Mucosal Cell Transporters and Enzymes for Enhancing Oral Drug Absorption

Summary

Principal Investigator: Gordon L Amidon
Abstract: DESCRIPTION (provided by applicant): In this proposal we apply the novel mechanistic oral absorption strategy we have developed to the important category of polar antiviral drugs. Progress during the previous grant period has demonstrated that the transport and activation strategy can be remarkable successful. Further, we have identified the novel activating enzyme, Valacyclovirase and determined the X-ray structure of this novel enzyme that activates valacyclovir and valganciclovir. Most significantly, the X-ray structure and our studies to date with nucleoside prodrugs indicates that this enzyme has a broad leaving group pocket (drug side), indicating that VACVase maybe a versatile prodrug activating enzyme. In this proposal we focus on demonstrating that this mechanistic oral absorption strategy can improve the oral absorption of the very important antiviral drugs, zanamivir and guanidino oseltamivir carboxylate. We will develop a specific substrate and analogues to fully explore the specificty and enzymology of VACVase as a prodrug target. We show very exciting preliminary results that the prodrugs of zanamivir and a highly potent oseltamivir carboxylate analogue have more than 10 fold higher epithelial cell permeability than the parent drug and further that these prodrugs are activated by VACVase. The specific aims of this proposal are: 1. Synthesize a range of prodrugs of the antiviral drugs, zanamivir and guanidino oseltamivir carboxylate and the specific substrate, Val-HPG, varying the pro-group and the linker and evaluate both chemical and enzymatic stability. 2. Determine the mucosal transport and activation of the prodrugs, evaluate the uptake in HeLa cells, transport permeability and activation in Caco-2 monolayers, and in vivo in the in situ perfused rat jejunum. Select at least 4 prodrugs for in vivo plasma level determination and bioavailability and pharmacokinetic analysis. 3. Determine valacyclovirase hydrolysis and its potential utility as a oral prodrug activating target through kinetics studies of specific substrate analogues for VACVase and determine the structure activity relationship (SAR) of valacyclovirase (VACVase) with the antiviral prodrugs and the specific substrate analogues. In summary, we will develop and extend a mechanistic strategy for improving oral bioavailability through membrane transporter and activating enzyme targeting and apply this strategy to important antiviral drugs. Further, the mechanistic strategy developed in this proposal can be applied to significantly increase the range of therapeutic agents that can be delivered via the oral route of administration. PUBLIC HEALTH RELEVANCE: Enhancing the oral absorption of drug candidates will greatly increase the number and efficacy of drugs available to effectively treat disease. Many potential drugs cannot be used because of poor oral absorption. This project will develop a strategy based on current influenza drugs that is mechanistically based on oral transport and activation in the intestinal tract. This will enable the development of new, more effective, drugs for treatment of viral infections.
Funding Period: 1986-09-01 - 2014-05-30
more information: NIH RePORT

Top Publications

  1. ncbi Nucleoside ester prodrug substrate specificity of liver carboxylesterase
    Christopher P Landowski
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, 48109, USA
    J Pharmacol Exp Ther 316:572-80. 2006
  2. ncbi Potential of amino acid/dipeptide monoester prodrugs of floxuridine in facilitating enhanced delivery of active drug to interior sites of tumors: a two-tier monolayer in vitro study
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, Michigan 48109, USA
    Pharm Res 28:2575-88. 2011
  3. pmc Enhancing the intestinal membrane permeability of zanamivir: a carrier mediated prodrug approach
    Sheeba Varghese Gupta
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, United States
    Mol Pharm 8:2358-67. 2011
  4. pmc The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, MI 48109, USA
    Molecules 17:3672-89. 2012
  5. pmc The fraction dose absorbed, in humans, and high jejunal human permeability relationship
    Arik Dahan
    Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
    Mol Pharm 9:1847-51. 2012
  6. pmc In silico prediction of drug dissolution and absorption with variation in intestinal pH for BCS class II weak acid drugs: ibuprofen and ketoprofen
    Yasuhiro Tsume
    College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 1065, USA
    Biopharm Drug Dispos 33:366-77. 2012
  7. pmc Selection of suitable prodrug candidates for in vivo studies via in vitro studies; the correlation of prodrug stability in between cell culture homogenates and human tissue homogenates
    Yasuhiro Tsume
    College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    J Pharm Pharm Sci 15:433-46. 2012
  8. pmc Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue
    Deepak Gupta
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
    Mol Pharm 10:512-22. 2013
  9. pmc Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification
    Tuba Incecayir
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    Mol Pharm 10:958-66. 2013
  10. pmc The development of orally administrable gemcitabine prodrugs with D-enantiomer amino acids: enhanced membrane permeability and enzymatic stability
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, MI, USA
    Eur J Pharm Biopharm 86:514-23. 2014

