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Investigations of Piperazine Derivatives as Intestinal Permeation Enhancers in Isolated Rat Intestinal Tissue Mucosae

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Abstract

A limiting factor for oral delivery of macromolecules is low intestinal epithelial permeability. 1-Phenylpiperazine (PPZ), 1-(4-methylphenyl) piperazine (1-4-MPPZ) and 1-methyl-4-phenylpiperazine (1-M-4-PPZ) have emerged as potential permeation enhancers (PEs) from a screen carried out by others in Caco-2 monolayers. Here, their efficacy, mechanism of action and potential for epithelial toxicity were further examined in Caco-2 cells and isolated rat intestinal mucosae. Using high-content analysis, PPZ and 1-4-MPPZ decreased mitochondrial membrane potential and increased plasma membrane potential in Caco-2 cells to a greater extent than 1-M-4-PPZ. The Papp of the paracellular marker, [14C]-mannitol, and of the peptide, [3H]-octreotide, was measured across rat colonic mucosae following apical addition of the three piperazines. PPZ and 1-4-MPPZ induced a concentration-dependent decrease in transepithelial electrical resistance (TEER) and an increase in the Papp of [14C]-mannitol without causing histological damage. 1-M-4-PPZ was without effect. The piperazines caused the Krebs-Henseleit buffer pH to become alkaline, which partially attenuated the increase in Papp of [14C]-mannitol caused by PPZ and 1-4-MPPZ. Only addition of 1-4-MPPZ increased the Papp of [3H]-octreotide. Pre-incubation of mucosae with two 5-HT4 receptor antagonists, a loop diuretic and a myosin light chain kinase inhibitor, reduced the permeation enhancement capacity of PPZ and 1-4-MPP for [14C]-mannitol. 1-4-MPPZ holds most promise as a PE, but intestinal physiology may also be impacted due to multiple mechanisms of action.

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Abbreviations

1-4-MPPZ:

1-(4-Methylphenyl)piperazine

1-M-4-PPZ:

1-Methyl-4-phenylpiperazine

BZP:

N-Benzylpiperazine

CPE:

Chemical permeation enhancer

FCCP:

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FITC:

Fluorescein isothiocyanate

I sc :

Short circuit current

KH:

Krebs-Henseleit buffer

MDMA:

3,4-Methylenedioxymethamphetamine

ML9:

1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine

MLCK:

Myosin light chain kinase

MMP:

Mitochondrial membrane potential

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium

Na/K/2Cl:

Sodium/potassium/chloride cotransporter

P app :

Apparent permeability coefficient

PKA:

Protein kinase A

PKC:

Protein kinase C

PMP:

Plasma membrane potential

PPZ:

1-Phenylpiperazine

TEER:

Transepithelial electrical resistance

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Funding

This study was funded by Science Foundation Ireland Grant 13/RC/2073, the CÚRAM Centre for Medical Devices and by the EU Regional Development Fund.

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  1. School of Veterinary Medicine and Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

    V. Stuettgen & D. J. Brayden

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  1. V. Stuettgen
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Correspondence to D. J. Brayden.

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Animals were used in accordance with UCD Animal Research Ethics Committee protocol for use of post-mortem tissue from animals in research (AREC 14-28-Brayden), in adherence with the “Principles of Laboratory Animal Care”, (NIH Publication #85-23, revised in 1985).

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Stuettgen, V., Brayden, D.J. Investigations of Piperazine Derivatives as Intestinal Permeation Enhancers in Isolated Rat Intestinal Tissue Mucosae. AAPS J 22, 33 (2020). https://doi.org/10.1208/s12248-020-0416-9

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