Single-Dose Pharmacokinetics of a Pleconaril (VP63843) Oral Solution and Effect of Food

Abstract

Pleconaril is an orally active broad-spectrum antipicornaviral agent which demonstrates excellent penetration into the central nervous system, liver, and nasal epithelium. We report the results of a randomized two-way crossover study designed to characterize the disposition of a single dose (200 mg) of pleconaril oral solution in fed and fasting humans. Twelve healthy adult subjects (18.7 to 39 years of age) participated in this study. Each subject received a single 200-mg dose of pleconaril oral solution, both coadministered with a standard English breakfast and following a 10-h predose fast. There was a minimum 7-day washout period between pleconaril doses. Repeated blood samples (n = 10) were obtained over 24 h postdose, and the pleconaril level in plasma was quantified by gas chromatography. Plasma concentration-versus-time data were curve fitted for each subject by using a nonlinear weighted least-squares algorithm, and pharmacokinetic parameters were determined from the polyexponential estimates. Pleconaril disposition was best characterized by a one-compartment open model with first-order absorption. The apparent bioavailability of pleconaril oral solution was significantly increased with the administration of food. The area under the concentration-time curve and maximum concentration of pleconaril in plasma achieved following the standard English breakfast (i.e., 9.08 ± 3.23 mg/liter · h and 1.14 ± 0.58 mg/liter, respectively) were 2.2- and 2.5-fold higher, respectively than those achieved in the fasting state (i.e., 4.08 ± 2.74 mg/liter · h and 0.46 ± 0.30 mg/liter, respectively). Mean plasma pleconaril concentrations 12 h after a single 200-mg oral dose (fed, 0.25 ± 0.2 mg/liter; fasting, 0.11 ± 0.10 mg/liter) in healthy adults remained greater than that required to inhibit more than 90% of the enteroviruses in cell culture (i.e., 0.07 mg/liter). To enhance the oral bioavailability of pleconaril, coadministration with a fat-containing meal is recommended.

Pleconaril, 3-[3,5-dimethyl-4[[3-(3-methyl-5-isoxazolyl)propyl]oly]phenyl]-5-(trifluoromethyl)-1,2,4-oxadiazole (Fig. 1), is an orally active broad-spectrum antipicornaviral agent with potential therapeutic application in the treatment of viral meningitis, upper respiratory disease including the common cold, and other enterovirus-associated infections. Pleconaril, like similar [(oxazolylphenoxy)alkyl]isoxazole compounds, inhibits viral replication at the site of viral attachment and uncoating. These compounds insert themselves into a hydrophobic pocket beneath the canyon sites on the icosahedral face of the virion, raising the floor of the canyon and altering the virion’s ability to bind to cellular receptors. Additionally, these agents increase the stability of the viral capsid against receptor- and pH-induced conformational changes which normally occur during the process of cellular entry. Thus, the compounds force the virion to resist changes which must occur for efficient disassembly and release of viral ribonucleic acid (5).

FIG. 1.

Chemical structure of pleconaril.

Preclinical studies have demonstrated that the bioavailability of pleconaril in a hard gelatin dosage form is markedly enhanced in the presence of food, with roughly a sevenfold difference in bioavailability between fed and fasting states (11). An oral liquid dosage form in a medium-chain triglyceride-based vehicle was developed in an attempt to minimize the effects of food intake and also to enhance the bioavailability of pleconaril. Additionally, the availability of an oral liquid formulation will expand the potential therapeutic application of this drug to include pediatric and geriatric patients, for whom such a formulation is critical. To ascertain the bioavailability of the pleconaril oral solution relative to food intake, we examined its pharmacokinetics in fasting and fed adults.

(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, September 1997.)

MATERIALS AND METHODS

Subjects.

Twelve healthy adults were enrolled in this open study of pleconaril pharmacokinetics. Subjects were eligible for enrollment if they met the following inclusion criteria: (i) 18 to 45 years of age, (ii) nonsmoking for >6 months, and (iii) body weight between 50 and 100 kg and within 15% of the ideal for height and frame according to Metropolitan Life Insurance Company averages (6). Additionally, female volunteers were required to be surgically sterile or to provide evidence of contraceptive use. Subjects were excluded from participation if any of the following criteria were met: a history or presence of a significant disease state as detected by medical history, physical examination, and/or laboratory tests; significant allergies (medication or other), allergic skin rash, or recent episode of asthma; consumption of any prescription drug within 2 weeks, any over-the-counter drug within 3 days, or any investigational drug as part of a research study within 90 days; or a history of treatment for alcoholism or drug abuse.

