Reduced drug susceptibility was reported in HIV infection shortly after antiretroviral agents were shown to be clinically effective [1,2]. Since the early design of these agents, the effective inhibition of the HIV-1 reverse transcriptase has remained a challenge, because of the continual emergence of drug-resistant mutants .
Virologic failure of non-nucleoside reverse transcriptase inhibitors (NNRTI) is characterized by rapid rebounds in HIV RNA levels and by the emergence of high-level phenotypic drug resistance . This can occur even after exposure to a single dose of a NNRTI, such as has been seen with nevirapine [5,6]. It has been shown that an entire viral population can shift from a susceptible to a resistant phenotype in 2–4 weeks further confirming that viral replication is a continuous, dynamic and rapid process [7–9] and is an important factor in the management of HIV.
TMC 125, a new, next generation NNRTI, has a diarylpyrimidine-based structure (Fig. 1) that interferes directly with the global hinge-bending mechanism that controls the co-operative motions of the transcriptase subdomains . However, the molecular flexibility of the diarylpyrimidine structure allows TMC 125 to accommodate efficiently mutational changes in the binding pocket even in the presence of significant mutations . TMC 125 has a 50% effective concentration (EC) of 1.4 nm and an EC90 of 2.9 nm against wild-type HIV. It has demonstrated extensive in vitro activity against both wild-type HIV and more than 1000 NNRTI-resistant HIV mutants. Over a period of 7 days, TMC 125 as monotherapy produced a pronounced decrease in HIV-1 RNA of 2.0 log10 in antiretroviral-naive patients  and its unique advantage appears to be its high level of activity against NNRTI-resistant virus. Thus we performed a short-term proof-of-concept study in patients failing initial NNRTI regimes, with confirmed phenotypic resistance to efavirenz.
Patients and interventions
Subjects were required to be at least 18 years of age with documented HIV-1 infection and virologic failure on an antiretroviral treatment regimen comprised of at least two nucleoside analogues and one NNRTI (either efavirenz or nevirapine). Virologic failure was defined as HIV-1 RNA > 2000 copies/ml while receiving the current NNRTI-containing treatment regimen, in a sample taken up to 1 month prior to enrolment. The patient's virus must have demonstrated resistance to efavirenz [10–500-fold, or 10–230-fold increase in resistance relative to wild type IIIB laboratory strain for the Antivirogram (Virco BVBA, Mechelen, Belgium) or VirtualPhenotype assay, respectively] as a marker of pre-existing NNRTI resistance.
In order to be eligible, recruited individuals agreed to not change their failing therapy prior to day 1. Exclusion criteria included: a history of alcohol or drug use judged to potentially compromise compliance; the use of disallowed concomitant therapy; receipt of an investigational drug within 30 days of trial drug administration; a life expectancy of < 6 months; a history of severe drug allergy or hypersensitivity; no further treatment options based on the viral resistance pattern determined after the moment of failing the current therapy and within 3 months prior to screening; current hepatitis B or C infection; or febrile illness within 72 h of study drug ingestion. Subjects were also excluded with the following laboratory values: serum creatinine > 2 × the upper limit of normal (ULN), pancreatic amylase or lipase > 1.3 × ULN, haemoglobin < 6.0 mM (9.6 g/dl), platelet count < 75 × 109 cells/l, anti-neutrophil cytoplasmic antibody < 1.0 × 109 cells/l, or alanine aminotransferase, aspartate aminotransferase or γ-glutamyl transpeptidase > 3 × ULN.
Patients received TMC 125 formulated in PEG 4000, at a dose of 900 mg twice daily for 7 days with witnessing of the morning dose. The twice-daily schedule was chosen to minimize pill burden with each administration (thalf TMC 125 = 30–40 h). Daily HIV-1 RNA measurements were performed, and 12-h pharmacokinetic profiles of TMC 125 were performed on day 1 and day 7 and the resistance test was also repeated on day 1. TMC 125 minimum concentration (Cmin) values were measured on days 2–6 inclusive. On day 8, the resistance test was repeated and an optimized regimen was started based upon the resistance profile at initial screening in accordance with treatment guildines .
