Stereoselective urinary excretion of formoterol and its glucuronide conjugate in human

Abstract

Aims

Formoterol is an inhaled β₂-adrenoceptor agonist used as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). Glucuronidation is an important route of metabolism in humans which occurs faster for (S; S)-formoterol in human liver microsomes. The aim of this study was to investigate the stereoselectivity of urinary excretion of formoterol and its glucuronide conjugate after oral dosing with rac-formoterol.

Methods

Seven nonsmoking volunteers (six males, one female) were included in the study. After an overnight fast, a single 60 µg oral dose of rac-formoterol fumarate dihydrate was ingested. Urine samples were collected at 1 h intervals for the first 4 h, and at 6, 8, 12 and 24 h after dosing. Formoterol enantiomers were analysed by chiral h.p.l.c. assay and formoterol glucuronides were determined as formoterol enantiomers after enzymatic cleavage with β-glucuronidase.

Results

The female subject displayed a different pattern of metabolism and statistical analysis was therefore limited to data for the six males. The median (range) of the total urinary excretion of formoterol was 37.8% (20.9–51.2%) of the dose. The medians (ranges) of the amounts of (R; R)- and (S; S)-formoterol and of (R; R)- and (S; S)-formoterol glucuronide excreted were 2.1 (1.0–2.9), 3.5 (2.6–3.8), 21.0 (13.1–31.0) and 10.3 (4.2–14.6)%, respectively, of the dose. Unchanged (S; S)-formoterol excretion was significantly greater than that of unchanged (R; R)-formoterol and (R; R)-formoterol glucuronide excretion was significantly greater than that of (S; S)-formoterol glucuronide. The total RR-formoterol (unchanged drug plus glucuronide) excreted was significantly greater than the total (S; S)-formoterol.

Conclusions

Our study demonstrates that the urinary excretion of formoterol in male humans after oral administration of rac-formoterol is stereoselective with preferential excretion of the active (R; R)-formoterol as unchanged drug and glucuronide. The different pattern of metabolism in the female subject provides impetus for further studies of the effect of gender on the stereoselective metabolism and pharmacokinetics of formoterol.

Introduction

Formoterol is a potent long-acting β₂-adrenoceptor agonist marketed for inhaled use as a racemic mixture of the active (R; R)- and inactive (S; S)-formoterol (rac-formoterol) [1, 2]. Non-chiral studies of the urinary excretion of formoterol have shown that the drug undergoes glucuronidation in humans after oral [3] and inhaled [4] doses. The glucuronidation of formoterol occurs mainly at the phenolic position, but a benzyl glucuronide is also formed [5]. Glucuronidation has also been studied using human liver microsomes and chiral assay has shown it to be stereoselective with large interindividual variation [6].

So far, pharmacokinetic studies of formoterol enantiomers have been limited by the very low plasma concentrations (< 100 pg ml⁻¹) associated with even high doses of the drug [7, 8]. However, the higher concentrations found in urine are amenable to chiral assay and urine studies have shown that excretion of formoterol enantiomers after inhalation of rac-formoterol is stereoselective [9, 10]. Although in clinical use salmeterol is administered exclusively via inhalation, a large proportion of an inhaled dose is inevitably swallowed and absorbed into the systemic circulation from the gastrointestinal tract. Data relating to oral administration are therefore relevant to the inhaled route and avoid the complexity associated with absorption from two distinct sites. The aim of the present study was to provide further information on the urinary excretion of the enantiomers of formoterol and its glucuronide conjugate after oral dosing with rac-formoterol.

Methods

Materials

(R; R)-, (S; S)- and rac-formoterol fumarate dihydrate were kindly donated by Ciba-Geigy Ltd (Basel, Switzerland). rac-Formoterol for clinical use was from Foradil (12 µg formoterol fumarate dihydrate) capsules (Novartis NZ Ltd, Auckland). H.p.l.c. grade 2-propanol, AR grade ethyl acetate, sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate and sodium hydrogen carbonate were obtained from BDH (Poole, UK). β-Glucuronidase (EC 3.2.1.31, Type H-1 from Helix pomatia) was purchased from Sigma Chemical Company (St Louis, MO, USA). EDTA-Na₂ was from May & Baker (Dagenham, UK). Distilled, deionized water was produced by a Milli-Q Reagent Water System (Millipore, MA, USA). Solid-phase extraction (SPE) cartridges (Extract-Clean, 500 mg/2.8 ml silica packing) were obtained from Alltech Associates (Deerfield, IL, USA).

