In resource-rich countries, planned or ‘structured’ treatment interruptions (STI) have been evaluated as a strategy in salvage therapy, primary infection and chronic infection, with mixed results. However, stopping antiretroviral therapy (ART) is common in many other clinical situations: for example, acute drug toxicity, short-course ART to prevent perinatal HIV transmission, to allow treatment of opportunistic infections and, particularly, in resource-limited settings when an individual runs out of money to buy drugs or drug supplies are interrupted. Whatever the reason, whenever an ART regimen containing drugs with different half-lives or low genetic barriers to resistance is stopped, there is a risk of drug-resistant virus emerging. Development of drug resistance (mainly to non-nucleoside reverse transcriptase inhibitors; NNRTI) led to the early termination of two trials assessing cycles of STI for 7  and 28  days, and the risk of resistance with simultaneous discontinuation of all components of a nevirapine-containing regimen has also been highlighted .
Data emerging from Africa have suggested that treatment discontinuation rates are high [4,5], particularly for reasons of drug toxicity, drug interactions and cost. There are few data addressing how ART should be stopped safely in such situations. The current study examines the plasma elimination of nevirapine in patients undergoing an STI as part of a large trial in Uganda and Zimbabwe.
DART (Development of AntiRetroviral Therapy in Africa) is a randomized controlled trial evaluating ART management strategies in 3316 symptomatic previously untreated HIV-infected adults with CD4 cell counts < 200 cells/μl in Uganda and Zimbabwe. The main comparison is clinical monitoring only versus laboratory plus clinical monitoring; the second comparison was STI (12 weeks on, 12 weeks off ART) versus continuous ART in those achieving CD4 cell counts ≥ 300 cells/μl after 48 or 72 weeks of continuous ART. All patients initiated triple drug ART with coformulated zidovudine/lamivudine (Combivir) plus tenofovir, nevirapine or abacavir.
The first 21 patients from Uganda receiving nevirapine-containing ART and randomized to STI consented to participate in a pharmacokinetic substudy prior to interrupting ART, with the primary objective of measuring nevirapine elimination (planned sample size 20 patients). ART was discontinued according to best available evidence and current practice as a ‘staggered stop’ with nucleoside reverse transcriptase inhibitors (NRTI: lamivudine plus zidovudine/stavudine) continued for 7 days after stopping nevirapine [www.bhiva.org]. Both DART and this pharmacokinetic substudy were approved by local ethics committees, and all patients provided written, informed consent.
Venous blood (5 ml) was sampled at baseline (nevirapine discontinuation), and at 1, 2, 3 and 4 weeks after stopping nevirapine. All samples were taken at clinic visits: the baseline sample was taken > 4 h after last intake. Plasma was stored at −20°C before shipment to Liverpool for analysis. Details of age, gender, body weight, liver function tests and CD4 cell count at nevirapine discontinuation were collected. HIV viral load and hepatitis B status were not routinely assessed within DART.
Nevirapine was measured using validated high performance liquid chromatography with UV detection. Briefly, drug was extracted from heat-inactivated plasma (200 μl) using dichloromethane after the addition of internal standard (bromazepam; Sigma, St Louis, Missouri, USA). The organic layer was evaporated to dryness and reconstituted in mobile phase (27% ammonium formate buffer, pH 5.0 plus 73% acetonitrile) prior to analysis. Nevirapine recovery using this method was > 95%. Nevirapine and standard were resolved by high performance liquid chromatography (Kontron Instruments, Watford, UK) with peak areas quantified using the Chromeleon (version 6.5) data acquisition system (Dionex Corporation, Sunnyvale, California, USA). The lower limit of quantification (LLQ) of nevirapine was taken as the lowest point on the standard curves (100 ng/ml). The limit of detection (the observation of a distinct peak on the chromatogram) lies below this, and from in-house serial dilution data is approximately 20 ng/ml. Intra- and inter-assay variability (coefficient of variation) at 800 ng/ml (quality control standards aimed at low levels) were both 4.7%. The laboratory participates in an external quality assurance programme (KKGT, The Hague, the Netherlands). The therapeutic range was defined as 3400–8000 ng/ml (www.hivpharmacology.com).
