Plasma concentrations of disodium cromoglycate after various inhalation methods in healthy subjects

Abstract

Aims

To compare the plasma concentrations of disodium cromoglycate (DSCG) following various inhalation procedures in healthy volunteers.

Methods

Nine healthy subjects inhaled 2 mg of aerosol, 20 mg of nebuliser solution only, 20 mg of nebuliser solution mixed with isotonic saline, or 20 mg of nebuliser solution mixed with saline and procaterol, a β₂-adenoceptor agonist, on separate occasions 2–3 weeks apart. Plasma concentrations of DSCG were determined by high-performance liquid chromatography (h.p.l.c.).

Results

The peak plasma concentrations of DSCG were 1.5±0.7 (range 0.4–2.4) ng ml⁻¹ in the aerosol group, 8.8±6.2 (range 5.3–19.9) ng ml⁻¹ in the nebuliser solution only group, 17.2±16.3 (range 5.0–38.6) ng ml⁻¹ in the nebuliser solution plus isotonic saline group, and 24.5±11.9 (range 10.2–44.9) ng ml⁻¹ in the nebuliser solution plus saline and procaterol group. Thus subjects who used the nebuliser had markedly higher plasma concentrations of DSCG than subjects who used the aerosol inhaler.

Conclusions

These findings may have important implications for the evaluation of inhalation treatment with DSCG for bronchial asthma.

Introduction

Disodium cromoglycate (DSCG, Intal®) has been used to treat bronchial asthma for about 30 years [1, 2]. DSCG was previously administered by a nebuliser or an inhalation device known as Spinhaler®, but is currently available in an aerosol inhaler (Aerosol®, metered dose inhaler) or as a nebulized solution mixed with saline [3] and/or a β₂-adrenergic receptor agonist [4]. However, data on the plasma concentrations of DSCG result up from the various inhalation methods in children and adults are limited because of a lack of sensitive analytical methods. Therefore, reliable pharmacokinetic data for selecting the optimal method of inhalation are not available. We determined the plasma concentrations of DSCG resulting from various inhalation methods using an improved high-performance liquid chromatography (h.p.l.c.) method [5, 6].

Methods

Subjects and treatment

We studied nine healthy Japanese volunteers (7 males aged 31–36 years, weight: 60–78 kg; 2 females aged 32–37 years, weight: 45–52 kg). There were no significant differences in the distribution of subjects by age, gender, or body size among the aerosol group, the nebuliser solution only group, the nebuliser solution plus isotonic saline group, and the nebuliser solution plus saline and the β₂-adrenoceptor agonist, procaterol group. Written informed consent was obtained from all subjects. Routine biochemical and haematological parameters were within normal ranges. No subjects had any symptoms of allergic disorders. Subjects followed a normal diet and were not receiving any other medications. They were trained in the use of the nebuliser or aerosol inhaler to minimize errors in drug inhalation technique and gargled immediately after inhalation to limit the absorption of DSCG from the oral mucosa. Each subject inhaled DSCG as an aerosol (n = 6), a nebuliser solution only (n = 5), a nebuliser solution plus isotonic saline (n = 8), and a nebuliser solution plus saline and procaterol (n = 6). In the aerosol group, the subjects inhaled the usual clinical dose of two puffs (1 mg puff⁻¹) as follows: with the mouthpiece of the aerosol inhaler inserted in the mouth, the subject performed forced expiration and then a deep forceful inspiration to inhale the drug, holding the breath for 5 s. After an interval of 10 s, the second puff was inhaled. In the nebuliser solution groups, subjects inhaled 2 ml of DSCG (10 mg ml⁻¹) only, 2 ml of DSCG plus 5 ml of isotonic saline, or 2 ml of DSCG plus 5 ml of isotonic saline and 0.3 ml of procaterol (0.1 mg ml⁻¹) through an ultrasonic nebuliser (NE-U07, Omron, Tokyo, Japan). On each occasion the drug solution was inhaled using tidal breathing with a face mask until the nebulized mist was not visible to the naked eye. No attempt was made to regulate or measure the frequency or depth of inhalation. The individual tests with the different DSCG formulations were separated by 2–3 weeks for each subject.

