Intrapulmonary Pharmacokinetics of Laninamivir, a Neuraminidase Inhibitor, after a Single Nebulized Administration of Laninamivir Octanoate in Healthy Japanese Subjects

ABSTRACT

A single dose of laninamivir octanoate (LO) inhaled using a dry powder inhaler (DPI) is effective for the treatment and prophylaxis of influenza. Nebulizers are an option for pediatric and elderly patients who may have difficulty in using a DPI. A single-center, open-label study was conducted to evaluate the plasma and intrapulmonary pharmacokinetics (PK) of laninamivir after a single nebulized administration of LO in healthy male Japanese subjects for identifying a safe and effective dosage regimen for a nebulizer. A single dose of LO (40 to 320 mg) was administered using a nebulizer, and plasma concentrations of LO and laninamivir were analyzed up to 168 h after inhalation by validated liquid chromatography-tandem mass spectrometry methods. Subgroups of 6 subjects each underwent bronchoalveolar lavage at specified time intervals over 4 to 168 h following a single nebulized administration of LO (160 mg), and the concentrations in epithelial lining fluid (ELF) were calculated by the urea diffusion method. PK parameters were determined by noncompartment analysis. Inhaled nebulized LO was found to be safe and well tolerated up to the highest dose evaluated (320 mg). Plasma laninamivir concentrations increased almost dose proportionally. Laninamivir concentrations in ELF exceeded the 50% inhibitory concentrations for viral neuraminidase up to 168 h after the nebulized inhalation of 160 mg LO. Thus, similarly to the DPI, ELF concentration profiles of laninamivir after a single nebulized administration support its long-lasting effect against influenza virus infection. This study has been registered at JAPIC Clinical Trials Information (http://www.clinicaltrials.jp/) under registration no. JAPIC CTI-152996.

INTRODUCTION

Laninamivir octanoate (LO), an octanoyl prodrug of laninamivir, potently inhibits neuraminidase activity in various influenza A and B viruses, including subtypes N1 to N9, A(H1N1)pdm09, highly pathogenic avian influenza H5N1 viruses, and oseltamivir-resistant viruses (1, 2). A single dose of LO inhaled using the dry powder inhaler (DPI) is effective and well tolerated in adults and children for treatment (3, 4) and prophylaxis (5, 6) of influenza. It has been approved for the treatment and prophylaxis of influenza in Japan.

The evaluation of pharmacokinetics (PK) of laninamivir after the inhalation of a single dose of LO (40 mg) using a DPI revealed a long plasma half-life in healthy young adults (64.7 to 74.4 h) and renally impaired subjects (53.2 to 57.0 h) (7–9). Intrapulmonary pharmacokinetics of laninamivir administered using a DPI investigated by bronchoalveolar lavage (BAL) revealed that laninamivir concentration in epithelial lining fluid (ELF) exceeded the 50% inhibitory concentration (IC₅₀) for viral neuraminidases up to 240 h after inhalation (10). Laninamivir was retained in the trachea and lungs over long periods after a single intranasal/intratracheal administration of LO in mice and rats (11, 12). This prolonged retention of laninamivir in the respiratory tissues was explained by a consecutive series of LO uptake, hydrolysis, and limited efflux of laninamivir in mice (13). Both S-formylglutathione hydrolase (esterase D) and acyl-protein thioesterase 1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue (14). In addition, laninamivir bound to viral neuraminidase more stably in vitro than other neuraminidase inhibitors, including oseltamivir, zanamivir, and peramivir (15). These PK and binding characteristics support its potential as a long-acting neuraminidase inhibitor and its efficacy against influenza virus infection following a single treatment.

As nebulizers do not require patient coordination between inhalation and actuation, they are useful for pediatric, elderly, ventilated, and unconscious patients or those who are unable to use DPIs (16). In addition, nebulizers can deliver larger doses than those delivered by other aerosol devices; however, this will require a longer administration time.

The purpose of this study was to evaluate the PK of laninamivir after a single nebulized administration of LO in healthy adult subjects for identifying a safe and effective nebulizer regimen for those who may have difficulty in using DPIs.

(Selected results from this study were presented as abstracts at the 13th Congress of the European Association for Clinical Pharmacology and Therapeutics, Prague, Czech Republic, 2017 [17, 18].)

RESULTS

Part A—dose escalation plasma PK study.