This study aimed to estimate the time until nevirapine levels fell below thresholds favouring the emergence of resistance mutations (the zone of selective pressure) in order to inform the length of NRTI cover in a ‘staggered stop’ strategy. No consensus exists for defining such a zone of selective pressure. Definition is further complicated by the low genetic barrier of nevirapine to development of high-level resistance, as even very low drug concentrations might provide sufficient pressure to drive acquisition of resistance. Previous studies evaluating both clinical  and laboratory  HIV-1 isolates have reported a median inhibitory concentration of 40–100 nmol/l (corresponding to a plasma concentration of 10–20 ng/ml, depending on whether correction for plasma protein binding is performed) in ‘wild-type’ virus lacking any recognized mutation associated with decreased nevirapine sensitivity. Clinical studies of nevirapine have suggested an efficacy threshold of 3400 ng/ml [6,8], below which viral replication leading to treatment failure was more frequent. In this study, the zone of selective pressure was defined as 20–3400 ng/ml, in keeping with two previous studies [3,9], although a third utilized a higher level of 200 ng/ml as the lower limit . Given the lack of consensus, 100 and 200 ng/ml were also considered as lower limits to the zone of selective pressure.
Accurate estimation of nevirapine elimination half-life was not possible because of the large proportion of subjects who achieved undetectable plasma concentrations 1–2 weeks after stopping the drug. In common with others , duration in the zone of selective pressure with a lower threshold (20 ng/ml) below our LLQ (100 ng/ml) was estimated by inspecting chromatograms and assigning a nominal value of 20 ng/ml (‘limit of detection’) for the first undetectable result for each patient with no chromatogram peak < 100 ng/ml. Upper estimates for elimination half-life and time in the zone of selective pressure for nevirapine were then calculated based on linear regression of the log10 plasma nevirapine level on week (half-life = −log10(2)/slope).
Of 21 subjects recruited, none reported missing any ART doses in the last 4 days on self-completed adherence questionnaires. However, one subject had no detectable nevirapine in any plasma sample, and a further subject had a nevirapine concentration just above quantification (156 ng/ml) at baseline and no detectable nevirapine in other samples; both were excluded from all further analyses. In the remaining 19 subjects [5 male, 14 female; median age 35 years (range, 23–61)], median CD4 cell count was 341 cells/μl (range, 301–692); median body weight was 61 kg (range, 52–77); median body mass index was 23 (range, 18–28); and median alanine transaminase was 18 IU/l (range, 10–97). All patients had received 52 weeks of prior therapy with nevirapine (200 mg twice daily), initiated with zidovudine/lamivudine (Combivir); one patient had substituted stavudine for zidovudine at week 16 because of anaemia.
The geometric mean nevirapine concentration at baseline was 6421 ng/ml [95% confidence interval (CI), 5603–7359; range, 3724–9473]. One week after discontinuation, 15 (83%) of 18 subjects with samples available had detectable plasma nevirapine (ranging from below the LLQ of 100 ng/ml to 555 ng/ml); 11 (61%) were above the LLQ, but only five (28%) were > 200 ng/ml (Fig. 1). By 2 weeks, only 5 (26%) of the 19 subjects had detectable nevirapine in plasma [only one (5%) above the LLQ at 415 ng/ml], and by 3 and 4 weeks, no subject had detectable plasma nevirapine. Although numbers were small, the only patient with plasma nevirapine concentration above the LLQ at week 2 was a female patient, as were four of the five patients with nevirapine > 200 ng/ml at week 1 (exact P = 1.0 men versus women).