Before starting each test, a cannula was inserted into a forearm vein for blood sampling. Blood samples for drug determination were obtained prior to inhalation and at 5, 10, 30, 60, 120, and 480 min after inhalation. Blood samples were drawn into tubes containing EDTA, and were then centrifuged at 3000 rev min⁻¹ to separate the plasma which was stored in polystyrene tubes at −20° C until assayed. A solution consisting of 1.0 ml of plasma, 2.0 ml of distilled water (Nacalai Tesque, Inc., Kyoto, Japan), 0.5 ml of 10 N HCl (Nacalai), and 5.0 ml of ethyl acetate (Nacalai) was pipetted into a 10 ml glass-stoppered centrifuge tube. The mixture was shaken for 10 min and centrifuged at 2700 rev min⁻¹ for 10 min. A 4.0 ml aliquot of the supernatant organic phase and 0.2 ml of Titrisol® (Merck, Darmstadt, Germany), a buffer solution with a pH of 4.0, were placed in another 10 ml glass-stoppered tube, and the mixture was shaken andcentrifuged at 2700 rev min⁻¹ for 10 min. The aqueous phase was dried with an evaporator, dissolved in 50 μl of Titrisol® and 20 μl aliquots used for the h.p.l.c. analysis.

Reversed-phase h.p.l.c. was performed at 40° C using a composite system that consisted of a pump (LC-4AD, Shimadzu, Kyoto, Japan), a variable-wavelength u.v. detector (SPD-6 A, Shimadzu), and a column oven (CTO-6 A, Shimadzu). The chromatograms were recorded and integrated with a chromatographic data system (C-R4 A, Shimadzu). The stainless-steel column (250×4.0 mm internal diameter) was packed with LiChrosorb NH₂ (particle size 5 μm, GL-Science, Tokyo, Japan). A guard column (10×4.0 mm internal diameter) was packed with LiChrosorb CN (particle size 5 μm, GL-Science). The analyses were performed with a mobile phase composed of distilled water, 10 g KH₂PO₄ (Nacalai) and 0.5 ml H₃PO₄ (Katayama Chemical Ind., Osaka, Japan) per 1450 ml acetonitrile (Nacalai) (35:65, vol.%). The flow rate was set at 2.0 ml min⁻¹, and the detection wavelength was 254 nm.

Calibration curve

The plasma concentration of DSCG was calculated using a calibration curve, which was constructed by plotting the relationship between the plasma concentration and the peak area of DSCG. Five sets of plasma samples containing 0, 2.5, 5, 10, 25, and 50 ng ml⁻¹ of DSCG were prepared.

Pharmacokinetic parameters

The area under the curve of plasma drug concentration vs time curve was calculated by the linear trapezoidal rule up to 480 min (AUC 0,480 min). The peak plasma concentration (C_max) was obtained by inspection of the data.

Statistics

Results are presented as the mean±s.d. (or range) as appropriate. Differences between paired groups were assessed by the unpaired Student's t-test for variables with equal value distributions. The Mann–Whitney U-test was used for other analyses. Post-hoc comparisons among groups were made using analysis of variance with Fisher's test for multiple comparisons. Correlations were calculated by Spearman's rank correlation test. A level of P less than 0.05 was accepted as statistically significant.

Results

The chromatograms of a standard sample with 50 ng ml⁻¹ DSCG and an extracted plasma sample from a subject who inhaled DSCG via an aerosol inhaler showed no interference from other u.v.-absorbing substances. The retention time of DSCG was 4.8±0.4 min. The lower limit of detection for DSCG was 0.1 ng ml⁻¹. The efficiency of extraction of DSCG from plasma was 92.7±9.5% at a plasma concentration of 10 ng ml⁻¹. The coefficients of variation (CV; n = 5) for intra-assay and interassay were 3.3% and 4.1%, respectively.

The mean C_max of DSCG was significantly higher in the nebuliser solution only group (8.8±6.2 ng ml⁻¹) than in the aerosol group (1.5±0.7 ng ml⁻¹). With both methods, C_max ocurred 30 min after inhalation and the drug was detectable 480 min after inhalation in all subjects (Figure 1). The AUC(0, 480 min) was also significantly higher in the nebuliser solution only group (2680±2180; range 1180–5910 ng ml⁻¹ min) than in the aerosol group (246±117; range 112–432 ng ml⁻¹ min).

