Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine

Abstract

Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC r ) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV.Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC r RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC r substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC r background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC r substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC r HBV background, leading to reduced ETV susceptibility and treatment failure.Nearly 400 million people are chronically infected with hepatitis B virus (HBV) worldwide (16,19). After prolonged infections, often lasting decades, patients frequently develop severe liver disease that can lead to cirrhosis and hepatocellular carcinoma. Chronically infected patients also serve as sources of HBV transmission. There are currently three approved therapies for chronic HBV infections: interferon, lamivudine (3TC; ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine), and adefovir-dipivoxil, the prodrug of adefovir [ADV; 9-(2-phosphonylmethoxyethyl)adenine] (18). Interferon is administered subcutaneously and is associated with numerous adverse events, some of which can be severe, and a sustained antiviral response in only 30 to 40% of treated patients (30). 3TC treatment, administered orally, is effective in reducing viral loads but results in the frequent emergence of drug-resistant HBV due to substitutions at the Tyr-Met-Asp-Asp (YMDD) nucleotide binding site motif of viral DNA polymerase. Data from four large clinical trials revealed 3TC resistance (3TC r ) mutations in 24, 42, 53, and 70% of patients after 1, 2, 3, and 4 years of therapy, respectively (15). Treatment with ADV, recently approved by the U.S. Food and Drug Administration,...

Entecavir for the treatment of lamivudine-refractory chronic hepatitis B patients in China

“…None of these 13 patients experienced an ALT flare in association with the virologic breakthrough. Previous studies have shown that among patients harboring lamivudine-resistance mutations rtL180M and rtM204V, an additional substitution at residue rtT184, rtS202, or rtM250 may be associated with reduced susceptibility to entecavir [25]. Genotypic analyses on paired baseline and on-treatment samples from the 13 patients in the current study found no emerging substitutions at residues rtT184, rtS202, or rtM250, suggesting that the observed virologic breakthroughs were not due to the emergence of genotypic resistance to entecavir.…”

“…Entecavir's potent suppression of viral replication and high genetic barrier helps to minimize the emergence of virologic resistance. Tenney and coworkers have shown that resistance to entecavir requires the presence of lamivudine-resistance mutations rtL180M and/or rtM204V plus additional substitution in the HBV reverse transcriptase at positions rtT184, rtS202, or rtM250 [25]. In this study, there was no evidence of emerging substitutions associated with virologic resistance to entecavir.…”

“…1 log 10 copies/ml above the patient's nadir on entecavir therapy as determined by two sequential HBV DNA measurements by PCR assay at least 2 weeks apart or last on-treatment measurement. HBV DNA was extracted from patient samples, PCR amplified, and amino acids 1-344 of the reverse transcriptase sequenced as described elsewhere [25].…”

Resistance mutations of hepatitis B virus in entecavir‐refractory patients

“…To date, a limited number of ETV‐resistant mutations have been reported. They include the LAM‐resistant‐associated amino acid mutations at rt180 and rt204 and require additional mutations at rt169, rt184, rt202, or rt250 8, 12. Mutations of rtI163V and rtA186T in addition to the LAM‐resistant mutations have been reported to confer ETV resistance 13…”

Two cases of development of entecavir resistance during entecavir treatment for nucleoside-naive chronic hepatitis B

“…In the case of ETV, it has been reported that resistance to the drug requires at least one of three substitutions in HBV RT, that is, rtT184, rtS202, and rtM250, as well as LVDr-related substitutions rtL180M and M204V [24]. Phenotypic analyses of samples associated with virologic breakthrough confirmed that ETV susceptibility correlates with the spectrum of these additional substitutions conferring genotypic resistance and the increased level of circulating HBV DNA [25].…”