Forty participants received LO, and all participants were included in the pharmacokinetic analyses. The subjects' age, weight, and body mass index (mean ± standard deviation [SD]) were 25.5 ± 5.5 years, 62.9 ± 5.7 kg, and 21.3 ± 1.5 kg/m², respectively. The plasma LO and laninamivir concentration profiles after a single nebulized administration of LO are presented in Fig. 1, and the pharmacokinetic parameters are summarized in Table 1. LO appeared rapidly in the plasma after administration, with median values of the time to maximum concentration (T_max) ranging from 2.0 to 2.5 h, and the plasma concentration decreased with the mean apparent elimination half-life (t_1/2) from 1.84 to 55.1 h across the doses, which was affected by the lower limit of quantitation (1 ng/ml) of the plasma sample analysis. The median T_max of laninamivir was 4.0 to 6.0 h, and laninamivir concentration decreased slowly after maximum concentration (C_max) was achieved, with the mean t_1/2 from 58.3 to 165.8 h. The C_max and area under the concentration (AUC)-time curve up to infinity (AUC_inf) for LO and laninamivir increased almost dose proportionally over 40 to 320 mg (Fig. 2).

FIG 1.

(A and B) Mean plasma laninamivir octanoate (A) and laninamivir (B) concentration profiles following a single inhalation of laninamivir octanoate using a nebulizer in healthy subjects. (C) Mean plasma concentration of laninamivir octanoate up to 24 h after administration. Symbols indicate the mean ± SD from 8 subjects.

TABLE 1.

Pharmacokinetic parameters of laninamivir octanoate and laninamivir in plasma after a single inhalation of laninamivir octanoate using a nebulizer in healthy subjects (part A)^a

Drug and parameter (n = 8 for all)	40 mg	80 mg	160 mg	240 mg	320 mg
Laninamivir octanoate
Cmax (ng/ml)	40.2 (9.8)	55.9 (20.6)	77.1 (7.7)	134.8 (36.2)	193.9 (62.0)
AUClast (ng · h/ml)	186.2 (49.4)	318.0 (143.8)	474.9 (57.2)	1,030 (390)	1,402 (315)
AUCinf (ng · h/ml)	193.1 (47.9)	325.3 (144.2)	485.2 (56.6)	1,102 (450)	1,504 (346)
Tmax (h)	2.0 (1.5, 3.0)	2.0 (0.5, 3.0)	2.5 (2.0, 3.0)	2.0 (1.5, 4.0)	2.5 (0.5, 6.0)
t1/2 (h)	1.84 (0.15)	3.11 (1.14)	4.01 (1.94)	34.1 (37.7)	55.1 (22.1)
CL/F (liters/h)	218.0 (51.5)	305.5 (174.2)	333.7 (39.3)	250.5 (100.5)	225.1 (63.3)
Vz/F (liters)	582.3 (164.3)	1,230 (494)	1,869 (780)	8,911 (8,439)	16,750 (7,707)
Laninamivir
Cmax (ng/ml)	10.8 (4.4)	14.3 (4.5)	26.6 (1.6)	40.0 (11.8)	54.8 (11.8)
AUClast (ng · h/ml)	256.5 (132.1)	524.8 (225.0)	1,040 (217)	1,735 (598)	2,597 (582)
AUCinf (ng · h/ml)	371.1 (159.4)	810.9 (396.4)	1,629 (609)	3,164 (2,043)	5,059 (2,650)
Tmax (h)	4.0 (4.0, 6.0)	6.0 (4.0, 6.0)	6.0 (6.0, 6.0)	6.0 (4.0, 6.0)	6.0 (4.0, 8.0)
t1/2 (h)	58.3 (20.7)	95.0 (42.8)	115.6 (46.1)	144.6 (64.4)	165.8 (78.6)

^aData are shown as mean (SD), except those for T_max, which are shown as median (minimum, maximum). C_max, maximum concentration; AUC_last, area under the concentration-time curve up to the time of the last measurable concentration data; AUC_inf, AUC values extrapolated to infinity; T_max, time to C_max; t_1/2, half-life; CL/F, apparent total body clearance; V_z/F, apparent volume of distribution.

FIG 2.

