Plasma and urine pharmacokinetics of two formulations of dexamethasone in greyhound dogs

Dexamethasone, formulated as sodium phosphate and as phenylpropionate combined with sodium phosphate, was administered subcutaneously to six greyhounds. Plasma and urine were collected for up to 240 h and analysed with a limit of quantification (LOQ) of at least 100 pg/ml for dexamethasone. Dexamethasone, formulated as sodium phosphate, terminal half- life was 10.4 h in plasma and approximately 16 h in urine, and at 96 h, plasma hydrocortisone concentrations returned to background with dexamethasone levels around the LOQ. Dexamethasone, formulated as phenylpropionate combined with sodium phosphate, terminal half- life, was 25.6 h in plasma and approximately 26 h in urine, and at 96 h, plasma hydrocortisone concentrations returned to background with dexamethasone levels in three of the six greyhounds around the LOQ. Critical assessment of the pharmacokinetic and pharmacodynamic data indicated how it might be utilized for medication control in racing greyhounds.


| INTRODUC TI ON
Dexamethasone, a synthetic corticosteroid, is a commonly used veterinary medicine. Injectable preparations require formulation, often as esters, to take account of the very limited solubility of glucocorticoids in water and to provide a range of formulations that extend duration of action (Bishop, 2000). For example, dexamethasone sodium phosphate is resorbed rapidly from the injection site, thus ensuring a rapid onset of activity, whereas dexamethasone phenylpropionate is absorbed more slowly from the injection site, thus ensuring a more prolonged duration of activity (Australian Pesticides & Veterinary Medicines Authority, 2002).
There are limited data on the pharmacokinetics of dexamethasone in dogs. 1 mg/kg of dexamethasone in alcohol or as isonicotinate was given intravenously or intramuscularly, respectively, to five mixed breed dogs (Toutain et al., 1983). Dexamethasone in plasma was measured for 10 h after administration, and the limit of quantification (LOQ) was 2 ng/ml. 1 mg of an undefined formulation of dexamethasone was injected intramuscularly into 25 greyhounds, and urine samples collected for up to 96 h (Hill et al., 1997) with dexamethasone being detected for 24 h.
For medication control of drugs for animals used in sport, urine is the sample matrix of choice (Morris, 2014). To inform risk assessment for medication control, information on contemporaneous plasma (as plasma levels drive effects) and urine levels, after clinical glucuronidase. Samples were then extracted on mixed-mode C8-SCX columns (Bond Elut-Certify, 130 mg, 3 ml, Agilent, CA, USA) previously conditioned with methanol (2 ml) and water (2 ml). After urine loading, the column was washed with water (4 ml) then acetic acid (1 M, 2 ml) for pH adjustment and dried with nitrogen at 200 ml/sec for 10 min. The acid/neutral fraction was eluted with dichloromethane/ethyl acetate (4:1, 2 ml) and then washed with sodium hydroxide/sodium chloride (1 M/0.15 M, 0.5 ml). The organic layer was removed and evaporated under nitrogen at 50°C and reconstituted in formic acid/ammonium formate (0.1%) and methanol (50:50) and submitted for LCMS analysis.

analysis.
Urine and plasma extracts were analysed by liquid chromatography-mass spectrometry using a Shimadzu 8060 triple quadruple mass spectrometer (Shimadzu Corp., Kyoto, Japan) coupled to a Nexera LC-30AD (Shimadzu Corp., Kyoto, Japan) liquid chromatography. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Electrospray ionization was carried out with heater block, interface and DL temperatures of 300°C, 300°C and 275°C, respectively. The nebulizer, heating and drying gas flow rates were 3, 10 and 5 L/min, respectively.
For urine, dexamethasone was monitored in positive ion mode using the de-fluorinated in-source fragment of 373.1 as the precursor ion and daughter ions m/z 147.1 (for quantification) and m/z 171.1 (for identification). The internal standard, dexamethasone-d4, was monitored using the transition m/z 377.1 >m/z 149.1.
Hydrocortisone was monitored in positive ion mode using the precursor ion m/z 363.1 and daughter ions m/z 121.1 (for quantification) and m/z 309.1 (for identification). The internal standard, hydrocortisone-d4, was monitored using the transition m/z 364.1 >m/z 121.1.
For plasma, dexamethasone was monitored in negative ion mode using the formate adduct of 437.2 as the precursor ion and daughter ion of m/z 361.3. The internal standard, dexamethasone-d4, was monitored using the transition m/z 441.2 >m/z 363.2. Hydrocortisone was monitored using the same transitions as the urine method.
Chromatographic separation was achieved using a Poroshell 120 EC-C18 column (3 mm × 50 mm, 2.7 μm particle size) (Agilent Technologies, CA, USA). The mobile phase consisted of formic acid (0.1%) and ammonium formate (0.1%) (A) and methanol (B). The initial composition was 50% B, which was held for 0.3 min and then ramped to reach 98% B at 2.5 min. This was held for 1.5 min before being returned to 50% B and equilibrated for 1.5 min.
Calibration ranges, correlation coefficients, limits of quantification (LOQ), and detection (LOD) and inter-batch variability of precision and accuracy for all methods are listed in Table 1.
The PK profiles were analysed with a non-compartmental anal-

