- Email: [email protected]
Plasma Dexamethasone Concentration in Relation to Glucose Response in the Horse
Accepted Manuscript Plasma dexamethasone concentration in relation to glucose response in the horse Carl Ekstrand, Ulrika Falkenö, Peter Kallings, Harold Tvedten, Inger Lilliehöök
PII:
S0737-0806(18)30413-1
DOI:
https://doi.org/10.1016/j.jevs.2018.11.008
Reference:
YJEVS 2630
To appear in:
Journal of Equine Veterinary Science
Received Date: 15 May 2018 Revised Date:
23 November 2018
Accepted Date: 23 November 2018
Please cite this article as: Ekstrand C, Falkenö U, Kallings P, Tvedten H, Lilliehöök I, Plasma dexamethasone concentration in relation to glucose response in the horse, Journal of Equine Veterinary Science (2018), doi: https://doi.org/10.1016/j.jevs.2018.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Original Article
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Plasma dexamethasone concentration in relation to glucose response in the horse
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Carl Ekstranda,*, Ulrika Falkenöb, Peter Kallingsd,e, Harold Tvedtenb,c, Inger Lilliehöökb,c
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a
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Address for correspondence:
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Swedish University of Agricultural Sciences
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Department of Biomedical Sciences and Veterinary Public Health
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P.O. Box 7028 SE-750 07 Uppsala
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Sweden
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*Corresponding author
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Tel.: +46-(0)18 - 67 31 71
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Fax: +46-(0)18 - 67 35 32
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E-mail: [email protected]
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Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Div. of Pharmacology and Toxicology, Uppsala, Sweden b Swedish University of Agricultural Sciences, University Animal Hospital, Clinical Pathology Laboratory, Uppsala, Sweden c Swedish University of Agricultural Sciences, Department of Clinical Sciences, Div. of Clinical Chemistry, Uppsala, Sweden d Swedish-Norwegian Foundation for Equine Research, Stockholm, Sweden e Swedish Trotting Association, Stockholm, Sweden
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ACCEPTED MANUSCRIPT Abstract
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Therapeutic agents capable of altering the performance of horses are monitored in racing and
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equestrian sports to guarantee horse welfare, fair competition and integrity of the sport.
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Dexamethasone is a common glucocorticoid drug for treating horses. Among other effects,
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dexamethasone is gluconeogenic and increases blood glucose. In this study, plasma samples from two
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dexamethasone exposure studies were analysed for glucose using an automated clinical chemistry
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analyzer. In study one, dexamethasone-21-isonicotinate was administered to six horses at the dose 30
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µg/kg intramuscularly. In study two, dexamethasone 21-phosphate disodium salt was administered
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intravenously to six horses as a bolus dose followed by 3 hours of infusion (bolus + infusion) at four
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different doses (placebo, 0.1+0.07µg/kg, 1+0.7µg/kg and 10+7µg/kg). Plasma dexamethasone
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concentrations were linked to plasma glucose concentrations by means of a turnover model. The
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median (range) pharmacodynamic parameters for glucose-response for the two studies were as
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follows: The EC50-value was 0.84 µg/L (0.47-1.50) and 0.85µg/L (0.75-2.45) respectively, the
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fractional turnover rate of response was 0.18 per h (0.07-0.27) and 0.25 per h (0.17.0.48) and the
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unaffected baseline of response was 4.20 mmol/L (4.10-6.60) and 5.42 mmol/L (5.22-5.96). These
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results may be used as input to future studies of the anti-inflammatory response of dexamethasone.
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The results also enables calculations of irrelevant plasma concentrations to determine whether the
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presence of a drug is a trace from legitimate medication or not. Therefore, this study provides further
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evidence for dexamethasone screening limits which protects the integrity of the sport and the welfare
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of the horse.
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Keywords: glucocorticoids, anti-doping, turnover model, pharmacodynamics, medication-control, equine
ACCEPTED MANUSCRIPT Introduction
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Dexamethasone is a glucocorticoid commonly used in horses for anti-inflammatory and immune
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suppressive effects. Race horses could have variable joint trauma from the stress of extensive training
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which causes pain. Symptoms of arthrosis are reduced by use of anti-inflammatory drugs like
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dexamethasone. However, there is a balance between what is needed to return a horse to health and
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what may be used to improve performance in competitions. It is of great importance that horses are
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healthy and fit to perform, but it is inappropriate that they are given medications that enhance their
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performance at the time of competition or mask symptoms of injury (that might get worse if the horse
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compete with medication). Rigid medication regulations and drug testing programs protect the
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integrity of equine sports and guarantee fair competition. Racing and Equestrian regulatory
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jurisdictions separate legitimate drugs (i.e. drugs necessary for the treatment of ill or injured horses)
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from banned substances (i.e. doping agents, e.g. anabolic steroids and erythropoietin). Both
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Fédération Equestre Internationale (FEI) and International Federation of Horseracing Authorities
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(IFHA, via article six in the International Agreement on Breeding, Racing and Wagering) prohibit
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participation in competition if performance is altered by legitimate medication.[1, 2] Technical
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improvements in analytical techniques have lowered the thresholds for detection of legitimate drugs in
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biological fluids [3, 4]. Consequently, traces of legitimate substances can be detected more remote
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from the time of medication at concentrations without pharmacological relevance, i.e. the medication
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has ceased to have an effect on performance. Hence, it is important to identify at what concentrations
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a medication can have a pharmacological effect that may affect the actual performance in order to
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separate those from traces of legitimate medication.
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One method to separate therapeutic concentrations from e.g. traces of legitimate medication is the use
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of screening limits (SL) in anti-doping procedures [3, 5-8]. A SL is an analytical sensitivity level for
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the anti-doping laboratory were the drug concentration is considered relevant to report or irrelevant,
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i.e. it does not affect or alter the performance of the horse. One prerequisite for SLs is a direct and
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reversible relation between plasma concentration and response [5, 6]. Plasma drug-concentration is
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recognised as the driving concentration of local concentrations in the biophase [9, 10]. There is
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ACCEPTED MANUSCRIPT recently published evidence that cortisol response (suppression) relates to plasma dexamethasone
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concentration [11, 12]. Unfortunately, there is no evidence that cortisol response is useful for
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estimation of neither the therapeutic response of glucocorticoids nor alteration of sport-horses’
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performance. Evidence is thus needed to demonstrate which plasma levels of dexamethasone have a
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biological effect in horses. One biological response to glucocorticoid exposure is elevation of plasma
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glucose concentration [13-16]. This hyperglycaemic response is well correlated to the anti-rumatic
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effect in man and liver glycogen disposition were earlier used in screening for new glucocorticoid
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compounds [17, 18]. The desired therapeutic response to glucocorticoid treatment may also be
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increased plasma glucose concentrations, for example when treating ketosis in cattle [19-22]. The aim
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of this study was to quantify the plasma glucose response at various plasma dexamethasone
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concentrations. Identification of which plasma dexamethasone concentrations give a gluconeogenic
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effect should contribute to the scientific evidence for the choice of dexamethasone screening limits. A
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second aim was to determine the potency of dexamethasone to aid in design of future research.
