Advertisement

Pharmaceutical Research

, Volume 33, Issue 10, pp 2506–2516 | Cite as

An Assessment of the Permeation Enhancer, 1-phenyl-piperazine (PPZ), on Paracellular Flux Across Rat Intestinal Mucosae in Ussing Chambers

  • V. A. Bzik
  • D. J. Brayden
Research Paper

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.

KEY WORDS

epithelial tight junctions intestinal permeation enhancers oral peptides phenyl piperazine Ussing chambers 

LIST OF 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

Notes

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).

REFERENCES

  1. 1.
    Whitehead K, Karr N, Mitragotri S. Safe and effective permeation enhancers for oral drug delivery. Pharm Res. 2008;25(8):1782–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Whitehead K, Mitragotri S. Mechanistic analysis of chemical permeation enhancers for oral drug delivery. Pharm Res. 2008;25(6):1412–29.CrossRefPubMedGoogle Scholar
  3. 3.
    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.CrossRefPubMedGoogle Scholar
  4. 4.
    Martin RJ. Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum somatic muscle. Br J Pharmacol. 1982;77(2):255–65.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    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.CrossRefPubMedGoogle Scholar
  6. 6.
    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.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefPubMedGoogle Scholar
  8. 8.
    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.CrossRefPubMedGoogle Scholar
  9. 9.
    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.CrossRefPubMedGoogle Scholar
  10. 10.
    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.Google Scholar
  11. 11.
    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.CrossRefPubMedGoogle Scholar
  12. 12.
    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.CrossRefPubMedGoogle Scholar
  13. 13.
    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.CrossRefPubMedGoogle Scholar
  14. 14.
    Borman RA, Burleigh DE. Human colonic mucosa possesses a mixed population of 5-HT receptors. Eur J Pharmacol. 1996;309(3):271–4.CrossRefPubMedGoogle Scholar
  15. 15.
    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.CrossRefPubMedGoogle Scholar
  16. 16.
    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.PubMedGoogle Scholar
  17. 17.
    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.CrossRefPubMedGoogle Scholar
  18. 18.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Al-Tabakha MM. Future prospect of insulin inhalation for diabetic patients: The case of Afrezza versus Exubera. J Control Release. 2015;215:25–238.CrossRefPubMedGoogle Scholar
  20. 20.
    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.CrossRefPubMedGoogle Scholar
  21. 21.
    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.CrossRefPubMedGoogle Scholar
  22. 22.
    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.CrossRefPubMedGoogle Scholar
  23. 23.
    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.CrossRefPubMedGoogle Scholar
  24. 24.
    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.CrossRefGoogle Scholar
  25. 25.
    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.CrossRefPubMedGoogle Scholar
  26. 26.
    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.CrossRefPubMedGoogle Scholar
  27. 27.
    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.CrossRefGoogle Scholar
  28. 28.
    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.Google Scholar
  29. 29.
    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.Google Scholar
  30. 30.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    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.CrossRefPubMedGoogle Scholar
  32. 32.
    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.CrossRefPubMedGoogle Scholar
  33. 33.
    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.CrossRefPubMedGoogle Scholar
  34. 34.
    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.PubMedGoogle Scholar
  35. 35.
    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.CrossRefPubMedGoogle Scholar
  36. 36.
    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.CrossRefPubMedGoogle Scholar
  37. 37.
    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.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.School of Veterinary Medicine and Conway InstituteUniversity College DublinBelfield, Dublin 4Ireland

Personalised recommendations