| RE SULTS
The elimination profile in six greyhounds after dexamethasone sodium phosphate administration (DXD-Dexadreson ® formulation) in plasma and urine are shown in Figure 1 (first 24 h shown in Appendix S1) and the pharmacokinetic parameters are in Table 2.
TA B L E 1 Calibration ranges, limits of quantification (LOQ), limits of detection (LOD), correlation coefficients (R 2 ), inter-batch variability of precision and accuracy for the analysis of dexamethasone and hydrocortisone in plasma and urine

| DISCUSS ION
The data produced in this study were compatible with the much more limited existing data that have informed clinical usage for many years (Hill et al., 1997;Toutain et al., 1983): For example, Toutain et al., 1984 determined a clearance of 6.4 ml/min/kg for IV administration of dexamethasone to dogs. This study herein usefully extends the duration of plasma data, adds extended urine data, as well as an indication of one component of the pharmacodynamic effect by measuring the reduction in endogenous hydrocortisone. As such, it addressed the primary and secondary objectives set out for this study and makes this information available for wider scientific and clinical use. Moreover, the data from this study can be combined with existing and future data and analysed using a population approach such as Non-Linear Mixed Effects (NLME) methodology (Schoemaker & Cohen, 1996). NLME is appropriate for analysing unbalanced data sets collected with analytical techniques of different sensitivity, but having generated similar data above the LOQ.
Such an aggregation of data will solve the question of low statistical power of individual data sets and will facilitate harmonization between regulatory jurisdictions.
The data from this study also provide scientific information for the first stage, risk assessment (Toutain, 2010), of medication control. There are two established methodologies to derive medication control parameters from such administration studies (Toutain, 2013). The first methodology used in most horse and greyhound regulatory jurisdictions is to use the data to estimate the effective plasma concentration (EPC), irrelevant plasma concentration (IPC), irrelevant urine concentration (IUC) and so derive a screening limit (SL) and detection time (DT) (Toutain & Lassourd, 2002). The second methodology used in US horseracing is similar to that used for determining drug residues in meat for human safety such that, with a risk of ⩽5%, that at least 95% of the animal population is under the specified drug level as for example used for dexamethasone (European

