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Telbivudine Has No In Vitro Activity Against Laboratory and Clinical HIV-1, Including 5 Clades and Drug Resistant Clinical Isolates
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Reported by Jules Levin
Croi 2009 Feb 8-12 Montreal
C Avila,1 Karwowska S,2 ChingHa Lai2 ,Thomas G Evans2
1Novartis Pharma AG, Basel, Switzerland; 2Novartis Institute for Biomedical Research, Cambridge, MA, US
ABSTRACT
Background: Therapy with most approved oral anti-HBV agents poses the risk of inducing HIV resistance and treatment options are limited for treatment of Chronic Hepatitis B (CHB) in HIV-HBV co-infected patients, where treatment for CHB maybe indicated, but not concurrent treatment for HIV infection. This study reports the in vitro anti-HIV activity of telbivudine using a robust, validated HIV phenotypic assay and a comprehensive collection of HIV isolates including drug resistant HIV, using efavirenz and entecavir as a positive control.
Methods: Several in vitro experiments were performed to determine anti-HIV activity of telbivudine against strains BH-10 and NL4-3 in cells treated for 3-7 days, using efavirenz as a control. Anti HIV-1 activity was also determined against a drug sensitive reference HIV-1 strain (CNDO), a multidrug resistant strain (MDRC4), eight different wild-type HIV-1 clinical isolates representing different geographic locations and viral subtypes (A, B, BF, C and D), and 2 HIV multi-drug resistant isolates to either telbivudine or entecavir (as a control), using the PhenoSense HIV assay (VR001, Monogram Sciences) by measuring viral replication 48 hrs after infection.
Results: Telbivudine did not exhibit in vitro anti-HIV activity (IC50 values >600 μM) against any of the selected laboratory or clinical HIV isolates using a validated, sensitive HIV phenotypic assay. In contrast both efavirenz and entecavir exhibited antiviral activity against the clinical HIV isolates. For efavirenz, IC50 values ranged from 0.0009 to 0.0021 μM and for entecavir from 7.62 to 15.09 μM with the IC50 increased >8-fold in HIV isolates harboring the M184V mutation.
Conclusions: This in vitro report confirms that telbivudine shows no in vitro antiviral effect against a broad range of wild-type and multi-drug resistant HIV isolates at IC50 values >600 μM, a value well above physiologically relevant concentrations and known to inhibit HBV activity in vitro (EC50 value = 200 nM). In contrast, entecavir activity against HIV was confirmed against drug sensitive and drug resistant clinical HIV-1 isolates. These results support further investigation in a clinical trial setting, of the efficacy and safety of telbivudine, for treatment of CHB in HIV-HBV co-infected patients who do not concurrently require anti-HIV treatment and for whom there are few available treatment options.
INTRODUCTION
Treatment of hepatitis B (HBV) in patients co-infected with human immunodeficiency virus (HIV) is an important health issue due to increased morbidity and mortality.
5-10% of HIV-infected patients in the United States are co-infected with HBV (Goedert et al., 2004; Solomon et al., 2004) and may be higher in other parts of the world (Lukhwareni et al., 2009).
The nucleos(t)ide analogues (lamivudine, emtricitabine and tenofovir) have dual activity against HIV and HBV and can be used as part of combination treatment targeting both HBV and HIV for co-infected patients (Dore et al., 1999; 2004; Wolters et al., 2002; Benhamou et al., 2006).
However, few options exist for patients who require treatment for HBV, but not for HIV, as treatment with most approved HBV agents have a real (lamivudine, tenofovir) risk of inducing HIV resistance.
Telbivudine (LdT), a synthetic thymidine nucleoside analogue, approved for the treatment of patients with chronic HBV infection in over 87 countries, has specific HBV-polymerase inhibitor activity and previously reported no measurable in vitro antiviral activity against a panel of 15 DNA and RNA viruses, including HIV (Standring et al., 2001).
Here we report in vitro anti-HIV activity of telbivudine, using an additional robust, validated HIV phenotypic assay (PhenoSense HIV assay) and a comprehensive collection of HIV isolates, including drug resistant HIV, using either efavirenz or entecavir (ETV) as positive controls.
METHODS
HIV strains
HIV strains from an HIV library (Monogram Biosciences) were selected.
Anti-HIV activity of telbivudine was determined against BH-10 and NL4-3 HIV strains, a multidrug resistant strain (MDRC4), 8 different wild-type HIV-1 clinical isolates representing different geographic locations and viral subtypes (A, B, BF, C and D), and 2 HIV multi-drug resistant isolates (Figure 1) using the established PhenoSense assay.
Genotyping and virus stock
The reverse transcriptase (RT) and protease (PR) coding regions derived from HIV isolates were amplified from viral RNA by RT-PCR. The amplified product plus the luciferase indicator gene were inserted into a resistance test vector (RVT). To produce a virus stock for the drug susceptibility assay, human embryonic kidney cells (HEK 293) were co-transfected with a RTV plasmid DNA and an expression vector encoding the envelop proteins of amphotropic murine leukemia virus.
Following transfection, pseudotyped virus particles were harvested and used to infect fresh target cells.
PhenoSense HIV drug susceptibility assay
PhenoSense HIV assay (VR001, Monogram Biosciences) is a phenotypic drug susceptibility assay for HIV-1. The assay determines drug susceptibility of HIV virus from plasma samples containing 500 or more copies of HIV-1 RNA/mL. This assay is used clinically to detect emergence of resistant HIV (Lin et al., 2008)
Susceptibility of HIV with RT and PR coding regions derived from selected isolates to telbivudine and lamivudine was assessed.
Drugs were serially diluted in DMEM with 10% FBS and added to naive HEK-293 for overnight incubation. No observable cytotoxicity was found in treated cells at drug concentrations up to 600 μM for LdT and 100 μM for ETV when compared to the cell density of untreated cells.
Drug-treated target cells were infected by pseudotyped viruses containing the RT and PR regions derived from HIV isolates or a drug sensitive control HIV-1 strain (CNDO). The ability of virus to replicate was assessed 48 hours post-infection by measuring luciferase activity in target cells.
Testing of LdT and ETV against different HIV viruses and the control CNDO virus was performed in a single experiment.
Statistical analysis
Results are expressed as IC50 values and fold-change values (IC50 patient/IC50 CNDO). Data was analyzed by plotting the percent inhibition (y-axis) against log10 drug concentration (x-axis) for a selected HIV virus and a control virus strain (CNDO). Drug susceptibility curve was used to determine the drug concentration to achieve 50% inhibition (IC50).
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