Anti-HIV and Anti-HBV Activity and Resistance Profile of 2′,3′-Dideoxy-3′-Thiacytidine (3TC) and Its Arylphosphoramidate Derivative CF 1109

Abstract

A novel membrane-soluble prodrug of the 5-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2-deoxycytidine kinase-deficient CEM/dCK 0 cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration Ç 0.02 mM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells. ᭧ 1996 Academic Press, Inc.The most recently approved 2,3-dideoxynucleoside (ddN) derivative for treatment of HIV infections is 2,3-dideoxy-3-thiacytidine (3TC, lamivudine) (Fig. 1). This drug is also subject of clinical trials in human hepatitis B virus (HBV)-infected patients. In contrast with the other approved (b-D) ddN derivatives has 3TC the b-L(0) isomeric conformation. 3TC needs to be converted to its 5-triphosphate derivative by cellular enzymes to be inhibitory to the reverse transcriptase (RT) of HIV, and is endowed with low if any cytotoxicity (1-5). However, the first activation (phosphorylation) step is catalysed by cytosolic 2-deoxycytidine kinase (dCK) which has a relatively poor affinity for 3TC (K i /K m Ç 50) resulting in a moderate efficiency of conversion of 3TC to its 5-monophosphate derivative (6). In addition, a potential drawback of 3TC is the rapid emergence of drug-resistant HIV-1 strains (7-11). Indeed, mutant virus strains that emerged in the presence of 3TC were shown to contain an amino acid change from methionine (Met) to valine (Val) or isoleucine (Ile) at position 184 of the RT. Such virus is highly resistant to the inhibitory effects of 3TC in cell culture (7-11), and is reported to show low-level cross-resistance to 2,3-dideoxycytidine (ddC) (Fig. 1) and 2,3-dideoxyinosine (ddI).In an attempt to circumvent the first intracellular phosphorylation step of 3TC, we synthesized the arylphosphoramidate derivative of 3TC containing a methylester of L-alanine linked to the 5-phosphate of 3TC-MP through its primary amino group (Fig. 1). The corresponding 2,3-didehydro-2,3-dideoxythymidine (d4T, stavudine) analogue (So324) was previously shown to be able to deliver directly the 5-monophosphate derivative of d4T into intact cells and to efficiently bypass the first (thymidine kinase-directed) phosphorylation of d4T (12,13). The antiviral potency and resistance profile of Cf 1109 is described compared to 3TC.

Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy

“…However, the emergence of lamivudine-resistant HBV may limit the efficacy of lamivudine monotherapy in some patients. [23][24][25][26][27][28][29][30] To date, YMDD mutant have not been detected in lamivudine-naive patients. In the present study, we attempted to detect YMDD mutant before the development of HBV-DNA breakthrough.…”

“…[11][12][13][14][15][16][17][18][19][20][21][22] However, the emergence of lamivudineresistant HBV strains was initially noticed in patients who received orthotopic liver transplantation and immunosuppressive therapy. [23][24][25][26][27][28] Such resistant viruses show a characteristic mutation of the 550th amino acid methionine in the YMDD motif of DNA polymerase to isoleucine (YIDD mutant) or valine (YVDD mutant). Honkoop et al 29 recently showed the emergence of such mutant viruses in 5 of 14 (36%) immunocompetent patients who were treated with lamivudine over 26 weeks.…”

Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy

“…10 The emergence of lamivudine-resistant mutants, however, casts a new challenge for such strategy. [11][12][13][14][15][16][17][18][19][20][21] The resistant viruses were initially noticed in patients under immunosuppressive therapy but were later found in immunocompetent patients. Although such mutants were initially considered replication defective, 22 we have recently shown that acute exacerbations occurred frequently and hepatic decompensation may develop in patients with lamivudine-resistant mutants during long-term lamivudine treatment.…”

“…12 The methionine at codon 552 was either replaced by an isoleucine (M552I) or a valine (M552V). [13][14][15][16][17] In addition, the M552V mutation was frequently accompanied by a leucine 528-to-methionine (L528M) substitution.…”

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.…”