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Materials and Methods
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Plasma glucose was analysed in samples from two previously described dexamethasone exposure
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studies [11, 12]. In study one dose (DEX IM), dexamethasone-21-isonicotinate (Vorenvet vet. 1
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mg/mL, Boehringer Ingelheim Vetmedica, Malmö, Sweden,) was administered intramuscularly
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(IM) at the dose 30 µg/kg to six Standardbred horses (geldings), 3-16 years old and weighing 420-545
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kg. Heparin plasma samples from day 0 (pre-dose), 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 14, 20 and 28 days were
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analysed. In study two (DEX IV), Dexamethasone 21-phosphate disodium salt (Dexadreson 2 mg/mL,
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Intervet AB, Sollentuna, Sweden) was administered as an intravenous (IV) bolus dose followed by
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three hours constant rate infusion (bolus + infusion) at four different dose levels: control (saline), 0.1
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+ 0.07 µg/kg, 1 + 0.7 µg/kg and 10 + 7 µg/kg to six standardbred horses (four mares and two
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geldings) 6-20 years old and weighing 430-584 kg. The doses were given to the horses in a
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randomized order. Heparin plasma samples from hour 0 (pre-dose), 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36
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and 48 hours were analysed from all dose-levels. The study protocols were both approved by Ethics
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Committees for Animal Experiments, Stockholm/Uppsala, Sweden (C232/8, C333/11) and are more
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thoroughly described in the original publications [11, 12].
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Analytical methods for drug analyses in plasma
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Total plasma dexamethasone concentrations were analysed and quantified using Ultra High
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Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS). The method
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showed linearity over a concentration range of 0.025-10 µg/L. The analytical method was described in
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detail by Ekstrand et al. [11].
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Plasma glucose concentrations was determined with a hexokinase method and an automatic chemistry
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instrument, Architect c4000 both from Abbott Laboratories, Abbott Park, IL, US.
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Disposition of dexamethasone and glucose response
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Dexamethasone plasma disposition was characterised by means of compartmental modelling (Fig. 1A,
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2A). In the DEX IM study the disposition of dexamethasone was described as =
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∙[
∙(
−
)
∙(
)
]
(1)
Where Cp denote the dexamethasone plasma concentration, A is a pharmacokinetic macro parameter,
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k, ka, t and tlag denote the elimination- and absorption rate constants, the time and the lag-time
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respectively.
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In the DEX IV study, the disposition of dexamethasone in the central compartment was described as =
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∙
+
∙
−
∙
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∙
(2)
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where Cp and Ct denote the dexamethasone concentration in the central and peripheral compartment,
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respectively, Vc is the central volume of distribution, Inf is the dose infused, Cl is the clearance of
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dexamethasone and Cld is the inter-compartmental distribution.
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The peripheral compartment was described as ∙
=
∙
−
∙
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(3)
where Vt is the peripheral volume of distribution.
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Dexamethasone was assumed to directly stimulate the glucose turnover rate described as =1+!
"# $
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(4)
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where S(Cp) and EC50 are the stimulatory drug mechanism function and the dexamethasone plasma
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concentration at 50 % of maximum response, respectively. The turnover of glucose with the
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stimulatory drug mechanism incorporated was in the DEX IM study described by
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%
= &'( ∙ [1 + !
"# $
] − &)* ∙ +
(5)
%
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where
, kin and kout are the rate of change of response the turnover rate and the first-order fractional
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turnover rate respectively.
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The turnover of glucose with the stimulatory drug mechanism incorporated was in the DEX IV study
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described by ,
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%-
%0
= &'( ∙ [1 + !
"# $
] − &)* ∙ +/
= &)* ∙ (+/ − +1 )
%0
(6)
where
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turnover model. R1 and R2 denote the target interaction compartment and glucose response in plasma,
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respectively. Initial parameter estimates were derived graphically for the pharmacodynamic
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parameters. A constant (absolute) error (weighting) model was used for regression of glucose
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response-time data performed using WinNonlin 4.0.1 (Certara, St. Louis, Missouri, U.S.A).
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Statistical analyses
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Statistical analyses was performed by means of analysis of variance (ANOVA) using a non-linear
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mixed-effects model. In the DEX IM study time was used as fixed effect and horse as random effect.
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An ad hoc analyses (Dunnett´s test) was performed in order to compare glucose concentrations after
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dexamethasone administration with the pre-administration glucose concentrations. In the DEX IV
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study time and dose were used as fixed effects and horse as random effect. Glucose concentrations
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were compared between doses (contrasts) for every time-point. Glucose concentrations after
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dexamethasone administration were also compared for each dose-level to pre-administration
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concentrations by use of Dunnett´s test as described for the DEX IM study. Statistical significance
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was considered when P < .05. All statistical analyses were performed using the statistical software R
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version 3.4.4 (The R Foundation for Statistical Computing, Vienna, Austria).
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, are the rate of change of response in respectively compartment 1 and 2 of the
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and
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ACCEPTED MANUSCRIPT Results
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In the DEX IM study, plasma glucose concentrations increased after dexamethasone administration.
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Maximal plasma glucose concentration was observed at 0-36 hours after maximal dexamethasone
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plasma concentration. The glucose response was positively related to dexamethasone concentration;
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increased dexamethasone plasma concentration increased the glucose response. The greatest glucose
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response was observed in the horse with the greatest dexamethasone peak concentration and the
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lowest glucose response was observed in the horse with the lowest observed dexamethasone peak
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concentration. Plasma glucose concentrations gradually returned to baseline as plasma dexamethasone
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concentrations declined. Glucose concentrations were significantly increased compared at 24 h (P <
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.0001), 48 h (P < .001) and 72 h (P = .0274) compared with 0 h. Dexamethasone was still quantifiable
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in plasma up to 13 days (LOQ 0.025 µg/L). Mean observed and model predicted dexamethasone and
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glucose response data are shown in Fig. 2.
