J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12150
Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration M. E. WIEDER* S. W. PAINE
†
P. R. HINCKS*
Wieder, M. E., Paine, S. W., Hincks, P. R., Pearce, C. M., Scarth, J., Hillyer, L. Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration. J. vet. Pharmacol. Therap. doi: 10.1111/jvp. 12150.
C. M. PEARCE* J. SCARTH* & L. HILLYER ‡ *LGC, Fordham, Cambridgeshire, UK; †The University of Nottingham, Sutton Bonington, Nottinghamshire, UK; ‡British Horseracing Authority, London, UK
Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post last administration, lie below the LLOQ of the method and so cannot be used for plasma PK analysis. Urine PK analysis suggests a half-life consistent with the pharmacological effect duration. An estimate of the urine average concentration at steady-state was collected by averaging the concentration measurements in the dosing period from 12 to 0 h relative to the last administered dose. The value was averaged across the six horses and used to estimate an effective urine concentration as a marker of effective lung concentration. The value estimated was 9.6 ng/mL and from this a number of detection times were calculated using a range of safety factors. (Paper received 21 October 2013; accepted for publication 9 June 2014) Martina E. Wieder, LGC, Fordham, Cambridgeshire CB7 5WW, UK. E-mail: [email protected]
INTRODUCTION Salbutamol, also known as albuterol, is a short-acting preferential b-adrenargic receptor agonist drug, prescribed under the ‘cascade’ in the United Kingdom and other countries for the treatment of recurrent airway obstruction (RAO) in horses. RAO is a common equine pulmonary disease causing a loss of lung function (Matera et al., 2011) and also performance in racehorses (Allen et al., 2006), with some similarities to human asthma and chronic obstructive pulmonary disease. Salbutamol mimics the effects of adrenaline on b2-receptors, part of the sympathetic nervous system. When activated by bagonists, receptors stimulate the enzyme adenylate cyclase causing increased production of cyclic adenosine monophosphate which leads to relaxation of the smooth muscle, resulting in bronchodilation (Lynes, 2007). Salbutamol has been reported to improve parameters of pulmonary function by approximately 70% in horses during an episode of airway obstruction (Rush, 2002). Salbutamol is administered by the inhalation route in two main ways: by continuous nebulisation or use of a pressurized metered-dose inhaler (pMDI). Drug therapy delivered by © 2014 John Wiley & Sons Ltd
inhalation has been an established practice over many years and is an attractive route of delivery over systemic parenteral alternatives because lower more effective doses may be used with fewer side effects. A reason for using the inhaled administration route is due to salbutamol showing no evidence of a pulmonary metabolism (Ward et al., 2000). Optimizing delivery to the lung presents a challenge however as a result of a number of factors, such as the variation in lung-related anatomical characteristics across species and the impact of disease on lung physiology (Cooper et al., 2012). It is reported that b2-agonist administration via a nebuliser is the standard treatment for acute exacerbation especially in young children. However, there are some limitations for the use of nebulisation, for example its inconvenience, high cost and uncontrolled particle sizes (Direkwatanachai et al., 2011). Significantly in treating racehorses in training, cross-contamination is another limitation; detectable concentrations have been found in samples from horses treated using an inhalation mask, untreated horses and persons administering the drug (Kalimo, 2004). Devices for such administrations have been greatly improved in recent years to reduce the problem, but it remains advisable that they are used by experienced personnel. The pMDI is a convenient 1
2 M. E. Wieder et al.
device to use for quick relief of acute airway obstruction, but there may be problems between actuation and inhalation (Lin & Hseih, 1995). The advantages of using a MDI system, over nebulisation, with horses include rapid administration, consistent dose delivery and minimal risk of pulmonary contamination by environmental micro-organisms (Rush, 2002). The advantage over oral treatment (e.g. clenbuterol which is far more economic) is its efficacy (Cooper et al., 2012). The recommended dosage regimen for salbutamol administered via pMDI to horse is 500–1000 mg with a frequency of 4–6 h (Duvivier et al., 1999). This is consistent with a study carried out by Bertin et al. (2011), who have shown that a dose of 500 lg of salbutamol administered to 10 horses via two different MDI inhalers gives optimal efficacy against RAO. For the study reported here, the MDI technique was employed. Previous research investigating the detection of salbutamol by LC-MS/MS has been carried out (Van Vyncht et al., 1996; Josefsson & Sabanovic, 2006). LC-MS methods have been developed for the determination of salbutamol in human urine with limits of quantification (LOQ) of 10 and 15 ng/mL (Zhang et al., 2006; Mareck et al., 2011) and for salbutamol enantiomers in human plasma with a limit of detection (LOD) of 0.25 ng/mL (Jacobson et al., 2003). In another study, salbutamol was extracted from human urine using SPE and analysed by GC-MS with a LOQ of 0.2 ng/mL (Van Rhijn et al., 1995). A number of studies have reported the concentration of salbutamol in plasma and urine following the administration of the drug to horses by nebulisation. The detection of salbutamol and salmeterol after therapeutic treatment for chronic obstructive pulmonary disease (COPD), using enzyme linked immunosorbent assay (ELISA) has been investigated (Van Eenoo et al., 2002). In this work, salbutamol was detected for 24 h after administration with a LOQ of 0.5 ng/mL. The administration of salbutamol sulphate by nebulisation to healthy horses was also reported by Dumasia et al. (2000). Here, postadministration urine samples were analysed by GC-MS and LC-MS and salbutamol concentrations calculated semi-quantitatively to produce urine clearance profiles. Salbutamol was detected in hydrolyzed urine for at least 54 h (last void collected) at a concentration of 2 ng/mL. The presence of b2 agonists in horses on raceday, is prohibited under the rules of British horseracing and most international racing jurisdictions, which state that horses must race free from concentrations of medication that are clinically effective. The use of therapeutic medications to treat racehorses for welfare purposes out of competition is however legitimate. Therefore, to deal with the race-day scenario where a horse races having received medication in its training programme, the term ‘free of any medication’ has to be defined. One such definition can be a drug concentration in plasma that is less than the concentration required for a significant therapeutic effect. Toutain and Lassourd (2002), proposed an approach based upon the above definition that also takes into account the variation in both PK and pharmacodynamic (PD) parameters for a population of horses. An estimate of the irrelevant plasma concentration (IPC) is based upon a pharmacologically