A medical history, physical examination, and clinical laboratory tests (complete blood count, serum chemistry panel, liver function tests, urinalysis, human immunodeficiency virus screen, and hepatitis B virus screen) were obtained prior to enrollment. This study was approved by an independent institutional review board, and all subjects signed a written informed consent prior to enrollment.

Study design.

The study consisted of a randomized, two-way crossover evaluation of the pharmacokinetics of the pleconaril oral solution (ViroPharma Inc., Malvern, Pa.) in the fed and fasting states. All participants were housed in the clinical facility for a period of 10 h prior to administration of the study drug and through the 24-h sample collection period. Subjects received a single oral 200-mg dose of pleconaril solution (40 mg/ml) with 240 ml of water either 30 min after consuming a standard English (high-fat) breakfast (two eggs, two strips of bacon, two slices of toast, two pats of butter, and 240 ml of milk) or following a minimum 10-h fast. Following pleconaril administration in each arm of the study, all subjects fasted for an additional 4 h. Subjects completed a minimum 7-day washout period between doses, and the study was subsequently repeated under the reverse conditions. Subjects were not permitted to consume ethanol within 48 h of pleconaril administration.

Sample collection.

Venous blood samples (5 ml each) for determination of pleconaril concentration were collected from an indwelling venous catheter in glass tubes containing EDTA. Samples were collected immediately prior to drug administration and at 1, 2, 3, 4, 5, 6, 9, 12, and 24 h following the dose. Plasma was separated by centrifugation (1,500 × g for 10 min at 4°C) and stored in polypropylene tubes at −20°C until analysis, which was performed within 90 days from the time of collection.

Analytical procedures.

Plasma samples were allowed to thaw at room temperature, and 100 μl was transferred to a 13- by 100-mm screw-cap tube with 50 μl of internal standard. Hexane (2 ml) was added, and the sample was allowed to mix for 10 min on a reciprocating shaker. Samples were subsequently centrifuged at 2,000 × g for 10 min, and the organic phase was transferred to a 13- by 100-mm screw-cap tube and evaporated to dryness under nitrogen at 40°C. Dry samples were reconstituted with 0.1 ml of methanol and transferred to analytical vials for subsequent analysis by gas chromatography with electron capture detection. One microliter of sample was injected through a split injector onto a DB-17 column (15 m by 0.32 mm [inside diameter]; 0.25-μm film thickness) at an oven temperature of 210°C. Pleconaril and the internal standard eluted at 2.8 and 8.0 min, respectively (7).

A seven-point standard curve of the peak ratio of active compound to internal standard (WIN 66407) was prepared daily and was used to calculate all plasma pleconaril concentrations. The limit of detection for the assay was set at the low standard, 49 ng/ml. The analytical method demonstrated linearity at plasma pleconaril concentrations over a range of 49 to 1,976 ng/ml (r > 0.99). Intraday assay variability ranged from 3.8 to 6.2%, and interday assay variability was consistently ≤10.2% for concentrations between 49 and 1,976 ng/ml (7). All assays were performed in duplicate by an independent laboratory (Phoenix International Life Sciences, Inc., Montreal, Quebec, Canada), and the mean plasma concentrations were used for the pharmacokinetic analysis.

Pharmacokinetic and statistical analysis.

Pharmacokinetic and statistical analyses were conducted with Kinetica, version 2.0 (Micropharm International, Paris, France). Plasma drug concentration-versus-time data were curve fitted by using a peeling algorithm to generate initial polyexponential parameter estimates. Final estimates were determined from an iterative, nonlinear, weighted, least-squares regression algorithm with reciprocal (1/Y_calc) weighting. Model-dependent pharmacokinetic parameters were calculated from final polyexponential parameter estimates. Final model selection was performed after application of the Akaike information criterion (12) and examination of the coefficients of variation for the parameters estimated from a given model.