Because sampling was only undertaken weekly, it was only possible to estimate the trajectories of decay from early sampling and by assigning an arbitrary value of 20 ng/ml (the limit of detection) to the first undetectable sample for each patient. Using these conservative estimates, the median times to thresholds of 200, 100 and 20 ng/ml were 7.6 [interquartile range (IQR), 7.0–10.1], 9.3 (IQR, 8.7–13.0) and 13.2 (IQR, 12.3–18.4) days, respectively. Using 20 ng/ml as the lower threshold for the zone of selective pressure for resistance, 3/19 (16%) and 14/19 (74%) had estimated plasma nevirapine below this limit by 7 and 14 days, respectively; 3/19 (16%) and 17/19 (89%) had estimated plasma nevirapine < 100 ng/ml by 7 and 14 days, respectively; and 4/19 (21%) and 18/19 (94%) were < 200 ng/ml. The median upper estimate of half-life was 39 h (IQR, 38–42). The single patient with substantially slower decay of plasma nevirapine was comparable with other patients in terms of baseline characteristics and was not taking hormonal contraceptives or any other medications known to affect nevirapine metabolism.
Potent anti-HIV activity relies on the continued presence of all components of the ART regimen: resistance develops more readily when replicating virus is exposed to only one drug. Consequently, ‘asymmetrical’ regimens containing drugs with differing elimination kinetics (as measured by terminal elimination half-life) result in functional monotherapy if all drugs are discontinued at the same time and so are potentially vulnerable to the development of resistance. NNRTI drugs, such as efavirenz and nevirapine, appear to be particularly susceptible, both having longer half-lives than other antiretroviral drugs and a low genetic barrier to resistance.
Current UK guidelines recommend continuing the dual NRTI components of the regimen for 7 days after discontinuing NNRTI or covering this period with a protease inhibitor (www.bhiva.org). The 7 day period has been arbitrarily determined using limited pharmacokinetic data derived from Caucasians. There is a growing awareness that marked differences exist between ethnic groups that cannot be accounted for by differences in body weight or gender; these are probably related to genetic variation in drug-metabolizing enzymes or drug transporters. A lower clearance of efavirenz has been observed in Africans , possibly related to the P450 2B6 genotype. Fewer data exist for nevirapine, but lower clearance in Africans has also been reported .
Most data on elimination of nevirapine derive from single-dose administration to healthy volunteers or HIV-positive mothers during labour. Data from HIV-positive patients discontinuing nevirapine after reaching steady state are scarce, and there are even fewer pharmacokinetic data in African populations. Here we have observed much more rapid nevirapine elimination following discontinuation in African patients after 52 weeks of therapy than the previously reported elimination half-lives of approximately 60 h in healthy volunteers [10,13] and HIV-positive women postpartum  who received single dose nevirapine. However, our results are consistent with the shorter elimination half-lives observed in small numbers of HIV-positive patients during chronic dosing with nevirapine , although our upper estimate of median half-life of 39 h is slightly longer than observed in previous studies (e.g. 24 h by Mackie et al.). This difference in drug elimination between single dose and steady state is almost certainly a result of the induction of hepatic cytochrome P450 enzymes by nevirapine, which also increases its own clearance. Increased frequency of sampling is needed to determine whether there are genuine ethnic differences in nevirapine half-life when dosed at steady state. Of note, we enrolled only four men in our study, which precludes accurate identification of any gender differences.
It is important to note that estimates of optimum period of staggered therapy are sensitive to the lower threshold used to define the zone of selective pressure for resistance, and no consensus yet exists for this. Further, extending the period of dual NRTI cover increases the risk of developing NRTI mutations in those with faster nevirapine elimination: M184V in particular for patients receiving lamivudine. Resistance testing 4 weeks after nevirapine discontinuation (3 weeks after NRTI discontinuation) is planned. In the absence of definitive data, we suggest that the optimum period of staggered therapy for patients discontinuing nevirapine (at steady state) is 7–10 days when covered with NRTI drugs with short to moderate intracellular half lives, such as zidovudine, lamivudine, stavudine, abacavir and didanosine, or with currently licensed (ritonavir-boosted) protease inhibitors.
