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Profiling the Role of Deacylation-Reacylation in the Lymphatic Transport of a Triglyceride-Mimetic Prodrug



Recent studies have demonstrated the potential for a triglyceride (TG) mimetic prodrug to promote the delivery of mycophenolic acid (MPA) to the lymphatic system. Here, the metabolic pathways that facilitate the lymphatic transport of the TG prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) were examined to better inform the design of next generation prodrugs.


In vitro hydrolysis experiments in simulated intestinal conditions and in vivo rat lymphatic transport experiments were conducted in the presence and absence of orlistat and A922500 (inhibitors of lipolysis and TG re-esterification, respectively), to evaluate the importance of 2-MPA-TG digestion and re-esterification of 2-MPA-MG (the 2-monoglyceride derivative) in promoting lymphatic transport.


2-MPA-TG was rapidly hydrolysed to 2-MPA-MG on incubation with fresh bile and pancreatic fluid (BPF), but not in simulated gastric fluid, heat-inactivated BPF or BPF + orlistat. Orlistat markedly decreased lymphatic transport and systemic exposure of 2-MPA-TG derivatives suggesting that inhibition of pancreatic lipase hindered luminal digestion and absorption of the prodrug. A922500 also significantly decreased lymphatic transport of 2-MPA-TG but redirected MPA to the portal blood, suggesting that hindered re-acylation of 2-MPA-MG resulted in intracellular degradation.


Incorporation into TG deacylation-reacylation pathways is a critical determinant of the utility of lymph directed TG-mimetic prodrugs.

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Bile and pancreatic fluid




Diacylglycerol acyltransferease


Diacylglycerol transacylase


Fatty acid






Monoacylglycerol acyltransferase


Mycophenolic acid


Molecular weight


Simulated gastric fluid




  1. White KL, Nguyen G, Charman WN, Edwards GA, Faassen WA, Porter CJH. Lymphatic transport of methylnortestosterone undecanoate (MU) and the bioavailability of methylnortestosterone are highly sensitive to the mass of coadministered lipid after oral administration of MU. J Pharmacol Exp Ther. 2009;331(2):700–9.

    Article  CAS  PubMed  Google Scholar 

  2. Shackleford DM, Faassen WA, Houwing N, Lass H, Edwards GA, Porter CJH, et al. Contribution of lymphatically transported testosterone undecanoate to the systemic exposure of testosterone after oral administration of two andriol formulations in conscious lymph duct-cannulated dogs. J Pharmacol Exp Ther. 2003;306(3):925–33.

    Article  CAS  PubMed  Google Scholar 

  3. Trevaskis NL, Tso P, Rider T, Charman WN, Porter CJH, Jandacek R. Tissue uptake of DDT is independent of chylomicron metabolism. Arch Toxicol. 2006;80(4):196–200.

    Article  CAS  PubMed  Google Scholar 

  4. Caliph SM, Cao E, Bulitta JB, Hu L, Han S, Porter CJH, et al. The impact of lymphatic transport on the systemic disposition of lipophylic drugs. J Pharm Sci. 2013;102(7):2395–408.

    Article  CAS  PubMed  Google Scholar 

  5. McAllaster JD, Cohen MS. Role of the lymphatics in cancer metastasis and chemotherapy applications. Adv Drug Deliv Rev. 2011;63(10–11):867–75.

    Article  CAS  PubMed  Google Scholar 

  6. Trevaskis NL, Charman WN, Porter CJH. Targeted drug delivery to lymphocytes: a route to site-specific immunomodulation? Mol Pharm. 2010;7(6):2297–309.

    Article  CAS  PubMed  Google Scholar 

  7. Dane KY, Nembrini C, Tomei AA, Eby JK, O’Neil CP, Velluto D, et al. Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival. J Control Release. 2011;156(2):154–60.

    Article  CAS  PubMed  Google Scholar 

  8. Porter CJH, Trevaskis NL, Charman WN. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat Rev Drug Discov. 2007;6(3):231–48.