Mean and individual plasma pharmacokinetic parameters (C_max and AUC_inf) of laninamivir octanoate (A and B) and laninamivir (C and D) following a single inhalation of laninamivir octanoate using a nebulizer in healthy subjects. Each symbol represents an individual concentration, and the bar indicates the mean and SD from 8 subjects.

Part B—bronchoalveolar lavage study.

A total of 24 subjects were enrolled and evaluated. The subjects' age, weight, and body mass index (mean ± SD) were 24.9 ± 4.3 years, 61.3 ± 6.8 kg, and 20.6 ± 1.3 kg/m², respectively. Mean and individual concentrations of LO and laninamivir in ELF after a single inhaled administration of 160 mg LO are shown in Fig. 3, and mean concentrations of LO and laninamivir in the plasma, ELF, and alveolar macrophages (AM) are shown in Fig. 4. Drug concentrations in ELF and AM in each group are summarized in Table 2. The concentrations of LO and laninamivir in ELF were measurable in all samples examined. The C_maxs of LO and laninamivir in ELF were 10.30 and 1.46 μg/ml, respectively, and laninamivir in ELF decreased with a long t_1/2 (Table 3). Mean AUC up to 3.5 h after the inhalation (AUC_{3.5 h}) did not differ for LO and laninamivir among the time points of BAL (LO, 194 to 247 ng · h/ml, and laninamivir, 32.8 to 48.2 ng · h/ml). Both LO and laninamivir concentrations in ELF and AM were much higher than those in plasma (Fig. 4) and lasted for 168 h after dosing, with a longer t_1/2 than that in plasma.

FIG 3.

Mean and individual concentrations of laninamivir octanoate (A) and laninamivir (B) in epithelial lining fluid after a single inhalation of 160 mg laninamivir octanoate using a nebulizer in healthy subjects. Each symbol represents an individual concentration, and the line indicates the mean concentration.

FIG 4.

Mean concentrations of laninamivir octanoate (A) and laninamivir (B) in plasma, epithelial lining fluid, and alveolar macrophages after a single inhalation of 160 mg laninamivir octanoate using a nebulizer in healthy subjects. Symbols are the mean ± SD from 8 subjects.

TABLE 2.

Laninamivir octanoate and laninamivir concentrations in epithelial lining fluid and alveolar macrophages at each time point^a

Time (h)	Laninamivir octanoate		Laninamivir
	ELF (μg/ml)	AM (μg/ml)	ELF (μg/ml)	AM (μg/ml)
4	10.25 (2.77)	3,143 (2,238)	1.46 (0.35)	125 (61)
24	3.11 (1.99)	3,860 (2,251)	1.10 (0.60)	480 (256)
72	0.43 (0.31)	805 (460)	0.59 (0.33)	296 (151)
168	0.20 (0.06)	228 (119)	0.64 (0.14)	277 (129)

^aData are expressed as the mean (SD). ELF, epithelial lining fluid; AM, alveolar macrophages.

TABLE 3.

Pharmacokinetic parameters of laninamivir octanoate and laninamivir after a single inhalation of 160 mg laninamivir octanoate using a nebulizer in healthy subjects (part B)^a

Drug and sample type	Cmax (μg/ml)	Tmax (h)	AUClast (μg · h/ml)	AUCinf (μg · h/ml)	t1/2 (h)
Laninamivir octanoate
Plasma	0.0825 (0.0046)	2.0	1.07 (0.07)	NC	NC
ELF	10.30 (0.85)	4.0	269 (31)	281	39.9
AM	3,860 (919)	24.0	238,000 (35,800)	NC	NC
Laninamivir
Plasma	0.0240 (0.0014)	3.5	1.18 (0.07)	1.57	86.5
ELF	1.46 (0.14)	4.0	128 (13)	329	219
AM	480 (105)	24.0	52,400 (6,240)	NC	NC

^aPharmacokinetic parameters were estimated using a sparse sampling option in WinNonlin and represented as mean (standard error) for C_max and AUC_last. ELF, epithelial lining fluid; AM, alveolar macrophages; NC, not calculated; C_max, maximum concentration; T_max, time to C_max; AUC_last, area under the concentration-time curve up to the time of the last measurable concentration data; AUC_inf, AUC values extrapolated to infinity; t_1/2, half-life.

Safety.