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In the DEX IV study only the highest dexamethasone dose resulted in an increase in glucose response.
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Plasma glucose concentrations was significantly increased at 4 h (P = .0001), 5 h (P < .0001), 6 h (P <
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.0001), 9 h (P = .0001), 12 h (P = .0065), 18 h (P = .001) and 24 h (P = .0455) after dexamethasone
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administration compared with control treatment. Plasma glucose concentrations peaked 6-12 hours
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after the start of dexamethasone infusion, and then declined as plasma dexamethasone concentrations
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decreased. Plasma glucose concentrations were increased at 4 h (P = .001), 5 h (P < .0001), 6 h (P <
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.0001), 9 h (P = .0002), 12 h (P = .0025), 18 h (P = .022) and 36 h (P = .0179) compared with the pre-
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administration samples. Dexamethasone was still quantifiable at 24 hours (LOQ 0.025µg/L) in all
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horses. Mean observed and model predicted dexamethasone and glucose response data are shown in
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Fig. 3.
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The EC50, kout, and R0 (baseline) of glucose was estimated in all horses. The EC50 parameter varied
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over a ten-fold range in the DEX IM study and four-fold in the DEX IV study. The kout parameter
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varied four-fold in the IM study and over a three-fold range in the IV study. The R0 parameter showed
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low variability within studies and approximately 20 % difference between studies. The parameter
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ACCEPTED MANUSCRIPT estimates are shown in table 1. With the drug present the parameters corresponding to the baseline-
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parameter in the unaffected system were in median (range) 5.42 mmol/L (5.22-5. 57), 5.40 mmol/L
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(5.19-5.50) and 5.80 mmol/L (5.49-6.06) for the low, intermediate and high dose, respectively, in the
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DEX IV study. The regressed plasma dexamethasone-time and response-time courses are shown in
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Fig. 4. Both regressed plasma dexamethasone concentration-time courses and the regressed plasma
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glucose response-time courses show larger variation between individuals in DEX IM compared to the
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DEX IV study.
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ACCEPTED MANUSCRIPT Discussion
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In this study the use of a turnover model allowed quantification of glucose response data. The time
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courses from both a single dose administered IM and multiple IV doses could be characterised by
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means of pharmacokinetic and pharmacodynamic analyses. The results show a plasma dexamethasone
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concentration – glucose response relation. The use of a slow release pharmaceutical product in the
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DEX IM study caused dexamethasone plasma exposure characterised by low peak in plasma
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dexamethasone concentration above the lower limit of quantification (LOQ) characterised by a
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relatively mildly increased plasma dexamethasone concentration of several days duration compared to
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the DEX IV study (Figs. 2 and 3). The use of an intravenous solution in DEX IV gave a pattern of
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high immediate concentrations but more rapid decrease below LOQ with lower variation between the
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horses compared to the DEX IM study. The plasma glucose time courses corresponded well to plasma
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dexamethasone time-courses in both studies (Fig. 4). The pharmacodynamic parameters were in
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general estimated with acceptable precision (Table 1). The potency (EC50)-value for glucose response
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presented here is also ten-fold higher than the potency value for cortisol response in the horse [11,
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12]. The variation in parameter estimates in this study is similar compared to parameter variation in
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other studies. For instance the potency (EC50)-value varies four to ten-fold in this study. This is
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consistent with the ten-fold variation in different potency-values previously reported for both horses
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and dogs and the kout-parameter in this study showed lower between animal variation compared with
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that for cortisol response in horses or the response to nimesulide in dogs [12, 23, 24]. The R0 was
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4.20 and 5.42 in the DEX IM and the DEX IV study, respectively. Both values are consistent with
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previously reported pre-race or pre-experimental baseline plasma glucose concentrations [13, 25-27].
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It is also within the range of the internal laboratory reference value for plasma glucose (3.6-6.5
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mmol/L) where the samples were analysed.
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In this study, the glucose response was predicted by means of a turnover model with stimulatory
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function for production of the response. Glucocorticoids exert their plasma glucose response by
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increasing the rate of gluconeogenesis and decreasing the tissue uptake of glucose. Decreased uptake
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is by means of decreased tissue insulin sensitivity [28, 29]. Glucose homeostasis is strongly associated
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ACCEPTED MANUSCRIPT with other hormones e.g. insulin and glucagon. Plasma insulin is elevated in glucocorticoid treated
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horses [13]. The most elegant studies modelling glucose response to drug exposure has included a
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moderator of glucose response, e.g. insulin concentrations [30, 31]. In those studies, the moderator
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has allowed separation of different mechanisms in plasma glucose regulation resulting in more precise
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parameter estimates. The model in this study did not include any moderator of glucose dynamics other
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than dexamethasone exposure. As a consequence, the potency (EC50)-values presented here is of
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lower value for glucose regulation. However, from an anti-doping perspective the data are useful
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since the focus is on the drug response. The model predicted response-time courses are acceptable
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even though the model constantly underestimated the peaks of the response. Conclusively, the results
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presented in this study should mainly function as input to future research and anti-doping procedures
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although the weaknesses might be considered when interpreting the results and choosing an
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uncertainty factor in the screening limit decision process.
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Both endogenous and exogenous glucocorticoids increase endurance in rats and humans [32-36]. It is
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known that carbohydrate supplementation during exercise prevents reduction in performance capacity
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in humans and that glucocorticoids have a gluconeogenetic effect, for instance by upregulation of
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gluconeogenetic enzymes [37-43] . During prolonged exercise (e.g. endurance sports) plasma glucose
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concentrations decrease and fatigue develops [26, 27, 44]. If gluconeogenesis is inhibited these
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changes develop quicker [45]. Supplementary carbohydrates during exercise maintain blood glucose
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concentrations, attenuate decrease in both maximum muscle contraction force and technical skills and
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delay the development of fatigue [37-41]. The gluconeogenetic formation of glucose from lactate has
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also been described as one of the main pathways for lactate disposal [46]. There is no currently
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available evidence that pre-exercise blood glucose elevation increases performance in horses. On the
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contrary, there is conflicting evidence that pre-exercise carbohydrate feeding that increases blood
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glucose concentrations may result in more severe hypoglycaemia during exercise [25, 47-49].