effective plasma drug concentration divided by an appropriate safety factor. Also, with knowledge of urine PK parameters an irrelevant urine concentration (IUC) can also be estimated. The IPC and IUC can be used for the purpose of deriving drug screening limits, which, in turn, can be used in conjunction with drug clearance profiles to obtain appropriate detection times for the matrices concerned. The detection time is the time post last therapeutic administration when the drug concentration in plasma or urine drops below the IPC/IUC. In association with the European Horserace Scientific Liaison Committee (EHSLC), the British Horseracing Authority (BHA) develops advice in the form of detection times for commonly used therapeutic medications. By a detection time to form the basis of a withdrawal time, veterinary surgeons working with racehorses can help trainers avoid ‘positive’ results on race days following legitimate treatments (BHA, 2013). For inhaled drugs, such as b2 agonists, the site of therapeutic effect is localized in the lungs and therefore relatively small doses are typical. Circulating concentrations are, consequently, often very low and prepare for challenging drug detection. The objectives of this study were to develop a fully quantitative method for the detection of salbutamol and to determine excretion profiles in both equine urine and blood plasma following the administration of the drug via the inhalation route. Additionally, PK parameters would be derived and used to generate detection time advice for the administration of salbutamol to racehorses. MATERIALS AND METHODS Chemicals and reagents Salbutamol sulphate salt was purchased as Ventolin Evohaler from GSK (Middlesex, UK) and salbutamol-d3 was supplied by the National Measurement Institute (London, UK). Acetic acid, acetonitrile, ethyl acetate, methanol, propan-2-ol and sodium hydroxide were supplied by Fisher Scientific (Leicestershire, UK). Ammonium acetate, pancreatin and b-glucuronidase (helix pomatia) were obtained from Sigma-Aldrich (Dorset, UK). Ammonia solution 34% was supplied by Eurolab Supplies (Warwickshire, UK) and hydrochloric acid by VWR (Leicestershire, UK). All water used was purified by a EuRO 80â purification unit which includes deionization (Triple Red, Buckinghamshire, UK). Administration and sample collection Ventolin Evohaler was administered by inhalation via MDI to six healthy male thoroughbred horses with no history or current clinical signs of RAO (ages 4–8 years, body weights 442– 489 kg). Each horse received a dose of 0.5 mg (comprising 5 9 100 lg actuations per dose) every 4 h during the day for 2 days (total dose 4 mg over eight doses). Urine and blood samples were collected prior to administration, and postadministration samples were collected up to 92 h. Approximately 25 mL of blood was collected from each horse at each recorded © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 3
sampling time point. In total, 43 plasma samples were collected per horse; two predose, 28 during the dosing period and 13 postdose. Immediately after collection, the blood was centrifuged, plasma was separated, and aliquots were dispensed into different vials. All urine void times and volumes were also recorded, and all samples were stored at 20 °C until required for analysis. In total between 13 and 32, urine samples were collected per horse during the administration period. The work was carried out under the auspices of the Animals (Scientific Procedures) Act 1986 and also approved by the BHA’s own internal Ethics Committee. Plasma sample preparation and extraction Ten nanogram per millilitres salbutamol-d3 [Internal Standard (IS)] and helix pomatia solution (100 lL) were added to each plasma sample (1 mL). The sample was incubated overnight at 37 °C, after which 50 mM ammonium bicarbonate pH 10 (1 mL) was added. The samples were prepared by SPE on Oasis HLBâ 60 mg3 mL cartridges (Waters, Elstree, UK). The cartridges were conditioned with methanol (1 mL) and water (1 mL) before the hydrolyzed sample was added. The cartridge was washed with water (1 mL), and the compounds of interest were eluted with methanol (2 9 500 lL). The elution solvent was evaporated to dryness under nitrogen at ambient temperature and reconstituted in 0.1% acetic acid: propan-2-ol (90:10, v/v) (100 lL). For each analysis batch, salbutamol calibration curves in blank pooled plasma were prepared in duplicate at concentrations between 10 and 500 pg/mL. Salbutamol quality control samples were also prepared in duplicate at 10, 15, 200 and 400 pg/mL. Urine sample preparation and extraction Ten nanogram per millilitres salbutamol-d3 (IS) 1 M acetate buffer pH 4.7 (1 mL), helix pomatia solution (100 lL) and pancreatin (100 lL) were added to each urine sample (2 mL). The sample was incubated overnight at 37 °C before 50 mM ammonium bicarbonate pH 10 (1 mL) was added. The samples were prepared by SPE on Strata X-Câ 60 mg3 mL cartridges (Phenomenex, Macclesfield, UK). The cartridges were conditioned with methanol (3 mL) and water (3 mL). The hydrolyzed sample was applied and the cartridge sequentially washed with 0.1 M acetate buffer pH 9.0 (3 mL), 0.1 M hydrochloric acid (3 mL) and methanol (3 mL). The compounds were eluted in ethyl acetate: propan-2-ol: ammonia (80:17:3, v/v) (2 9 1 mL) and the elution solvent was evaporated to dryness under nitrogen at ambient temperature and reconstituted in 0.1% acetic acid: propan-2-ol (95:5, v/v) (100 lL). For each analysis batch, salbutamol calibration curves in blank pooled urine were prepared in duplicate at concentrations between 10 and 500 pg/mL. Salbutamol quality control © 2014 John Wiley & Sons Ltd
samples were also prepared in duplicate at 10, 15, 200 and 400 pg/mL. UPLC-MS/MS Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was carried out on an API 5500 Qtrapâ mass spectrometer (ABI, Warrington, UK) interfaced to an Acquityâ ultra performance liquid chromatograph (Waters). Chromatographic separation was achieved using an HSS T3â column (2.1 9 100 mm 1.8 lm particle size) (Waters). The column temperature was maintained at 60 °C. Elution was performed at a flow rate of 700 lL/min with solvents 0.1% acetic acid in acetonitrile (mobile phase A) and 0.1% acetic acid (aq v/v; mobile phase B). A gradient was employed starting at 5% A at 0 min increasing to 99% at 5.0 min. The gradient was maintained at 99% A for 2 min followed by re-equilibration at 5% A for 1 min. The ion source was operated in the electrospray ionization positive mode at 450 °C. The curtain gas was 30 units, nebuliser gas 50 units, turbo gas 40 units and the ionspray was 5.5 kV. Ten microlitre of sample was injected onto the LC-MS/MS system. Data were acquired in the selected reaction monitoring (SRM) mode. The SRM transitions corresponding to m/z 240/ 148 for salbutamol and m/z 243/151 for salbutamol-d3 were used for quantification. Plasma and urine chromatograms of salbutamol following administration were analysed using commercial software (Analyst 1.5.1; AB Sciex, Warrington, Cheshire, UK).
