Optimization of the Local Inhibition of Intestinal Adenosine Deaminase (ADA) by eryfftro-9-(2-Hydroxy-3-nonyl)adenine: Enhanced Oral Delivery of an ADA-Activated Prodrug for Anti-HIV Therapy

Abstract

Previous in situ perfusion studies in rat ileal segments have demonstrated that high concentrations (>40 microg/mL) of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a semitight binding inhibitor of adenosine deaminase (ADA), are effective in completely inhibiting the intestinal metabolism of 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), an ADA activated prodrug of the anti-HIV agent 2', 3'-dideoxyinosine (ddI) designed for improved targeting to the central nervous system. However, the intestinal absorption of EHNA results in complete inhibition of the ADA activity in the mesenteric blood draining the isolated intestinal segment being perfused and may lead to complete inhibition of ADA present in the systemic circulation and other sites, an unacceptable outcome since bioconversion in the target tissue is required for prodrug efficacy. This study examines the feasibility of locally inhibiting ADA present in the intestinal wall using EHNA to increase the intestinal absorption of 6-Cl-ddP. Transport experiments conducted in isolated ileal segments from mesenteric cannulated rats using perfusate containing prodrug and various concentrations of EHNA demonstrated that a 0.1 microg/mL logarithmic mean lumenal concentration of EHNA was effective in increasing the intestinal bioavailability of Cl-ddP to > 90%. Intestinal uptake parameters for EHNA and pharmacokinetic parameters generated in vivo in chronically catheterized rats given intravenous infusions ranging from 12.5 to 310 microg/kg/min were used to demonstrate that <10% of systemic ADA would be inhibited at steady state using the optimal perfusate concentration of EHNA. Thus, in continuous perfusions it is possible to increase the intestinal bioavailability of 6-Cl-ddP to >90% with minimal (<10%) inhibition of systemic ADA. Local inhibition of enzymes may be an effective strategy to increase the oral bioavailability of tissue enzyme-activated prodrugs or other drugs which may also be substrates for intestinal enzymes.

Absorption and Intestinal Metabolism of Purine Dideoxynucleosides and an Adenosine Deaminase-Activated Prodrug of 2′,3′-Dideoxyinosine in the Mesenteric Vein Cannulated Rat Ileum

“…In this study, partial inhibition was obtained with DCF but full inhibition was obtained with EHNA. Further, the apparent K i of ADA present in the intestinal epithelium was determined in in situ perfusions in mesenteric cannulated rat ileal segments to be <5 nM using EHNA, 61 which strongly suggests that the ADA responsible for bioconversion of 6-Cl-ddP in the intestinal wall is ADA 1 . This is also consistent with the fact that ADA 1 constitutes 90-100% of the total ADA which is present intracellularly.…”

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells

“…To corroborate this conclusion, we next examined the DNA incorporation of [ 15 N 5 ]-dA using the Ada inhibitor. By treating 293T cells with an Ada inhibitor erythro -9-(2-hydroxy-3-nonyl)­adenine (EHNA), , the signals of [ 15 N 4 ]-dA and [ 15 N 4 ]-dG in genomic DNA decreased by 75.0%–96.4% and 75.4%–96.8% (Figure B), respectively. Meanwhile, we observed the presence of [ 15 N 5 ]-dA in genomic DNA, but the signal of [ 15 N 5 ]-dA is less than 5% of that for dA.…”

5′-O-D-ValylaraA, A Potential Prodrug for Improving Oral Bioavailability of the Antiviral Agent Vidarabine

“…At this low level (1 uM), the 2′-deoxycoformycin alone had no effect on virus replication; however, with a Ki of ~50 nM as an ADA inhibitor, its effect is likely at the level of adenosine deaminase. [20] The D-valyl ester prodrugs of vidarabine showed EC50 values comparable to vidarabine alone. It is unclear at this stage whether the antiviral activity of the prodrugs resulted from the prodrug itself or vidarabine derived by the hydrolysis of the amino acid ester moiety.…”

“…Significant levels of this enzyme are found in the intestinal mucosa,[20,21] which limits the effectiveness of oral vidarabine delivery. Previous studies suggested that 5′-hydroxy substituted vidarabine analogs were resistant to adenosine deaminase.…”