Since its introduction in 1996, highly active antiretroviral therapy (HAART) has been credited with a marked and sustained reduction in AIDS-related death and disease in the United States. As with any pharmacotherapeutic agent, drug delivery to the site of action is absolutely required to elicit a favorable response. A significant obstacle to complete eradication of the HIV virus from the patient's body is the existence of biological barriers creating sanctuaries where the virus remains free from drug exposure. Two such barriers exist, ensuring the virus'survival and possible development of a resistant phenotype: the blood- brain barrier (BBB) and the blood-testis barrier (BTB). While there are no current methods that fully recapitulate the BBB to study the transport of drugs, we have developed a novel means of studying the intact BTB. The BTB protects the developing germ cells from the effects of potentially disruptive chemical interactions as well as immunological influences. Although this normal, protective function of the BTB is physiologically beneficial for reproduction, it can also provide a sanctuary for the HIV virus during antiretroviral therapy. This sanctuary site for HIV is particularly significant because the presence of HIV virus in the ejaculate of infected men is the major means of transmission to uninfected persons. Therefore, even while a patient is being effectively treated with a cocktail of antiretroviral drugs, the selective exclusion of the drugs at the BTB increases the viral load of the ejaculate and potential for transmission. Indeed, protease inhibitors and non-nucleoside reverse transcriptase inhibitors are actively excluded at the BTB and do not reach therapeutic concentrations in the testis. Alternatively, nucleoside analog drugs (NSAs) offer a unique opportunity to examine the characteristics of the BTB as well as the possibility of utilizing endogenous transporters as a means of circumventing the BTB since a number of NSAs are capable of accumulating in the semen of treated men. As the major component of the blood-testis barrier, Sertoli cells separate the adluminal compartment of the seminiferous tubules (STs) from the rest of the body by the formation of tight junctions below developing germ cells. In order to reach the adluminal space, drugs must first avoid barrier-function transporters before subsequently passing through Sertoli cells either by passive diffusion or carrier-mediated transport. Inward-facing transporter proteins, located at the basolateral and luminal membranes, allow specific compounds that cannot pass by passive diffusion to cross the BTB via transepithelial secretion. Selective movement of compounds across Sertoli cells therefore comprises the physiologic or functional aspect of the BTB. Therefore, we hypothesize that specific transporters are present at the basolateral and luminal membranes of Sertoli cells that allow specific NSA drugs to penetrate the BTB. The following aims have been designed to test this hypothesis: 1. Determine the constitutive expression and cellular localization of the major drug transporter proteins at the blood-testis barrier, as well as penetration of NSAs and PIs into the testis. 2. Determine the ability of selected inward-facing transporters (particularly Ent1, Ent2, Mate1 and Mate2) to support transepithelial secretion of NSAs and PIs. 3. Determine the functional capacity and molecular specificity of isolated Sertoli cells and intact STs to transport NSAs (didanosine, azidothymidine, abacavir, emtricitabine, tenofovir) and PIs (lopinavir and indinavir).The major emphasis of the current proposal is to focus on mechanisms by which therapeutics can successfully utilize endogenous transepithelial transporters to accumulate in the adluminal compartment, even within the context of the functional physiologic barrier.

Public Health Relevance

Even when effectively managed by antiretroviral therapy, patients can transmit HIV virus because the blood-testis barrier provides a sanctuary where the virus can hide from the drugs. This project seeks to determine the molecular mechanisms whereby specific nucleoside analog drugs can cross the blood-testis barrier and the possibility of utilizing endogenous transporter systems as a means of circumventing the blood-testis barrier. This mechanism could be utilized in the development of new drugs to reduce the risk of transmission by infected patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083927-03
Application #
8278026
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Zhang, Hao
Project Start
2010-06-17
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$374,963
Indirect Cost
$127,463
Name
University of Arizona
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Merrell, Matthew D; Nyagode, Beatrice A; Clarke, John D et al. (2014) Selective and cytokine-dependent regulation of hepatic transporters and bile acid homeostasis during infectious colitis in mice. Drug Metab Dispos 42:596-602
Kyriakides, Michael; Hardwick, Rhiannon N; Jin, Zhaosheng et al. (2014) Systems level metabolic phenotype of methotrexate administration in the context of non-alcoholic steatohepatitis in the rat. Toxicol Sci 142:105-16
Klein, David M; Wright, Stephen H; Cherrington, Nathan J (2014) Localization of multidrug resistance-associated proteins along the blood-testis barrier in rat, macaque, and human testis. Drug Metab Dispos 42:89-93
Canet, Mark J; Hardwick, Rhiannon N; Lake, April D et al. (2014) Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models. Drug Metab Dispos 42:586-95
Canet, Mark J; Cherrington, Nathan J (2014) Drug disposition alterations in liver disease: extrahepatic effects in cholestasis and nonalcoholic steatohepatitis. Expert Opin Drug Metab Toxicol 10:1209-19
Clarke, John D; Novak, Petr; Lake, April D et al. (2014) Characterization of hepatocellular carcinoma related genes and metabolites in human nonalcoholic fatty liver disease. Dig Dis Sci 59:365-74
Clarke, John D; Hardwick, Rhiannon N; Lake, April D et al. (2014) Experimental nonalcoholic steatohepatitis increases exposure to simvastatin hydroxy acid by decreasing hepatic organic anion transporting polypeptide expression. J Pharmacol Exp Ther 348:452-8
Clarke, John D; Hardwick, Rhiannon N; Lake, April D et al. (2014) Synergistic interaction between genetics and disease on pravastatin disposition. J Hepatol 61:139-47
Hardwick, Rhiannon N; Clarke, John D; Lake, April D et al. (2014) Increased susceptibility to methotrexate-induced toxicity in nonalcoholic steatohepatitis. Toxicol Sci 142:45-55
Clarke, John D; Sharapova, Tatiana; Lake, April D et al. (2014) Circulating microRNA 122 in the methionine and choline-deficient mouse model of non-alcoholic steatohepatitis. J Appl Toxicol 34:726-32

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