Agency for the Evaluation of Medicinal Products & Committee for
Veterinary Medicinal Products, 1997). It is important to be clear on the methodology used, for example, the latter approach can tend be F I G U R E 2 Mean dexamethasone (filled symbols) and hydrocortisone (open symbols) concentration in plasma (circle symbols) and urine (square symbols) after administration of dexamethasone sodium phosphate and dexamethasone phenylpropionate (DXF) to six greyhounds more permissive in terms of drug levels, which will be an important factor in the second stage of medication control; risk management by regulators informed by scientific advice (Toutain, 2013).
A considerable body of published medication control parameters using the former methodology (EPC, IPC, IUC, SL, DT) is being utilized by regulators in horse racing and now in greyhound racing.
However, using this approach can be questioned in some situations.
With drugs that act both locally and systemically, such as glucocorticoids, there is no single concentration value which covers every site of action and the use of an IPC/IUC may be challenged (Toutain, 2010), although it has already been used in respect of dexamethasone isonicotinate in racehorses (Ekstrand et al., 2015). Non-genomic effects of glucocorticoids do not require protein synthesis and occur within seconds to minutes of GR activation. For example, when released from the inactive GR protein complex, the non-receptor tyrosine kinase protein c-Src activates signalling cascades that inhibit phospholipase A2 activity, phosphorylate annexin 1 and impair the release of arachidonic acid. Slower genomic effects of glucocorticoids involve binding to GR followed by translocation to the nucleus. Once inside the nucleus, GR binds directly to glucocorticoid-responsive elements (GREs) and stimulates target gene expression followed by protein synthesis, and therefore, there can be a delay in some effects.
However, most of the anti-inflammatory effects of glucocorticoids appear to result from an important negative regulatory mechanism called transrepression, in which GR directly interferes with the transcriptional activation of key inflammatory proteins.
Therefore, if pharmacodynamic parameters are considered rather than pharmacokinetic alone, this can significantly affect a determination of medication control information (Knych et al., 2020).
However, with corticoids having a complex array of PD effects, there is a question with regard to which effects should be considered. For instance, in the herein study it is clear that inhibition of hydrocortisone in plasma occurs rapidly (statistically significant at 2 h). This has also been shown in horses where significant inhibition of hydrocortisone was observed in a few hours (Ekstrand et al., 2015;Knych et al., 2020).
Initial suppression of hydrocortisone is rapid because dexamethasone causes incremental inhibition for as long as dexamethasone plasma concentrations are greater than the IC 50. However, the maximum observed suppression of hydrocortisone occurs much later than the time of occurrence of maximum dexamethasone plasma concentrations (time =0) because after hydrocortisone suppression reaches a maximum the return to baseline is then a function of both the rate of hydrocortisone production and dexamethasone elimination. Therefore, the response lasts beyond the presence of effective dexamethasone concentrations because of the time needed for the system to equilibrate (Sharma & Jusko, 1998).
While statistically significant differences from baseline were only observed between 2 and 24 h, six dogs may not be statistically powerful enough, given the number of comparisons prepared in the post hoc Dunnett's test, to reject the null hypothesis for later time points.
Moreover, caution must be applied as 'not statistically different' is insufficient to conclude equivalence to baseline. The limited statistical power of this trial (6 dogs) prevents the conclusion that the hydrocortisone plasma concentrations at ≥48 h are significantly lower than baseline values. On the other hand, a conclusion that the difference is 'statistically significant' (eg 24 h) means there is strong evidence that the difference is not zero, but it will not be known whether the difference is large enough to be clinically or scientifically indistinguishable. With a limitation of 6 dogs, a simple a priori contrast approach could be applied to hydrocortisone levels using a t test comparison at a specified day (e.g. day 3 or 4) relative to pre-dose control. However, t tests between control and day 3 or 4 were not statistically signif- Given that the major anti-inflammatory effects and inhibition of hydrocortisone production by dexamethasone are rapid processes and that clinical indications in racing greyhounds relate to the administration of either DXD or DXF to greyhounds for their systemic anti-inflammatory effects, for example, its use for medial tibial periostitis, there is a strong case that in these circumstances the Toutain method has validity for medication control in racing greyhounds as it is in racing horses (Ekstrand et al., 2015).
In conclusion, this study makes available for dexamethasone, in widely available commercial formulations, extended plasma data, adds extended urine data, as well as providing an indication of one component of the pharmacodynamic effect by measuring the reduction in endogenous hydrocortisone. Greyhound racing regulators also now have the option of using this risk assessment information for risk management to decide an analytical cut-off value for screening of medications (Toutain, 2010). Further information on risk management using the methodology described by Toutain and Lassourd (2002) is provided in the supplementary online information.

ACK N OWLED G EM ENTS
None. contributed to regulatory interpretation. Sally Colgan was the principal investigator for the in-vivo study. All authors contributed to and reviewed the manuscript and are accountable for its contents.

A N I M A L WE LFA R E A N D E TH I C S S TATE M E NT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have adhered to international standards for the protection of animals used for scientific purposes. Ethics approval (TRIM 15/699(156)) was collected from the Secretary's Animal Care and Ethics Committee of the NSW Department of Primary Industry.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Toutain, P. L., & Lassourd, V. (2002). Pharmacokinetic/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse.