Prototype trial design for rapid dose selection of antiretroviral drugs: an example using emtricitabine (Coviracil)

Franck S. Rousseaua,*, James O. Kahnb, Melanie Thompsonc, Donna Mildvand, David Sheppe, Jean-Pierre Sommadossif, John Delehantyg, Jeffrey N. Simpsona, Laurene H. Wanga, Joseph B. Quinna, Charles Wakeforda and Charles van der Horsth

a Triangle Pharmaceuticals, Inc., 4611 University Drive, PO Box 50530, Durham, NC 27717-0530; b University of California, San Francisco, CA; c AIDS Research Consortium, Atlanta, GA; d Beth Israel Medical Center, New York, NY; e North Shore University Hospital, Manhasset, NY; f University of Alabama, Birmingham, AL; g Trimeris, Inc., Durham, NC; h University of North Carolina, Chapel Hill, NC, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antiretroviral monotherapy for initial drug characterization risks the selection of resistant virus, yet monotherapy is the only setting where many fundamental properties of a new drug can be reliably determined. Using data on viral replication kinetics and dynamics, we designed an accelerated (14 day) open-label study of single agent emtricitabine (formerly known as FTC)—a nucleoside reverse transcriptase inhibitor—to select a dosing regimen for further therapeutic study. Five regimens (25 mg bd, 100 mg od, 200 mg od, 100 mg bd and 200 mg bd) were evaluated in HIV-1-infected subjects over a 14 day dosing period to determine the optimal dose and pharmacokinetics. Serial blood samples for virological, pharmacokinetic and intracellular FTC-triphosphate measurements were drawn frequently. A dose–response relationship for the antiviral activity of emtricitabine was established, with total daily doses of 200 mg or more producing the greatest median HIV-1 viral load suppression: 1.72–1.92 log10. Based on virological outcomes, dose–response analysis and intracellular triphosphate levels, a once-daily dose of 200 mg was selected for further long-term clinical study. Adverse events possibly related to emtricitabine were unremarkable. The antiviral activity of emtricitabine correlated well with intracellular FTC-triphosphate concentrations. This study design is a safe, useful tool for early dose selection for drugs with potent antiretroviral activity and linear pharmacokinetics.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Despite substantial progress in the treatment of HIV infection with combination chemotherapy, new antivirals are needed to improve the activity, safety, tolerability and simplicity of treatment regimens. Development of these new agents requires early demonstration of antiviral activity, desirable pharmacokinetics and a dose–response relationship that can only be established reliably in monotherapy trials.

Defining these parameters in the dose selection process would be a challenge if a compound were administered as part of triple drug therapy. If drug exposure is suboptimal, the risk of resistance is great whether the drug is given as monotherapy or in combination. A monotherapy trial with a proper study design has several advantages in aiding dose selection. The potential for selection of resistant virus is limited to a single agent in a monotherapy trial, whereas failure in combination drug trials may lead to resistance to multiple drugs. Intrinsic potency is most easily determined in monotherapy, as are basic facets of safety and tolerability. Within a combination, it is difficult to dissect the activity and safety of one single agent in a small-scale dose selection trial. Larger number of subjects and lengthy study periods are often required to select between doses of a given drug when used in combination regimens.1 High rates of drug resistance were observed in the early monotherapy trials, due primarily to the lack of accurate and sensitive viral load assays that supported intensive monitoring of HIV-1 RNA over a short study period. The purpose of this trial was to capitalize on the sensitive virological assays now available to improve upon the design of monotherapy trials for dose selection of antivirals with linear pharmacokinetic characteristics in HIV-1-infected adults. The antiretroviral nucleoside analogue emtricitabine (formerly FTC) was chosen as a candidate compound to evaluate the prototype trial design.