RESULTS AND DISCUSSION For this work, salbutamol was analysed as a racemic compound and not the individual enantiomers. Quantitative analysis of salbutamol was carried out using a standard calibration line and quality control samples. The method was validated on an API 5500 Qtrapâ mass spectrometer using multiple batches extracted on different days by different analysts. Concentrations were calculated as area ratios of the target drug/IS. The calibration curves (analyte concentration/IS concentration vs. analyte peak area/IS peak area) demonstrated a linear relationship over the range 10–500 pg/mL, and the coefficient of determination (R2) of the calibration curve for all batches was 0.99 or greater. The analysis of salbutamol in this study involved separate investigations in plasma and urine. Plasma The precision of the method was evaluated within a single run (intra-assay) and in different runs (interassay). The intra-assay variation of salbutamol in plasma was determined to be 10% of the expected concentration and the interassay variation 10%. The LLOQ for salbutamol in plasma was determined to be 10 pg/mL and the LOD to be 3 pg/mL. Figures 1 and 2 show the plasma excretion profiles of six horses. Salbutamol
4 M. E. Wieder et al.
Fig. 1. Elimination profile for salbutamol in the plasma of six horses during and after a series of eight administrations of salbutamol by inhalation at a nominal dose of 0.5 mg per dose; plasma concentrations (pg/mL) vs. time (h) are plotted using a linear scale.
was detected in all horses with maximum concentrations between 21 and 150 pg/mL. The profiles were obtained by analysing samples using the validated method for plasma. Urine The intra-assay variation of salbutamol in urine was determined to be 12% of the expected concentration and the interassay variation 10%. The LLOQ for salbutamol in urine was determined to be 10 pg/mL and the LOD to be 5 pg/mL. Figure 3 shows the excretion profiles of six horses. The profiles were obtained by analysing samples using the validated method for urine. Log plots were used as a means of visualizing data that were spread over several orders of magnitude. Salbutamol was detected in all horses with maximum concentrations between 3.4 and 35 ng/mL. Pharmacokinetics As the analysis used racemic salbutamol, the pharmacokinetic and detection time calculations are based on the exact product and not individual enantiomers. For drugs that act systemically, the average concentration of drug in plasma at steady-state resulting from a therapeutic dose can be considered as the EC50. The Toutain model for estimating an IPC assumes that a sigmoid Emax model exists between the PD response and plasma drug concentration. In the Toutain model, the EC50 is reduced by a factor of 50 giving
an EC2 which is considered to be clinically nonsignificant. The EC2 is further reduced by an uncertainty factor that takes account of variability in both the PK and PD within a population of horses. Overall, a safety factor of 500 is applied to the EC50 to produce an IPC. This approach is robust for systemically administered drugs because there is a direct relationship between plasma concentration and pharmacological effect. Furthermore, urine concentrations can be used as marker concentrations for plasma as they, too, are part of the same relationship. This is not the case for a local administration for which plasma and/or urine concentrations are only a marker of exposure rather than the driving force controlling the biophase concentration. For locally acting therapies, such as the inhaled delivery of salbutamol, the use of drug concentrations in plasma or urine as markers of concentration at the site of action, and therefore of a localized pharmacological effect, is not straight-forward. Most importantly, a relationship between plasma or urine concentration and local concentration must be established before an IPC/IUC-based screening limit can be calculated. This may only be achieved with full knowledge of local as well as systemic PK parameters, however, under certain circumstances it may be possible to elucidate a plasma or urine concentration that can act as a reasonable marker of an effective local concentration, following a therapeutic dose. An EC50 as determined from equine isolated bronchi cannot be related to the effective plasma marker concentration or irrelevant marker plasma concentration as the lung concentration © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 5
Fig. 2. Elimination profile for salbutamol in plasma from six horses during and after a series of eight administrations of salbutamol by inhalation at a nominal dose of 0.5 mg per dose; plasma concentrations (pg/mL) vs. time (h) are plotted using a linear scale from the first administration up until 20 h after the last salbutamol administration ( 40 h to 20 h); administration times are indicated by arrows.