The treatment-emergent adverse events (TEAEs) in part A and part B have been listed in Tables 4 and 5, respectively. All TEAEs were mild and transient. Among the 64 participants who received LO, 19 experienced TEAEs. Two TEAEs were judged to be drug related in part A. One subject in the 40-mg group had blood in the urine, and 1 subject in the 80-mg group had increased blood uric acid levels. No TEAE was judged to be drug related in part B. The elevation in C-reactive protein (CRP) levels in 10 participants considered to be related to BAL was reported in part B (Table 5). No notable mean changes from the baseline were recorded in the vital signs or clinical laboratory variables, and none of the individual participant values outside the laboratory reference ranges were considered to be clinically significant.

TABLE 4.

Number of subjects with treatment-emergent adverse events in part A

System organ class (preferred terma)	No. (%) of subjects at doseb:
	40 mg	80 mg	160 mg	240 mg	320 mg	All
Total no. of treatment-emergent adverse events	2 (25.0)	1 (12.5)	2 (25.0)	1 (12.5)	2 (25.0)	8 (20.0)
Infections and infestations	1 (12.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)	2 (5.0)
Nasopharyngitis	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)	1 (2.5)
Enteritis, infectious	1 (12.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.5)
Musculoskeletal and connective tissue disorders	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)	1 (2.5)
Back pain	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)	1 (2.5)
Investigations	1 (12.5)	1 (12.5)	2 (25.0)	1 (12.5)	0 (0.0)	5 (12.5)
Blood creatine phosphokinase increased	0 (0.0)	0 (0.0)	1 (12.5)	0 (0.0)	0 (0.0)	1 (2.5)
Blood triglycerides increased	0 (0.0)	0 (0.0)	1 (12.5)	0 (0.0)	0 (0.0)	1 (2.5)
Blood uric acid increased	0 (0.0)	1 (12.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.5)
C-reactive protein increased	0 (0.0)	0 (0.0)	1 (12.5)	0 (0.0)	0 (0.0)	1 (2.5)
Blood urine present	1 (12.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.5)
White blood cell count increased	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)	0 (0.0)	1 (2.5)
Blood alkaline phosphatase increased	0 (0.0)	0 (0.0)	1 (12.5)	0 (0.0)	0 (0.0)	1 (2.5)

^aTerminology is according to MedDRA version 18.1.

^bn = 8 subjects for each dose, with a total of 40 subjects (All).

TABLE 5.

Number of subjects with treatment-emergent adverse events in part B

System organ class (preferred terma)	No. (%) of subjects (n = 24) with events:
	Caused by BAL	Unrelated to BAL
Total no. of treatment-emergent adverse events	11 (45.8)	1 (4.2)
Nervous system disorders	1 (4.2)	0 (0.0)
Headache	1 (4.2)	0 (0.0)
General disorders and administration site conditions	1 (4.2)	0 (0.0)
Chest pain	1 (4.2)	0 (0.0)
Pyrexia	1 (4.2)	0 (0.0)
Investigations	10 (41.7)	1 (4.2)
C-reactive protein increased	10 (41.7)	0 (0.0)
White blood cell count increased	1 (4.2)	0 (0.0)
Forced vital capacity decreased	0 (0.0)	1 (4.2)

^aTerminology is according to MedDRA version 18.1.

DISCUSSION

A single administration of 40 to 320 mg of LO inhaled using a nebulizer was safe and well tolerated in healthy adult subjects. A high intrapulmonary concentration of laninamivir was observed at 4 h after a single nebulized dose of 160 mg LO, which lasted for 7 days. The concentration of laninamivir (molecular weight, 346.34) in ELF exceeded the mean in vitro 50% inhibitory concentration (IC₅₀) for influenza viral neuraminidases [1.70, 3.98, and 14.86 nM for subtype A(H1N1)pdm09, A(H3N2), and B neuraminidases, respectively] in the 2013–2014 season (19). As the plasma protein binding of laninamivir was less than 0.1% (7) and the albumin concentration in ELF was more than 10-fold lower than that in plasma (20), the observed laninamivir concentration in ELF could be deemed to be similar to the unbound laninamivir concentration in ELF. The mean laninamivir concentration in ELF at 168 h was 0.64 μg/ml, which is more than 100 times the IC₅₀ for influenza virus neuraminidases mentioned above. These PK profiles support LO as a long-acting neuraminidase inhibitor after nebulized administration.