Emtricitabine is a new deoxycytidine nucleoside reverse transcriptase inhibitor (2'3'-dideoxy-5-fluoro-3'-thiacytidine, Coviracil) structurally similar to lamivudine but with a median five-fold greater in vitro activity against HIV-1.2 FTC-5'-triphosphate, the active intracellular moiety, possesses very strong affinity for HIV-1 reverse transcriptase. We hypothesized that a 14 day monotherapy study in HIV-1-infected individuals would be adequate to safely determine antiviral activity and intracellular pharmacology and to define the dose for therapeutic trials, and would minimize the potential for selection of mutations associated with phenotypic resistance. Our hypothesis was based on two observations: (i) the very high turnover of HIV replication, which would allow potent antivirals to demonstrate suppression within a few days;36 and (ii) the appearance of mutant virus only after c. 2 weeks of treatment under selective pressure by lamivudine monotherapy.7


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subject selection

This study was open to men and women 18–70 years of age with HIV-1 infection. Eligibility required a CD4+ cell count of at least 200 cells/mm3 and a viral load of at least 5000 copies of HIV-1 RNA per millilitre of plasma (copies/mL). Previous treatment with lamivudine or abacavir was not permitted. Men and women of reproductive potential had to agree to use an effective method of contraception during the study. The protocol was reviewed and approved by the investigational review board at each study site, and all subjects gave written informed consent.

Study design

The study was designed as an open-label, sequential, dose-ranging trial in HIV-1-infected male and female subjects. Five cohorts, with eight or nine subjects per cohort, were evaluated over a 14 day dosing period using the following emtricitabine dose regimens: 25 mg bd, 200 mg od, 100 mg bd, 100 mg od and 200 mg bd. Before initiation of the next dosing cohort, at least four subjects in the preceding cohort had to complete the entire 14 days of therapy without significant toxicity. All subjects were followed for an additional 14 days after cessation of treatment. Frequent samples were obtained for evaluation of plasma viral load, plasma pharmacokinetics and intracellular pharmacology.

Safety and HIV-1 resistance monitoring

On days 1, 8 and 15, each subject underwent a complete physical examination, an update of his or her medical status and clinical laboratory safety tests. Subjects were asked about adverse events at each clinic visit during the dosing period (up to day 14), at the end of treatment (day 15) and on days 21 and 28. Any subject with an adverse event persisting beyond day 28 was monitored until the event resolved. When viral rebound (i.e. >0.25 log10 increase in plasma HIV-1 RNA from nadir) occurred, HIV genotype analysis was carried out using Affymetrix GeneChip technology (Affymetrix, Inc., Santa Clara, CA, USA) on samples collected before dosing on day 1 and after 14 days of therapy (day 15). If mutant virus was detected on day 15, samples from earlier time points were also genotyped. In addition, HIV genotype analysis was carried out retrospectively on day 15 samples from all other subjects. Again, if mutant virus was detected on day 15, samples from earlier time points were genotyped.

Plasma and intracellular pharmacokinetics

Full-profile plasma pharmacokinetics were evaluated over a 12 h interval after the first dose of emtricitabine on days 1 and 10 (steady state) after the morning dose. Blood for determination of plasma emtricitabine concentration was collected at 0.5, 1, 2, 3, 4, 6, 8 and 12 h post-dose. On days of pharmacokinetic sampling, subjects received emtricitabine after an overnight fast. Plasma samples were analysed for emtricitabine concentration using an HPLC-mass spectrometry method after solid-phase extraction. The lower limit of quantification of emtricitabine in plasma was 5 ng/mL. Pharmacokinetic analysis was conducted using noncompartmental methods. Blood samples for intracellular FTC-triphosphate levels in peripheral blood mononuclear cells (PBMCs) were drawn on day 1 at 1, 3, 6, 9 and 12 h after the first dose and on day 12 at 1 and 4 h after the morning dose. PBMCs were first isolated by Ficoll–Hypaque density gradient. FTC-phosphates in PBMCs were then extracted with 60% methanol at –70°C, separated by HPLC and hydrolysed to emtricitabine, which was then quantified by HPLC with UV detection at 280 nm.8