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However the doping of sport horses could aim at both increasing performance (so called positive
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doping) and decreasing the performance (so called negative doping) [50]. The effects on performance
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described above and the correlation between increased blood-glucose levels and anti-rheumatic
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ACCEPTED MANUSCRIPT response in man [18] strongly suggest that plasma glucose concentrations are relevant for the anti-
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doping control in sport-horses. The pharmacodynamics parameters presented in this study increase the
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evidence-base for dexamethasone screening limits. Unfortunately, IFHA has not yet published an
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international SL for dexamethasone in plasma but there is an international SL for dexamethasone in
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urine (0.2 µg/L) [51]. In urine, dexamethasone concentration is approximately ten-fold higher
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compared to the concurrent concentration in plasma [11]. Since the plasma/urine ratio is not a robust
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factor [5] a conservative attitude when transforming irrelevant plasma concentrations to irrelevant
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urine concentrations may be appropriate. Toutain & Lassourd [5] established that the effective plasma
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concentration can be calculated by dividing the standard dose (per dosing interval) with the clearance
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(per dosing interval). Using data collected from an experimental study by Soma et al. [52], the dose
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50 µg/kg and the clearance 0.5 L · h ⁄ kg result in the effective plasma concentration 4 µg/L in horses.
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Applying an uncertainty factor 500 to the effective plasma concentration 4 µg/L creates an irrelevant
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plasma concentration (IPC) that translates into the SL 0.008 µg/L. This uncertainty factor consist of a
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factor 50 that transforms an effective concentration to an irrelevant concentration and a factor 10 that
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takes inter-individual variability into account [5]. The value 0.008 µg/L is below the EC50-value for
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glucose response presented in this study and lower than the median IC50-values for inhibiting cortisol
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secretion into plasma presented elsewhere [11, 12]. The use of the uncertainty factor 50 (that
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according to Toutain & Lassourd [5]convert a concentration close to a IC50-value to an irrelevant
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concentration) on the lowest IC50-value presented in this study produce the irrelevant concentration
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0.006µg/L which is consistent with 0.008 µg/L. Applying the ten-fold ratio between plasma and urine,
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the irrelevant plasma concentrations suggested in this study result in the irrelevant urine concentration
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0.08 µg/L. That can be compared with 0.2 µg/L given by IFHA. Obviously, 0.008 µg/L plasma and
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0.08 µg/L urine are concentrations without biologically relevant effect that does not alter the
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performance of horses and lower concentrations can be considered as traces of legitimate medication.
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Acknowledgment
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This work was supported by the Swedish-Norwegian Foundation for Equine Research and the Nordic
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Equine Medication and Anti-Doping Committee. Preliminary results were presented as an abstract, an
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oral presentation and in conference proceedings of the 20th International Conference of Racing
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Analysts and Veterinarians (ICRAV), Mauritius, 20th-28th September 2014.
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References 1. Fédération Equestre Internationale. 2018. Treatment and Prohibited Substances. http://inside.fei.org/fei/your-role/veterinarians/cleansport. Accessed 2018 04 16. 2. International Association of Horseracing Authorites. 2018. International Agreement on Breeding, Racing and Wagering. Available from: http://www.horseracingintfed.com/resources/2016Agreement.pdf. Accessed 2018 04 16. 3. Barragry, T., Doping and drug detection times in horses: New data for therapeutic agents. Irish Veterinary Journal, 2006. 59(7): p. 394-398. 4. Webbon, P., Medication: a way forward from zero tolerance to irrelevant plasma concentrations. Equine Vet J, 2002. 34(3): p. 220-1. 5. Toutain, P.L. and V. Lassourd, Pharmacokinetic/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse. Equine Veterinary Journal, 2002. 34(3): p. 242-9. 6. Toutain, P.L., Veterinary medicines and competition animals: the question of medication versus doping control. Handbook of experimental pharmacology, 2010(199): p. 315-39. 7. European Horseracing Science and Liason Committée. 2018, The Science behind this work. https://www.ehslc.com/detection-times/the-science-behind-this-work. Accessed 2018 04 16. 8. International Association of Horseracing Authorites. 2018. Screening limits in Plasma. http://www.horseracingintfed.com/default.asp?section=IABRW&area=6. Accessed 2018 04 16. 9. Gabrielsson, J. and A.R. Green, Quantitative pharmacology or pharmacokinetic pharmacodynamic integration should be a vital component in integrative pharmacology. J Pharmacol Exp Ther, 2009. 331(3): p. 767-74. 10. Wright, D.F., H.R. Winter, and S.B. Duffull, Understanding the time course of pharmacological effect: a PKPD approach. Br J Clin Pharmacol, 2011. 71(6): p. 815-23.