Figure 1.

Plasma concentrations of DSCG (mean±s.d.) in the nebuliser solution only group (•, n = 5), and the aerosol group (▴, n = 6). *P<0.05, +P<0.01 vs the nebuliser solution only group.

Amongst the nebuliser groups, the mean C_max of nebulized DSCG varied considerably and was 8.8±6.2 (range 5.3–19.9) ng ml⁻¹ in the nebuliser solution only group, 17.2±16.3 (range 5.0–38.6) ng ml⁻¹ in the nebuliser solution plus isotonic saline group, and 24.5±11.9 (range 10.2–44.9) ng ml⁻¹ in the nebuliser solution plus saline and procaterol group. Thus the C_max was highest in the nebuliser plus saline and procaterol group, but the difference was not significant (P = 0.08). Although the plasma concentrations of DSCG tended to be higher in the nebuliser plus saline group than in the nebuliser solution only group, the difference was not significant (Figure 2). The AUC(0, 480 min) of nebulized DSCG also varied considerably and was 2680±2180 (range 1180–5910) ng ml⁻¹ min in the nebuliser solution only group, 4950±4400 (range 1150–14200) ng ml⁻¹ min in the nebuliser solution plus isotonic saline group, and 4830±2840 (range 2410–10100) ng ml⁻¹ min in the nebuliser solution plus saline and procaterol group. Thus AUC(0, 480 min) was somewhat higher in the nebuliser plus saline and procaterol group, but the difference was again not significant.

Figure 2.

Plasma concentrations of DSCG (mea±s.d.) in the nebuliser solution only group (•, n = 5), the DSCG with saline group (▪, n = 8), and the DSCG with saline and procaterol group (▴, n = 6). *P<0.05 vs DSCG with saline and procaterol group.

Discussion

The clinical dose of DSCG has not been determined using pharmacokinetic data from patients but is adjusted according to clinical response due to the lack of readily available accurate methods for measuring clinically relevant plasma concentrations of DSCG. We have improved the h.p.l.c. method established by Curry and coworkers [6] by incorporating an evaporation technique into the extraction process. This method is simpler than radioimmunoassay [7, 8] but is just as sensitive, and the measurements can be completed in less than 6 min.

In the present study, plasma concentrations of DSCG were higher in the nebuliser inhalation groups than in the aerosol group. The low inspiratory flow during tidal breathing results in deeper penetration and better absorption of the drug through the bronchial epithelium, to its target site of action [9]. A poor aerosol inhalation technique may result in the adhesion of a greater amount of the drug to the throat and a decrease in the quality of drug that is delivered to the lungs [10]. Thus, the usual clinical dose in the aerosol (two puffs containing 2 mg) may be smaller than that in the nebuliser solution (20 mg) for acquisition of a similar therapeutic effect.

Although not statistically significant, the plasma concentrations of DSCG were somewhat higher in the nebuliser solution plus saline group than in the nebuliser solution only group. The increased osmotic pressure provided by the saline mixture may result in a greater absorption of drug by the lungs. The plasma concentrations of DSCG were highest in the nebuliser solution plus saline and procaterol group, but not statistically significant. This method resulted in a greater deposition of DSCG in the bronchial epithelium, presumably due to the bronchodilator effect of procaterol, a β₂-adrenergic receptor agonist.

There are few studies on the relationship between the plasma concentration of DSCG and its prophylactic effects on patients with bronchial asthma. Patel et al. reported that aerosol and nebulized DSCG had a maximum efficacy rate of 66% with 30 min plasma concentrations of 4 ng ml⁻¹ and above in adult patients with exercise-induced asthma [11]. The present findings suggest that the standard aerosol dose may not be sufficient to achieve this plasma concentration. Yahav et al. suggested that the clinical situation in asthmatic children administered DSCG by a Spinhaler® was not substantially improved when their peak serum DSCG concentration was less than about 15 ng ml⁻¹ [12]. We suggest that plasma concentrations of DSCG should be measured in patients who do not respond to DSCG treatment. If their plasma concentrations of DSCG are low, once adequate compliance and inhaler technique has been confirmed, increasing the clinical dose of DSCG or switching the patient to another DSCG formulation should be considered.