H.p.l.c. assay

Formoterol enantiomers were analysed by chiral h.p.l.c. on a CHIRAL-AGP (α₁-acid glycoprotein) column (ChromTech AB, Hagersten, Sweden) with electrochemical detection as described by Butter et al.[11]. The h.p.l.c. system consisted of a Shimadzu LC-10AD pump (Shimadzu Corporation, Kyoto, Japan), a manual injector fitted with a 50 µl loop (Rheodyne 7125, Cotati, CA, USA),aCHIRAL-AGP10 × 3.0 mmguardcolumn and a CHIRAL-AGP 100 × 4.0 mm analytical column. Detection was via an ESA coulometric electrochemical detector with a Model 5020 guard cell operated at 1000 mVandaModel5011dual-electrodeanalytical cell (ESA, Inc., Bedford, MA, USA) with detectors 1 and 2 set at 300 and 630 mV, respectively. The signal from detector 2 was processed by a Hitachi D-2500 Chromato-Integrator (Hitachi Ltd, Tokyo, Japan) to obtain peak heights. The mobile phase of 0.05 m sodium phosphate buffer containing 1.5% 2-propanol and 50 µm EDTA-Na₂ pH 6.8 was filtered through a 0.45 µm filter and degassed by sonication under vacuum before use. The flow rate was 0.9 ml min⁻¹ and the system was operated at ambient temperature. Under these conditions (R; R)- and (S; S)-formoterol eluted at 13.0 and 17.0 min, respectively.Formoterolglucuronidesweredetermined as formoterol enantiomers after enzymatic cleavage with β-glucuronidase.

Subjects

Seven nonsmoking volunteers (six males, one female; age 22–55 years, body weight 65.0–80.5 kg) participated in the study. Subjects were not receiving any regular medication or suffering from any disease. Written informed consent to take part in this study was obtained from each volunteer. The study was approved by the Southern Regional Health Authority Ethics Committee (Otago).

Protocol

After an overnight fast, urine was voided and a single 60 µg dose of rac-formoterol fumarate dihydrate (molecular weight = 420.5, equivalent to 142.7 nmol formoterol) was ingested with 200 ml tap water. The dose is within the recommended range for oral administration [1] and no side-effects were observed. Subjects remained fasting for a further 3 h. Urine was collected at 1 h intervals for the first 4 h, and at 6, 8, 12 and 24 h after dosing. The volume of urine passed at each time was recorded, and an aliquot from each sample was stored at −80 °C until analysis. To maintain urine output the subjects drank 200 ml of tap water after each urine collection for the first 4 h.

Sample analysis

Sample preparation of urine was carried out using a modification of the method of Butter et al.[11]. The SPE cartridges were preconditioned by washing with 3 ml of methanol followed by 3 ml of ethyl acetate. Urine (1 ml) was made alkaline with 0.5 g sodium hydrogen carbonate and extracted with 4 ml of ethyl acetate. Aliquots of the organic phase (3.0 ml) were carefully transferred to preconditioned SPE cartridges. The cartridges were washed with 1 ml of ethyl acetate followed by 10 ml of 5% 2-propanol in water and were then centrifuged to dryness. Formoterol was eluted from the columns with 3 ml of methanol. The eluates were evaporated to dryness, residues dissolved in 100 µl aliquots of mobile phase and 40 µl analysed by chiral h.p.l.c.

Glucuronide conjugates were hydrolysed by incubation with β-glucuronidase as described previously [6]. Samples of urine (1 ml) were treated with 1 ml aliquots of 0.1 m acetate buffer pH 5.0 containing β-glucuronidase (4000 units) in a shaking waterbath at 37 °C for 20 h. Formoterol enantiomers were shown to be stable under these conditions. The incubation samples were then analysed for formoterol as described above and the concentrations of unchanged formoterol enantiomers were subtracted to obtain the concentrations of glucuronide conjugates.

Standard curves for formoterol enantiomers were freshly prepared for each analytical run by spiking drug freeurinewithrac-formoterolfumaratedihydratein the range 10.0–80.0 nmol l⁻¹. The spiked urine standards werethenanalysedasdescribedabove.Thestandardcurves for enantiomers of formoterol were linear (r > 0.99) over the calibration range and the recoveries were > 80%. Intra- and interday coefficients of variation of the assay were <10%. The lower limit of quantification for both (R; R)-formoterol and (S; S)-formoterol was 0.4 nmol l⁻¹ at a signal-to-noise ratio of 3 : 1. This is comparable with the limit of quantification of 0.3 nmol l⁻¹ determined for a similar chiral assay developed by Lecaillon et al. [10].