Research Grants

Detail Information

Publications24

  1. ncbi Nucleoside ester prodrug substrate specificity of liver carboxylesterase
    Christopher P Landowski
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, 48109, USA
    J Pharmacol Exp Ther 316:572-80. 2006
    ..The low relative hydrolysis rates of isoleucyl, aspartyl, and lysyl prodrugs may facilitate prolonged systemic disposition of the nucleoside analogs for improved therapeutic action...
  2. ncbi Potential of amino acid/dipeptide monoester prodrugs of floxuridine in facilitating enhanced delivery of active drug to interior sites of tumors: a two-tier monolayer in vitro study
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, Michigan 48109, USA
    Pharm Res 28:2575-88. 2011
    ....
  3. pmc Enhancing the intestinal membrane permeability of zanamivir: a carrier mediated prodrug approach
    Sheeba Varghese Gupta
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, United States
    Mol Pharm 8:2358-67. 2011
    ....
  4. pmc The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, MI 48109, USA
    Molecules 17:3672-89. 2012
    ..Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs...
  5. pmc The fraction dose absorbed, in humans, and high jejunal human permeability relationship
    Arik Dahan
    Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
    Mol Pharm 9:1847-51. 2012
    ..In this situation, the use of multiple permeability experimental methods, including the use of metabolism, which except for luminal degradation requires absorption, is prudent and encouraged...
  6. pmc In silico prediction of drug dissolution and absorption with variation in intestinal pH for BCS class II weak acid drugs: ibuprofen and ketoprofen
    Yasuhiro Tsume
    College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 1065, USA
    Biopharm Drug Dispos 33:366-77. 2012
    ..Simulation software may be a very useful tool to aid the selection of dissolution media that may be useful in setting an in vitro bioequivalence dissolution standard...
  7. pmc Selection of suitable prodrug candidates for in vivo studies via in vitro studies; the correlation of prodrug stability in between cell culture homogenates and human tissue homogenates
    Yasuhiro Tsume
    College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    J Pharm Pharm Sci 15:433-46. 2012
    ..To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues...
  8. pmc Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue
    Deepak Gupta
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
    Mol Pharm 10:512-22. 2013
    ....
  9. pmc Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification
    Tuba Incecayir
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    Mol Pharm 10:958-66. 2013
    ..However, the concentration-dependent permeability of labetalol in mice due to P-gp and the inhibition study with verapamil in Caco-2 cells indicated that labetalol is not an ideal reference standard for BCS classification...
  10. pmc The development of orally administrable gemcitabine prodrugs with D-enantiomer amino acids: enhanced membrane permeability and enzymatic stability
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, MI, USA
    Eur J Pharm Biopharm 86:514-23. 2014
    ....
  11. pmc The achievement of mass balance by simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil, 5-dihydrouracil, α-fluoro-β-ureidopropionate, α-fluoro-β-alanine using LC-MS
    Yasuhiro Tsume
    Department of Pharmaceutical Science, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 1065, USA
    J Chromatogr B Analyt Technol Biomed Life Sci 879:915-20. 2011
    ..6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies...
  12. pmc Specificity of a prodrug-activating enzyme hVACVase: the leaving group effect
    Jing Sun
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 1065, United States
    Mol Pharm 7:2362-8. 2010
    ..It is proposed that hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate toward amide prodrugs...