HIV-1 RNA analysis

Blood samples for viral load analysis were collected on day 1, immediately before the first dose and at 2, 6 and 12 h post-dose; samples were also drawn before the morning dose on days 2, 3, 5, 8, 9, 10, 12 and 15. Plasma levels of HIV-1 RNA were determined by Roche Amplicor PCR assay (Roche Diagnostics, Branchburg, NJ, USA) with a lower limit of quantification of 400 copies/mL. Three measures of antiviral activity were employed: average area under the viral load–time curve minus baseline (AAUCMB) to day 15, reduction from baseline at day 15 and maximum reduction from baseline. Medians were tabulated by dose group. In addition, viral load decay slopes were evaluated at days 2, 3, 5 and 8. The viral load decay slope for a given day, n, was defined as the absolute value of the minimum measure of [0, log10 (HIV-1 RNA copies/mL at day n) – log10 (HIV-1 RNA copies/mL at baseline)] divided by hours from baseline and multiplied by the natural log conversion factor [ln(10) = 2.3]. With respect to all of the above measures of antiviral activity, the Spearman rank test was used to assess the dose response among emtricitabine doses. Linear regression models were fitted to assess the relationship between early changes in viral load (e.g. viral load decay slope at day 3) and end-of-study results (e.g. change from baseline at day 15).

Dose–response analysis

A dose–response relationship of emtricitabine was analysed using the pharmacological simple Emax model as follows: effect = (Emax x dose)/(ED50 + dose), where Emax is the maximal antiviral activity, and ED50 is the dose required to produce 50% of the maximal antiviral activity. The effect parameter used for antiviral activity was median AAUCMB to day 15 and the dose parameter was the total daily dose of emtricitabine. AAUCMB was chosen as the parameter for antiviral activity as it accounted for all antiviral activity data to day 15, including any rebound in viral load levels.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Forty-one subjects enrolled in this study: nine received emtricitabine 25 mg bd, and eight each received the other four emtricitabine regimens. Treatment groups were comparable with respect to baseline demographics and characteristics, which are summarized by dosing cohort in Table 1Go. Two subjects in the 25 mg bd cohort and one volunteer in the 200 mg bd cohort harboured HIV-1 at baseline that had a valine substitution at codon 184 of the reverse transcriptase gene (M184V mutation). Those subjects were excluded from the virological analysis, in accordance with the protocol.


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Table 1. Subject characteristics at study entry by dosing cohort
 
HIV-1 RNA suppression, pharmacokinetics and dose response

All doses produced substantial suppression of HIV-1 viraemia (Figure 1Go). In general, at daily doses of 200 mg or more, median viral load reduction was highest after 14 days of monotherapy and ranged from 1.72 to 1.92 log10 (Table 2Go). Statistically significant correlations with dose were found with respect to HIV-1 RNA reduction at day 15 and maximum HIV-1 RNA reduction. Correlation between dose and AAUCMB for plasma HIV-1 RNA to day 15 approached significance (P = 0.055, Table 2Go). Although there was a statistically significant correlation between dose and viral load decay slope on days 3 and 8 (Table 2Go), viral load decay slopes at days 2 and 5 were not consistent with respect to differentiating between emtricitabine doses. This lack of consistency was due primarily to the high antiviral potency of emtricitabine. As shown in the dose–response curve (Figure 2Go), the antiviral activity of emtricitabine reaches a plateau as dose increases, with little difference in the activity between the 200 and 400 mg daily doses. The Emax model predicted that maximal antiviral activity (median AAUCMB) is estimated to be 1.34 log10 and ED50 to be 12.7 mg. This analysis confirms that emtricitabine is a potent antiviral agent, with the lowest dose evaluated in this study (50 mg/day) being four-fold above the ED50. The antiviral activity of emtricitabine at a dose of 200 mg/day would have already achieved close to 95% of the maximal antiviral activity with little additional (c. 3%) activity observed at the 400 mg/day dose (by doubling the dose). As the 100 mg daily dose of emtricitabine produces antiviral activity that is <90% of the maximal activity, the 200 mg daily dose was chosen for use in therapeutic efficacy trials.



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Figure 1. Median viral load suppression from baseline according to emtricitabine dosing cohort. Cohort symbols: {diamond}, 25 mg bd; {blacksquare}, 100 mg od; {square}, 100 mg bd; {blacktriangleup}, 200 mg od; {triangleup}, 200 mg bd.