Data analysis

Thecumulativeamountsexcretedinurineaspercent of dose (Ae), highest observed urinary excretion rates (ER_max) and the corresponding times (t_maxR) were noted directly from the data. Elimination rate constants (k) for formoterol enantiomers and their glucuronides were calculated by linear regression of semilogarithmic excretion rate-time curves (time = midpoint of the collection interval) between 2 h and 8 h. Corresponding elimination half-lives (t_1/2(2,8 h)) were calculated from t_1/2(2,8 h) = 0.693/k. Data are presented as median and range unless otherwise indicated. Differences in the amounts of enantiomers and glucuronides recovered in the urine were tested using the Wilcoxon paired signed rank test with a significance level of P < 0.05.

Results

The amounts of formoterol enantiomers and their glucuronide conjugates excreted in urine for the seven subjects are shown in Figure 1. With the exception of the female volunteer (number 7), concentrations of formoterolinurinesamplescollectedafter8 hwerebelow the limit of quantification. This reflects the fact that the female subject excreted glucuronide conjugate much slower than the males. The relative proportion of glucuronides excreted by the female (SS > RR) was also opposite to that of the males (RR > SS). Data analysis was therefore limited to the six males.

Figure 1.

Amounts of formoterol enantiomers and their glucuronide conjugates excreted in urine over 8 h following an oral dose of rac-formoterol (60 µg formoterol fumarate dihydrate) to seven volunteers (subjects 1–6 males, subject 7 female). ▪ (R; R)-formoterol, □ (S; S)-formoterol, (R; R)-glucuronide, (S; S)-glucuronide

The urinary excretion of enantiomers and glucuronidesineachcollectionintervalupto8 hisgiven in Table 1 and the cumulative excretion of the four compounds is shown in Figure 2. The total formoterol (unchanged formoterol plus glucuronide) excreted in the urine was 37.8% (20.9–51.2%) of the dose of which 5.6% (3.5–6.6%) was excreted unchanged. The excretion of unchanged (R; R)- and (S; S)-formoterol and of (R; R)- and (S; S)-formoterol glucuronide as percentages of the total formoterol excreted over 8 h were 5.1% (4.5–6.3%), 9.5% (6.9–12.3%), 59.4% (51.7–65.2%) and 26.0% (18.1–34.7),respectively.Theexcretionofunchanged (S; S)-formoterol was significantly greater than that of unchanged(R;R)-formoterolandtheexcretionof (R; R)-formoterol glucuronide was significantly greater than that of (S; S)-formoterol glucuronide. The total RR-formoterol (unchanged formoterol plus glucuronide) in urine was significantly greater than the total (S; S)-formoterol. The Ae values for the four compounds and other pharmacokinetic parameters are listed in Table 2. The highest observed urinary excretion rates (ER_max) for (R; R)- and (S; S)-formoterol were at 1.5 (0.5–2.5) h. The t_1/2(2,8 h) values for (R; R)- and (S; S)-formoterol were 2.9 (1.0–5.8) and 2.7 (1.1–3.2) h, respectively.

Table 1.

Amounts of formoterol enantiomers and their glucuronide conjugates in urine for each collection interval over 8 h following an oral dose of rac-formoterol (60 µg formoterol fumarate dihydrate). (Data are median and range for six male volunteers).

	(R; R)-formoterol (nmol)		(S; S)-formoterol (nmol)		(R; R)-glucuronide (nmol)		(S; S)-glucuronide (nmol)		Total formoterol (nmol)
Time (h)	Median	Range	Median	Range	Median	Range	Median	Range	Median	Range
0–1	0.39	0.18–1.26	0.95	0.43–2.12	6.68	1.82–17.0	3.02	0.65–9.44	11.6	3.08–29.9
1–2	0.74	0.34–0.86	1.25	0.94–1.48	10.4	4.03–18.1	4.77	1.66–8.18	17.6	6.98–28.1
2–3	0.45	0.24–0.54	0.64	0.46–0.91	5.60	3.51–7.26	2.54	1.49–3.91	9.34	6.96–11.7
3–4	0.24	0.15–0.58	0.51	0.39–0.62	3.00	1.15–4.50	1.08	0.52–1.69	4.72	2.87–6.83
4–6	0.39	0.18–0.91	0.69	0.24–1.06	3.61	0.39–5.70	1.28	0.20–1.90	5.94	1.37–9.58
6–8	0.37	0.17–1.00	0.59	0.29–0.93	0.86	0.35–2.69	0.52	0.15–2.23	2.17	1.25–6.15
Total (nmol)	2.95	1.39–4.12	4.98	3.65–5.36	29.9	18.7–44.3	14.7	6.04–20.9	53.9	29.8–73.1

Figure 2.

