Microbiology

For additional information on Mechanism of Action, Antiviral Activity, Resistance and Cross Resistance, please consult the SUSTIVA, EMTRIVA and VIREAD prescribing information.

Mechanism of Action

Efavirenz: Efavirenz is a non-nucleoside reverse transcriptase inhibitor of HIV-1. Efavirenz activity is mediated predominantly by noncompetitive inhibition of HIV-1 reverse transcriptase (RT). HIV-2 RT and human cellular DNA polymerases α, β, γ, and δ are not inhibited by efavirenz.

Emtricitabine: Emtricitabine, a synthetic nucleoside analog of cytidine, is phosphorylated by cellular enzymes to form emtricitabine 5'-triphosphate. Emtricitabine 5'-triphosphate inhibits the activity of the HIV-1 RT by competing with the natural substrate deoxycytidine 5'-triphosphate and by being incorporated into nascent viral DNA which results in chain termination. Emtricitabine 5′-triphosphate is a weak inhibitor of mammalian DNA polymerase α, β, ε, and mitochondrial DNA polymerase γ.

Tenofovir disoproxil fumarate: Tenofovir DF is an acyclic nucleoside phosphonate diester analog of adenosine monophosphate. Tenofovir DF requires initial diester hydrolysis for conversion to tenofovir and subsequent phosphorylations by cellular enzymes to form tenofovir diphosphate. Tenofovir diphosphate inhibits the activity of HIV-1 RT by competing with the natural substrate deoxyadenosine 5′-triphosphate and, after incorporation into DNA, by DNA chain termination. Tenofovir diphosphate is a weak inhibitor of mammalian DNA polymerases α, β, and mitochondrial DNA polymerase γ.

Antiviral Activity

Efavirenz, emtricitabine, and tenofovir disoproxil fumarate: In combination studies evaluating the antiviral activity in cell culture of emtricitabine and efavirenz together, efavirenz and tenofovir together, and emtricitabine and tenofovir together, additive to synergistic antiviral effects were observed.

Efavirenz: The concentration of efavirenz inhibiting replication of wild-type laboratory adapted strains and clinical isolates in cell culture by 90–95% (EC90-95) ranged from 1.7−25 nM in lymphoblastoid cell lines, peripheral blood mononuclear cells, and macrophage/monocyte cultures. Efavirenz demonstrated additive antiviral activity against HIV-1 in cell culture when combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (delavirdine and nevirapine), nucleoside reverse transcriptase inhibitors (NRTIs) (abacavir, didanosine, lamivudine, stavudine, zalcitabine, and zidovudine), protease inhibitors (PIs) (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir), and the fusion inhibitor enfuvirtide. Efavirenz demonstrated additive to antagonistic antiviral activity in cell culture with atazanavir. Efavirenz demonstrated antiviral activity against most non-clade B isolates (subtypes A, AE, AG, C, D, F, G, J, and N), but had reduced antiviral activity against group O viruses. Efavirenz is not active against HIV-2.

Emtricitabine: The antiviral activity in cell culture of emtricitabine against laboratory and clinical isolates of HIV was assessed in lymphoblastoid cell lines, the MAGI-CCR5 cell line, and peripheral blood mononuclear cells. The 50% effective concentration (EC₅₀) values for emtricitabine were in the range of 0.0013–0.64 μM (0.0003–0.158 μg/mL). In drug combination studies of emtricitabine with NRTIs (abacavir, lamivudine, stavudine, zalcitabine, and zidovudine), NNRTIs (delavirdine, efavirenz, and nevirapine), and PIs (amprenavir, nelfinavir, ritonavir, and saquinavir), additive to synergistic effects were observed. Emtricitabine displayed antiviral activity in cell culture against HIV-1 clades A, B, C, D, E, F, and G (EC50 values ranged from 0.007– 0.075 μM) and showed strain specific activity against HIV-2 (EC50 values ranged from 0.007–1.5 μM).

Tenofovir disoproxil fumarate: The antiviral activity in cell culture of tenofovir against laboratory and clinical isolates of HIV-1 was assessed in lymphoblastoid cell lines, primary monocyte/macrophage cells and peripheral blood lymphocytes. The EC50 values for tenofovir were in the range of 0.04–8.5 μM. In drug combination studies of tenofovir with NRTIs (abacavir, didanosine, lamivudine, stavudine, zalcitabine, and zidovudine), NNRTIs (delavirdine, efavirenz, and nevirapine), and PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir), additive to synergistic effects were observed. Tenofovir displayed antiviral activity in cell culture against HIV-1 clades A, B, C, D, E, F, G and O (EC₅₀ values ranged from 0.5–2.2 μM) and showed strain specific activity against HIV-2 (EC₅₀ values ranged from 1.6 μM to 4.9 μM).

Resistance

Efavirenz, emtricitabine, and tenofovir disoproxil fumarate: HIV-1 isolates with reduced susceptibility to the combination of emtricitabine and tenofovir have been selected in cell culture and in clinical studies. Genotypic analysis of these isolates identified the M184V/I and/or K65R amino acid substitutions in the viral RT.