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Ekstrand, C., et al., Plasma concentration-dependent suppression of endogenous hydrocortisone in the horse after intramuscular administration of dexamethasone-21isonicotinate. Journal of Veterinary Pharmacology and Therapeutics, 2015. 38(3): p. 235-42. Ekstrand, C., et al., A quantitative approach to analysing cortisol response in the horse. J Vet Pharmacol Ther, 2016. 39(3): p. 255-263. Tiley, H.A., R.J. Geor, and L.J. McCutcheon, Effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses. American journal of veterinary research, 2007. 68(7): p. 753-9. Soma, L.R., et al., Pharmacokinetics of dexamethasone with pharmacokinetic/pharmacodynamic model of the effect of dexamethasone on endogenous hydrocortisone and cortisone in the horse. Journal of Veterinary Pharmacology and Therapeutics, 2005. 28(1): p. 71-80. Abraham, G., et al., Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: association with topical dexamethasone usage. Veterinary journal, 2011. 188(3): p. 307-12. Cartmill, J.A., et al., Leptin secretion in horses: effects of dexamethasone, gender, and testosterone. Domest Anim Endocrinol, 2006. 31(2): p. 197-210. Ringler, I. and W.E. Dulin, Potency relationships of various C-21 steroids as measured by two methods of liver glycogen deposition. Proc Soc Exp Biol Med, 1962. 110: p. 869-72. Ringler, I., et al., Biological potencies of chemically modified adrenocorticosteroids in rat and man. Metabolism, 1964. 13: p. 37-44. Andersson, L. and T. Olsson, The effect of two glucocorticoids on plasma glucose and milk production in healthy cows and the therapeutic effect in ketosis. Nordisk Veterinaermedicin, 1984. 36(1-2): p. 13-8. Wierda, A., et al., Effects of two glucocorticoids on milk yield and biochemical measurements in healthy and ketotic cows. Vet Rec, 1987. 120(13): p. 297-9. Shpigel, N.Y., et al., Use of corticosteroids alone or combined with glucose to treat ketosis in dairy cows. J Am Vet Med Assoc, 1996. 208(10): p. 1702-4. Braun, R.K., E.N. Bergman, and T.F. Albert, Effects of various synthetic glucocorticoids on milk production and blood glucose and ketone body concentrations in normal and ketotic cows. Journal of the American Veterinary Medical Association, 1970. 157(7): p. 941-6. Toutain, P.L., et al., Plasma concentrations and therapeutic efficacy of phenylbutazone and flunixin meglumine in the horse: pharmacokinetic/pharmacodynamic modelling. J Vet Pharmacol Ther, 1994. 17(6): p. 459-69. Toutain, P.L., et al., A pharmacokinetic/pharmacodynamic approach vs. a dose titration for the determination of a dosage regimen: the case of nimesulide, a Cox-2 selective nonsteroidal anti-inflammatory drug in the dog. J Vet Pharmacol Ther, 2001. 24(1): p. 43-55. Stull, C.L. and A.V. Rodiek, Stress and glycemic responses to postprandial interval and feed components in exercising horses. Journal of Equine Veterinary Science, 1995. 15(9): p. 382386. Essen-Gustavsson, B., K. Karlstrom, and A. Lindholm, Fibre types, enzyme activities and substrate utilisation in skeletal muscles of horses competing in endurance rides. Equine Vet J, 1984. 16(3): p. 197-202. Larsson, J., et al., Physiological parameters of endurance horses pre- compared to post-race, correlated with performance: a two race study from scandinavia. ISRN veterinary science, 2013. 2013: p. 684353. Baxter, J.D. and P.H. Forsham, Tissue effects of glucocorticoids. Am J Med, 1972. 53(5): p. 573-89. Pagano, G., et al., An in vivo and in vitro study of the mechanism of prednisone-induced insulin resistance in healthy subjects. J Clin Invest, 1983. 72(5): p. 1814-20. Cao, Y., W. Gao, and W.J. Jusko, Pharmacokinetic/pharmacodynamic modeling of GLP-1 in healthy rats. Pharmaceutical research, 2012. 29(4): p. 1078-86. Jin, J.Y. and W.J. Jusko, Pharmacodynamics of Glucose Regulation by Methylprednisolone. II. Normal Rats. Biopharmaceutics & Drug Disposition, 2009. 30(1): p. 35-48.
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Gorostiaga, E.M., S.M. Czerwinski, and R.C. Hickson, Acute glucocorticoid effects on glycogen utilization, O2 uptake, and endurance. J Appl Physiol (1985), 1988. 64(3): p. 1098106. Sellers, T.L., et al., Effect of the exercise-induced increase in glucocorticoids on endurance in the rat. J Appl Physiol (1985), 1988. 65(1): p. 173-8. Le Panse, B., et al., Short-term glucocorticoid intake improves exercise endurance in healthy recreationally trained women. Eur J Appl Physiol, 2009. 107(4): p. 437-43. Arlettaz, A., et al., Effects of short-term prednisolone intake during submaximal exercise. Medicine and Science in Sports and Exercise, 2007. 39(9): p. 1672-8. Viru, M., et al., Glucocorticoids in metabolic control during exercise: glycogen metabolism. J Sports Med Phys Fitness, 1994. 34(4): p. 377-82. Coyle, E.F., et al., Carbohydrate feeding during prolonged strenuous exercise can delay fatigue. J Appl Physiol Respir Environ Exerc Physiol, 1983. 55(1 Pt 1): p. 230-5. Farris, J.W., et al., Effect of tryptophan and of glucose on exercise capacity of horses. J Appl Physiol (1985), 1998. 85(3): p. 807-16. Harper, L.D., et al., Physiological and performance effects of carbohydrate gels consumed prior to the extra-time period of prolonged simulated soccer match-play. J Sci Med Sport, 2015. Nybo, L., CNS fatigue and prolonged exercise: effect of glucose supplementation. Med Sci Sports Exerc, 2003. 35(4): p. 589-94. Patterson, S.D. and S.C. Gray, Carbohydrate-gel supplementation and endurance performance during intermittent high-intensity shuttle running. Int J Sport Nutr Exerc Metab, 2007. 17(5): p. 445-55. Frijters, R., et al., Prednisolone-induced differential gene expression in mouse liver carrying wild type or a dimerization-defective glucocorticoid receptor. BMC Genomics, 2010. 11: p. 359. Yoon, J.C., et al., Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature, 2001. 413(6852): p. 131-8. Lucke, J.N. and G.M. Hall, Biochemical changes in horses during a 50-mile endurance ride. Vet Rec, 1978. 102(16): p. 356-8. John-Alder, H.B., R.M. McAllister, and R.L. Terjung, Reduced running endurance in gluconeogenesis-inhibited rats. Am J Physiol, 1986. 251(1 Pt 2): p. R137-42. Jitrapakdee, S., Transcription factors and coactivators controlling nutrient and hormonal regulation of hepatic gluconeogenesis. Int J Biochem Cell Biol, 2012. 44(1): p. 33-45. Lawrence, L., et al., Feeding staus affects glucose-metabolism in exercising horses. Journal of Nutrition, 1993. 123(12): p. 2152-2157. Bullimore, S.R., et al., Carbohydrate supplementation of horses during endurance exercise: Comparison of fructose and glucose. Journal of Nutrition, 2000. 130(7): p. 1760-1765. Vervuert, I., M. Coenen, and A. Bichmann, Comparison of the effects of fructose and glucose supplementation on metabolic responses in resting and exercising horses. Journal of Veterinary Medicine Series a-Physiology Pathology Clinical Medicine, 2004. 51(4): p. 171177. Wong, J.K. and T.S. Wan, Doping control analyses in horseracing: a clinician's guide. Vet J, 2014. 200(1): p. 8-16. International Association of Horseracing Authorites. 2018. Screening limits in Urine. http://www.ifhaonline.org/default.asp?section=IABRW&area=1. Accessed 2018 04 16. Soma, L.R., et al., Pharmacokinetics of dexamethasone following intra-articular, intravenous, intramuscular, and oral administration in horses and its effects on endogenous hydrocortisone. Journal of Veterinary Pharmacology and Therapeutics, 2013. 36(2): p. 18191.
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Table 1. Pharmacodynamic parameters and their precision (relative standard deviation).