Individual values for the maximum concentration of pleconaril in plasma (C_max) and the time to the maximum concentration of pleconaril in plasma (T_max) were derived from the pharmacokinetic model. The area under the plasma concentration-versus-time curve from 0 to 24 h (AUC_0–24) was determined by the log-linear trapezoidal rule. Extrapolation of AUC_0–∞ was calculated by the summation of AUC_0–24 + Cp₂₄/λ, where Cp₂₄ is the plasma pleconaril concentration at 24 h and λ is the apparent terminal elimination rate constant. Total body clearance (CL) and steady-state volume of distribution (V_ss) were calculated from the AUC_0–∞ by correcting the apparent CL (CL/F) and V_ss (V_ss/F) for the relative bioavailability expressed by the ratio of AUC_Fasting to AUC_Fed. Statistical differences between pharmacokinetic parameters from the two arms were compared by using 90% confidence intervals for log-transformed values, analysis of variance, and two one-tailed t tests. The significance limit accepted for all statistical analyses was P = 0.05.

RESULTS

Twelve subjects completed the study, and their demographic data (means ± standard deviations [SD]) are as follows: age, 26.1 ± 5.3 years; weight, 72.6 ± 14.2 kg; gender, five males and seven females. The administration of pleconaril was well tolerated in all subjects, as no drug-associated adverse events attributed to pleconaril were reported by any of the participants during the study period. Additionally, no subject complained of poor palatability of the oral suspension formulation. Poststudy physical examination and laboratory values were within normal limits for all subjects and did not deviate significantly from prestudy values.

The plasma pleconaril concentration-versus-time data over a 24-h postdose period following a single oral dose in the fasting or fed state were best characterized by a one-compartment open model with first-order absorption. Composite plasma drug concentration-versus-time profiles from all study subjects for both the fed and fasting states are presented in Fig. 2.

FIG. 2.

Composite concentration-versus-time data for fed and fasting subjects. mcg, micrograms.

The mean (±SD) pharmacokinetic parameters for the two groups are provided in Table 1. The administration of the pleconaril solution in the presence of food significantly enhanced the extent of bioavailability for the drug. The mean AUC and C_max were increased 2.2- and 2.5-fold, respectively, in the presence of food. However, neither T_max nor absorption half-life values for the study periods were significantly different. Similarly, CL and V_ss, when adjusted for relative bioavailability, did not significantly differ between fed and fasting states. Mean plasma pleconaril concentrations remained measurable in both groups for 12 h after drug administration, although they were more than twofold higher in subjects in the fed state, and for 24 h after drug administration in the fed group.

TABLE 1.

Pharmacokinetic parameters for fed and fasting subjects^a

Subject group	AUC (mg/liter · h) [0.45]b	Cmax (mg/liter) [0.40]c	C12 (mg/liter) [0.44]d	C24 (mg/liter) [0.45]e	Tmax (h)	t1/2 abs (h)	t1/2 elim (h)	CL (liters/h/kg)	Vss (liters/kg)
Fed	9.08 ± 3.23	1.14 ± 0.58	0.253 ± 0.197	0.094 ± 0.048	4.68 ± 2.29	1.45 ± 0.70	6.74 ± 2.43	0.34 ± 0.12	1.94 ± 1.54
Fasting	4.08 ± 2.74 (<0.0001)	0.46 ± 0.30 (0.003)	0.112 ± 0.103 (0.033)	0.042 ± 0.048 (0.001)	3.89 ± 1.78 (NS)	1.16 ± 1.02 (NS)	4.38 ± 1.90 (0.019)	0.34 ± 0.12 (NS)	1.54 ± 3.41 (NS)

^aValues are means ± SD. Values in brackets are ratios of parameter values for the fasting group to those for the fed group. Values in parentheses are P values derived from a comparison of the fed and fasting groups; NS, not significant. C₁₂ and C₂₄, plasma concentrations at 12 and 24 h, respectively; t_{1/2 abs}, absorption half-life; t_{1/2 elim}, elimination half-life. 

^b90% confidence interval (CI₉₀), 0.24 to 0.58. 

^cCI₉₀, 0.31 to 0.64. 

^dCI₉₀, 0.02 to 0.87. 

^eCI₉₀, 0.13 to 0.78. 

DISCUSSION

Nonpolio enteroviruses (NPEV) are estimated to cause at least 10 to 15 million illnesses each year (9). The majority of these illnesses are minor; however, serious infections including aseptic meningitis, encephalitis, pericarditis, and myocarditis do result (2, 8). Currently, no enterovirus-specific antiviral agents are available, and management of patients with infection is primarily supportive (3). Intravenous immunoglobulin has been utilized successfully in acute and chronic infections; however, the response may be variable (1, 4). With new developments such as PCR techniques which increase the rapidity and sensitivity of diagnosis, early institution of specific antiviral therapy against enterovirus may play a potentially important role in managing acute, severe febrile illness and chronic infections as well as their sequelae (10).