All patients were planned to have their treatment optimized upon completion of the study. An optional HIV-1 resistance test and HIV-1 RNA measurement were also available within 1 month of study completion. The study was approved by the local ethics committee/investigation review board at all participating centres, and all patients voluntarily provided written informed consent in accordance with the declaration of Helsinki.
Tolerability, durability and safety were assessed by following the development of clinical and laboratory adverse events. Clinically significant changes in safety laboratory values, other diagnostic tests (except for changes in resistance pattern or HIV-related events), and inter-current illnesses were considered to be adverse events. During the 7-day treatment period patients attended the clinic twice a day for assessments. A final visit was performed on day 8, 12 h after the last drug intake. HIV-1 RNA (Amplicor HIV-1 Monitor version 1.5, Roche Diagnostics, Branchburg, New Jersey, USA) was assessed on three occasions in the week preceding the first TMC 125 intake, while receiving a failing regimen with either nevirapine or efavirenz, daily from day 1 to day 8 of TMC 125 therapy, and once on day 10 and 12. If HIV-1 RNA levels were < 5000 copies/ml plasma, repeat measurements using the Amplicor HIV-1 Monitor version 1.5 with the sensitive sample preparation method (lower limit of quantification 400 copies/ml) were performed. Ultrasensitive viral load assays were not carried out as it was not anticipated that viral loads would be fully suppressed.
Immunological, electrocardiogram, vital signs, blood haematology, biochemistry, and urinalysis assessments were performed on days 1, 4 and 8. Samples for TMC 125 drug level measurement were collected on days 2 to 6 just before drug intake. The pharmacokinetic parameters Cmax, tmax, and AUC12 h were evaluated for TMC 125 during 12-h sampling on days 1 and 8 and Css,av, tmax, and fluctuation index (FI) were evaluated during 12-h sampling on day 8. TMC 125 Cmin values were measured from days 2 to 7. Resistance patterns were determined for samples obtained at study entry and day 8 using genotypic (VirtualPhenotype) and phenotypic analyses (Antivirogram).
The primary outcome measure was the mean HIV-1 RNA viral load decay rate for the whole treatment period. The secondary objectives were mean viral load change from baseline, viral load response in terms of number of patient achieving 0.5 log10, 1 log10 or < 400 copies/mL. Difference in average (DAVG) for the change in viral load over the whole treatment period and nadir and time to nadir viral load were also measured. Changes in CD4 cell count and CD4 percentage, safety and tolerability, and the pharmacokinetics of TMC 125 were also assessed.
As this was an exploratory proof-of-concept study, no formal power calculations were made. The decay in viral load was estimated by means of a mixed effect model (random intercept and random slope) on the changes in log10 of viral load. Baseline values of the log of viral load were included in the model as covariate. Values below the assay detection limit of 400 copies/ml were scored 200 copies/ml for the purpose of analysis.
Sixteen patients were included in the study, and one of these discontinued after day 3 because of non-compliance with the protocol. All subjects were male, mean age 38.9 years (median 38 years; range, 32–53 years), the mean viral load was 15 849 copies/ml (median, 12 106 copies/ml; range, 2401–75 200 copies/ml) and the mean CD4 cell count was 464 × 106 cells/l (median, 408 × 106 cells/l; range, 41 × 106–1103 × 106 cells/l) (Table 1). The majority of patients (81%) was receiving nevirapine as the NNRTI in their failing baseline antiretroviral regimen.