is not known. However, Cooper et al. (2012) have shown for a series of inhaled drugs, including salbutamol, that lung PK terminal half-life is equal to the corresponding plasma PK terminal half-life when administered via the intratracheal route to rat and dog. This establishes that a fixed lung to plasma concentration ratio will exist at steady-state. Also, salbutamol has been shown to follow a sigmoid Emax model in human patients (Lalonde et al., 1999) and a sigmoid Emax model has also been established for the action of salbutamol in isolated human bronchi (Naline et al., 2007). Therefore, the Toutain model can be applied to the average steady-state concentration of salbutamol within the lung resulting from a therapeutic dose which in turn can be indirectly monitored by an effective plasma or urine marker concentration. The recommended dosing frequency for pMDI administration of salbutamol to horses is 4–6 h (Duvivier et al., 1999). This is entirely consistent with a clinical study in humans whereby efficacy levels (mean change in FEV1) dropped by 50% after 4 h from a pMDI therapeutic administration of salbutamol (Van Noord et al., 1996). This is not surprising as evidence suggests that the lung PK of inhaled drugs is mainly controlled by lung retention of drug rather than systemic clearance and therefore PK terminal half-life will be similar across species (Cooper et al., 2012). © 2014 John Wiley & Sons Ltd
Analysis of plasma samples taken post last administration of salbutamol gave responses that fell below the LLOQ of the method and so these could not be used for PK purposes or act as reliable markers of lung concentration. However, the analysis of urine samples from the same administration gave analyte responses that remained above the LLOQ out to approximately 100 h, post last administration. It will be proposed below that salbutamol concentrations in urine can act as markers for lung concentrations and therefore an IUC can represent a marker for an irrelevant lung concentration. In this way, a detection time in urine can be suggested on a rational basis. Urine concentration as a marker for effective lung concentration The elimination curves for salbutamol in urine (Fig. 3) show an exponential decay out to 40 h, beyond which there is an indication of the beginnings of a second elimination phase, having a lower elimination rate constant. Salbutamol requires dosing every 4 h for sustained efficacy and based upon an Emax PK/PD model suggests the associated effective lung exposure has a half-life of 70 >70
curves means that average concentrations during this period can act as marker concentrations for the average biophase lung concentrations at steady-state. Therefore, if a safety factor is applied to the overall average urine concentration at steadystate then the resulting urine concentration can be used as the marker for an irrelevant lung biophase concentration. Ordinarily, the average steady-state concentration for a specific delivery device can be obtained from the corresponding area under the curve (AUC) associated with the dosing interval at steady-state. The AUC is not available as the dosing interval is only 4 h, and there are only a few concentration measurements within the dosing interval. However, it is clear from the urine curves that steady-state levels have been reached in each horse, and an estimate of the effective urine marker concentration (EUMC) can be obtained by averaging the concentration measurements in the dosing period from 12 to 0 h relative to the last administered dose. The average concentration has been determined for each horse, and then the average value across the six horses has been used to estimate the EUMC for
lung at 9.6 ng/mL. A range of detection times can be estimated by dividing the EUMC by a range of safety factors and application to the full urine PK profile (Table 1). Table 1 shows that the larger the safety factor applied to the EUMC the longer the resulting Detection Time. The authors would like to point out that the detection times in Table 1 are specific to the Ventolin Evohaler where the particle size of salbutamol sulphate is quality controlled. Other delivery methods of salbutamol such as nebulisation solutions may result in different detection times to the Evohaler. Furthermore, the disease state of the lungs may alter both the PK and PD of salbutamol thus leading to further variability. The safety factor used for screening purposes and the resulting detection time is agreed by horseracing regulators based upon the above considerations. The EHSLC and its members, after taking into account all influencing parameters recommend a detection time of 96 h for the pMDI administration of salbutamol. Methodologies have been described that allow the detection of inhaled salbutamol in urine out to 100 h for the thoroughbred racehorse. Drug clearance curves have been obtained for six horses following pMDI administrations of salbutamol and an approach postulated, based upon the level of significance of the pharmacological effect, which estimates drug screening concentrations and detection times.
ACKNOWLEDGMENTS Animal administrations were performed on behalf of and funded by the BHA. Particular thanks are due to the BHA staff © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 7
and students at the British Racing School who cared for and sampled horses involved in this work.
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sion variability. Journal of Pharmacokinetics and Biopharmaceutics, 27, 67–83. Lin, Y.-Z. & Hseih, K.-H. (1995) Metered dose inhaler and nebuliser in acute asthma. Archives of Disease in Childhood, 72, 214–218. Lynes, D. (2007). Pharmacological Management of Chronic Disease. Management of COPD in primary and secondary care, pp. 40–41. M + K Update Ltd, Keswick, UK. Mareck, U., Guddat, S., Schwenke, A., Beuck, S., Geyer, H., Flenker, U., Elers, J., Backer, V., Thevis, M. & Scahnzer, W. (2011) Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry. Drug Testing and Analysis, 3, 820–827. Matera, M.G., Calzetta, L., Rogliani, P., Barbado, F., Page, C.P. & Cazzola, M. (2011) Evaluation of the effects of the R- and S-enantiomers of salbutamol on equine isolated bronchi. Pulmonary Pharmacolology and Therapeutics, 24, 221–226. Naline, E., Trifilieff, A., Fairhurst, R.A., Advenier, C. & Molimard, M. (2007) Effect of indacaterol, a novel long-acting b2-agonist, on isolated human bronchi. European Respiratory Journal, 29, 575–581. Rush, B.R. (2002) Treatment of inflammatory airway disease: aerosol delivery devices and medications. AAEP Proceedings, 48, 218–227. Toutain, P.L. & Lassourd, V. (2002) Pharmacokinetics/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse. Equine Veterinary Journal, 34, 242–249. Van Eenoo, P., Deventer, K. & Delbeke, F.T. (2002). Excretion studies with salbutamol and salmeterol after inhalation. Proceedings of the14th International Conference of Racing Analysts and Veterinarians, pp. 364–367, R + W (Newmarket) Publications, Newmarket. Van Noord, J.A., Smeets, J.J., Raaijmakers, J.A.M., Bommer, A.M. & Maesen, F.P.V. (1996) Salmeterol versus formoterol in patients with moderately severe asthma: onset and duration of action. European Respiratory Journal, 9, 1684–1688. Van Rhijn, J.A., Heskamp, H.H., Essers, M.L., Van de Wetering, H.J., Kleijnen, H.C.H. & Roos, A.H. (1995) Possibilities for confirmatory analysis of some b-agonists using two different derivatives simultaneously. Journal of Chromatography B, 665, 395–398. Van Vyncht, G., Preece, S., Gaspar, P., Maghuin-Rogister, G. & DePauw, E. (1996) Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of b-agonists in biological matrices. Journal of Chromatography A, 750, 43–49. Ward, J.K., Dow, J., Dallow, N., Eynott, P., Milleri, S. & Ventresca, G.P. (2000) Enantiomeric disposition of inhaled, intravenous and oral racemic-salbutamol in man- no evidence of enantioselective lung metabolism. British Journal of Clinical Pharmacology, 49, 15– 22. Zhang, J., Xu, Y., Di, X. & Wu, M. (2006) Quantification of salbutamol in human urine by liquid chromatography- electrospray ionization mass spectrometry. Journal of Chromatography B, 831, 328–332.
Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration M. E. WIEDER* S. W. PAINE
†
P. R. HINCKS*
Wieder, M. E., Paine, S. W., Hincks, P. R., Pearce, C. M., Scarth, J., Hillyer, L. Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration. J. vet. Pharmacol. Therap. doi: 10.1111/jvp. 12150.
C. M. PEARCE* J. SCARTH* & L. HILLYER ‡ *LGC, Fordham, Cambridgeshire, UK; †The University of Nottingham, Sutton Bonington, Nottinghamshire, UK; ‡British Horseracing Authority, London, UK
Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post last administration, lie below the LLOQ of the method and so cannot be used for plasma PK analysis. Urine PK analysis suggests a half-life consistent with the pharmacological effect duration. An estimate of the urine average concentration at steady-state was collected by averaging the concentration measurements in the dosing period from 12 to 0 h relative to the last administered dose. The value was averaged across the six horses and used to estimate an effective urine concentration as a marker of effective lung concentration. The value estimated was 9.6 ng/mL and from this a number of detection times were calculated using a range of safety factors. (Paper received 21 October 2013; accepted for publication 9 June 2014) Martina E. Wieder, LGC, Fordham, Cambridgeshire CB7 5WW, UK. E-mail: [email protected]
INTRODUCTION Salbutamol, also known as albuterol, is a short-acting preferential b-adrenargic receptor agonist drug, prescribed under the ‘cascade’ in the United Kingdom and other countries for the treatment of recurrent airway obstruction (RAO) in horses. RAO is a common equine pulmonary disease causing a loss of lung function (Matera et al., 2011) and also performance in racehorses (Allen et al., 2006), with some similarities to human asthma and chronic obstructive pulmonary disease. Salbutamol mimics the effects of adrenaline on b2-receptors, part of the sympathetic nervous system. When activated by bagonists, receptors stimulate the enzyme adenylate cyclase causing increased production of cyclic adenosine monophosphate which leads to relaxation of the smooth muscle, resulting in bronchodilation (Lynes, 2007). Salbutamol has been reported to improve parameters of pulmonary function by approximately 70% in horses during an episode of airway obstruction (Rush, 2002). Salbutamol is administered by the inhalation route in two main ways: by continuous nebulisation or use of a pressurized metered-dose inhaler (pMDI). Drug therapy delivered by © 2014 John Wiley & Sons Ltd
inhalation has been an established practice over many years and is an attractive route of delivery over systemic parenteral alternatives because lower more effective doses may be used with fewer side effects. A reason for using the inhaled administration route is due to salbutamol showing no evidence of a pulmonary metabolism (Ward et al., 2000). Optimizing delivery to the lung presents a challenge however as a result of a number of factors, such as the variation in lung-related anatomical characteristics across species and the impact of disease on lung physiology (Cooper et al., 2012). It is reported that b2-agonist administration via a nebuliser is the standard treatment for acute exacerbation especially in young children. However, there are some limitations for the use of nebulisation, for example its inconvenience, high cost and uncontrolled particle sizes (Direkwatanachai et al., 2011). Significantly in treating racehorses in training, cross-contamination is another limitation; detectable concentrations have been found in samples from horses treated using an inhalation mask, untreated horses and persons administering the drug (Kalimo, 2004). Devices for such administrations have been greatly improved in recent years to reduce the problem, but it remains advisable that they are used by experienced personnel. The pMDI is a convenient 1
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device to use for quick relief of acute airway obstruction, but there may be problems between actuation and inhalation (Lin & Hseih, 1995). The advantages of using a MDI system, over nebulisation, with horses include rapid administration, consistent dose delivery and minimal risk of pulmonary contamination by environmental micro-organisms (Rush, 2002). The advantage over oral treatment (e.g. clenbuterol which is far more economic) is its efficacy (Cooper et al., 2012). The recommended dosage regimen for salbutamol administered via pMDI to horse is 500–1000 mg with a frequency of 4–6 h (Duvivier et al., 1999). This is consistent with a study carried out by Bertin et al. (2011), who have shown that a dose of 500 lg of salbutamol administered to 10 horses via two different MDI inhalers gives optimal efficacy against RAO. For the study reported here, the MDI technique was employed. Previous research investigating the detection of salbutamol by LC-MS/MS has been carried out (Van Vyncht et al., 1996; Josefsson & Sabanovic, 2006). LC-MS methods have been developed for the determination of salbutamol in human urine with limits of quantification (LOQ) of 10 and 15 ng/mL (Zhang et al., 2006; Mareck et al., 2011) and for salbutamol enantiomers in human plasma with a limit of detection (LOD) of 0.25 ng/mL (Jacobson et al., 2003). In another