  13. ncbi N-methylpurine DNA glycosylase and 8-oxoguanine dna glycosylase metabolize the antiviral nucleoside 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole
    Philip L Lorenzi
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor MI 48109 1065, USA
    Drug Metab Dispos 34:1070-7. 2006
    ..Thus, in silico observations were in perfect agreement with experimental observations. These findings implicate DNA glycosylases in drug metabolism...
  14. ncbi A provisional biopharmaceutical classification of the top 200 oral drug products in the United States, Great Britain, Spain, and Japan
    Toshihide Takagi
    College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    Mol Pharm 3:631-43. 2006
    ....
  15. ncbi Proline prodrug of melphalan targeted to prolidase, a prodrug activating enzyme overexpressed in melanoma
    Sachin Mittal
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
    Pharm Res 24:1290-8. 2007
    ....
  16. ncbi Proline prodrug of melphalan, prophalan-L, demonstrates high therapeutic index in a murine melanoma model
    Sachin Mittal
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, An Arbor, MI 48109 1065, USA
    Eur J Pharm Biopharm 67:752-8. 2007
    ..An excellent correlation between GI50 and tumor reduction efficacy was observed for the tested drugs (r2=0.95). Prophalan-L thus demonstrates higher therapeutic index than melphalan in the murine melanoma model...
  17. pmc Molecular basis of prodrug activation by human valacyclovirase, an alpha-amino acid ester hydrolase
    Longsheng Lai
    Department of Pharmaceutical Sciences and Center for Molecular Drug Targeting, University of Michigan, Ann Arbor, MI 48109 1065, USA
    J Biol Chem 283:9318-27. 2008
    ..Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies...
  18. pmc Enhanced cancer cell growth inhibition by dipeptide prodrugs of floxuridine: increased transporter affinity and metabolic stability
    Yasuhiro Tsume
    Department of Pharmaceutical Science, University of Michigan, Ann Arbor, Michigan 48109, USA
    Mol Pharm 5:717-27. 2008
    ....
  19. ncbi Enhanced absorption and growth inhibition with amino acid monoester prodrugs of floxuridine by targeting hPEPT1 transporters
    Yasuhiro Tsume
    Department of Pharmaceutical Science, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109 1065, USA
    Molecules 13:1441-54. 2008
    ..This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy...
  20. ncbi Interferon-gamma increases expression of the di/tri-peptide transporter, h-PEPT1, and dipeptide transport in cultured human intestinal monolayers
    David R Foster
    Department of Pharmacy Practice, Purdue University School of Pharmacy and Pharmaceutical Sciences, West Lafayette, IN, United States
    Pharmacol Res 59:215-20. 2009
    ..These findings imply that intestinal absorption of peptides and peptidomimetic drugs may be increased in certain inflammatory conditions...
  21. pmc Toward an in vivo dissolution methodology: a comparison of phosphate and bicarbonate buffers
    Jennifer J Sheng
    College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 1065, USA
    Mol Pharm 6:29-39. 2009
    ....
  22. pmc Enhancing the intestinal absorption of molecules containing the polar guanidino functionality: a double-targeted prodrug approach
    Jing Sun
    College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 1065, USA
    J Med Chem 53:624-32. 2010
    ..In conclusion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino analogues via targeting hPEPT1 for transport and hVACVase for activation...
  23. pmc The Biopharmaceutics Classification System: subclasses for in vivo predictive dissolution (IPD) methodology and IVIVC
    Yasuhiro Tsume
    College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 1065, United States
    Eur J Pharm Sci 57:152-63. 2014
    ....

Research Grants30

  1. Prodrugs of Neuraminidase Inhibitors for Increased Oral Bioavailability
    John M Hilfinger; Fiscal Year: 2013
    ..Ultimately, this approach may increase the number of potent anti-virus compounds that are available for therapeutic and prophylactic treatment of influenza. ..