Zidovudine and Ursodeoxycholic Acid Conjugation: Design of a New Prodrug Potentially Able To Bypass the Active Efflux Transport Systems of the Central Nervous System

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 compartments (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.

Development and characterization of PLGA nanoparticles as delivery systems of a prodrug of zidovudine obtained by its conjugation with ursodeoxycholic acid

“…The prodrug UDCA-AZT (solubility in water ¼ 0.0030 AE 0.0001 mg/mL À 4.75 AE 0.19 10 À6 M; solubility in the mixture water and methanol 70:30 v/v ¼ 0.058 AE 0.002 mg/ mL À 9.0 AE 0.3 10 À5 M) was synthesized as previously described (Dalpiaz et al, 2012). PLGA 50:50, Purasorb Õ PDLG 5004 (Mw $30 000 Da) was kindly gifted by Purac Biochem (The Netherlands).…”

“…The employment of methanol as co-solvent was necessary to increase the low solubility of the prodrug in water. In particular, the solubility value of UDCAÀAZT in water was 0.0030 AE 0.0001 mg/mL (4.75 AE 0.19 Â 10 À6 M), whereas in the mixture of water and methanol (70:30 v/v), it was 0.058 AE 0.002 mg/mL (9.0 AE 0.3 Â 10 À5 M) (Dalpiaz et al, 2012). For this reason, also the release studies of UDCA-AZT from the NPs were performed in the same water/methanol mixture (see below).…”

“…We have chosen the rat liver homogenates because the hydrolysis rate of the prodrug is relatively fast (Dalpiaz et al, 2012). The average recovery AE SD of UDCA-AZT and AZT from rat liver homogenates were 83.2 AE 2.9% and 85.5% AE 3.4%, respectively.…”

“…We have recently evidenced that the ester conjugation of AZT with UDCA allows obtaining a prodrug (UDCA-AZT; Figure 1) that is able to elude the AET systems (Dalpiaz et al, 2012). This prodrug appears, therefore, an interesting substrate for encapsulation studies in PLGA nanoparticulate systems potentially able to induce its delivery in specific sites of the body.…”

“…NPs were then characterized by evaluating their prodrug loading, their morphology and size and their ability to control the UDCA-AZT release. Finally, in the aim to verify the ability of the NP to stabilize the prodrug in physiological environments, we have considered that rat liver homogenates induce high instability of UDCA-AZT, causing its hydrolysis in few minutes (Dalpiaz et al, 2012); we have therefore employed rat liver homogenates to compare the hydrolysis rate of the prodrug in the free and encapsulated forms.…”