study, salbutamol was extracted from human urine using SPE and analysed by GC-MS with a LOQ of 0.2 ng/mL (Van Rhijn et al., 1995). A number of studies have reported the concentration of salbutamol in plasma and urine following the administration of the drug to horses by nebulisation. The detection of salbutamol and salmeterol after therapeutic treatment for chronic obstructive pulmonary disease (COPD), using enzyme linked immunosorbent assay (ELISA) has been investigated (Van Eenoo et al., 2002). In this work, salbutamol was detected for 24 h after administration with a LOQ of 0.5 ng/mL. The administration of salbutamol sulphate by nebulisation to healthy horses was also reported by Dumasia et al. (2000). Here, postadministration urine samples were analysed by GC-MS and LC-MS and salbutamol concentrations calculated semi-quantitatively to produce urine clearance profiles. Salbutamol was detected in hydrolyzed urine for at least 54 h (last void collected) at a concentration of 2 ng/mL. The presence of b2 agonists in horses on raceday, is prohibited under the rules of British horseracing and most international racing jurisdictions, which state that horses must race free from concentrations of medication that are clinically effective. The use of therapeutic medications to treat racehorses for welfare purposes out of competition is however legitimate. Therefore, to deal with the race-day scenario where a horse races having received medication in its training programme, the term ‘free of any medication’ has to be defined. One such definition can be a drug concentration in plasma that is less than the concentration required for a significant therapeutic effect. Toutain and Lassourd (2002), proposed an approach based upon the above definition that also takes into account the variation in both PK and pharmacodynamic (PD) parameters for a population of horses. An estimate of the irrelevant plasma concentration (IPC) is based upon a pharmacologically
effective plasma drug concentration divided by an appropriate safety factor. Also, with knowledge of urine PK parameters an irrelevant urine concentration (IUC) can also be estimated. The IPC and IUC can be used for the purpose of deriving drug screening limits, which, in turn, can be used in conjunction with drug clearance profiles to obtain appropriate detection times for the matrices concerned. The detection time is the time post last therapeutic administration when the drug concentration in plasma or urine drops below the IPC/IUC. In association with the European Horserace Scientific Liaison Committee (EHSLC), the British Horseracing Authority (BHA) develops advice in the form of detection times for commonly used therapeutic medications. By a detection time to form the basis of a withdrawal time, veterinary surgeons working with racehorses can help trainers avoid ‘positive’ results on race days following legitimate treatments (BHA, 2013). For inhaled drugs, such as b2 agonists, the site of therapeutic effect is localized in the lungs and therefore relatively small doses are typical. Circulating concentrations are, consequently, often very low and prepare for challenging drug detection. The objectives of this study were to develop a fully quantitative method for the detection of salbutamol and to determine excretion profiles in both equine urine and blood plasma following the administration of the drug via the inhalation route. Additionally, PK parameters would be derived and used to generate detection time advice for the administration of salbutamol to racehorses. MATERIALS AND METHODS Chemicals and reagents Salbutamol sulphate salt was purchased as Ventolin Evohaler from GSK (Middlesex, UK) and salbutamol-d3 was supplied by the National Measurement Institute (London, UK). Acetic acid, acetonitrile, ethyl acetate, methanol, propan-2-ol and sodium hydroxide were supplied by Fisher Scientific (Leicestershire, UK). Ammonium acetate, pancreatin and b-glucuronidase (helix pomatia) were obtained from Sigma-Aldrich (Dorset, UK). Ammonia solution 34% was supplied by Eurolab Supplies (Warwickshire, UK) and hydrochloric acid by VWR (Leicestershire, UK). All water used was purified by a EuRO 80â purification unit which includes deionization (Triple Red, Buckinghamshire, UK). Administration and sample collection Ventolin Evohaler was administered by inhalation via MDI to six healthy male thoroughbred horses with no history or current clinical signs of RAO (ages 4–8 years, body weights 442– 489 kg). Each horse received a dose of 0.5 mg (comprising 5 9 100 lg actuations per dose) every 4 h during the day for 2 days (total dose 4 mg over eight doses). Urine and blood samples were collected prior to administration, and postadministration samples were collected up to 92 h. Approximately 25 mL of blood was collected from each horse at each recorded © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 3