The primary parameter, the viral load decay rate per day, was 0.13 log10 RNA copies/ml per day for the 8-day treatment period, which was statistically significant (P < 0.001). A median decrease of 0.89 log10 RNA copies/ml (mean, −0.86; range, −1.71 to −0.18 log10 RNA copies/ml) was seen after 8 days of treatment. From day 3 onwards, the changes in mean log viral load versus baseline were statistically significant (day 3, P < 0.05; day 4 onwards, P < 0.001). From day 4 onwards, all patients demonstrated significant decreases in HIV-1 viral load, including the one patient with K103N/K.
Graphical presentations of the actual values and changes from baseline in viral load are presented in Figs 2a and b. The DAVG (time-averaged difference) for the change in viral load over the treatment period was −0.46 log10 RNA copies/ml. The mean nadir was 3.20 log10 RNA copies/ml and eight out of 16 subjects reached the nadir on day 8. Twelve (75%) subjects had a decrease of at least 0.5 log10 and seven (44%) subjects had a decrease of at least 1 log10 in viral load at any time point. Two subjects (12.5%) had a viral load of < 400 RNA copies/ml at any time point. Mean CD4 cell count and CD4 percentage showed no statistically significant changes from baseline.
The genotypic mutations at baseline are shown in Table 2. There were an average of two NNRTI-associated mutations: three patients had three mutations and one patient had four mutations (101E/Q+ 179I+181C+190A). All patients had nucleoside reverse transcriptase inhibitors mutations and all except one had protease mutations (Table 2). The Antivirogram (Table 3) demonstrates phenotypic resistance to all three NNRTI that are currently in use and also shows that patients had virus that was phenotypically sensitive to TMC 125 at the start of the study.
Drug susceptibility profiles (phenotype) and mutational patterns (genotype), were studied at screening, baseline and at end of therapy in 15 of 16 NNRTI-experienced patients. Comparing baseline and end of therapy, one case of a new NNRTI-associated resistance mutation was detected (K103N/K) in one subject. The appearance of this mutation was not associated with a change in fold resistance to TMC 125 or to any of the other NNRTI. Subsequent analyses have shown that the K103N/K mutation was also found in other plasma samples from this patient including two pre-treatment samples. The frequency of the virus strain containing this mutation was close to the detection limit of the assay, and the occurrence at end of therapy therefore does not represent selection of mutant virus.
At baseline, there was no correlation between the fold resistance values to efavirenz and TMC 125. The median fold resistance values to efavirenz and TMC 125 were 116.1 and 2.2, respectively.
Full pharmacokinetic profiles were available from 13 subjects on day 1 and 12 subjects on day 8 (Fig. 3). Based on pre-dose concentrations, steady state was reached between 5 and 6 days of treatment. Mean trough level on day 8 was 200 ng/ml (range, 47–828 ng/ml). Mean maximum plasma concentration, typically attained at 4 h post-dose, was 390 ng/ml (range, 110–1270 ng/ml) on day 8. A lag time in absorption of about 1 h was seen in seven out of 14 subjects. The range in parameter estimates indicate inter-individual variability in this study. The mean elimination half-life after the last administration on day 8 was 36 h.
During the 8-day treatment phase, 11 subjects (68.8%) reported at least one adverse event. The most frequently reported adverse events were diarrhoea (31.3%) and headache (25%). These adverse events were reported as mild or moderate in severity. No rashes were seen. No severe adverse events were reported. One subject had increased triglyceride values on screening (3.085 mmol/l; normal range, 0–2.5 mmol/l); increased triglycerides was reported as an adverse events on day 8 and relationship to TMC 125 was considered to be doubtful. No consistent changes in blood chemistry, haematology or urinalysis were observed. No clinically relevant changes in vital signs or electrocardiograms were observed (Table 4).
Within one week of therapy, TMC 125 significantly reduced HIV-1 viral loads in patients with NNRTI resistance. It has been established that resistance to NNRTI can develop rapidly after initiation of NNRTI-containing therapy. It is also known that failure of treatment with one currently available NNRTI leads to broad cross-resistance to the currently licensed agents in this drug class [14,15]. These resistant strains can be present both in treatment-experienced patients and in patients naive to therapy whose primary infection has been caused by resistant HIV strains.
