Decomposition Pathways of the Mono- and Bis(Pivaloyloxymethyl) Esters of Azidothymidine 5′-Monophosphate in Cell Extract and in Tissue Culture Medium: An Application of the ‘on-line ISRP-Cleaning’ HPLC Technique

Abstract

Bis(pivaloyloxymethyl) azidothymidine 5'-monophosphate (piv 2-AZTMP) was designed as a cell membrane-permeable precursor of AZTMP. We have reported previously that when incubated with CEM cells deficient in thymidine kinase, piv 2-AZTMP gives rise to intracellular AZTMP and the corresponding diphosphate (AZTDP) and triphosphate (AZTTP). Under similar conditions, no intracellular nucleotides were formed with AZT. However, the mechanism bỹ ich piv 2-AZTMP is converted to AZTMP has not been established. To address this question, we have used the recently developed 'on-line ISRP-cleaning' HPLC technique to investigate the stability and metabolic fate of piv 2-AZTMP (1) in RPMI 1640 medium, (2) in RPMI containing 10% heat-inactivated fetal calf serum, and (3) in CEM cell extracts. Similar studies were conducted starting with mono(pivaloyloxymethyl) azidothymidine 5'-monophosphate (piv 1-AZTMP).From the kinetics of these reactions, it appears that piv 2-AZTMP is slowly hydrolyzed to piv 1-AZTMP in RPMI and that the metabolic sequence in cell extract and in tissue culture medium is clearly: piv 2-AZTMP~piv1-AZTMP~AZTMP~AZT. The rate constants are quite different in these three media. Although it is evident that the first step in the metabolism of piv 2-AZTMP is catalysed by carboxy-. *For correspondence. Tel. +67545873; Fax. +67 0420 29. late esterase, the enzyme(s) responsible for the second step, piv1-AZTMP~AZTMP, is less apparent, as carboxylate esterases and/or phosphodiesterases can be taken in account. However, analysis of the kinetic data strongly suggests that carboxylate esterase does not playa significant role and that this second step is mediated by phosphodiesterases. Collectively, these studies demonstrate, that piv 2-AZTMP is an effective prodrug of AZTMP. They also establish that piv1-AZTMP is an intermediate in this process, and define the sequence of the overall metabolic reaction. With this increased understanding of the metabolism of piv 2-AZTMP, it should be possible rationally to design analogues with optimal structural and pharmacological properties for use in vivo.

Comparison of Cytotoxicity of Mononucleoside Phosphotriester Derivatives Bearing Biolabile Phosphate Protecting Groups in Normal Human Bone Marrow Progenitor Cells

“…Consequently, a mononucleoside phosphotriester derivative of AZT cannot exhibit a higher in-vitro anti-HIV activity than that of AZT in cells in which the cytosolic thymidine kinase activity is expressed such as PHA-stimulated PBMCs, thereby explaining the similar inhibiting capacity of the bis(MeSATE) ester 1 and the bis(POM) ester 3 derivatives as AZT in PBMCs. The slight decrease in anti-HIV-1 activity observed with these two pronuc1eotides may be related to a faster intracellular accumulation of AZTMP by the parent drug (Pompon et al, 1994;Lefebvre et al, 1995). It was demonstrated that an accumulation of this metabolite causes a potent inhibition of thymidylate kinase resulting in a blockage of the subsequent phosphorylation to 3'-azido-2',3'-deoxythymidine 5'-diphosphate (AZTOP) (Furman et al, 1986;Harrington et el., 1987;Frick et al, 1988).…”

“…) involved, in the first step, an enzyme-mediated activation process leading to new phosphotriesters which were chemically unstable and which decompose spontaneously to afford the phosphodiester intermediates. In the second step, these phosphodiesters will then be converted into AZTMP following a similar mechanism and/or by action of phosphodiesterases (Puech et el., 1993;Pompon et al, 1994;Lefebvre et el., 1995). The hydrolysis of the pronucleotides 1-3 result in the formation of the 5'-mononucleotide and also small organic molecules as byproducts (Fig.…”

“…1. ) which incorporate the S-(2-hydroxyethylsulphidyl)-2-thioethyl (OTE) and pivaloyloxymethyl (PaM) (Pompon et al, 1994) groups as other biolabile phosphate protections, respectively (Table 1).…”

Selective Intracellular Activation of a Novel Prodrug of the Human Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir Leads to Preferential Distribution and Accumulation in Lymphatic Tissue

“…MT-2 cell extract was prepared from MT-2 cells according to a previously published procedure (21). Extract (80 l) was transferred into a screw-cap centrifuge tube and incubated at 37°C for 5 min.…”

Improved antiviral activity of the aryloxymethoxyalaninyl phosphoramidate (APA) prodrug of abacavir (ABC) is due to the formation of markedly increased carbovir 5′‐triphosphate metabolite levels

“…Examples of a relatively poor conversion rate by the activating enzyme other than ABC are stavudine (d4T) by thymidine kinase (TK) [7,8], zalcitabine (ddC) by 2 0 -deoxycytidine kinase [9,10], and didanosine (ddI) by 5 0 -nucleotidase [4,5]. Therefore, several attempts have been undertaken to deliver activated (phosphorylated) nucleoside analogues directly into the virusinfected target cells [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. One of these approaches has proven very successful when applied on thymidine analogues such as d4T.…”