The effects of ritonavir and lopinavir/ritonavir on the pharmacokinetics of a novel CCR5 antagonist, aplaviroc, in healthy subjects

Abstract

Aims

This study assessed the effects of the CYP3A inhibitors lopinavir/ritonavir (LPV/r) on the steady-state pharmacokinetics (PK) of aplaviroc (APL), a CYP3A4 substrate, in healthy subjects.

Methods

In Part 1, APL PK was determined in eight subjects who received a single oral 50-mg APL test dose with/without a single dose of 100 mg ritonavir (RTV). Part 2 was conducted as an open-label, single-sequence, three-period repeat dose study in a cohort of 24 subjects. Subjects received APL 400 mg every 12 h (b.i.d.) for 7 days (Period 1), LPV/r 400/100 mg b.i.d. for 14 days (Period 2) and APL 400 mg + LPV/r 400/100 mg b.i.d. for 7 days (Period 3). All doses were administered with a moderate fat meal. PK sampling occurred on day 7 of Periods 1 and 3 and day 14 of Period 2.

Results

In Part 1, a single RTV dose increased the APL AUC_0–∞ by 2.1-fold [90% confidence interval (CI) 1.9, 2.4]. Repeat dose coadministration of APL with LPV/r increased APL exposures to a greater extent with the geometric least squares mean ratios (90% CI) being 7.7 (6.4, 9.3), 6.2 (4.8, 8.1) and 7.1 (5.6, 9.0) for the APL AUC, C_max, and C_min, respectively. No change in LPV AUC or C_max and a small increase in RTV AUC and C_max (28% and 32%) were observed. The combination of APL and LPV/r was well tolerated and adverse events were mild in severity with self-limiting gastrointestinal complaints most commonly reported.

Conclusions

Coadministration of APL and LPV/r was well tolerated and resulted in significantly increased APL plasma concentrations.

Introduction

Aplaviroc (APL; 873140) is a novel spirodiketopiperazine chemokine receptor 5 (CCR5) antagonist that has demonstrated potent in vitro and in vivo activity against human immunodeficiency virus (HIV). In contrast to currently marketed antiretroviral drugs, CCR5 antagonists target a host receptor, rather than a viral receptor or enzyme. CCR5 antagonists block the binding of the HIV envelope glycoprotein, gp120, to CCR5, a coreceptor on human T lymphocytes that is required for viral entry and infection [1]. In vitro studies have shown that APL has potent in vitro antiviral activity with an IC₅₀ in the low nanomolar range against CCR5-tropic HIV-1 strains and that it has a slow offset rate from CCR5 with no appreciable dissociation in over 150 h in isolated membrane preparations [2, 3].

The pharmacokinetics (PK) and metabolism of APL were investigated in a series of oral and intravenous studies in mice, rats and monkeys (unpublished data). In rats and monkeys APL was extensively metabolized to oxidative and glucuronide metabolites, with negligible urinary excretion (<1% dose) observed. The relatively low oral bioavailability, ranging from 4 to 20% across species, was attributed to extensive first-pass gastrointestinal and/or hepatic metabolism as the fraction absorbed in rats was estimated to be 50–60%. In vitro metabolism studies in microsomes prepared from cells stably expressing individual human CYP450 cDNA showed the highest turnover with CYP3A4. Studies with pooled human liver microsomes and specific CYP450 isozyme inhibitors also suggested that CYP3A was the major CYP450 involved in APL metabolism, with some minor involvement of CYP2C19. Various cell-based transport assays showed that APL was a P-glycoprotein (Pgp) substrate with high permeability (P_{app A→B} = 38.4 nm s⁻¹; _{Papp B→A} = 199 nm s⁻¹). APL exposures were identical in Pgp knockout and wild-type mice, suggesting that Pgp did not limit the in vivo oral absorption of APL. APL was 92% bound in human plasma.