Pleconaril is a novel antiviral which demonstrates potent activity against the rhinovirus and enterovirus members of the Picornaviridae. In vitro 50% tissue culture infectious dose assays of pleconaril against 15 NPEV clinical isolates have been conducted, and the 50% inhibitory concentrations (IC₅₀) for these serotypes are contained in Table 2. The concentration of pleconaril required to inhibit >90% of the 215 clinical isolates of the NPEV serotypes tested was 0.07 μg/ml (data not shown) (11). In the present study, this concentration was exceeded at 12 h following a single 200-mg oral pleconaril dose to subjects in the fed and fasting states, and in most subjects at 24 h in the fed state (Table 2). Additionally, animal studies using radiolabeled [¹⁴C]pleconaril demonstrated concentrations in the liver, nasal epithelium, brain, and plasma of 6.1 to 17.5, 4.2, 2.8, and 0.7 mg/l, respectively, 2 h following oral administration of the pleconaril solution. These data suggest that following oral administration pleconaril penetrates tissue where viral replication likely occurs, at concentrations severalfold in excess of those observed in the plasma (11).

TABLE 2.

In vitro antiviral activity of pleconaril against selected picornaviral clinical isolates (11)

Virus type (no. of clinical isolates tested)	IC50 (μg/ml)
	Median	Range
Coxsackievirus A9 (14)	0.006	0.002–0.058
Coxsackievirus B1 (13)	0.006	0.003–0.017
Coxsackievirus B2 (14)	0.005	0.002–0.717
Coxsackievirus B3 (15)	0.017	0.01–0.195
Coxsackievirus B4 (10)	0.062	0.053–0.115
Coxsackievirus B5 (16)	0.007	0.004–0.40
Echovirus 3 (15)	0.050	0.007–0.463
Echovirus 4 (18)	0.009	0.002–0.206
Echovirus 5 (8)	0.04	0.01–0.10
Echovirus 6 (19)	0.005	0.002–0.014
Echovirus 7 (11)	0.014	0.005–1.26
Echovirus 9 (9)	0.024	0.004–0.057
Echovirus 11 (20)	0.002	0.0006–0.007
Echovirus 24 (12)	0.01	0.0009–0.80
Echovirus 30 (21)	0.009	0.002–0.042

Following a single oral 200-mg dose of pleconaril in solution administered to healthy adults, the pharmacokinetics of the drug in plasma were best described by a simple one-compartment model with first-order absorption. This is in contrast to the polyexponential postpeak decay previously observed following single-dose administration of a capsule formulation of the drug in preclinical trials conducted in a manner similar to that of the study presented herein (11). The mean apparent elimination half-life (fed subjects, 6.7 ± 2.4 h; fasting subjects, 4.4 ± 1.9 h) of pleconaril following a single dose of solution was substantially lower than that previously observed (24.8 ± 12.3 h) after a single oral dose of the capsule formulation (11). The apparent difference may represent formulation-dependent changes in the absorption profile; however, it more likely represents the consequence of truncated postpeak sampling in the present study, which was necessary to accurately resolve both a distribution and an apparent terminal elimination phase from the curve-fitting procedure used.

The results of this study suggest that the extent of pleconaril absorption is significantly enhanced by the presence of food. However, the rate of absorption appears to remain unaffected as evidenced by an insignificant change in T_max. Although apparent CL and V_ss values were elevated in the fasting state, calculation of each parameter is AUC dependent, and correcting for changes in the extent of absorption results in apparent CL and V_ss values that are similar regardless of prandial state.

Finally, plasma pleconaril concentrations 12 h after a single 200-mg dose of oral solution fed and fasting subjects remain approximately 3.6- and 1.6-fold greater, respectively, than that which is required to inhibit 90% of NPEV in cell culture (Table 2). Accordingly, the data from the present study of pleconaril in adults suggest that a twice-daily dosing schedule for the oral solution formulation administered with a fat-containing meal would be appropriate in future clinical trials designed to assess the therapeutic efficacy and safety of this novel antiviral agent.