The fact that no significant increase in CD4 cell count or percentage was seen in this study is not surprising considering the short duration of the study and the relative lack of immunological compromise in these study subjects as evidenced by the baseline CD4 cell count. Furthermore, it can be suggested that pre-existing mutations in the NNRTI binding pocket reduced drug potency although as yet there are no data from site-directed mutagenesis or other studies to support this theory. In this study, there was no relationship in this study between trough concentrations at day 7 of TMC 125 and the fall in viral load, which is consistent with in vitro data demonstrating that these drug levels in all cases were in excess of those required to inhibit 90% of resistant viruses.
The findings of this initial, proof-of-concept study suggest that TMC 125 may be used following failure with current NNRTI (Table 5), assuming that its effectiveness is confirmed in further, larger studies. In addition, the use of TMC 125 might logically be extended for use in NNRTI-naive patients, provided that the resistance, tolerability and safety profiles continue to be favourable. Clearly the data presented here suggest that TMC 125 a new, next-generation NNRTI has potent antiviral efficacy even in the presence of mutations conferring high level phenotypic resistance to efavirenz and nevirapine.
We would like to thank Karen Manson for her extensive help with this manuscript.
1. Loeb DD, Swanstrom R, Everitt L, Manchester M, Stamper SE, Hutchison CA, 3rd. Complete mutagenesis of the HIV-1 protease.Nature
2. Wainberg MA, Gu Z, Gao Q, et al
. Clinical correlates and molecular basis of HIV drug resistance.J Acquir Immune Defic Syndr
3. Hammer SM. Increasing choices for HIV therapy.N Engl J Med
4. Deeks SG. International perspectives on antiretroviral resistance. Nonnucleoside reverse transcriptase inhibitor resistance.J Acquir Immune Defic Syndr
5. Cunningham CK, Chaix ML, Rekacewicz C, et al
. Development of resistance mutations in women receiving standard antiretroviral therapy who received intrapartum nevirapine to prevent perinatal human immunodeficiency virus type 1 transmission: a substudy of pediatric AIDS clinical trials group protocol 316.J Infect Dis
6. Eshleman SH, Jackson JB. Nevirapine resistance after single dose prophylaxis.AIDS Rev
7. Munoz JL, Parks WP, Wolinsky SM, Korber BT, Hutto C. HIV-1 reverse transcriptase. A diversity generator and quasispecies regulator.Ann N Y Acad Sci
8. Preston BD, Poiesz BJ, Loeb LA. Fidelity of HIV-1 reverse transcriptase.Science
9. Brenner BG, Turner D, Wainberg MA. HIV-1 drug resistance: can we overcome?Expert Opin Biol Ther
10. Temiz NA, Bahar I. Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase.Proteins
11. Udier-Blagovic M, Tirado-Rives J, Jorgensen WL. Validation of a model for the complex of HIV-1 reverse transcriptase with nonnucleoside inhibitor TMC125.J Am Chem Soc
12. Gruzdev et al
., AIDS Res Hum Retroviruses
13. Yeni PG, Hammer SM, Carpenter CC, et al
. Antiretroviral treatment for adult HIV infection in 2002: updated recommendations of the International AIDS Society-USA Panel.JAMA
14. Antinori A, Zaccarelli M, Cingolani A, et al
. Cross-resistance among nonnucleoside reverse transcriptase inhibitors limits recycling efavirenz after nevirapine failure.AIDS Res Hum Retroviruses
15. Hanna GJ, D'Aquila RT. Clinical use of genotypic and phenotypic drug resistance testing to monitor antiretroviral chemotherapy.Clin Infect Dis
Keywords:© 2003 Lippincott Williams & Wilkins, Inc.
HIV; resistance; NNRTI; TMC-125; pharmacokinetic