We thank all the patients and staff from all the centres participating in the DART trial.
Members of the DART trial team: H. Grosskurth, P. Munderi, K. Wangati, H. Byomire, B. Amuron, D. Nsibambi; R. Kasirye, E. Zalwango, M. Nakazibwe, B. Kikaire, G. Nassuna, R. Massa, M. Aber, M. Namyalo, A. Zalwango, L. Generous, P. Khauka, N. Rutikarayo, W. Nakahima, A. Mugisha, J. Nakiyingi-Miiro, P. Hughes (MRC/UVRI Uganda Research Unit on AIDS, Entebbe, Uganda); P. Mugyenyi, C. Kityo, F. Ssali, D. Tumukunde, T. Otim, L. Namale, A. Mukose, A. Muhwezi, G. Kabuye, G. Mulindwa, D. Atwine, H. Kyomugisha, A. Drasiku, C. Tumusiime, J. Sabiiti, C. Zawedde, J. Komugyena, J. Okiror, R. Byaruhanga, P. Ocitti, T. Bakainyaga Grace, H. Katabira, G. Barungi, D. Masiira, A. Atwine, S. Murungi, J. Tukamushaba, L. Mugarura, P. Mwesigwa (Joint Clinical Research Centre, Kampala, Uganda); A. Latif, J. Hakim, A. Reid, A. Jamu, S. Makota, T. Mupudzi, G. Musoro, N. Ngorima, M. Pascoe, F. Taziwa, L. Chakonza, E. Chidziva, H. Chirairo, S. Chitsungo, F. Mapinge, A. Mawora, C. Muvirimi, G. Tinago, J. Chimanzi, J. Machingura, C. Maweni, S. Mutsai, R. Warara, M. Matongo, N. Mdege, S. Mudzingwa, M. Jangano, I. Machingura, K. Moyo, L. Vere, E. Chigwedere, M. Phiri (University of Zimbabwe, Harare, Zimbabwe); E. Katabira, J. Oyugi, A. Ronald, A. Kambungu, J. Martin, R. Nalumenya, R. Nairubi, E. Bulume, M. Teopista, C. Twijukye, F. Sematala, H. Byakwaga (Academic Alliance, Mulago Hospital, Uganda); A. Coutinho, B. Etukoit (The AIDS Support Organisation, Uganda); C. Gilks, K. Boocock, C. Puddephatt, D. Winogron (Imperial College, London); J. Darbyshire, D. M. Gibb, A. Burke, D. Bray, A. Babiker, A. S. Walker, H. Wilkes, M. Rauchenberger, S. Sheehan, L. Peto, K. Taylor (MRC Clinical Trials Unit, London).
Trial Steering Committee: I. Weller (Chair), A. Babiker (Trial Statistician), S. Bahendeka, M. Bassett, A. Chogo Wapakhabulo, J. Darbyshire, B. Gazzard, C. Gilks, H. Grosskurth, J. Hakim, A. Latif, O. Mugurungi, P. Mugyenyi, E. Loeliger (observer), J. Leith (observer), P. Naidoo (observer), M. Palmer (observer), J. Rooney (observer), J.-M. Steens (observer).
Data and Safety Monitoring Committee: A. McLaren (Chair), C. Hill, J. Matenga, A. Pozniak, D. Serwadda.
Sponsorship: DART is funded by the UK Medical Research Council, the UK Department for International Development (DFID), and the Rockefeller Foundation. GlaxoSmithKline, Gilead and Boehringer-Ingelheim donated first-line drugs.
1. Ananworanich J, Nuesch R, Le Braz M, Chetchotisakd P, Vibhagool A, Wicharuk S, et al
. Failures of 1 week on, 1 week off antiretroviral therapies in a randomized trial. AIDS 2003; 17:F33–F37.