Single and repeat oral dose escalation studies have been conducted in healthy subjects. APL exhibited dose-proportional PK in the 200–800 mg twice daily (b.i.d.) dose range, with a t_1/2 of 2.5–3 h [4]. A clinical food–effect study revealed that both moderate (30%) fat and high (53%) fat meals significantly increased the APL AUC by 1.6-fold and 1.7-fold, respectively. Based on these results, APL was administered with food in all subsequent studies to improve oral bioavailability [5]. In a 10-day dose-ranging monotherapy study in HIV-infected patients, APL demonstrated potent antiviral activity with a mean 1.6 log reduction in HIV-1 RNA at the highest dose tested (600 mg b.i.d.) and a short-term safety profile which supported further clinical development [6]. Based on the exposure–antiviral effect relationship, an AUC_0–24 of 1900 ng h⁻¹ ml⁻¹ was selected as a target exposure for future clinical trials [7].

Kaletra™ (Abbott Laboratories, Abbott Park, IL, USA) is the fixed dose combination of lopinavir/ritonavir (LPV/r), two HIV protease inhibitor drugs that are also substrates and inhibitors of CYP450 enzymes. Ritonavir (RTV) is a very potent CYP3A4 inhibitor with K_i values in the 5–70 nm range. A subtherapeutic dose of RTV is coadministered with LPV to inhibit metabolism and ‘boost’ LPV concentrations into the therapeutic range. RTV and LPV also inhibit CYP2D6, 2C9, 2C19 and 2B6, although the inhibitory potency for these enzymes is ∼10–100-fold less than that for CYP3A [8]. RTV and LPV are also inducers of some drug-metabolizing enzymes and transporters, including CYP3A4, CYP2C9, UGT and Pgp, via activation of the transcription factor PXR [9, 10]. The net effect of RTV administration represents the balance between the inhibition and induction effects on the elimination pathways of the coadministered drug. For drugs that are predominantly metabolized by CYP3A, the net effect of RTV or LPV/r coadministration is inhibition of metabolism and increases in exposures ranging from 2- to 30-fold for various substrates have been reported in clinical studies [8, 9]. In rat studies, coadministration of APL with various protease inhibitor drugs increased the oral AUC_0–∞ of APL, with the increase ranging from 1.7-fold for saquinavir to 7.7-fold for RTV.

Combination antiretroviral therapy is the standard of care for HIV-infected patients and early evaluation of potential drug interactions is critical. Preclinical studies indicated that CYP3A was an important pathway of APL elimination; therefore, studies to examine the effects of CYP3A inhibitors on APL PK were needed to provide dosing recommendations for Phase 2b/3 clinical trials. The primary objective of the present clinical study was to examine the effect of repeat dose LPV/r on the PK and safety of APL in healthy male and female subjects. The effect of a single RTV dose on a single low dose of APL was determined prior to the repeat dose study in order to estimate the potential magnitude of inhibition and select a safe dose regimen for the LPV/r study.

Methods

Study design

This was a phase I open label study divided into two parts: Part 1 was conducted as a single-sequence, two-period single dose study of APL with/without RTV in eight subjects; and Part 2 was conducted as a single-sequence, three-period repeat dose study of APL with/without LPV/r in a different group of 24 subjects. The study was approved by the MDS Pharma Services Institutional Review Board (Phoenix, AZ, USA) and all subjects gave written informed consent.

All subjects underwent a screening evaluation within 30 days prior to dosing for Part 1 and Part 2. Inclusion criteria included healthy male and female subjects age 18–55 with no significant abnormalities on medical and laboratory evaluations. Women were allowed to enroll if they were not of child-bearing potential. Subjects could not receive any vitamins, dietary supplements, prescription or nonprescription medications for 7–14 days (depending on the enzyme induction potential) or five half-lives (whichever was longer) before the first dose of study drug and for the duration of the trial. Subjects were asked not to consume grapefruit-related products for 7 days prior to the start of dosing until collection of the final PK sample. Other exclusion criteria included a history of alcohol or illicit drug use, history of regular tobacco- or nicotine-containing products within 3 months of screening visit, blood donation of >500 ml in the preceding 56-day period, hepatitis B virus, hepatitis C virus or HIV positivity and any clinically significant disease or abnormality.

