT1002), FCIGRL-NH2 (Pep1), Ac-FCIGRL (Pep2), PBS (negative control), and Triton X-100 (positive control) at 1 hour incubation time in Caco-2 cell monolayers.Notes: The control for PBS viability (%) is LDH activity of PBS at 0 hours incubation time. Each data point represents the mean ± SEM (n=3).Abbreviations: LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; SEM, standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383222&req=5

f5-dddt-9-1815: The LDH assay for FCIGRL (AT1002), FCIGRL-NH2 (Pep1), Ac-FCIGRL (Pep2), PBS (negative control), and Triton X-100 (positive control) at 1 hour incubation time in Caco-2 cell monolayers.Notes: The control for PBS viability (%) is LDH activity of PBS at 0 hours incubation time. Each data point represents the mean ± SEM (n=3).Abbreviations: LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; SEM, standard error of the mean.
Mentions: An LDH assay was used to examine the toxic effects of terminally modified AT1002 peptides on epithelial cells. An increase in LDH released from the cytosol of cells resulted from an increase in dead or plasma membrane–damaged cells. Caco-2 cell monolayers were incubated with AT1002, Pep1, and Pep2 in PBS for 1 hour. The viability of cells was not significantly different for each peptide compared to the PBS negative control (Figure 5). These results suggest that the terminally modified peptides of AT1002 did not produce a cytotoxic response to Caco-2 cells during the incubation time. AT1002 was also not cytotoxic, which correlates with our previous results.17

Bottom Line: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002.In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration-time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone.Thus, Pep1 increased the stability or possibly reduced the instability of AT1002, which resulted in an increased permeation-enhancing effect of AT1002.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Engineering, Soonchunhyang University, Asan, Republic of Korea.

ABSTRACT

Background: The identification of permeation enhancers has gained interest in the development of drug delivery systems. A six-mer peptide, H-FCIGRL-OH (AT1002), is a tight junction modulator with promising permeation-enhancing activity. AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. However, AT1002 is not stable in neutral pH or after incubation under physiological conditions, which is necessary to fully uncover its permeation-enhancing effect. Thus, we increased the stability or mitigated the instability of AT1002 by modifying its terminal amino acids and evaluated its subsequent biological activity.

Methods: C-terminal-amidated (FCIGRL-NH2, Pep1) and N-terminal-acetylated (Ac-FCIGRL, Pep2) peptides were analyzed by liquid chromatography-mass spectrometry. We further assessed cytotoxicity on cell monolayers, as well as the permeation-enhancing activity following nasal administration of the paracellular marker mannitol.

Results: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002. In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration-time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone. In contrast, no increase in mannitol concentration was shown with mannitol/AT1002 or mannitol/Pep2 compared to the control. Thus, Pep1 increased the stability or possibly reduced the instability of AT1002, which resulted in an increased permeation-enhancing effect of AT1002.

Conclusion: These results suggest the potential usefulness of C-terminal-amidated AT1002 in enhancing nasal drug delivery, which may lead to the development of a practical drug delivery technology for drugs with low bioavailability.

No MeSH data available.


Related in: MedlinePlus