    Article  CAS  PubMed  Google Scholar 

  9. Trevaskis NL, Charman WN, Porter CJH. Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update. Adv Drug Deliv Rev. 2008;60(6):702–16.

    Article  CAS  PubMed  Google Scholar 

  10. Mansbach CM, Siddiqi SA. The biogenesis of chylomicrons. Annu Rev Physiol. 2010;72:315–33.

    Article  CAS  PubMed  Google Scholar 

  11. Trevaskis NL, Shanker RM, Charman WN, Porter CJH. The Mechanism of lymphatic access of two cholesteryl ester transfer protein inhibitors (CP524,515 and CP532,623) and evaluation of their impact on lymph lipoprotein profiles. Pharm Res. 2010;27(9):1949–64.

    Article  CAS  PubMed  Google Scholar 

  12. Trevaskis NL, Shackleford DM, Charman WN, Edwards GA, Gardin A, Appel-Dingemanse S, et al. Intestinal lymphatic transport enhances the post-prandial oral bioavailability of a novel cannabinoid receptor agonist via avoidance of first-pass metabolism. Pharm Res. 2009;26(6):1486–95.

    Article  CAS  PubMed  Google Scholar 

  13. Garzonaburbeh A, Poupaert JH, Claesen M, Dumont P, Atassi G. 1,3-dipalmitoylglycerol ester of chlorambucil as a lymphotropic, orally administrable anti-neoplastic agent. J Med Chem. 1983;26(8):1200–3.

    Article  CAS  Google Scholar 

  14. Han S, Quach T, Hu L, Wahab A, Charman WN, Stella VJ, et al. Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies. J Control Release. 2014;177:1–10.

    Article  CAS  PubMed  Google Scholar 

  15. Leak LV. Permeability of lymphatic capillaries. J Cell Biol. 1971;50(2):300–23.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Dixon JB, Raghunathan S, Swartz MA. A tissue-engineered model of the intestinal lacteal for evaluating lipid transport by lymphatics. Biotechnol Bioeng. 2009;103(6):1224–35.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Trevaskis NL, Porter CJH, Charman WN. Bile increases intestinal lymphatic drug transport in the fasted rat. Pharm Res. 2005;22(11):1863–70.

    Article  CAS  PubMed  Google Scholar 

  18. Johnson BM, Chen WQ, Borchardt RT, Charman WN, Porter CJH. A kinetic evaluation of the absorption, efflux, and metabolism of verapamil in the autoperfused rat jejunum. J Pharmacol Exp Ther. 2003;305(1):151–8.

    Article  CAS  PubMed  Google Scholar 

  19. Waynforth HB, Flecknell PA. Bile duct catheterisation. Experimental and surgical technique in the rat (2nd edition) Academic Press. 1992:206–10.

  20. The United States Pharmacopoeia, Inc., Rockville, MD. 2000.

  21. Yau W-P, Vathsala A, Lou H-X, Zhou S, Chan E. Mechanism-based enterohepatic circulation model of mycophenolic acid and its glucuronide metabolite: assessment of impact of cyclosporine dose in asian renal transplant patients. J Clin Pharmacol. 2009;49(6):684–99.

    Article  CAS  PubMed  Google Scholar 

  22. Saitoh H, Kobayashi M, Oda M, Nakasato K, Kobayashi M, Tadano K. Characterization of intestinal absorption and enterohepatic circulation of mycophenolic acid and its 7-O-glucuronide in rats. Drug Metab Pharmacokinet. 2006;21(5):406–13.

    Article  CAS  PubMed  Google Scholar 

  23. Paris GY, Garmaise DL, Cimon DG, Swett L, Carter GW, Young P. Glycerides as prodrugs. 1. synthesis and anti-inflammatory activity of 1,3-bis(alkanoyl)-2-(O-acetylsalicyloyl)glycerides (aspirin triglycerides). J Med Chem. 1979;22(6):683–7.