In Part 1, the effects of a single dose of RTV 100 mg on the PK of a single dose of APL 50 mg (5 × 10 mg tablets) were evaluated in eight subjects (cohort 1). All doses of study drug were administered 5 min after consumption of the following moderate fat (30% fat/669 kcal) breakfast: apple juice (170 g), melons (1 cup), scrambled eggs (57 g), Canadian bacon (28 g), toasted white bread (2 slices), margarine (10 g), jelly (2 Tbsp), 2% milk (1 cup). On day 1, a single 50-mg dose of APL was administered and blood samples (2 ml) for APL PK analysis were collected prior to dosing and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16 and 24 h after the morning dose. Subjects initiated a 1-day wash-out period on day 2. On day 3, subjects were concomitantly dosed with APL 50 mg and RTV 100 mg. Blood samples for APL and RTV PK were collected prior to dosing and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 24, 36 and 48 h postdose. All PK blood samples were centrifuged within 1 h of collection and the plasma was stored at −20 °C or below until analysis. Vital signs were collected daily, resting 12-lead electrocardiograms (ECG) were performed on days 1 and 3 and clinical laboratory assessments (clinical chemistry, haematology and coagulation panels) were obtained on days 1 and 5.

The safety and PK results from Part 1 were used to select a dose regimen for the subsequent LPV/r repeat dose evaluation. In Part 2, the effect of LPV/r on the PK of APL was investigated in 24 subjects (cohort 2) in a single-sequence, open-label, three-period design. There was no wash-out between the periods. A different, higher strength APL formulation (200-mg tablet) was used in Part 2. Subjects were dosed according to the following schedule:

• Period 1

  APL 400 mg b.i.d. for 7 days

• Period 2

  LPV/r (LPV 400 mg/RTV 100 mg) b.i.d. for 14 days

• Period 3

  LPV/r (LPV 400 mg/RTV 100 mg) b.i.d. + APL 400 mg b.i.d. for 7 days

Subjects self-administered study drugs as outpatients during Periods 1 and 2 but were admitted to the study centre on the evening before serial pharmacokinetic sampling days (day 7 of Period 1 and day 14 of Period 2) for observed dosing. Subjects were inpatient for the duration of Period 3 (APL and LPV/r). All doses of study drug were administered after a moderate fat (30% fat) meal. During the outpatient treatment periods, subjects were given examples of 30% fat meals and asked to record dose and meal compliance on a diary card. On PK sampling days, all doses were administered by clinic staff immediately following the 30% fat breakfast described in Part 1. Identical breakfasts were served on PK days to minimize potential effects of different meal content on APL PK. Serial blood samples were obtained for measurement of APL, LPV and RTV concentrations prior to dosing and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10 and 12 h after the morning dose on day 7 of Period 1, day 14 of Period 2 and days 1 and 7 of Period 3. Additional samples were collected at 16 h and 24 h after the morning dose on day 7 of Period 3 (last APL dose) and prior to dosing on the mornings of days 3, 4, 5 and 6 of Period 3 for determination of trough concentrations. All PK samples were processed within 1 h of collection and plasma was stored at −20 °C or below until analysis. Safety and tolerability were assessed throughout the study by adverse events (AEs), clinical and laboratory evaluations (clinical chemistry, haematology and coagulation panels), vital signs, and 12-lead ECGs.

Bioanalytical methods

APL and RTV plasma concentrations were determined by a validated high-performance liquid chromatography-tandem mass spectrometric detection (HPLC/MS/MS) method at GlaxoSmithKline (Research Triangle Park, NC, USA). APL and RTV were extracted from 50 µl of plasma using 150 µl of acetonitrile containing isotopically labelled internal standards, ²H₄-873140 and ²H₂¹⁵N₁¹³C₃-ritonavir. The sample was briefly mixed and centrifuged for 5 min at 3000 g. Then 120 µl of the supernatant was added to 120 µl of 0.1% formic acid and mixed. A small aliquot (typically 5 µl) of the extracted sample was injected into a 50 × 2.1 mm (inside diameter) Polaris C₁₈ 3-µm column (Varian, Inc., Palo Alto, CA, USA). The mobile phase consisted of two eluents, mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile). APL and RTV were eluted from the column using a linear gradient from 35 to 70% B over 1 min at a flow rate of 0.37 ml min⁻¹. A Sciex API4000 (Applied Biosystems/MDS Sciex, Concord, ON, Canada) with a TurboIonSpray™ interface was used to monitor the precursor to product ion transitions (m/z 578–227 and 582–231 for APL and its internal standard; 721–140 and 727–140 for RTV and its internal standard) to ensure high selectivity for the compounds. A calibration plot of analyte/internal standard peak area ratio vs. APL or RTV concentration was constructed and a weighted 1/x² linear regression was applied to the data. The calibration range of the method was 0.5–500 ng ml⁻¹ for APL and 1–1000 ng ml⁻¹ for RTV. The calibration lines were used to determine concentrations of APL and RTV in study samples and quality control samples (prepared at concentrations of 1, 35 and 425 ng ml⁻¹ for APL and 2, 70 and 850 ng ml⁻¹ for RTV). Performance of the method, as assessed from APL and RTV determinations in quality control samples, showed that the average within-run precision (coefficient of variation) was ≤7.0% and 7.8% for APL and RTV, respectively. The between-run precision (coefficient of variation) was ≤1.3% and 0.8% for APL and RTV, respectively.