sampling time point. In total, 43 plasma samples were collected per horse; two predose, 28 during the dosing period and 13 postdose. Immediately after collection, the blood was centrifuged, plasma was separated, and aliquots were dispensed into different vials. All urine void times and volumes were also recorded, and all samples were stored at 20 °C until required for analysis. In total between 13 and 32, urine samples were collected per horse during the administration period. The work was carried out under the auspices of the Animals (Scientific Procedures) Act 1986 and also approved by the BHA’s own internal Ethics Committee. Plasma sample preparation and extraction Ten nanogram per millilitres salbutamol-d3 [Internal Standard (IS)] and helix pomatia solution (100 lL) were added to each plasma sample (1 mL). The sample was incubated overnight at 37 °C, after which 50 mM ammonium bicarbonate pH 10 (1 mL) was added. The samples were prepared by SPE on Oasis HLBâ 60 mg3 mL cartridges (Waters, Elstree, UK). The cartridges were conditioned with methanol (1 mL) and water (1 mL) before the hydrolyzed sample was added. The cartridge was washed with water (1 mL), and the compounds of interest were eluted with methanol (2 9 500 lL). The elution solvent was evaporated to dryness under nitrogen at ambient temperature and reconstituted in 0.1% acetic acid: propan-2-ol (90:10, v/v) (100 lL). For each analysis batch, salbutamol calibration curves in blank pooled plasma were prepared in duplicate at concentrations between 10 and 500 pg/mL. Salbutamol quality control samples were also prepared in duplicate at 10, 15, 200 and 400 pg/mL. Urine sample preparation and extraction Ten nanogram per millilitres salbutamol-d3 (IS) 1 M acetate buffer pH 4.7 (1 mL), helix pomatia solution (100 lL) and pancreatin (100 lL) were added to each urine sample (2 mL). The sample was incubated overnight at 37 °C before 50 mM ammonium bicarbonate pH 10 (1 mL) was added. The samples were prepared by SPE on Strata X-Câ 60 mg3 mL cartridges (Phenomenex, Macclesfield, UK). The cartridges were conditioned with methanol (3 mL) and water (3 mL). The hydrolyzed sample was applied and the cartridge sequentially washed with 0.1 M acetate buffer pH 9.0 (3 mL), 0.1 M hydrochloric acid (3 mL) and methanol (3 mL). The compounds were eluted in ethyl acetate: propan-2-ol: ammonia (80:17:3, v/v) (2 9 1 mL) and the elution solvent was evaporated to dryness under nitrogen at ambient temperature and reconstituted in 0.1% acetic acid: propan-2-ol (95:5, v/v) (100 lL). For each analysis batch, salbutamol calibration curves in blank pooled urine were prepared in duplicate at concentrations between 10 and 500 pg/mL. Salbutamol quality control © 2014 John Wiley & Sons Ltd
samples were also prepared in duplicate at 10, 15, 200 and 400 pg/mL. UPLC-MS/MS Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was carried out on an API 5500 Qtrapâ mass spectrometer (ABI, Warrington, UK) interfaced to an Acquityâ ultra performance liquid chromatograph (Waters). Chromatographic separation was achieved using an HSS T3â column (2.1 9 100 mm 1.8 lm particle size) (Waters). The column temperature was maintained at 60 °C. Elution was performed at a flow rate of 700 lL/min with solvents 0.1% acetic acid in acetonitrile (mobile phase A) and 0.1% acetic acid (aq v/v; mobile phase B). A gradient was employed starting at 5% A at 0 min increasing to 99% at 5.0 min. The gradient was maintained at 99% A for 2 min followed by re-equilibration at 5% A for 1 min. The ion source was operated in the electrospray ionization positive mode at 450 °C. The curtain gas was 30 units, nebuliser gas 50 units, turbo gas 40 units and the ionspray was 5.5 kV. Ten microlitre of sample was injected onto the LC-MS/MS system. Data were acquired in the selected reaction monitoring (SRM) mode. The SRM transitions corresponding to m/z 240/ 148 for salbutamol and m/z 243/151 for salbutamol-d3 were used for quantification. Plasma and urine chromatograms of salbutamol following administration were analysed using commercial software (Analyst 1.5.1; AB Sciex, Warrington, Cheshire, UK).
RESULTS AND DISCUSSION For this work, salbutamol was analysed as a racemic compound and not the individual enantiomers. Quantitative analysis of salbutamol was carried out using a standard calibration line and quality control samples. The method was validated on an API 5500 Qtrapâ mass spectrometer using multiple batches extracted on different days by different analysts. Concentrations were calculated as area ratios of the target drug/IS. The calibration curves (analyte concentration/IS concentration vs. analyte peak area/IS peak area) demonstrated a linear relationship over the range 10–500 pg/mL, and the coefficient of determination (R2) of the calibration curve for all batches was 0.99 or greater. The analysis of salbutamol in this study involved separate investigations in plasma and urine. Plasma The precision of the method was evaluated within a single run (intra-assay) and in different runs (interassay). The intra-assay variation of salbutamol in plasma was determined to be 10% of the expected concentration and the interassay variation 10%. The LLOQ for salbutamol in plasma was determined to be 10 pg/mL and the LOD to be 3 pg/mL. Figures 1 and 2 show the plasma excretion profiles of six horses. Salbutamol
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Fig. 1. Elimination profile for salbutamol in the plasma of six horses during and after a series of eight administrations of salbutamol by inhalation at a nominal dose of 0.5 mg per dose; plasma concentrations (pg/mL) vs. time (h) are plotted using a linear scale.