While a nebulizer is an option and has been a commonly used aerosol device, there is no established method to extrapolate the therapeutic dose for a nebulizer from that of a DPI. A standardized in vitro method using a cascade impactor can be used to evaluate the particle size distribution of drug substances, as the particle size is a key factor in pulmonary drug delivery (21). In this study, the nebulized formulation of 160 mg LO was designed such that the drug delivery was similar to that of 40 mg LO using DPI by an in vitro method (A. Ito and S. Yada, unpublished data). The drug concentration in the therapeutic target tissue is another marker to consider for setting an appropriate nebulizer dose. The concentration of laninamivir in ELF after inhalation of 40 mg LO using a DPI in healthy adults was previously evaluated to demonstrate the distribution of laninamivir in the lungs and to consider its long-acting efficacy based on its pulmonary PK (10). The mean C_max (standard error [SE]) of laninamivir in ELF after administration of 40 mg LO with a DPI was 2.40 (1.67) μg/ml, and laninamivir in ELF decreased with a long half-life (241 h). The mean concentration (SD) of laninamivir in ELF at 168 h after administration with a DPI was 0.18 (0.14) μg/ml. In this study, the C_max of laninamivir in ELF after the nebulized administration of 160 mg LO was almost comparable (1.46 [0.14] μg/ml) to the value obtained from the DPI, and the laninamivir concentration in ELF decreased with a similar long half-life (219 h). The laninamivir concentration in ELF at 168 h after the nebulized administration was 0.64 (0.14) μg/ml, which was relatively higher than that obtained with DPI administration. The estimated AUC up to the last measurable concentration (AUC_last) in ELF after administration with a nebulizer was higher than that after administration with a DPI (128 versus 69.4 ng · h/ml), although ELF sampling was performed over a longer period after dosing with a DPI (240 h) than with a nebulizer (168 h). Though the ELF concentration would not necessarily be a definitive marker for the selection of therapeutic dose for the treatment and prophylaxis of influenza using neuraminidase inhibitors, these data indicate that a single nebulized administration of 160 mg LO may be effective in adult patients for treatment and prophylaxis of influenza, as demonstrated by administration of 40 mg with a DPI. With these data, a phase 3 study is under way in Japan to evaluate the efficacy and safety of 160 mg nebulized LO in the treatment of influenza virus infection among adults and children (not less than 10 years of age) (JAPIC CTI-163397).

The plasma concentrations of LO peaked relatively slowly after nebulized administration (median T_max, 2 to 2.5 h) compared with DPI administration (median T_max, 0.25 h) (8). The T_max of laninamivir also showed a similar tendency. The C_maxs of LO and laninamivir after the nebulized administration of 160 mg LO were lower than that with 40 mg LO using a DPI, and the mean AUC_inf of LO decreased by almost half (1,023 versus 485.2 ng · h/ml). However, the mean AUC_inf of laninamivir did not change with the formulation (1,379 versus 1,629 ng · h/ml). The reasons for this difference in PK between the 2 formulations remain unclear. Lung surfactant, which covers the lung epithelium, can have active roles, including drug dissolution by solubilization, diffusion, and partitioning, in drug delivery of inhaled drugs (22). Therefore, LO inhaled by nebulization would provide different conditions for the lung surfactant from the dry powder conditions, which may affect the absorption from the lung into systemic circulation and cause a delay in C_max, in addition to the change in metabolic rate of LO to laninamivir. Whether these differences in PK would have a clinically meaningful impact on the efficacy will be clarified in the ongoing phase 3 study.

The mean plasma C_max and AUC increased almost dose proportionally after nebulized administration and have large interindividual variations in the PK parameters among the higher-nebulized-dose groups (240 and 320 mg). As the higher volume of nebulized formulation was necessary for the higher doses (2 ml for 40- to 160-mg dose, 3 ml for 240-mg dose, and 4 ml for 320-mg dose), the longer inhalation time for the higher doses (the planned administration times for the nebulized 40- to 160-mg, 240-mg, and 320-mg doses were 5, 7.5, and 10 min, respectively) may cause some difficulties to subjects, who are required to maintain the same sitting position and to breathe consistently until the completion of dose administration, which would result in changes in the PK. This factor may cause larger interindividual variations among the higher-dose groups.