LPV plasma concentrations were determined in a separate validated HPLC/MS/MS assay by Covance Bioanalytical Services (Indianapolis, IN, USA). Lopinavir and the internal standard (reserpine) were extracted from 100-µl aliquots of human plasma using a solvent (methyl-t-butyl ether) extraction procedure. The organic phase was separated and evaporated to dryness with reconstitution of the remaining residue in buffered mobile phase. The extracts were quantified by Turbo IonSpray liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the positive ion mode. The HPLC conditions utilized a Genesis C₁₈ column (3 µm, 50 × 4.6 mm) with an isocratic mobile phase of 85 : 15, 0.05% trifluoroacetic acid (TFA) in acetonitrile : 0.05% TFA in water, at a flow rate of 0.3 ml min⁻¹. A Sciex API3000 mass spectrometer was used to monitor the precursor to product ion transition (m/z 629.5–447.2) for lopinavir. Calculated lopinavir concentrations were based on peak area ratios of lopinavir to reserpine, concentrations being determined using a least-squares linear regression with 1/x² as a weighting factor. The assay demonstrated a lower limit of quantification of 20 ng ml⁻¹ for a 100-µl aliquot of lopinavir and the calibration line was linear over the range 20–20 000 ng ml⁻¹. Quality control samples (QC), prepared at three different analyte concentrations (60, 6000, 14 000 ng ml⁻¹) and stored with the study samples, were analysed with each batch of samples against separately prepared calibration standards. Performance of the method, as assessed from determination of lopinavir in the QC samples, showed that average precision and bias were better than 9.0% and 7.1%, respectively.

Pharmacokinetic analysis

Plasma PK parameters for APL, LPV and RTV were estimated by standard noncompartmental methods with WinNonlin Professional v4.1 (Pharsight Corp., Mountain View, CA, USA). The maximum concentration (C_max), the time to reach maximum concentration (T_max) and the concentration at the end of a dosing interval of length τ (C_τ) were obtained from the observed data. The area under the concentration–time profile from time 0 to the time of the last measurable concentration (AUC_0–t) and the AUC from time 0 to infinity (AUC_0–∞) were calculated for APL and RTV in Part 1 and the AUC from time 0 to the end of the dosing interval (AUC_0–τ) was calculated for APL, RTV and LPV in Part 2. The AUC parameters were calculated using the linear up-log down trapezoidal method. The apparent elimination phase rate constant (λ_z) was estimated using only those data points judged to describe the terminal log-linear decline. The λ_z and other parameters that rely on λ_z (e.g. T_1/2 and AUC_0–∞) were accepted only if a minimum of three data points were used to estimate λ_z and the duration of time over which λ_z was estimated was at least twice the subsequent estimate T_1/2. Actual elapsed time from dosing was used for all individual pharmacokinetic analyses.