was detected in all horses with maximum concentrations between 21 and 150 pg/mL. The profiles were obtained by analysing samples using the validated method for plasma. Urine The intra-assay variation of salbutamol in urine was determined to be 12% of the expected concentration and the interassay variation 10%. The LLOQ for salbutamol in urine was determined to be 10 pg/mL and the LOD to be 5 pg/mL. Figure 3 shows the excretion profiles of six horses. The profiles were obtained by analysing samples using the validated method for urine. Log plots were used as a means of visualizing data that were spread over several orders of magnitude. Salbutamol was detected in all horses with maximum concentrations between 3.4 and 35 ng/mL. Pharmacokinetics As the analysis used racemic salbutamol, the pharmacokinetic and detection time calculations are based on the exact product and not individual enantiomers. For drugs that act systemically, the average concentration of drug in plasma at steady-state resulting from a therapeutic dose can be considered as the EC50. The Toutain model for estimating an IPC assumes that a sigmoid Emax model exists between the PD response and plasma drug concentration. In the Toutain model, the EC50 is reduced by a factor of 50 giving
an EC2 which is considered to be clinically nonsignificant. The EC2 is further reduced by an uncertainty factor that takes account of variability in both the PK and PD within a population of horses. Overall, a safety factor of 500 is applied to the EC50 to produce an IPC. This approach is robust for systemically administered drugs because there is a direct relationship between plasma concentration and pharmacological effect. Furthermore, urine concentrations can be used as marker concentrations for plasma as they, too, are part of the same relationship. This is not the case for a local administration for which plasma and/or urine concentrations are only a marker of exposure rather than the driving force controlling the biophase concentration. For locally acting therapies, such as the inhaled delivery of salbutamol, the use of drug concentrations in plasma or urine as markers of concentration at the site of action, and therefore of a localized pharmacological effect, is not straight-forward. Most importantly, a relationship between plasma or urine concentration and local concentration must be established before an IPC/IUC-based screening limit can be calculated. This may only be achieved with full knowledge of local as well as systemic PK parameters, however, under certain circumstances it may be possible to elucidate a plasma or urine concentration that can act as a reasonable marker of an effective local concentration, following a therapeutic dose. An EC50 as determined from equine isolated bronchi cannot be related to the effective plasma marker concentration or irrelevant marker plasma concentration as the lung concentration © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 5
Fig. 2. Elimination profile for salbutamol in plasma from six horses during and after a series of eight administrations of salbutamol by inhalation at a nominal dose of 0.5 mg per dose; plasma concentrations (pg/mL) vs. time (h) are plotted using a linear scale from the first administration up until 20 h after the last salbutamol administration ( 40 h to 20 h); administration times are indicated by arrows.
is not known. However, Cooper et al. (2012) have shown for a series of inhaled drugs, including salbutamol, that lung PK terminal half-life is equal to the corresponding plasma PK terminal half-life when administered via the intratracheal route to rat and dog. This establishes that a fixed lung to plasma concentration ratio will exist at steady-state. Also, salbutamol has been shown to follow a sigmoid Emax model in human patients (Lalonde et al., 1999) and a sigmoid Emax model has also been established for the action of salbutamol in isolated human bronchi (Naline et al., 2007). Therefore, the Toutain model can be applied to the average steady-state concentration of salbutamol within the lung resulting from a therapeutic dose which in turn can be indirectly monitored by an effective plasma or urine marker concentration. The recommended dosing frequency for pMDI administration of salbutamol to horses is 4–6 h (Duvivier et al., 1999). This is entirely consistent with a clinical study in humans whereby efficacy levels (mean change in FEV1) dropped by 50% after 4 h from a pMDI therapeutic administration of salbutamol (Van Noord et al., 1996). This is not surprising as evidence suggests that the lung PK of inhaled drugs is mainly controlled by lung retention of drug rather than systemic clearance and therefore PK terminal half-life will be similar across species (Cooper et al., 2012). © 2014 John Wiley & Sons Ltd
Analysis of plasma samples taken post last administration of salbutamol gave responses that fell below the LLOQ of the method and so these could not be used for PK purposes or act as reliable markers of lung concentration. However, the analysis of urine samples from the same administration gave analyte responses that remained above the LLOQ out to approximately 100 h, post last administration. It will be proposed below that salbutamol concentrations in urine can act as markers for lung concentrations and therefore an IUC can represent a marker for an irrelevant lung concentration. In this way, a detection time in urine can be suggested on a rational basis. Urine concentration as a marker for effective lung concentration The elimination curves for salbutamol in urine (Fig. 3) show an exponential decay out to 40 h, beyond which there is an indication of the beginnings of a second elimination phase, having a lower elimination rate constant. Salbutamol requires dosing every 4 h for sustained efficacy and based upon an Emax PK/PD model suggests the associated effective lung exposure has a half-life of 70 >70
curves means that average concentrations during this period can act as marker concentrations for the average biophase lung concentrations at steady-state. Therefore, if a safety factor is applied to the overall average urine concentration at steadystate then the resulting urine concentration can be used as the marker for an irrelevant lung biophase concentration. Ordinarily, the average steady-state concentration for a specific delivery device can be obtained from the corresponding area under the curve (AUC) associated with the dosing interval at steady-state. The AUC is not available as the dosing interval is only 4 h, and there are only a few concentration measurements within the dosing interval. However, it is clear from the urine curves that steady-state levels have been reached in each horse, and an estimate of the effective urine marker concentration (EUMC) can be obtained by averaging the concentration measurements in the dosing period from 12 to 0 h relative to the last administered dose. The average concentration has been determined for each horse, and then the average value across the six horses has been used to estimate the EUMC for
lung at 9.6 ng/mL. A range of detection times can be estimated by dividing the EUMC by a range of safety factors and application to the full urine PK profile (Table 1). Table 1 shows that the larger the safety factor applied to the EUMC the longer the resulting Detection Time. The authors would like to point out that the detection times in Table 1 are specific to the Ventolin Evohaler where the particle size of salbutamol sulphate is quality controlled. Other delivery methods of salbutamol such as nebulisation solutions may result in different detection times to the Evohaler. Furthermore, the disease state of the lungs may alter both the PK and PD of salbutamol thus leading to further variability. The safety factor used for screening purposes and the resulting detection time is agreed by horseracing regulators based upon the above considerations. The EHSLC and its members, after taking into account all influencing parameters recommend a detection time of 96 h for the pMDI administration of salbutamol. Methodologies have been described that allow the detection of inhaled salbutamol in urine out to 100 h for the thoroughbred racehorse. Drug clearance curves have been obtained for six horses following pMDI administrations of salbutamol and an approach postulated, based upon the level of significance of the pharmacological effect, which estimates drug screening concentrations and detection times.
ACKNOWLEDGMENTS Animal administrations were performed on behalf of and funded by the BHA. Particular thanks are due to the BHA staff © 2014 John Wiley & Sons Ltd
Detection and pharmacokinetics of salbutamol in horses 7
and students at the British Racing School who cared for and sampled horses involved in this work.
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