ABSTRACT
Purpose
1-phenyl piperazine (PPZ) emerged from a Caco-2 monolayer screen as having high enhancement potential due to a capacity to increase permeation without significant toxicity. Our aim was to further explore the efficacy and toxicity of PPZ in rat ileal and colonic mucosae in order to assess its true translation potential.
Methods
Intestinal mucosae were mounted in Ussing chambers and apparent permeability coefficient (Papp) values of [14C]-mannitol and FITC-dextran 4 kDa (FD-4) and transepithelial electrical resistance (TEER) values were obtained following apical addition of PPZ (0.6–60 mM). Exposed issues were assessed for toxicity by histopathology and lactate dehydrogenase (LDH) release. Mucosal recovery after exposure was also assessed using TEER readings.
Results
PPZ reversibly increased the Papp of both agents across rat ileal and distal colonic mucosae in concentration–dependent fashion, accompanied by TEER reduction, with acceptable levels of tissue damage. The complex mechanism of tight junction opening was part mediated by myosin light chain kinase, stimulation of transepithelial electrogenic chloride secretion, and involved activation of 5-HT4 receptors.
Conclusions
PPZ is an efficacious and benign intestinal permeation enhancer in tissue mucosae. However, its active pharmacology suggest that potential for further development in an oral formulation for poorly permeable molecules will be difficult.
Similar content being viewed by others
Abbreviations
- C10 :
-
Sodium salt of capric acid
- CFTR:
-
Cystic fibrosis transmembrane regulator
- DKFP:
-
Diketofumaryl piperazine
- FD:
-
FITC-dextran
- FITC:
-
Fluorescein isothiocyanate
- FSK:
-
Forskolin
- HBSS:
-
Hank’s balanced salt solution
- IBMX:
-
3-isobutyl-1-methyl-xanthine
- Isc:
-
Short circuit current
- KH:
-
Krebs-Henseleit solution
- LDH:
-
Lactate dehydrogenase
- ML9:
-
1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine]
- MLCK:
-
Myosin light chain kinase
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium
- Na/K/2Cl:
-
Sodium/potassium/chloride cotransporter
- Papp:
-
Apparent permeability coefficient
- PKA:
-
Protein kinase A
- PKC:
-
Protein kinase C
- PPZ:
-
1-phenyl piperazine
- TEER:
-
Transepithelial electrical resistance
REFERENCES
Whitehead K, Karr N, Mitragotri S. Safe and effective permeation enhancers for oral drug delivery. Pharm Res. 2008;25(8):1782–8.
Whitehead K, Mitragotri S. Mechanistic analysis of chemical permeation enhancers for oral drug delivery. Pharm Res. 2008;25(6):1412–29.
Lamson NG, Cusimano G, Suri K, Zhang A, Whitehead KA. The pH of piperazine derivative solutions predicts their utility as transepithelial permeation enhancers. Mol Pharm. 2016;13:578–85.
Martin RJ. Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum somatic muscle. Br J Pharmacol. 1982;77(2):255–65.
Neves G, Menegatti R, Antonio CB, Grazziottin LR, Vieira RO, Rates SM, et al. Searching for multi-target antipsychotics: discovery of orally active heterocyclic N-phenylpiperazine ligands of D2-like and 5-HT1A receptors. Bioorg Med Chem. 2010;18(5):1925–35.
Dilly S, Graulich A, Liegeois JF. Molecular modeling study of 4-phenylpiperazine and 4-phenyl-1,2,3,6-tetrahydropyridine derivatives: a new step towards the design of high-affinity 5-HT1A ligands. Bioorg Med Chem Lett. 2010;20(3):1118–23.
Di Fabio R, Griffante C, Alvaro G, Pentassuglia G, Pizzi DA, Donati D, et al. Discovery process and pharmacological characterization of 2-(S)-(4-fluoro-2-methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethylphenyl)ethyl]methylamide (vestipitant) as a potent, selective, and orally active NK1 receptor antagonist. J Med Chem. 2009;52(10):3238–47.
Neves G, Kliemann M, Betti AH, Conrado DJ, Tasso L, Fraga CA, et al. Serotonergic neurotransmission mediates hypothermia induced by the N-phenylpiperazine antipsychotic prototypes LASSBio-579 and LASSBio-581. Pharmacol Biochem Behav. 2008;89(1):23–30.
Weerts A, Pattyn N, Van de Heyning P, Wuyts F. Evaluation of the effects of anti-motion sickness drugs on subjective sleepiness and cognitive performance of healthy males. J Psychopharmacol. 2013;28(7):655–64.
Gaginella TS. In: Gaginella TS, Galligan JJ, Gaginella TS, Galligan JJ, editors. Serotonin in the intestinal tract: A synopsis. 1st ed. New York: CRC Press; 1995.
Costedio MM, Hyman N, Mawe GM. Serotonin and its role in colonic function and in gastrointestinal disorders. Dis Colon Rectum. 2007;50(3):376–88.
Tsukamoto K, Ariga H, Mantyh C, Pappas TN, Yanagi H, Yamamura T, et al. Luminally-released serotonin stimulates colonic motility and accelerates colonic transit in rats. Am J Physiol Regul Integr Comp Physiol. 2007;293(1):R64–9.
Haga K, Asano K, Fukuda T, Kobayakawa T. The function of 5-HT3 receptors on colonic transit in rats. Obes Res. 1995;3 Suppl 5:801S–10S.
Borman RA, Burleigh DE. Human colonic mucosa possesses a mixed population of 5-HT receptors. Eur J Pharmacol. 1996;309(3):271–4.