    Article  CAS  PubMed  Google Scholar 

  24. Amory JK, Scriba GKE, Amory DW, Bremner WJ. Oral testosterone-triglyceride conjugate in rabbits: Single-dose pharmacokinetics and comparison with oral testosterone undecanoate. J Androl. 2003;24(5):716–20.

    CAS  PubMed  Google Scholar 

  25. Scriba GKE, Lambert DM, Poupaert JH. Bioavailability of phenytoin following oral administration of phenytoin-lipid conjugates to rats. J Pharm Pharmacol. 1995;47(11):945–8.

    Article  CAS  PubMed  Google Scholar 

  26. Jacob JN, Hesse GW, Shashoua VE. Synthesis, brain uptake, and pharmacological properties of a glyceryl lipid containing GABA and the GABA-T inhibitor Gamma-vinyl-GABA. J Med Chem. 1990;33(2):733–6.

    Article  CAS  PubMed  Google Scholar 

  27. Paris GY, Garmaise DL, Cimon DG, Swett L, Carter GW, Young P. Glycerides as prodrugs. 2. 1,3-dialkanoyl-2-(2-methyl-4-oxo-1,3-benzodioxan-2-yl)glycerides (cyclic aspirin triglycerides) as anti-inflammatory agents. J Med Chem. 1980;23(1):79–82.

    Article  CAS  PubMed  Google Scholar 

  28. Scriba GKE. Synthesis and in-vitro degradation of testosterone-lipid conjugates. Arch Pharm (Weinheim). 1995;328(3):271–6.

    Article  CAS  Google Scholar 

  29. Mu HL, Hoy CE. The digestion of dietary triacylglycerols. Prog Lipid Res. 2004;43(2):105–33.

    Article  CAS  PubMed  Google Scholar 

  30. Lalanne M, Khoury H, Deroussent A, Bosquet N, Benech H, Clayette P, et al. Metabolism evaluation of biomimetic prodrugs by in vitro models and mass spectrometry. Int J Pharm. 2009;379(2):235–43.

    Article  CAS  PubMed  Google Scholar 

  31. Gargouri Y, Chahinian H, Moreau H, Ransac S, Verger R. Inactivation of pancreatic and gastric lipases by THL and C-12-0-TNB - a kinetic study with emulsified tributyrjn. Biochim Biophys Acta. 1991;1085(3):322–8.

    Article  CAS  PubMed  Google Scholar 

  32. Hadvary P, Lengsfeld H, Wolfer H. Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin. Biochem J. 1988;256(2):357–61.

    PubMed Central  CAS  PubMed  Google Scholar 

  33. Poorkhalkali N, Lidmer AS, Lundberg LG, Dalrymple MA, Gibson Y, Taylor L, et al. Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL. Histochem Cell Biol. 1998;110(4):367–76.

    Article  CAS  PubMed  Google Scholar 

  34. Li X, Lindquist S, Lowe M, Noppa L, Hernell O. Bile salt-stimulated lipase and pancreatic lipase-related protein 2 are the dominating lipases in neonatal fat digestion in mice and rats. Pediatr Res. 2007;62(5):537–41.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Bosner MS, Gulick T, Riley DJS, Spilburg CA, Lange LG. Heparin-modulated binding of pancreatic lipase and uptake of hydrolyzed triglycerides in the intestine. J Biol Chem. 1989;264(34):20261–4.

    CAS  PubMed  Google Scholar 

  36. Filippatos TD, Gazi IF, Liberopoulos EN, Athyros VG, Elisaf MS, Tselepis AD, et al. The effect of orlistat and fenofibrate, alone or in combination, on small dense LDL and lipoprotein-associated phospholipase A(2) in obese patients with metabolic syndrome. Atherosclerosis. 2007;193(2):428–37.

    Article  CAS  PubMed  Google Scholar 

  37. Mahan JT, Heda GD, Rao RH, Mansbach CM. The intestine expresses pancreatic triacylglycerol lipase: regulation by dietary lipid. Am J Physiolo-Gastrointest Liver Physiol. 2001;280(6):G1187–G96.