In a clinical study of treatment-naïve patients (Study 934, see Description of Clinical Studies) resistance analysis was performed on HIV isolates from all virologic failure patients with >400 copies/mL of HIV-1 RNA at Week 48 or early discontinuations. Genotypic resistance to efavirenz, predominantly the K103N substitution, was the most common form of resistance that developed. Resistance to efavirenz occurred in 9/12 (75%) analyzed patients in the emtricitabine + tenofovir DF group and in 16/22 (73%) analyzed patients in the zidovudine/lamivudine fixed-dose combination group. The M184V amino acid substitution, associated with resistance to emtricitabine and lamivudine, was observed in 2/12 (17%) analyzed patient isolates in the emtricitabine + tenofovir DF group and in 7/22 (32%) analyzed patient isolates in the zidovudine/lamivudine group. Through 48 weeks of Study 934, no patients developed a detectable K65R mutation in their HIV as analyzed through standard genotypic analysis. Insufficient data are available to assess the development of the K65R mutation upon prolonged exposure to this regimen.

In a clinical study of treatment-naïve patients, isolates from 8 of 47 patients receiving tenofovir DF developed the K65R substitution through 144 weeks of therapy; 7 of these occurred in the first 48 weeks of treatment and one at Week 96. In treatment experienced patients, 14/304 (5%) of tenofovir DF treated patients with virologic failure through Week 96 showed >1.4 fold (median 2.7) reduced susceptibility to tenofovir. Genotypic analysis of the resistant isolates showed a mutation in the HIV-1 RT gene resulting in the K65R amino acid substitution.

Efavirenz: Clinical isolates with reduced susceptibility in cell culture to efavirenz have been obtained. The most frequently observed amino acid substitution in clinical studies with efavirenz is K103N (54%). One or more RT substitutions at amino acid positions 98, 100, 101, 103, 106, 108, 188, 190, 225, 227, and 230 were observed in patients failing treatment with efavirenz in combination with other antiretrovirals. Other resistance mutations observed to emerge commonly included L100I (7%), K101E/Q/R (14%), V108I (11%), G190S/T/A (7%), P225H (18%), and M230I/L (11%).

HIV-1 isolates with reduced susceptibility to efavirenz (>380-fold increase in EC90 value) emerged rapidly under selection in cell culture. Genotypic characterization of these viruses identified mutations resulting in single amino acid substitutions L100I or V179D, double substitutions L100I/V108I, and triple substitutions L100I/V179D/Y181C in RT.

Emtricitabine: Emtricitabine-resistant isolates of HIV have been selected in cell culture and in clinical studies. Genotypic analysis of these isolates showed that the reduced susceptibility to emtricitabine was associated with a mutation in the HIV RT gene at codon 184 which resulted in an amino acid substitution of methionine by valine or isoleucine (M184V/I).

Tenofovir disoproxil fumarate: HIV-1 isolates with reduced susceptibility to tenofovir have been selected in cell culture. These viruses expressed a K65R mutation in RT and showed a 2–4 fold reduction in susceptibility to tenofovir.

Cross-resistance

Efavirenz, emtricitabine, and tenofovir disoproxil fumarate: Cross-resistance has been recognized among NNRTIs. Cross resistance has also been recognized among certain NRTIs. The M184V/I and/or K65R substitutions selected in cell culture by the combination of emtricitabine and tenofovir are also observed in some HIV-1 isolates from subjects failing treatment with tenofovir in combination with either lamivudine or emtricitabine, and either abacavir or didanosine. Therefore, cross-resistance among these drugs may occur in patients whose virus harbors either or both of these amino acid substitutions.

Efavirenz: Clinical isolates previously characterized as efavirenz-resistant were also phenotypically resistant in cell culture to delavirdine and nevirapine compared to baseline. Delavirdine- and/or nevirapine-resistant clinical viral isolates with NNRTI resistance-associated substitutions (A98G, L100I, K101E/P, K103N/S, V106A, Y181X, Y188X, G190X, P225H, F227L, or M230L) showed reduced susceptibility to efavirenz in cell culture. Greater than 90% of NRTI-resistant isolates tested in cell culture retained susceptibility to efavirenz.

Emtricitabine: Emtricitabine-resistant isolates (M184V/I) were cross-resistant to lamivudine and zalcitabine but retained susceptibility in cell culture to didanosine, stavudine, tenofovir, zidovudine, and NNRTIs (delavirdine, efavirenz, and nevirapine). HIV-1 isolates containing the K65R substitution, selected in vivo by abacavir, didanosine, tenofovir, and zalcitabine, demonstrated reduced susceptibility to inhibition by emtricitabine. Viruses harboring mutations conferring reduced susceptibility to stavudine and zidovudine (M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E) or didanosine (L74V) remained sensitive to emtricitabine.

Tenofovir disoproxil fumarate: The K65R mutation selected by tenofovir is also selected in some HIV-1 infected patients treated with abacavir, didanosine, or zalcitabine. HIV-1 isolates with the K65R mutation also showed reduced susceptibility to emtricitabine and lamivudine. Therefore, cross-resistance among these drugs may occur in patients whose virus harbors the K65R mutation. HIV-1 isolates from patients (N=20) whose HIV-1 expressed a mean of 3 zidovudine-associated RT amino acid substitutions (M41L, D67N, K70R, L210W, T215Y/F, or K219Q/E/N) showed a 3.1-fold decrease in the susceptibility to tenofovir. Multinucleoside resistant HIV-1 with a T69S double insertion mutation in the RT showed reduced susceptibility to tenofovir.