Statistical analysis

Plasma concentrations and calculated PK parameters were summarized by treatment and time. Plasma APL, LPV and RTV PK parameters were log_e-transformed prior to statistical analysis. Analysis of variance (anova) was performed using SAS (Cary, NC, USA) Mixed Linear Models procedure to assess treatment comparisons for APL, LPV and RTV PK parameters in which treatment was considered a fixed effect and subject was considered a random effect. The primary comparisons were made for APL AUC_0–τ, C_max and C_τ when APL was administered alone (reference treatment; Period 1) or coadministered with LPV/r (test treatment; Period 3). Secondary comparisons included LPV and RTV AUC_0–τ, C_max and C_τ when LPV/r was administered alone (Period 2) or coadministered with APL (Period 3) and a comparison of APL single dose PK with and without RTV in Part 1. The ratio of geometric least squares (GLS) means and associated 90% confidence intervals (CIs) for the treatment comparisons were estimated for PK parameters. Safety parameters including AEs, clinical laboratory evaluations, vital signs and 12-lead ECGs were listed by subject and summarized by treatment.

Results

Subject accountability

A total of 32 subjects were enrolled; eight subjects (three males/five females) in cohort 1 and 24 subjects (16 males/eight females) in cohort 2. Thirty subjects (eight in Part 1 and 22 in Part 2) completed the study. Two subjects withdrew from Part 2; one withdrew consent and the other was withdrawn because of a positive drug screen. Cohort 1 included four caucasian and four Hispanic subjects; cohort 2 included 15 Hispanic, eight caucasian and one black subject. The mean (range) age was 39.4 (22–54) and 36.2 (18–55) years in Part 1 and Part 2, respectively, and the corresponding mean ± SD weight was 68.6 ± 11.7 and 72.6 ± 9.6 kg.

Single-dose study with RTV

A summary of the PK parameter estimates for APL 50 mg alone and after coadministration with RTV 100 mg is shown in Table 1. Coadministration of a single dose of RTV and APL resulted in an approximate 2.2-fold increase in both the APL plasma AUC_0–∞ and C_max compared with administration of APL 50 mg alone.

Table 1.

Part 1 – summary of single-dose plasma aplaviroc (APL) pharmacokinetic (PK) parameters^* and treatment comparisons^†

Parameter	Treatment A: APL 50 mg (n = 8)	Treatment B: APL 50 mg + RTV 100 mg (n = 8)	Treatment comparison B/A
AUC0–∞, ng h−1 ml−1	20.6 (16.6, 25.4)	43.7 (35.8, 53.3)	2.13 (1.88, 2.41)
Cmax, ng ml−1	6.59 (5.09, 8.54)	14.7 (12.2, 17.7)	2.22 (1.72, 2.87)
Tmax, h	1.75 (1.0–3.0)	2.01 (1.0–2.5)	NC
T1/2, h	3.03 (2.48, 3.71)	2.10 (1.79, 2.45)	NC

^*Geometric mean values (95% confidence interval) presented for all PK parameters except T_max (median, range).

^†Ratio of geometric least squares means (90% confidence interval) presented for treatment comparisons. NC, Not calculated.

The geometric mean AUC_0–∞ and C_max of RTV when coadministered with APL were 2970 ng h⁻¹ ml⁻¹ (95% CI 1949, 4526) and 319 ng ml⁻¹ (95% CI 208, 489), respectively. The median RTV T_max was 4.0 h with a range of 4–12 h. The geometric mean T_1/2 of ritonavir was 6.9 h.

Repeat-dose study with LPV/r

Median plasma concentration–time profiles of APL, LPV and RTV are shown in Figure 1. Coadministration of APL 400 mg b.i.d. with LPV/r 400/100 mg b.i.d. for 7 days resulted in significant increases in APL AUC_0–τ, C_max and C_τ by 7.7-, 6.2- and 7.1-fold, respectively, compared with administration of APL 400 mg b.i.d. for 7 days alone (Table 2). The apparent terminal elimination phase half-life of APL was modestly increased by concomitant LPV/r (2.67 h vs. 3.66 h); the difference appeared to be related to the extended 24-h PK sampling in Period 3 as the estimated T_1/2 based on just the APL concentrations during the 5–12-h postdose period was similar between Period 1 and Period 3. As shown in Table 2, the AUC_0–τ of APL increased by twofold from day 1 to day 7 of coadministration with LPV/r and C_max increased by 2.3-fold over this period, suggesting accumulation of APL during the 7 days of combined dosing with LPV/r.

Figure 1.