2. Dybul M, Nies-Kraske E, Daucher M, Hertogs K, Hallahan CW, Csako G, et al
. Long-cycle structured intermittent versus continuous highly active antiretroviral therapy for the treatment of chronic infection with human immunodeficiency virus: effects on drug toxicity and on immunologic and virologic parameters. J Infect Dis 2003; 188:388–396.
3. Mackie NE, Fidler S, Tamm N, Clarke JR, Back D, Weber JN, et al
. Clinical implications of stopping nevirapine-based antiretroviral therapy: relative pharmacokinetics and avoidance of drug resistance. HIV Med 2004; 5:180–184.
4. Muhindo R, Bwana M, Gupta R, Emenyonu N, Ragland K, Bangsberg D. Treatment change and discontinuation among HIV+ persons treated with fixed-dose generic stavudine, lamivudine, and nevirapine in Mbarara, Uganda
. 13th Conference on Retroviruses and Opportunistic Infections.
Denver, February 2006 [abstract 557].
5. Munderi P, on behalf of the DART Trial Team. Safety of nevirapine compared to abacavir on a background of zidovudine/lamivudine as first-line antiretroviral therapy: a randomised double-blind trial.13th Conference on Retroviruses and Opportunistic Infections
. Denver, February 2006 [abstract 109LB].
6. Havlir D, Cheeseman SH, McLaughlin M, Murphy R, Erice A, Spector SA, et al
. High-dose nevirapine: safety, pharmacokinetics, and antiviral effect in patients with human immunodeficiency virus infection. J Infect Dis 1995; 171:537–545.
7. Hazen RJ, Harvey RJ, St Clair MH, Ferris RG, Freeman GA, Tidwell JH, et al
. Anti-human immunodeficiency virus type 1 activity of the nonnucleoside reverse transcriptase inhibitor GW678248 in combination with other antiretrovirals against clinical isolate viruses and in vitro selection for resistance. Antimicrob Agents Chemother 2005; 49:4465–4473.
8. Veldkamp AI, Weverling GJ, Lange JM, Montaner JS, Reiss P, Cooper DA, et al
. High exposure to nevirapine in plasma is associated with an improved virological response in HIV-1-infected individuals. AIDS 2001; 15:1089–1095.
9. Cressey TR, Jourdain G, Lallemant MJ, Kunkeaw S, Jackson JB, Musoke P, et al
. Persistence of nevirapine exposure during the postpartum period after intrapartum single-dose nevirapine in addition to zidovudine prophylaxis for the prevention of mother-to-child transmission of HIV-1. J Acquir Immune Defic Syndr 2005; 38:283–288.
10. Muro E, Droste JA, Hofstede HT, Bosch M, Dolmans W, Burger DM. Nevirapine plasma concentrations are still detectable after more than 2 weeks in the majority of women receiving single-dose nevirapine: implications for intervention studies. J Acquir Immune Defic Syndr 2005; 39:419–421.
11. Ribaudo HJ, Haas DW, Tierney C, Kim RB, Wilkinson GR, Gulick RM, et al
. Pharmacogenetics of plasma efavirenz exposure after treatment discontinuation: an Adult AIDS Clinical Trials Group Study. Clin Infect Dis 2006; 42:401–407.
12. Kappelhoff BS, van Leth F, MacGregor TR, Lange J, Beijnen JH, Huitema AD, on behalf of the 2NN Study Group. Nevirapine and efavirenz pharmacokinetics and covariate analysis in the 2NN study. Antivir Ther 2005; 10:145–155.
13. Lamson MJ, Sabo JP, MacGregor TR, Pav JW, Rowland L, Hawi A, et al
. Single dose pharmacokinetics and bioavailability of nevirapine in healthy volunteers. Biopharm Drug Dispos 1999; 20:285–291.