    CAS  Google Scholar 

  38. Zhi JG, Melia AT, Eggers H, Joly R, Patel IH. Review of limited systemic absorption of orlistat, a lipase inhibitor, in healthy-human volunteers. J Clin Pharmacol. 1995;35(11):1103–8.

    Article  CAS  PubMed  Google Scholar 

  39. Yen C-LE, Stone SJ, Koliwad S, Harris C, Farese Jr RV. DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res. 2008;49(11):2283–301.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. King AJ, Segreti JA, Larson KJ, Souers AJ, Kym PR, Reilly RM, et al. Diacylglycerol Acyltransferase 1 Inhibition Lowers Serum Triglycerides in the Zucker Fatty Rat and the Hyperlipidemic Hamster. J Pharmacol Exp Ther. 2009;330(2):526–31.

    Article  CAS  PubMed  Google Scholar 

  41. Imai T, Taketani M, Shii M, Hosokawa M, Chiba K. Substrate specificity of carboxylesterase isozymes and their contribution to hydrolase activity in human liver and small intestine. Drug Metab Dispos. 2006;34(10):1734–41.

    Article  CAS  PubMed  Google Scholar 

  42. Ho SY, Delgado L, Storch J. Monoacylglycerol metabolism in human intestinal Caco-2 cells - evidence for metabolic compartmentation and hydrolysis. J Biol Chem. 2002;277(3):1816–23.

    Article  CAS  PubMed  Google Scholar 

  43. Robertson MD, Parkes M, Warren BF, Ferguson DJP, Jackson KG, Jewell DP, et al. Mobilisation of enterocyte fat stores by oral glucose in humans. Gut. 2003;52(6):834–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Fielding BA, Callow J, Owen RM, Samra JS, Matthews DR, Frayn KN. Postprandial lipemia: the origin of an early peak studied by specific dietary fatty acid intake during sequential meals. Am J Clin Nutr. 1996;63(1):36–41.

    CAS  PubMed  Google Scholar 

  45. Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B, et al. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nat Genet. 2000;25(1):87–90.

    Article  CAS  PubMed  Google Scholar 

  46. Buhman KK, Smith SJ, Stone SJ, Repa JJ, Wong JS, Knapp FF, et al. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis. J Biol Chem. 2002;277(28):25474–9.

    Article  CAS  PubMed  Google Scholar 

  47. McFie PJ, Stone SL, Banman SL, Stone SJ. Topological orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation. J Biol Chem. 2010;285(48):37377–87.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Stone SJ, Levin MC, Farese Jr RV. Membrane topology and identification of key functional amino acid residues of murine Acyl-CoA: diacylglycerol acyltransferase-2. J Biol Chem. 2006;281(52):40273–82.

    Article  CAS  PubMed  Google Scholar 

  49. Lehner R, Kuksis A. Triacylglycerol synthesis by an sn-1,2(2,3)-diacylglycerol transacylase from rat intestinal microsomes. J Biol Chem. 1993;268(12):8781–6.

    CAS  PubMed  Google Scholar 

  50. Coleman RA, Mashek DG. Mammalian triacylglycerol metabolism: synthesis, lipolysis, and signaling. Chem Rev. 2011;111(10):6359–86.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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This work was financially supported by the National Health and Medical Research Council of Australia and the Australian Research Council. The authors thank Dr. David Shackleford for assistance in PK analysis and Ms Gracia for technical assistance during sample collection and analysis.

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Correspondence to Natalie L. Trevaskis or Christopher J. H. Porter.

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Han, S., Hu, L., Quach, T. et al. Profiling the Role of Deacylation-Reacylation in the Lymphatic Transport of a Triglyceride-Mimetic Prodrug. Pharm Res 32, 1830–1844 (2015).

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  • DGAT
  • lipase
  • lymphatic transport
  • prodrug
  • triglyceride mimetic