High accumulation of LO and laninamivir in AM was observed after a single inhalation of LO (Fig. 4). A similarly high accumulation in AM was also observed in several human BAL studies after the oral administration of antibiotics (23–25). The nonspecific distribution in addition to transporter(s) might contribute to the high accumulation in AM. Although the mechanism of uptake of LO and laninamivir to AM is unclear, the high concentrations of LO and laninamivir in AM even after washing AM with ice-cold buffer to minimize nonspecific binding of drugs to AM would be caused by the strong binding to and/or uptake into the cells.

For children aged less than 10 years, a single dose of 20 mg LO inhaled using a DPI has been approved in Japan for the treatment and prophylaxis of influenza. However, DPIs are not generally recommended for use in children less than 4 years of age as they cannot generate sufficiently high airflow through the devices and may not be able to reliably use a DPI (26). As pediatric patients are different from adult patients with respect to airway anatomy and breathing patterns and there is no established method to extrapolate the therapeutic nebulized dose for children from that of adults, in vitro measures of aerosol particle size with breathing patterns are quite important parameters to consider in the case of children. As the drug delivery of a nebulized dose of 160 mg LO in children was similar to or higher than that of 20 mg LO using a DPI in vitro (A. Ito and S. Yada, unpublished data), another phase 3 study for children is under way in Japan to evaluate the efficacy and safety of nebulized 160 mg LO in the treatment of influenza virus (JAPIC CTI-163398).

Although BAL is a useful technique for the evaluation of the intrapulmonary PK of drugs, it is difficult to evaluate the local drug distribution in the respiratory tract. The local distribution and retention of the drug at the site of influenza virus infection are important factors in the efficacy consideration of a topical neuraminidase inhibitor such as LO or zanamivir. The inhaled LO, which contains particles of various sizes, would not be expected to distribute evenly in the respiratory tract. The ELF concentration evaluated by BAL reflects the mean concentration where the bronchofiberscope reached and BAL was performed. As the same BAL evaluation as that conducted with a DPI was technically performed in this study, the comparison of the ELF drug concentrations between a DPI and a nebulizer provides important information for the consideration of appropriate dose regimens for LO.

LO was well tolerated by all the subjects following the administration of a single nebulized dose. Even with the high exposure attained in the dose escalation plasma PK study, no clinically significant changes in the physical examination, vital signs, or laboratory measurements were observed during the course of the study.

Laninamivir distributed and lasted in the lungs for 7 days after a single nebulized dose of LO. The laninamivir concentration profile in ELF would support its long-lasting effect in treating patients with influenza virus infection with a single nebulized dose.

MATERIALS AND METHODS

Study design and subjects.

This open-label, single-dose study was conducted at Osaka Pharmacology Clinical Research Hospital (Osaka, Japan) from August 2015 to February 2016. The study protocol was approved by the institutional review boards of the study site. The study was conducted in accordance with the guidelines on good clinical practice and ethical standards for human experimentation established by the Declaration of Helsinki Principles. All subjects gave written informed consent. The clinical trial registry's URL and the registration number are as follows: http://www.clinicaltrials.jp/ and JAPIC CTI-152996.

Healthy male Japanese subjects between the ages of 20 and 45 years, with normal body mass index (18.5 to 25.0), were eligible for inclusion in the study. Subjects were excluded from this study if any of the following conditions existed: evidence of organ dysfunction or any clinically significant deviation from the normal range in physical examination, vital signs, electrocardiograms (ECG), or clinical laboratory findings; hypersensitivity to a neuraminidase inhibitor, tyloxapol, lidocaine, atropine, or midazolam; forced expiratory volume in 1 s percentage (FEV_1.0%) of <70% or other pulmonary function test abnormality; history of alcohol abuse or drug abuse; a smoking experience (part B only); positivity for hepatitis B virus surface antigen, hepatitis C virus antibody, syphilis, Treponema pallidum antibody, or HIV antibody; donation of more than 200 ml of blood within 28 days before the screening test, more than 400 ml within 84 days, or more than 1,200 ml within a year or use of apheresis within 14 days; previous participation in a CS-8958 clinical study; and inability to communicate satisfactorily with the investigator.

Drug administration.

A lyophilized formulation for nebulized administration of LO was supplied by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). LO was suspended in saline and inhaled as a suspension using a commercially available nebulizer, PARI LC Sprint and PARI BOY SX compressor (Pari GmbH, Starnberg, Germany). The subjects were instructed to inhale each dose using quiet tidal breathing. LO was administered in a sitting position, and the subjects were prohibited from resting in a supine position for 2 h after inhalation. Subjects fasted for more than 10 h before and 4 h after dosing in part A. Subjects fasted for more than 4 h before and 2 h after dosing and 4 h before and 2 h after BAL in part B.