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DEX IV
Kout (1/h)
R0 (mmol/L)
Horse
EC50 (µg/L)
Kout (1/h)
R0 (mmol/L)
1 2 3 4 5 6 Median Range
0.31 ± 97.4 0.74 ± 43.4 3.26 ± 113 0.73 ± 67.3 0.51 ± 47.1 0.34 ± 68.1 0.62 0.31 – 3.26
0.11 ± 87.6 0.18 ± 77.9 0.27 ± 641 0.21 ± 120 0.07 ± 51.4 0.08 ± 56.4 0.15 0.07 – 0.27
4.88 ± 11.2 4.20 ± 5.16 4.34 ± 5.23 4.19 ± 8.09 4.25 ± 5.02 4.31 ± 8.14 4.23 4.19 – 4.88
A B C D E F Median Range
2.11 ± 47.9 0.66 ± 45.2 2.36 ± 91.7 0.62 ± 43.6 0.72 ± 34.6 0.57 ± 44.6 0.69 0.57 – 2.36
0.48 ± 36.3 0.17 ± 21.6 0.28 ± 56.1 0.24 ± 18.7 0.26 ± 16.7 0.20 ± 19.4 0.25 0.17 – 0.48
5.37 ± 1.6 5.22 ± 1.5 5.96 ± 2.0 5.32 ± 1.7 5.46 ± 1.4 5.57 ± 1.5 5.42 5.22 – 5.96
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EC50 (µg/L)
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DEX IM Horse
EC50, kout and R0 are the plasma dexamethasone concentration at 50 % of the response, the fractional turnover rate and the unaffected baseline of response, respectively.
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Fig. 1. Schematic illustration of the dexamethasone disposition models for the DEX IM study (1A), the DEX IV study (2A) and the glucose turnover models from the DEX IM study (1B) and DEX IV study (2B) describing the dexamethasone-induced changes in glucose response in horses. In the upper plot, DEX IM, Cp, ka, and k denote the dose (administered intramuscularly), the dexamethasone plasma concentration, the absorption- and the elimination rate constants of dexamethasone in plasma respectively. In the lower plot, DEX IV represents the bolus + intravenous infusion regimen. Vc, Vt, Cl and Cld represent the central and peripheral volume of distribution, clearance and inter-compartmental distribution parameter, respectively. The plasma exposure (Equations 1, 2 and 3) then served to ‘drive’ the drug-mechanism function (Equation 4) acting on the turnover rate of glucose in respectively the DEX IM study (2A) and the DEX IV study (2B). S(C), kin, kout and R represent the stimulatory drug mechanism function, the turnover rate, the fractional turnover rate and the glucose response, respectively. One transit compartment (shaded) was used to capture the delayed onset of action on glucose response in the DEX IV study (2B).
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Fig. 2. Mean observed (filled circles) and mean predicted (lines) plasma concentrations of dexamethasone (upper plot, A) and glucose (lower plot, B). The plots only include data from ten days and not the entire sampling period (28 days). The concentration-time courses are produced after administration of 30 µg/kg dexamethasone-21-isonicotinate intramuscularly. * = statistically different compared with day 0.
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Fig. 3. Mean observed (symbols) and mean predicted (lines) plasma concentrations of dexamethasone (upper plot, A) and glucose (lower plot, B) showing that only the highest dose dexamethasone increased glucose concentrations in plasma. Dexamethasone 21-phosphate disodium salt was administered as an intra-venous (IV) bolus dose followed by three hours constant rate infusion (bolus + infusion) at four different dose-levels: saline control (empty circles), 0.1+0.07 µg/kg (squares), 1+0.7 µg/kg (triangles) and 10+7 µg/kg (filled circles). * = significant difference between treatment with the highest dose dexamethasone and treatment with saline.
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Fig. 4. Predicted plasma dexamethasone-time (upper plots) and glucose-time (lower plots) courses from six horses in the DEX IM (left plots) study and six horses in the DEX IV (right plots) study, respectively. Note that only predicted concentration-time profiles based on data from the 17µg/kg study occasion of the DEX IV study are included in the figure.
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ACCEPTED MANUSCRIPT Administration of dexamethasone increased plasma glucose concentration The potency-value for glucose response to dexamethasone ranged 0.47-2.45 µg/L
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Pharmacodynamic parameters strengthen medication-control in equine sports
PII:
S0737-0806(18)30413-1
DOI:
https://doi.org/10.1016/j.jevs.2018.11.008
Reference:
YJEVS 2630
To appear in:
Journal of Equine Veterinary Science
Received Date: 15 May 2018 Revised Date:
23 November 2018
Accepted Date: 23 November 2018
Please cite this article as: Ekstrand C, Falkenö U, Kallings P, Tvedten H, Lilliehöök I, Plasma dexamethasone concentration in relation to glucose response in the horse, Journal of Equine Veterinary Science (2018), doi: https://doi.org/10.1016/j.jevs.2018.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Original Article
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Plasma dexamethasone concentration in relation to glucose response in the horse
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Carl Ekstranda,*, Ulrika Falkenöb, Peter Kallingsd,e, Harold Tvedtenb,c, Inger Lilliehöökb,c
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a
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Address for correspondence:
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Swedish University of Agricultural Sciences
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Department of Biomedical Sciences and Veterinary Public Health
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P.O. Box 7028 SE-750 07 Uppsala
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Sweden
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*Corresponding author
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Tel.: +46-(0)18 - 67 31 71
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Fax: +46-(0)18 - 67 35 32
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E-mail: [email protected]
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Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Div. of Pharmacology and Toxicology, Uppsala, Sweden b Swedish University of Agricultural Sciences, University Animal Hospital, Clinical Pathology Laboratory, Uppsala, Sweden c Swedish University of Agricultural Sciences, Department of Clinical Sciences, Div. of Clinical Chemistry, Uppsala, Sweden d Swedish-Norwegian Foundation for Equine Research, Stockholm, Sweden e Swedish Trotting Association, Stockholm, Sweden
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ACCEPTED MANUSCRIPT Abstract
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Therapeutic agents capable of altering the performance of horses are monitored in racing and
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equestrian sports to guarantee horse welfare, fair competition and integrity of the sport.