Median cumulative amounts of formoterol enantiomers and their glucuronide conjugates excreted in urine over 8 h following an oral dose of rac-formoterol (60 µg formoterol fumarate dihydrate) to six male volunteers. ○ (R; R)-formoterol; • (S; S)-formoterol; □ (R; R)-formoterol glucuronide; ▪ (S; S)-formoterol glucuronide.

Table 2.

Pharmacokinetic parameters for urinary excretion of formoterol enantiomers and their glucuronide conjugates following an oral dose of rac-formoterol (60 µg formoterol fumarate dihydrate). (Data are median and range for six male volunteers).

	(R; R)-formoterol		(S; S)-formoterol		(R; R)-glucuronide		(S; S)-glucuronide
	Median	Range	Median	Range	Median	Range	Median	Range
ERmax (nmol h−1)	0.74	0.35–1.26	1.36	0.94–2.12	12.5	4.53–18.1	4.85	1.66–9.44
tmaxR (h)	1.5	0.5–2.5	1.5	0.5–2.5	1.5	0.5–2.5	1.5	0.5–2.5
t1/2(2,8 h) (h)	2.9	1.0–5.8	2.7	1.1–3.2	1.2	0.9–1.7	1.4	0.8–2.4
Ae (0–8 h) (% of dose)	2.07	0.97–2.89	3.49	2.56–3.76	21.0	13.1–31.0	10.3	4.23–14.6

In the female volunteer (number 7), concentrations of formoterol in urine were measurable until 24 h after oral administration but the total formoterol excreted over 24 h was only 14.9% of the dose. The amounts of (R; R)- and (S; S)-formoterol and of (R; R)- and (S; S)-formoterol glucuronide excreted were 2.3, 4.7, 2.6 and 5.4%, respectively, of the dose (15.3, 31.3, 17.4 and 36.0%, respectively, of the total formoterol excreted).

Discussion

This study provides further characterization of the urinary excretion of formoterol enantiomers and their glucuronide conjugates after oral administration to healthy volunteers. The total formoterol (unchanged drug plus glucuronides) excreted in urine of 37.8% of the dose was higher than that of 24–25% reported for single inhaled doses [4] but similar to that of approximately 35% reported after simultaneous oral and intravenous dosing of tritium labelled formoterol [5]. The excretion of unchanged drug of 5.6% of the dose was lower than that of 7–8% found after single inhaled doses of 12–120 µg formoterol fumarate [4, 10]. The higher excretion of unchanged (S; S)-formoterol (9.5% of the dose) compared to unchanged (R; R)-formoterol (5.1% of the dose) is consistent with previous studies [9, 10].

(R; R)-formoterol glucuronide was the major form of formoterol excreted in urine accounting for more than half (59.4%) of the total formoterol excreted. In fact the total (R; R)-formoterol (unchanged formoterol plus glucuronide) recovered in urine was nearly twice that of the total (S; S)-formoterol. These results clearly indicate that the metabolism and disposition of formoterol is stereoselective and that the clearance of the active (R; R)-formoterol is faster than that of its antipode. This is inconsistent with the results of our previous in vitro study showing (S; S)-formoterol is glucuronidated twice as fast as its antipode by human liver microsomes [6]. This inconsistency suggests the clearance of formoterol and its glucuronide involves other stereoselective pathways. For example, biliary excretion of formoterol which accounts for about 25% of the dose [5] may be stereoselective as has been demonstrated for some 2-arylpropionate anti-inflammatory drugs in rats [11]. In addition, stereo-selective renal clearance or gut wall metabolism may contribute to producing the final pattern of urinary excretion of formoterol enantiomers and their glucuronides observed in this study.

The highest observed urinary excretion rates (ER_max) for (R; R)- and (S; S)-formoterol in our study are similar to those determined by Lecaillon et al. but our t_1/2(2,8 h) values for (R; R)- and (S; S)-formoterol of 2.9 and 2.7 h, respectively, are much shorter than their terminal elimination half-lives of 13.9 and 12.3 h, respectively [10]. In fact Lecaillon et al. clearly show that urinary excretion of formoterol enantiomers is biphasic with a half-life of about 3 h for the initial urinary excretion phase. Our inability to observe this much slower terminal elimination phase is possibly related to the lower dose used. However, it is interesting to note that in a study of oral administration of formoterol to eight healthy males, plasma levels declined bi-exponentially in half the subjects and mono-exponentially in the other half [8].

In conclusion our study demonstrates stereoselective processes lead to relatively higher urinary excretion of (R; R)-formoterol in male humans after oral administration of rac-formoterol.

Ms Zhang thanks the University of Otago for a PhD Scholarship.