Median plasma aplaviroc (A) APL 400 mg q12h (Day 7) (•), APL 400 mg + LP/r 400/100 mg q12h (Day 1) (▿), APL 400 mg + LPV/r 400/100 mg q12h (Day 7) (○), lopinavir (B) LPV/r 400/100mg q12h (Day 14) (•), LPV/r 400/100 mg + APL 400 mg q12h (Day 1) (▿), LPV/r 400/100 mg + APL 400 mg q12h (Day 7) (○), and ritonavir (C) LPV/r 400/100mg q12h (Day 14) (•), LPV/r 400/100 mg + APL 400 mg q12h (Day 1) (,), LPV/r 400/100 mg + APL 400 mg q12h (Day 7) (○), concentration–time curves during each period in Part 2 repeat dose

Table 2.

Summary of repeat-dose plasma aplaviroc (APL) pharmacokinetic (PK) parameters^* and treatment comparisons

	APL 400 mg b.i.d.	APL 400 mg ± LPV/r 400/100 mg b.i.d.		Treatment comparisons†
APL PK parameter	Day 7, Period 1 (n = 22)	Day 1, Period 3 (n = 22)	Day 7, Period 3 (n = 22)	Day 7, Period 3 vs. day 7, Period 1
AUC0–τ, ng h−1 ml−1	808	3078	6230	7.71
	(645, 1013)	(2545, 3723)	(4832, 8031)	(6.39, 9.29)
Cmax, ng ml−1	300	800	1864	6.21
	(221, 408)	(635, 1009)	(1437, 2418)	(4.77, 8.10)
Cτ, ng ml−1	6.47	45.8	45.9	7.09
	(5.23, 8.01)	(30.8, 68.0)	(33.4, 62.9)	(5.56, 9.04)
Tmax, h	2.5	5.0	4.0	NC
	(1.5–5.0)	(2.0–5.0)	(1.5–5.0)
T1/2, h	2.67	1.84‡	3.66§	NC
	(2.44, 2.92)	(1.56, 2.17)	(3.36, 3.99)

^*Geometric mean values (95% confidence interval) presented for all PK parameters except T_max (median, range).

^†Ratio of geometric least squares means (90% confidence interval) presented for treatment comparisons.

^‡n = 18.

^§n = 21. LPV, Lopinavir.

Repeated coadministration of APL and LPV/r resulted in a modest 1.3-fold increase in RTV AUC_0–τ and C_max, but had no effect on LPV AUC_0–τ and C_max(Tables 3 and 4). The RTV and LPV C_τ values were reduced by 34% and 25%, respectively, when coadministered with APL.

Table 3.

Summary of repeat-dose plasma lopinavir (LPV) pharmacokinetic (PK) parameters^* and treatment comparisons

LPV PK parameter	LPV/r 400/100 mg b.i.d. Day 14, Period 2 (n = 22)	LPV/r 400/100 mg + APL 400 mg b.i.d. Day 7, Period 3 (n = 22)	Treatment comparison†(Period 3 vs. Period 2)
AUC0–τ, ng h−1 ml−1	98 114	95 576	0.97
	(87 560, 109 941)	(83 821, 108 979)	(0.88, 1.08)
Cmax, ng ml−1	12 198	11 773	0.97
	(10 830, 13 739)	(10 325, 13 424)	(0.86, 1.08)
Cτ, ng ml−1	6 927	5 226	0.75
	(5 783, 8 298)	(4 267, 6 401)	(0.64, 0.89)
Tmax, h	5.0	4.5	NC
	(3.1–12.0)	(0–8.0)

^*Geometric mean values (95% confidence interval) presented for all parameters except T_max (median, range).

^†Ratio of geometric least squares means (90% confidence interval) presented for treatment comparisons. APL, Aplaviroc.

Table 4.

