: Cytochrome P450 3A4 (CYP3A4), most abundantly presently both in the liver and upper intestinal enterocytes, limits the systemic bioavailability of anti-HIV agents. P-glycoprotein (P-gp), the MDR-1 gene product is also known to reduce the oral bioavailability of protease inhibitors. Moreover the existence of """"""""sanctuary"""""""" sites of HIV-1 may potentially endanger the efficacy of highly active antiretroviral therapy (HAART). High cellular expression of P-gp in brain capillary endothelial cells, P-gp and CYP3A4 in mature enterocytes and alveolar cells and their similar substrate specificity suggest that the function of these proteins may be complementary and may form a coordinated intestinal, blood-brain and pulmonary barrier. The purpose of this study is to classify all anti-AIDS drugs, currently used clinically, under a new proposed Absorption-Metabolism Classification System (AMCS). Under AMCS, four classes of drugs, A, B, C and D, will be proposed based on their specificity towards P-gp and/or CYP3A4. We propose to investigate the simultaneous P-gp mediated efflux and CYP3A4 mediated metabolism of the above mentioned 14 anti-AIDS compounds across l alpha, 2,5-di-OH vitamin D3 treated Caco-2 cell monolayers expressing high levels of P-gp and CYP3A4. Moreover, in some experiments, isolated hepatocytes containing liver CYP3A, representing first pass liver metabolism, wil1 also be included. Under AMCS we hypothesize that apparent order of oral bioavailability will be A > BEC > D. Our proposed classification system may prove to be an important tool in screening anti-AIDS compounds for their oral absorption potential. According to this hypothesis Class A compounds should not interact with any other class. There should be no interaction between compounds of class B and C, whereas Class B and C compounds are expected to interact with Class D compounds. Also, maximum interaction is anticipated within Class D compounds compared to interaction with Class B and Class C compound. Our proposed theoretical model may predict, a priori, the extent of drug absorption and more importantly drug interactions among four WCS classes. Therefore, the specific aims of this project are: 1) to develop an in vitro Caco-2 cell culture model expressing high levels of P-gp and CYP3A4 to a priori predict oral absorption potential and drug interactions among the four AMCS class drugs, to utilize the model in predicting first pass metabolism using human hepatocytes. Specific inhibitors of P-gp and CYP3A4 will be used to demonstrate the involvement of each of these proteins. In addition, the expression of these proteins will be determined by Western Blot and RT PCR. 2) to develop a quantitative model for oral drug absorption and interactions based on mathematical expression of passive influx, saturable efflux, and saturable CYP3A4 metabolism and to correlate model predicted effluxes with experimentally determined efflux values. This theoretical model will also predict the fluxes of each of the A, B, C, and D classes of drugs. The theoretical model will be first validated using model compound in each class: Tolbutamide - Class-A, Rhodamine 123 - Class-B, Carbamazepine - Class-C, and Cortisol - Class-D. 3) to study the transport and metabolism across two other in vitro cell culture models, bovine brain microvessels endothelial cell (BBMEC) and Calu-3 monolayers representing blood-brain and alveolar cells respectively. The brain parenchymal and alveolar lining - two sanctuary sites that are 'protected' from optimal antiretroviral drug access. 4) to study the effect of protein binding on the influx and efflux of model compounds, and anti-HIV drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM064320-01A2
Application #
6496490
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Okita, Richard T
Project Start
2002-04-01
Project End
2005-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$203,000
Indirect Cost
Name
University of Missouri Kansas City
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
800772162
City
Kansas City
State
MO
Country
United States
Zip Code
64110
Renukuntla, Jwala; Vadlapudi, Aswani Dutt; Patel, Ashaben et al. (2013) Approaches for enhancing oral bioavailability of peptides and proteins. Int J Pharm 447:75-93
Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt et al. (2012) Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism. Mol Pharm 9:1877-86
Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Mitra, Ashim K (2012) Sodium dependent multivitamin transporter (SMVT): a potential target for drug delivery. Curr Drug Targets 13:994-1003
Pal, Dhananjay; Kwatra, Deep; Minocha, Mukul et al. (2011) Efflux transporters- and cytochrome P-450-mediated interactions between drugs of abuse and antiretrovirals. Life Sci 88:959-71
Paturi, Durga Kalyani; Kwatra, Deep; Ananthula, Hari Krishna et al. (2010) Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3). Int J Pharm 384:32-8
Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J et al. (2010) Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids. Mol Pharm 7:412-20
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Luo, Shuanghui; Wang, Zhiying; Kansara, Viral et al. (2008) Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake. Int J Pharm 358:168-76
Jain, Ritesh; Duvvuri, Sridhar; Kansara, Viral et al. (2007) Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats. Int J Pharm 336:233-40
Agarwal, Sheetal; Pal, Dhananjay; Mitra, Ashim K (2007) Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor. Int J Pharm 339:139-47

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