 

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Table 2. Median viral load efficacy outcomes by dosing cohort
 


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Figure 2. Dose–response relationship of antiviral activity of emtricitabine using the Emax model. The effect parameter used for antiviral activity was median AAUCMB to day 15 and the dose parameter was the total daily dose of emtricitabine. The curve shows the model prediction; observed data points are indicated by filled circles.

 
The steady-state plasma emtricitabine concentrations were well above the mean in vitro IC90 of emtricitabine for HIV-1 (Figure 3Go), and steady-state plasma trough concentrations of emtricitabine in all subjects receiving the 200 mg od dose exceeded the mean in vitro IC90 by about four-fold on average. The plasma half-life of emtricitabine was estimated to be 8–10 h. FTC-triphosphate was readily detected in PBMCs within 1 h of the first dose of emtricitabine on day 1 (data not shown). The steady-state (day 12) FTC-triphosphate concentrations obtained at 1 and 4 h post-dose increased in a dose-related manner, reaching a plateau of c. 4 pmol/106 cells at emtricitabine daily doses of 200 mg or more (Figure 4Go). When compared with FTC-triphosphate levels after initial dosing on day 1, the 2.5-fold higher FTC-triphosphate levels at steady state correspond to an intracellular half-life of FTC-triphosphate of >20 h.



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Figure 3. Mean steady-state (day 10) plasma emtricitabine concentrations, by dosing cohort. Cohort symbols: {triangleup}, 200 mg bd; {blacktriangleup}, 200 mg od; {square}, 100 mg bd; {blacksquare}, 100 mg od; {diamond}, 25 mg bd.

 


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Figure 4. Median steady-state FTC-triphosphate concentrations in PBMCs, by dosing cohort. Key: {square}, 25 mg bd; {blacksquare},100 mg od; {blacksquare}, 100 mg bd; {blacksquare}, 200 mg od; {blacksquare}, 200 mg bd.

 
Thus, intracellular pharmacokinetics together with virological activity results and dose–response analysis support the use of a once-daily emtricitabine dose of 200 mg in future therapeutic trials.

Viral resistance and safety

Four of 38 evaluable subjects experienced viral rebound and had the M184V mutant virus on days 8 and 9 of therapy. The median baseline viral load of these individuals was 5.26 log10 copies/mL. At day 15, but not earlier, M184V mutations were also detected in the plasma of four other subjects. The median baseline viral load for these four subjects was 4.86 log10 copies/mL, and their day 15 viral load was 3.17 log10 copies/mL. Only one of these four subjects experienced viral load rebound. Of the remaining 30 evaluable subjects for whom no M184V mutation was observed, five subjects experienced viral load rebound. The median baseline viral load in these 30 subjects with wild-type virus was 4.49 log10 copies/mL.

All subjects received study drug and were included in the safety analysis. Emtricitabine was well tolerated at all dosing levels. No serious or severe adverse events related to emtricitabine were reported. Moderate events possibly related to emtricitabine were limited to the following: four cases of nausea, three of headache, and two each of diarrhoea, vomiting and pharyngitis. The incidence of adverse events did not appear to be dose related.

Correlation of initial viral load decay slope and outcome

The correlation of initial viral load decay slope and antiviral activity endpoints were analysed using a multiple linear regression model. After adjusting for baseline viral load, screening CD4+ cell count, and dose of emtricitabine, viral load decay slopes at days 2, 3, 5 and 8 were significantly predictive of viral load nadir, maximum viral load reduction, AAUCMB and viral load reduction at day 15 (Table 3Go). These results indicate that dose selection studies of shorter duration may be possible and could further reduce the potential for development of viral resistance.