Summary of repeat-dose plasma ritonavir (RTV) pharmacokinetic (PK) parameters^* and treatment comparisons^†

RTV PK parameter	LPV/r 400/100 mg b.i.d. Day 14, Period 2 (n = 22)	LPV/r 400/100 mg + APL 400 mg b.i.d. Day 7, Period 3 (n = 22)	Treatment comparison (Period 3 vs. Period 2)
AUC0–τ, ng h−1 ml−1	5876	7510	1.28
	(5066, 6815)	(6319, 8926)	(1.11, 1.47)
Cmax, ng ml−1	1090	1440	1.32
	(917, 1298)	(1138, 1824)	(1.09, 1.61)
Cτ, ng ml−1	332	220	0.66
	(259, 425)	(174, 278)	(0.53, 0.84)
Tmax, h	5.0	4.5	NC
	(1.5–7.0)	(0–5.0)
T1/2, h	3.90‡	3.69	NC
	(3.46, 4.40)	(3.42, 3.97)

^*Geometric mean values (95% confidence interval) presented for all parameters except T_max (median, range).

^†Ratio of geometric least squares means (90% confidence interval) presented for treatment comparisons.

^‡n = 12. LPV, Lopinavir.

Safety

There were no deaths, serious adverse events (SAEs), or grade 2 (moderate), 3 (severe) or 4 (life threatening) AEs reported. There were no clinically significant changes in diastolic or systolic blood pressure, heart rate or ECG interval in subjects treated with APL alone or APL with RTV or LPV/r. Four of eight subjects experienced mild AEs during Part 1 of the study including loose stool, headache, gastrointestinal pain, pruritus and erythema. For Part 2, the most common drug-related AEs are shown in Table 5. Seventeen of 24 subjects (71%) reported 127 AEs that were considered by the investigator to be related to study drug. The most commonly reported drug-related AEs were mild gastrointestinal events (loose stools, nausea, diarrhoea, vomiting, flatulence). All gastrointestinal events were graded as mild in intensity and generally resolved within 1–3 days of therapy.

Table 5.

Summary of most frequently reported drug-related clinical adverse events^* (≥10% of subjects)

	APL N = 24	LPV/r N = 23	APL + LPV/r N = 22
Any event	12/24 (50%)	11/23 (48%)	11/22 (50%)
Gastrointestinal
Loose stools	11/24 (46%)	4/23 (17%)	5/23 (23%)
Nausea	5/24 (21%)	4/23 (17%)	7/22 (32%)
Abdominal pain	1/24 (4%)	4/23 (17%)	1/22 (5%)
Vomiting	0/24 (0%)	0/23 (0%)	3/22 (14%)
Diarrhoea	1/24 (4%)	4/23 (17%)	0/22 (0%)
Metabolism and nutrition
Anorexia	0/24 (0%)	0/23 (0%)	4/22 (18%)

^*All adverse events classified as grade 1. APL, Aplaviroc; LPV, lopinavir.

Discussion

CCR5 antagonists represent a novel class of agents targeting viral entry and have the potential to provide new treatment options for HIV-infected patients [1]. The complexity of antiretroviral regimens requires that the drug interaction profile of new agents be characterized. This study has demonstrated a clinically significant interaction between APL and LPV/r. The significant increase in APL AUC and C_max is probably due to the inhibition of CYP3A enzymes in both the intestinal epithelium and liver, consistent with in vitro metabolism and rat drug interaction study results. Although the steady-state plasma AUC and C_max were increased 7.7- and 6.2-fold, the apparent plasma elimination half-life was not markedly altered. This observation suggests that the interaction is primarily an effect on first-pass intestinal and liver metabolism resulting in a significantly increased oral bioavailability, relative to the reduction in clearance. Similar observations were made in drug interaction studies of RTV with the CYP3A substrates, LPV and saquinavir. A predominant effect of RTV inhibition on AUC and C_max, rather than half-life, seems to be a characteristic of CYP3A substrates with a relatively low bioavailability and high clearance and hepatic extraction ratio [11]. An alternative explanation is that CYP3A is not a large contributor to systemic clearance. At present, the quantitative importance of various enzymes and transporters in APL elimination in humans is not known.