Part A—dose escalation study.

Subjects were randomly assigned to 5 groups of 8 subjects each to receive a single nebulized dose of 40, 80, 160, or 320 mg LO in each group.

Part B—BAL study.

Subjects were allocated to 4 groups of 6 subjects each according to the time of BAL. All subjects were administered a single nebulized dose of 160 mg LO. This dose was selected for the BAL study considering the in vitro particle size distribution using a DPI and a nebulizer, the plasma PK profiles from part A, and those obtained from the DPI. The results suggested that the effect of a single administration of 160 mg LO using a nebulizer was similar to that of 40 mg using a DPI. BAL was performed at 4, 24, 72, and 168 h after dosing.

Each subject underwent fiber-optic bronchoscopy with BAL once at their respective time after LO dosing. The blood pressure, respiratory rate, and heart rate of each subject were recorded before, at the completion of, and at approximately 1 h after bronchoscopy. Oxygenation was monitored by fingertip oximetry throughout the procedure. As a pretreatment, inhalation of 1% lidocaine through a nebulizer and injection of atropine sulfate were administered 15 min before fiber-optic bronchoscopy. Following this, regional anesthesia of the pharynx was performed with lidocaine through a Jackson-type spray apparatus; systemic sedation was not used. The bronchoscope was inserted up to the right middle lobe and wedged. BAL was performed by infusion of four 50-ml volumes of sterile saline into the subsegmental bronchus of the right middle lobe, and each specimen was immediately aspirated. The instillation of a 50-ml volume of saline and its aspiration were performed within 1 min. The volume of each aspirate from the instillations was immediately measured, and the aspirate was kept on ice. The aspirate of the first aliquot was maintained separately, and the aspirates of the remaining 3 aliquots were pooled.

The BAL fluid was filtered through sterile gauze and centrifuged at 400 × g for 5 min at 4°C. The supernatants were separated, and the volumes were measured. An aliquot of the supernatant was stored at −70°C for urea nitrogen measurement. Acidified 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) solution, used as an enzyme inhibitor to minimize LO degradation, was added to the remaining supernatants (1:40, vol/vol) and mixed to prepare BAL fluid samples. The sediment cells from the BAL fluid were washed twice with an aliquot of 1 ml of ice-cold phosphate-buffered saline (PBS) and centrifuged at 400 × g for 5 min at 4°C. Part of the cells was suspended in an aliquot of 1 ml of ice-cold PBS for determining the cell counts with a hemocytometer. A differential count of BAL cells was performed with a stained cytocentrifuged sample of BAL fluid. The remaining BAL cells and supernatants were stored at −70°C until analysis of LO and laninamivir concentrations.

Blood samples for LO, laninamivir, and urea concentrations.

Blood (5 ml) was collected into Vacutainers containing heparin as an anticoagulant at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 120, and 168 h after dosing in each treatment period in part A and at predose and 0.25, 2, and 3.5 h after dosing and 30 min before each time point of BAL in part B. After the addition of 0.125 ml of AEBSF to 5 ml of blood, followed by centrifugation at 1,700 × g for 10 min at 4°C, the plasma supernatants were stored at −20°C until the assay. After the completion of BAL, blood samples (2 ml) that were collected in Vacutainers containing heparin for determining urea concentration were centrifuged, and plasma was stored at −70°C until the assay.

Sample analysis.

LO and laninamivir concentrations in plasma were analyzed by solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive ionization mode (10) at Covance Laboratories Ltd. (North Yorkshire, United Kingdom). The deuterated forms of LO and laninamivir were used as the internal standards to measure the plasma concentrations of LO and laninamivir, respectively. The lower limit of quantitation for the plasma assay was 1 ng/ml, and the linear calibration range was 1 to 1,000 ng/ml. The interassay accuracies of the plasma quality control (QC) samples for LO and laninamivir were within 100.0 to 102.1% and 98.7 to 100.0% of nominal concentration, respectively, and the interassay precision was less than 10%. Coefficients of variation (CVs) of the intra-assay QC samples for LO and laninamivir in plasma were 2.1 to 4.7% and 2.8 to 11.5%, respectively.

