Zidovudine and Ursodeoxycholic Acid Conjugation: Design of a New Prodrug Potentially Able To Bypass the Active Efflux Transport Systems of the Central Nervous System Alessandro Dalpiaz, † Guglielmo Paganetto, ‡ Barbara Pavan,* ,‡ Marco Fogagnolo, § Alessandro Medici, ‡ Sarah Beggiato, ∥ and Daniela Perrone ‡ † Department of Pharmaceutical Sciences, ‡ Department of Biology, § Department of Chemistry, and ∥ Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy ABSTRACT: We have synthesized a new prodrug obtained by the 5′-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA−AZT is quickly hydrolyzed in rat plasma and whole blood (half-life <10 s). The same compound was hydrolyzed with slower rates in human plasma (half-life =7.53 ± 0.44 h) and whole blood (half- life =3.71 ± 0.16 h), allowing to control the AZT release. UDCA−AZT appeared hydrolyzed also in rat brain (half-life = 7.24 ± 0.45 min) and liver homogenates (half-life = 2.70 ± 0.14 min). In the aim to study the permeation properties of the UDCA−AZT across physiological barriers, we have used an established human retinal pigment epithelium (HRPE) cell line to obtain a polarized cell monolayer showing epithelial features. The bidirectional permeation of 30 μM AZT across this monolayer was regulated by apparent permeability coefficients (P E ) higher from the apical to basolateral compart- ments (P E = 209 ± 4 × 10 −5 cm/min) than in the opposite way (P E = 133 ± 8 × 10 −5 cm/min), in conformity with the in vivo behavior of AZT, actively effluxed from the CNS. The influx (P E = 39.1 ± 1.2 × 10 −5 cm/min) and efflux (P E = 31.3 ± 3.6 × 10 −5 cm/min) permeability coefficients of 30 μM UDCA− AZT were instead the same, suggesting the ability of the prodrug to avoid the AET systems and, potentially, to allow its accumulation in the CNS. The relatively low P E values of UDCA−AZT were associated with a partial hydrolysis during its permeation across the cell monolayer. KEYWORDS: zidovudine, ursodeoxycholic acid, prodrug, hydrolysis, human blood, brain homogenate, liver homogenate, HRPE cells, polarized cell monolayer, efflux studies ■ INTRODUCTION 3′-Azido-3′-deoxythymidine (zidovudine or AZT) is the first drug approved by FDA for the treatment of acquired immunodeficiency syndrome (AIDS) which is caused by infection with the human immunodeficiency virus (HIV). 1 AZT is a nucleoside reverse transcriptase inhibitor (NRTI), and it can be employed in therapeutic protocols that make use of multiple-drug combinations including NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs), and non-nucleoside reverse transcriptase inhibitors (NNRTIs). 1−3 AZT is also recognized as an effective agent against hepatitis, and it has been considered for the treatment of some cancers. 4,5 Despite its clinical relevance, several important unwanted effects and limitations are related to AZT therapies. In particular, this drug has a relatively short half-life in vivo (about 1 h); 6 as a consequence relatively high amounts of AZT must be administered frequently inducing strong side effects that are dose-dependent on bone marrow and lead to severe anemia and granulocytopenia. 7,8 Moreover, the development of systems for effective cytoplasmatic delivery of AZT should be necessary to increase its efficacy against intracellular liver infections. 4 Finally, AZT has poor ability to reach the brain from the bloodstream. 9 Unfortunately, HIV is able to easily invade the central nervous system (CNS), where it induces neurological disorders ranging from mild cognitive impairments to the severe AIDS dementia complex. 10,11 Even if anti-HIV agents can be efficacious in periphery, they are in general unable to reach the CNS that, therefore, becomes a sanctuary of the virus from which the periphery can be continuously reinfected. 12 Delivery of anti-HIV agents to viral sanctuary sites, such as the CNS, constitutes an essential step for viral Received: November 7, 2011 Revised: February 7, 2012 Accepted: February 22, 2012 Published: February 22, 2012 Article pubs.acs.org/molecularpharmaceutics © 2012 American Chemical Society 957 dx.doi.org/10.1021/mp200565g | Mol. Pharmaceutics 2012, 9, 957−968