Surprisingly, the increase in exposure of APL was substantially greater after repeated dosing of APL with LPV/r 400/100 mg b.i.d. (7.7-fold increase in AUC) than after a single dose of RTV 100 mg (2.2-fold increase in AUC). There are several possible explanations for this finding. First, LPV may contribute to the inhibition of APL metabolism. Although LPV is a less potent CYP3A inhibitor than RTV, the plasma concentrations exceed the K_i value, making it likely that LPV contributes to CYP3A inhibition at therapeutic doses. In addition, LPV has been shown to inhibit CYP2C19, Pgp and organic anion transporting polypeptides [9, 10], which are suspected to play a role in the disposition of APL. Second, different APL dose levels were used in the single-dose study and the repeat-dose study. Although dose proportionality in APL PK was expected in the 50–400-mg dose range studied [4], the degree of inhibition by RTV may vary at different APL doses. A third explanation may be related to the difference in duration of dosing. Both RTV and LPV are time-dependent inhibitors of CYP3A in vitro[12], suggesting that the degree of inhibition could increase with time and duration of dosing. However, this hypothesis seems to be refuted by other studies, which have shown that repeated dosing of RTV or a protease inhibitor/RTV combination results in induction of various enzymes and transporters [9, 10] which would increase metabolism and decrease exposures. The net effect of inhibition and induction appears to depend on the quantitative importance of CYP3A relative to other disposition pathways. For drugs in which CYP3A is the primary route of elimination, net inhibition of metabolism is usually observed because the potent RTV-mediated CYP3A inhibition overcomes CYP3A induction. However, the degree of inhibition, as measured by fold-increase in exposure, is usually less after repeat dose than after single dose because of the CYP3A induction. For example, single doses of RTV have been reported to increase the AUC of a single dose of the CYP3A substrate saquinavir 50–131-fold, whereas repeat doses of RTV increased the AUC of repeat-dose saquinavir only 17–23-fold [13, 14]. Although the identification and quantitative importance of various enzyme/transporter pathways in the elimination of APL are not currently known, involvement of CYP3A, CYP2C19, UGT and Pgp is suspected based on preclinical and in vitro studies. Induction of these enzymes or transporters would be expected to reduce the fold-increase in APL exposure with repeat dosing, an opposite effect to that observed.

Coadministration with APL had no clinically significant effect on the pharmacokinetics of LPV and RTV, which are primarily metabolized by CYP3A with some minor involvement of CYP2D6. These results were consistent with a clinical study using the CYP450 probe substrates in the Cooperstown 5+1 cocktail, which showed that coadministration of APL at exposures (geometric mean AUC_0–τ = 1031 ng h⁻¹ ml⁻¹ and C_max = 346 ng ml⁻¹) similar to that observed in this study resulted in a slight increase (23%) in the oral AUC of midazolam (a CYP3A probe), but had no effect on the PK of probe substrates for CYP1A2, 2D6, 2C9 and 2C19 [15, 16]. The results of both studies confirmed the results of in vitro human liver microsomal studies which showed moderate CYP3A inhibition, but at a relatively high IC₅₀ that was >30-fold greater than observed C_max values, and no inhibition of CYP2D6.

The geometric mean AUC and C_max achieved at steady state in this study with 400 mg b.i.d. of APL and LPV/r were approximately threefold higher than those observed on the first human repeat-dose escalation study and the 10-day monotherapy study [4, 6]. Despite the higher concentrations achieved, the combination of APL and LPV/r was well tolerated and there were no drug-related discontinuations. All AEs in this trial were reported as grade 1 (mild) and no subjects had treatment-emergent grade 2 or higher changes in liver function tests. Gastrointestinal events were most common and the incidence of these events was similar in the APL administered alone period and the APL coadministered with LPV/r period, suggesting that gastrointestinal events are not related to AUC but possibly to a local effect on the gut. When gastrointestinal events did occur, they were mild in nature and generally resolved within 1–3 days while the subjects remained on treatment.

In conclusion, this study has demonstrated that an APL dose regimen of 400 mg b.i.d. in combination with LPV/r should provide an APL AUC_0–24 in excess of the antiviral target (1900 ng h⁻¹ ml⁻¹) determined from the exposure–antiviral response relationship in the 10-day monotherapy study [16]. These PK results, together with the acceptable short-term safety profile, supported further investigation of this combination in Phase 2b/3 clinical trials. The difference in the degree of inhibition seen with single-dose RTV vs. repeat-dose LPV/r suggest that successful extrapolation of RTV inhibition to inhibition by other RTV-boosted protease inhibitor combinations may be challenging because the protease inhibitor drugs differ in their inhibitory and induction effects on multiple drug-metabolizing enzymes and transporters.