LO and laninamivir in BAL fluid and AM were extracted by deproteinization using methanol and determined by validated LC-MS/MS methods at Shin Nippon Biomedical Laboratories, Ltd. (Wakayama, Japan). Intraday, interday, and dilution reproducibility; freeze-thaw stability; and storage stability were evaluated, and the results met the predefined acceptance criteria. The lower limit of quantitation was 0.1 ng/ml, and the linear calibration range was 0.1 to 10 ng/ml. CVs of the intra-assay QC samples (AEBSF-spiked pulmonary surfactant in physiological saline) for LO and laninamivir were 3.8 to 4.7% and 2.7 to 3.1%, respectively, and those of the interassay samples were 3.2 to 4.1% and 2.4 to 4.7%, respectively.

The concentrations of urea in plasma and BAL supernatant were analyzed by a modified diagnostic kit (Urea N B; Wako Pure Chemical Industries, Ltd., Tokyo, Japan), which was validated and measured at Shin Nippon Biomedical Laboratories, Ltd. (Kagoshima, Japan). Plasma was diluted 200-fold with physiological saline before measurement. The absorbance of standards, samples, and QC samples was measured at 570 nm against the absorbance of the blank. QC samples with concentrations of 7.5, 15, and 35 mg/dl were run with every standard curve. Standard curves ranging from 5 to 50 mg/dl were linear (r > 0.99). CVs of the intra- and interassay QC samples for urea concentration were 0.5 to 4.9% and 0.5 to 7.1%, respectively.

Determination of ELF volume and drug concentration in ELF and AM.

The ELF volume was determined by the urea dilution method (20). As urea diffuses readily through the body, which results in identical concentrations in plasma and ELF, it was used as an endogenous marker of ELF dilution. The volume of ELF in BAL fluid (V_ELF) was derived using the equation V_ELF = V_BAL × (urea_BAL/urea_plasma), where V_BAL is the volume of aspirated BAL fluid, urea_BAL is the concentration of urea in supernatant BAL fluid, and urea_plasma is the concentration of urea in plasma. As erythrocytes were minimal (<1,000 cells/μl) in all BAL fluids in which erythrocyte levels were determined, the urea concentration was not corrected for possible contamination with urea from blood (27).

The drug concentrations in ELF (C_ELF) were determined as follows: C_ELF = C_BAL × (urea_plasma/urea_BAL), where C_BAL is the measured concentration of LO or laninamivir in the supernatant of BAL fluid. The calculation of C_ELF was performed with BAL fluids recovered from the first aliquot and the remaining aliquot.

The drug concentrations in AM (C_AM) were determined as follows: C_AM = A_AM/V_AM, where A_AM is the measured amount of LO or laninamivir in a cell suspension and V_AM is the volume of AM in a cell suspension. Cell count by type was performed to determine the number of AM. A reported AM volume of 2.42 μl/10⁶ cells was used for estimating the volume of AM in the suspension (20).

Pharmacokinetic analysis.

The pharmacokinetic parameters were calculated by noncompartment analysis using the computer software Phoenix WinNonlin (version 6.2; Certara G.K., Tokyo, Japan).

For part A, the C_max and T_max were obtained by observation. The apparent elimination t_1/2 was obtained by linear regression of 3 or more log-transformed data points in the terminal phase. The AUC_last was obtained by the linear trapezoidal method. For single-dose studies, the AUC values were extrapolated to infinity (AUC_inf) using the equation AUC_last + C_tz/λ_z, where C_tz is the last measurable concentration and λ_z is the terminal elimination rate.

For part B, AUC_{3.5 h} was obtained by the linear trapezoidal method. AUC_last, C_max, T_max, t_1/2, and standard errors for AUC_last and C_max were estimated using a sparse sampling option in WinNonlin.

Safety.

The safety was assessed by clinical evaluation (including physical examinations, vital signs, and 12-lead ECG) and laboratory measurements (including hematology, serum chemistry, and urinalysis). AEs were assessed by questioning and spontaneous reporting. A 12-lead ECG and chest X-rays were performed at screening, 1 day before the administration of LO, 1 day after BAL, and the end-of-study visit. Investigators evaluated all the clinical AEs in terms of intensity (mild, moderate, or severe), duration, severity, outcome, and relationship with the study drug.