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Table 3. Multiple regression analysis of early viral load decay slope and virological outcomesa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Selection of an optimal dose of an investigational drug for subsequent evaluation in large therapeutic trials is a critical step in the development of new antiretroviral agents as the consequences of using an inappropriate dose of a given antiviral agent in larger HIV trials of combination regimens are not trivial. If the dose of a particular agent is too low when used in combination, the risk of selection of virus resistant to that agent is high, and likewise the risk of developing resistance to other drugs in the combination regimen also becomes much higher. Favouring higher doses to ensure efficacy risks greater toxicity, a particularly undesirable outcome for the treatment of a chronic disease such as HIV infection. Furthermore, results from pharmacokinetic interaction studies are justified before initiation of combination studies to avoid unfavourable pharmacokinetic interactions that might compromise the activity of one or more of the components of the combination regimen. Given the potent activity of current triple drug regimens, the individual antiviral activity of different doses of a given drug in combination regimens at early time points can be masked by the collective potency of the entire regimen typically requiring longer term efficacy data (i.e. >16 weeks) to discriminate between doses.

We believe that the trial design described herein supports the rationale for using short-course monotherapy to define the minimal dose giving maximum antiviral activity, desirable pharmacodynamics and pharmacokinetics, and preliminary safety data in chronic viral diseases such as HIV and viral hepatitis, where clinically relevant surrogate markers are readily available. This trial design may also help to enhance the efficiency of the early screening process for new drug candidates in order to eliminate those with unfavourable pharmacokinetics or antiviral activity. Results of the study demonstrated that viral load decay slopes at days 2, 3, 5 and 8 were significantly predictive of viral load nadir, maximum viral load reduction, AAUCMB and viral load reduction at day 15. These results indicate that dose selection studies of even shorter duration may be possible and could further reduce the potential for development of viral resistance. Analysis of genotypic changes in HIV-1 RNA and incidence of viral rebound further support limiting monotherapy to 2 weeks or less and limiting recruitment to subjects with plasma HIV-1 RNA of <100 000 copies/mL at baseline.

Analysis of the results of antiviral activity and safety for all doses of emtricitabine supported the selection of emtricitabine 200 mg od for use in subsequent phase II and III clinical trials. Emtricitabine was well tolerated at all doses with no serious or severe emtricitabine-related adverse events. Evidence of a dose relationship with the incidence of adverse events was not observed. Whereas strong viral suppression occurred at all doses, it was greatest at doses of 200 mg or more per day. Saturation of intracellular levels of FTC-triphosphate correlated with the plateau of HIV-1 RNA suppression of nearly 2.0 log10 at doses of >=200 mg/day. Further analysis of the antiviral activity using a simple Emax model demonstrated that at an emtricitabine dose of 200 mg/day, c. 95% of the maximal antiviral activity had been observed. Doubling the dose to 400 mg/day resulted in little additional (c. 3%) antiviral activity.

In conclusion, this short study design was particularly useful for an antiretroviral agent such as emtricitabine that has straightforward pharmacokinetics (i.e. linear kinetics) and potent activity that manifests without lengthy metabolic activation. Other new antiretroviral agents are also being investigated using this abbreviated design. Further studies to assess the efficacy and safety of emtricitabine at the optimal dose of 200 mg od are underway.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The University of North Carolina, National Center for Research Resources (GCRC, RR00046), Chapel Hill, North Carolina, USA, provided logistical support for C.v.d.H. This study was also supported by Triangle Pharmaceuticals, Inc., Durham, NC, USA. This study was presented in part at the Fifth Conference on Retroviruses and Opportunistic Infections, Chicago, IL, February 1–5, 1998 [abstract LB9]; the Twelfth World AIDS Conference, Geneva, Switzerland, June 28 to July 3, 1998 (abstracts 12208 and 12468LB); the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, September 24–27, 1998 (abstract LB–2); and the Thirty-sixth Annual Meeting of the Infectious Diseases Society of America, Denver, CO, November 12–15, 1998 (abstracts 294 and 415).


    Notes
 
* Corresponding author. Tel: +1-919-493-5980; Fax: +1-919-493-5925. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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8 . Darque, A., Valette, G., Rousseau, F., Wang, L. H., Sommadossi, J. P. & Zhou, X. J. (1999). Quantitation of intracellular triphosphate of emtricitabine in peripheral blood mononuclear cells from human immunodeficiency virus-infected subjects. Antimicrobial Agents and Chemotherapy 43, 2245–50.[Abstract/Free Full Text]

Received 16 February 2001; returned 11 May 2001; revised 9 July 2001; accepted 25 July 2001