Kuramoto H, Kadowaki M, Sakamoto H, Yuasa K, Todo A, Shirai R. Distinct morphology of serotonin-containing enterochromaffin (EC) cells in the rat distal colon. Arch Histol Cytol. 2007;70(4):235–41.
Chen JX, Pan H, Rothman TP, Wade PR, Gershon MD. Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception. Am J Physiol. 1998;275(3 Pt 1):G433–48.
Ning Y, Zhu JX, Chan HC. Regulation of ion transport by 5-hydroxytryptamine in rat colon. Clin Exp Pharmacol Physiol. 2004;31(7):424–8.
Bunce KT, Elswood CJ, Ball MT. Investigation of the 5-hydroxytryptamine receptor mechanism mediating the short-circuit current response in rat colon. Br J Pharmacol. 1991;102(4):811–6.
Al-Tabakha MM. Future prospect of insulin inhalation for diabetic patients: The case of Afrezza versus Exubera. J Control Release. 2015;215:25–238.
Petersen SB, Nolan G, Maher S, Rahbek UL, Guldbrandt M, Brayden DJ. Evaluation of alkylmaltosides as intestinal permeation enhancers: comparison between rat intestinal mucosal sheets and Caco-2 monolayers. Eur J Pharm Sci. 2012;47(4):701–12.
Hubatsch I, Ragnarsson EG, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc. 2007;2(9):2111–9.
Feighery LM, Cochrane SW, Quinn T, Baird AW, O'Toole D, Owens SE, et al. Myosin light chain kinase inhibition: correction of increased intestinal epithelial permeability in vitro. Pharm Res. 2008;25(6):1377–86.
Yang N, Xue H, Guo H, Chen X, Zhu JX. Segmental heterogeneity of epithelial ion transport induced by stimulants in rat distal colon. Biol Pharm Bull. 2006;29(9):1825–9.
Fowler SB, Poon S, Muff R, Chiti F, Dobson CM, Zurdo J. Rational design of aggregation-resistant bioactive peptides: re-engineering human calcitonin, Proc. Natl Acad Sci U S A. 2005;102:10105–10.
Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993;303(2):474–82.
Maher S, Kennelly R, Bzik VA, Baird AW, Wang X, Winter D, et al. Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. I. Studies in isolated rat and human colonic mucosae. Eur J Pharm Sci. 2009;38(4):291–300.
Briske-Anderson MJ, Finley JW, Newman SM. The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc. Soc. Exp. Biology Med. 1997;214(3):248–257.28.Leonard M, Creed E, Brayden D, Baird AW. Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport. Pharm Res. 2000;17(10):1181–8.
Marušić M, Zupančič T, Hribar G, Komel R, Anderluh G, Caserman S. The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-β-d-maltopyranoside. Nature Biotechnol. 2013;30(5):507–15.
Ungell AL, Nylander S, Bergstrand S, Sjoberg A, Lennernass H. Membrane transport of drugs in different regions of the intestinal tract of the rat. Journal of Pharmaceutical Sciences 1998. 1998;87(3):360–6.
Hamilton MK, Boudry G, Lemay DG, Raybould HE. Changes in intestinal barrier function and gut microbiota in high-fat diet-fed rats are dynamic and region dependent. Am J Physiol Gastrointest Liver Physiol. 2015;308(10):G840–51.
Feighery L, Smyth A, Keely S, Baird AW, O'Connor WT, Callanan JJ, et al. Increased intestinal permeability in rats subjected to traumatic frontal lobe percussion brain injury. J Trauma. 2008;64(1):131–7.
Gill RK, Saksena S, Tyagi S, Alrefai WA, Malakooti J, Sarwar Z, et al. Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells. Gastroenterology. 2005;128(4):962–74.
Alcalde AI, Sorribas V, Rodriguez-Yoldi MJ, Lahuerta A. Study of serotonin interactions with brush border membrane of rabbit jejunum enterocytes. Eur J Pharmacol. 2000;403(1–2):9–15.
Karczewski J, Groot J. Molecular physiology and pathophysiology of tight junctions III. Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am J Physiol Gastrointest Liver Physiol. 2000;279(4):G660–5.
Nagatsu T, Suzuki H, Kiuchi K, Saitoh M, Hidaka H. Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells. Biochem Biophys Res Commun. 1987;143(3):1045–8.
Denizot J, Sivignon A, Barreau F, Darcha C, Chan HF, Stanners CP, et al. Adherent-invasive Escherichia coli induce claudin-2 expression and barrier defect in CEABAC10 mice and crohn's disease patients. Inflamm Bowel Dis. 2012;18(2):294–304.
Aguirre TA, Teijeiro-Osorio D, Rosa M, Coulter IS, Alonso MJ, Brayden DJ. Current status of selected oral peptide technologies in advanced preclinical development and in clinical trials. Adv Drug Deliv Rev. 2016. doi:10.1016/j.addr.2016.02.004.
ACKNOWLEDGMENTS AND DISCLOSURES
This study was co-funded by Science Foundation Ireland grant 07/SRC B1144. V.A. Bzik was recipient of a UCD Ad Astra Scholarship. An abstract of this study was presented at the CRS Annual Meeting; Copenhagen, Denmark (2009).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bzik, V.A., Brayden, D.J. An Assessment of the Permeation Enhancer, 1-phenyl-piperazine (PPZ), on Paracellular Flux Across Rat Intestinal Mucosae in Ussing Chambers. Pharm Res 33, 2506–2516 (2016). https://doi.org/10.1007/s11095-016-1975-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11095-016-1975-4