Enzymatic Assay for Measurement of Intracellular DXG Triphosphate Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1-Infected Patients

Abstract

DXG {[2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one} and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2,3-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Current therapy for the treatment of human immunodeficiency virus (HIV) infection involves the use of two 2Ј,3Ј-dideoxynucleoside (ddN) reverse transcriptase (RT) inhibitors in combination with nonnucleoside RT inhibitors and protease inhibitors. However, continued use of these compounds can lead to the development of resistant virus as well as decreased tolerability. Therefore, the production of novel agents that possess drug-resistant viral activity while incorporating favorable toxicity profiles is justified.DXG {[2R-cis]-2-amino-1,9-dihydro-9-(2-[hydroxymethyl)-1,3-dioxolan-4-yl]-6H-purin-6-one} and its prodrug, DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir), are novel ddNs displaying activity against HIV type 1 (HIV-1) (8,9,14,22). The chemical structures of these two compounds are shown in Fig. 1. The use of DAPD (and DXG) in antiretroviral therapy is advantageous, because cross-resistance with other ddNs is minimal. For example, studies have shown that zidovudine (ZDV)-resistant virus remained sensitive to DAPD and DXG, whereas both compounds are only marginally less potent against lamivudine (3TC)-resistant virus (3, 9). Furthermore, DAPD and DXG display synergy with other antiretroviral agents and have favorable safety profiles (8, 9), suggesting that these compounds are good candidates in salvage therapy regimens.DAPD is deaminated to DXG by the ubiquitous enzyme adenosine deaminase (18), which is then sequentially phosphorylated by intracellular kinases to its active anabolite, DXG-5Ј-triphosphate (DXGTP). In vitro studies have shown that DXGTP is much more potent than DAPDTP against HIV-1 RT (K i ϭ 0.019 M for DXGTP and 250 M for DAPDTP)

Effect of Ribavirin on Intracellular and Plasma Pharmacokinetics of Nucleoside Reverse Transcriptase Inhibitors in Patients with Human Immunodeficiency Virus-Hepatitis C Virus Coinfection: Results of a Randomized Clinical Study

“…Nevertheless, these findings are not unexpected and are consistent with data from other studies of the NRTIs by several research groups using different methodologies. Large interpatient variation in both intracellular phosphorylation and plasma exposure of NRTIs have been reported previously (4,17,24,28,31,33,34,38). In addition, wide variation also exists in the intracellular concentrations of endogenous nucleoside triphosphates (16,17,24,37).…”

“…The study design used a parallel comparison between the patients receiving peginterferon alfa-2a (40KD) plus RBV and patients receiving peginterferon alfa-2a (40KD) plus placebo. However, as it is known that the concentrations of intracellular NRTI phosphates are highly variable (4,17,24,28,31,33,34,38), baseline values prior to the study or placebo administration were also collected for each of the arms (to measure the differences in the two arms at baseline). Furthermore, this design was chosen as previous data have suggested that intracellular NRTI phosphate levels change longitudinally with time (17).…”

Lack of Pharmacokinetic Interaction between Amdoxovir and Reduced- and Standard-Dose Zidovudine in HIV-1-Infected Individuals

“…The decay half-life of the active metabolite DXG-TP was ϳ16 h in activated primary human lymphocytes and ϳ9 h (or 27 h if including a 48-h time point) in humans, suggesting that twice a day (b.i.d.) dosing should provide adequate therapeutic coverage (26,30). Cellular toxicity studies suggested that AMDX and DXG did not affect the levels of mitochondrial DNA in human hepatoma cells (HepG2 cells) treated for 14 days at 10 M, and there was no increase in lactic acid production in these cells (9).…”

Practical considerations for developing nucleoside reverse transcriptase inhibitors

“…Drug resistant HIV mutants susceptible to DXG in vitro include viruses containing M184V/I and thymidine analog mutations (TAMs: M41L, D67N, K70R, L210W, T215Y/F, K219Q/E), and the 69SS double insert [82]. The t 1/2 of DXG-TP was ~16 hr in activated primary human lymphocytes and ~9 hr (or 27 hr including a questionable 48 hr time point) in humans, suggesting that twice a day (bid) dosing should provide adequate therapeutic coverage [83]. AMDX and DXG did not affect the levels of mtDNA in human hepatoma cells (HepG2) treated for 14 days at 10 μM, and there was no increase in lactic acid production in these cells [84].…”