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Dexamethasone is a common glucocorticoid drug for treating horses. Among other effects,
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dexamethasone is gluconeogenic and increases blood glucose. In this study, plasma samples from two
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dexamethasone exposure studies were analysed for glucose using an automated clinical chemistry
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analyzer. In study one, dexamethasone-21-isonicotinate was administered to six horses at the dose 30
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µg/kg intramuscularly. In study two, dexamethasone 21-phosphate disodium salt was administered
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intravenously to six horses as a bolus dose followed by 3 hours of infusion (bolus + infusion) at four
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different doses (placebo, 0.1+0.07µg/kg, 1+0.7µg/kg and 10+7µg/kg). Plasma dexamethasone
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concentrations were linked to plasma glucose concentrations by means of a turnover model. The
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median (range) pharmacodynamic parameters for glucose-response for the two studies were as
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follows: The EC50-value was 0.84 µg/L (0.47-1.50) and 0.85µg/L (0.75-2.45) respectively, the
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fractional turnover rate of response was 0.18 per h (0.07-0.27) and 0.25 per h (0.17.0.48) and the
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unaffected baseline of response was 4.20 mmol/L (4.10-6.60) and 5.42 mmol/L (5.22-5.96). These
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results may be used as input to future studies of the anti-inflammatory response of dexamethasone.
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The results also enables calculations of irrelevant plasma concentrations to determine whether the
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presence of a drug is a trace from legitimate medication or not. Therefore, this study provides further
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evidence for dexamethasone screening limits which protects the integrity of the sport and the welfare
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of the horse.
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Keywords: glucocorticoids, anti-doping, turnover model, pharmacodynamics, medication-control, equine
ACCEPTED MANUSCRIPT Introduction
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Dexamethasone is a glucocorticoid commonly used in horses for anti-inflammatory and immune
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suppressive effects. Race horses could have variable joint trauma from the stress of extensive training
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which causes pain. Symptoms of arthrosis are reduced by use of anti-inflammatory drugs like
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dexamethasone. However, there is a balance between what is needed to return a horse to health and
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what may be used to improve performance in competitions. It is of great importance that horses are
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healthy and fit to perform, but it is inappropriate that they are given medications that enhance their
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performance at the time of competition or mask symptoms of injury (that might get worse if the horse
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compete with medication). Rigid medication regulations and drug testing programs protect the
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integrity of equine sports and guarantee fair competition. Racing and Equestrian regulatory
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jurisdictions separate legitimate drugs (i.e. drugs necessary for the treatment of ill or injured horses)
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from banned substances (i.e. doping agents, e.g. anabolic steroids and erythropoietin). Both
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Fédération Equestre Internationale (FEI) and International Federation of Horseracing Authorities
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(IFHA, via article six in the International Agreement on Breeding, Racing and Wagering) prohibit
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participation in competition if performance is altered by legitimate medication.[1, 2] Technical
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improvements in analytical techniques have lowered the thresholds for detection of legitimate drugs in
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biological fluids [3, 4]. Consequently, traces of legitimate substances can be detected more remote
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from the time of medication at concentrations without pharmacological relevance, i.e. the medication
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has ceased to have an effect on performance. Hence, it is important to identify at what concentrations
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a medication can have a pharmacological effect that may affect the actual performance in order to
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separate those from traces of legitimate medication.
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One method to separate therapeutic concentrations from e.g. traces of legitimate medication is the use
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of screening limits (SL) in anti-doping procedures [3, 5-8]. A SL is an analytical sensitivity level for
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the anti-doping laboratory were the drug concentration is considered relevant to report or irrelevant,
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i.e. it does not affect or alter the performance of the horse. One prerequisite for SLs is a direct and
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reversible relation between plasma concentration and response [5, 6]. Plasma drug-concentration is
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recognised as the driving concentration of local concentrations in the biophase [9, 10]. There is
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ACCEPTED MANUSCRIPT recently published evidence that cortisol response (suppression) relates to plasma dexamethasone
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concentration [11, 12]. Unfortunately, there is no evidence that cortisol response is useful for
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estimation of neither the therapeutic response of glucocorticoids nor alteration of sport-horses’
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performance. Evidence is thus needed to demonstrate which plasma levels of dexamethasone have a
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biological effect in horses. One biological response to glucocorticoid exposure is elevation of plasma
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glucose concentration [13-16]. This hyperglycaemic response is well correlated to the anti-rumatic
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effect in man and liver glycogen disposition were earlier used in screening for new glucocorticoid
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compounds [17, 18]. The desired therapeutic response to glucocorticoid treatment may also be
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increased plasma glucose concentrations, for example when treating ketosis in cattle [19-22]. The aim
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of this study was to quantify the plasma glucose response at various plasma dexamethasone
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concentrations. Identification of which plasma dexamethasone concentrations give a gluconeogenic
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effect should contribute to the scientific evidence for the choice of dexamethasone screening limits. A
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second aim was to determine the potency of dexamethasone to aid in design of future research.
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Materials and Methods
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Plasma glucose was analysed in samples from two previously described dexamethasone exposure
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studies [11, 12]. In study one dose (DEX IM), dexamethasone-21-isonicotinate (Vorenvet vet. 1
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mg/mL, Boehringer Ingelheim Vetmedica, Malmö, Sweden,) was administered intramuscularly
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(IM) at the dose 30 µg/kg to six Standardbred horses (geldings), 3-16 years old and weighing 420-545
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kg. Heparin plasma samples from day 0 (pre-dose), 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 14, 20 and 28 days were
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analysed. In study two (DEX IV), Dexamethasone 21-phosphate disodium salt (Dexadreson 2 mg/mL,
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Intervet AB, Sollentuna, Sweden) was administered as an intravenous (IV) bolus dose followed by
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three hours constant rate infusion (bolus + infusion) at four different dose levels: control (saline), 0.1
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+ 0.07 µg/kg, 1 + 0.7 µg/kg and 10 + 7 µg/kg to six standardbred horses (four mares and two
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geldings) 6-20 years old and weighing 430-584 kg. The doses were given to the horses in a
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randomized order. Heparin plasma samples from hour 0 (pre-dose), 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36
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and 48 hours were analysed from all dose-levels. The study protocols were both approved by Ethics
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Committees for Animal Experiments, Stockholm/Uppsala, Sweden (C232/8, C333/11) and are more
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thoroughly described in the original publications [11, 12].
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Analytical methods for drug analyses in plasma
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Total plasma dexamethasone concentrations were analysed and quantified using Ultra High
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Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS). The method
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showed linearity over a concentration range of 0.025-10 µg/L. The analytical method was described in
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detail by Ekstrand et al. [11].
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Plasma glucose concentrations was determined with a hexokinase method and an automatic chemistry
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instrument, Architect c4000 both from Abbott Laboratories, Abbott Park, IL, US.
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Disposition of dexamethasone and glucose response
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Dexamethasone plasma disposition was characterised by means of compartmental modelling (Fig. 1A,
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2A). In the DEX IM study the disposition of dexamethasone was described as =
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∙[
∙(
−
)
∙(
)
]
(1)
Where Cp denote the dexamethasone plasma concentration, A is a pharmacokinetic macro parameter,
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k, ka, t and tlag denote the elimination- and absorption rate constants, the time and the lag-time
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respectively.
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In the DEX IV study, the disposition of dexamethasone in the central compartment was described as =
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∙
+
∙
−
∙
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(2)
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where Cp and Ct denote the dexamethasone concentration in the central and peripheral compartment,
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respectively, Vc is the central volume of distribution, Inf is the dose infused, Cl is the clearance of
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dexamethasone and Cld is the inter-compartmental distribution.
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The peripheral compartment was described as ∙
=
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(3)
where Vt is the peripheral volume of distribution.
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Dexamethasone was assumed to directly stimulate the glucose turnover rate described as =1+!
"# $
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(4)
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where S(Cp) and EC50 are the stimulatory drug mechanism function and the dexamethasone plasma
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concentration at 50 % of maximum response, respectively. The turnover of glucose with the
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stimulatory drug mechanism incorporated was in the DEX IM study described by
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%
= &'( ∙ [1 + !
"# $
] − &)* ∙ +
(5)
%
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where
, kin and kout are the rate of change of response the turnover rate and the first-order fractional
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turnover rate respectively.
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The turnover of glucose with the stimulatory drug mechanism incorporated was in the DEX IV study
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described by ,
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%-
%0
= &'( ∙ [1 + !
"# $
] − &)* ∙ +/
= &)* ∙ (+/ − +1 )
%0
(6)
where
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turnover model. R1 and R2 denote the target interaction compartment and glucose response in plasma,
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respectively. Initial parameter estimates were derived graphically for the pharmacodynamic
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parameters. A constant (absolute) error (weighting) model was used for regression of glucose
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response-time data performed using WinNonlin 4.0.1 (Certara, St. Louis, Missouri, U.S.A).
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Statistical analyses
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Statistical analyses was performed by means of analysis of variance (ANOVA) using a non-linear
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mixed-effects model. In the DEX IM study time was used as fixed effect and horse as random effect.
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An ad hoc analyses (Dunnett´s test) was performed in order to compare glucose concentrations after
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dexamethasone administration with the pre-administration glucose concentrations. In the DEX IV
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study time and dose were used as fixed effects and horse as random effect. Glucose concentrations
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were compared between doses (contrasts) for every time-point. Glucose concentrations after
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dexamethasone administration were also compared for each dose-level to pre-administration
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concentrations by use of Dunnett´s test as described for the DEX IM study. Statistical significance
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was considered when P < .05. All statistical analyses were performed using the statistical software R
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version 3.4.4 (The R Foundation for Statistical Computing, Vienna, Austria).
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, are the rate of change of response in respectively compartment 1 and 2 of the
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ACCEPTED MANUSCRIPT Results
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In the DEX IM study, plasma glucose concentrations increased after dexamethasone administration.
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Maximal plasma glucose concentration was observed at 0-36 hours after maximal dexamethasone
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plasma concentration. The glucose response was positively related to dexamethasone concentration;
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increased dexamethasone plasma concentration increased the glucose response. The greatest glucose
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response was observed in the horse with the greatest dexamethasone peak concentration and the
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lowest glucose response was observed in the horse with the lowest observed dexamethasone peak
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concentration. Plasma glucose concentrations gradually returned to baseline as plasma dexamethasone
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concentrations declined. Glucose concentrations were significantly increased compared at 24 h (P <
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.0001), 48 h (P < .001) and 72 h (P = .0274) compared with 0 h. Dexamethasone was still quantifiable
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in plasma up to 13 days (LOQ 0.025 µg/L). Mean observed and model predicted dexamethasone and
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glucose response data are shown in Fig. 2.
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In the DEX IV study only the highest dexamethasone dose resulted in an increase in glucose response.
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Plasma glucose concentrations was significantly increased at 4 h (P = .0001), 5 h (P < .0001), 6 h (P <
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.0001), 9 h (P = .0001), 12 h (P = .0065), 18 h (P = .001) and 24 h (P = .0455) after dexamethasone
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administration compared with control treatment. Plasma glucose concentrations peaked 6-12 hours
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after the start of dexamethasone infusion, and then declined as plasma dexamethasone concentrations
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decreased. Plasma glucose concentrations were increased at 4 h (P = .001), 5 h (P < .0001), 6 h (P <
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.0001), 9 h (P = .0002), 12 h (P = .0025), 18 h (P = .022) and 36 h (P = .0179) compared with the pre-
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administration samples. Dexamethasone was still quantifiable at 24 hours (LOQ 0.025µg/L) in all
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horses. Mean observed and model predicted dexamethasone and glucose response data are shown in
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Fig. 3.
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The EC50, kout, and R0 (baseline) of glucose was estimated in all horses. The EC50 parameter varied
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over a ten-fold range in the DEX IM study and four-fold in the DEX IV study. The kout parameter
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varied four-fold in the IM study and over a three-fold range in the IV study. The R0 parameter showed
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low variability within studies and approximately 20 % difference between studies. The parameter
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ACCEPTED MANUSCRIPT estimates are shown in table 1. With the drug present the parameters corresponding to the baseline-
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parameter in the unaffected system were in median (range) 5.42 mmol/L (5.22-5. 57), 5.40 mmol/L
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(5.19-5.50) and 5.80 mmol/L (5.49-6.06) for the low, intermediate and high dose, respectively, in the
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DEX IV study. The regressed plasma dexamethasone-time and response-time courses are shown in
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Fig. 4. Both regressed plasma dexamethasone concentration-time courses and the regressed plasma
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glucose response-time courses show larger variation between individuals in DEX IM compared to the
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DEX IV study.
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In this study the use of a turnover model allowed quantification of glucose response data. The time
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courses from both a single dose administered IM and multiple IV doses could be characterised by
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means of pharmacokinetic and pharmacodynamic analyses. The results show a plasma dexamethasone
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concentration – glucose response relation. The use of a slow release pharmaceutical product in the
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DEX IM study caused dexamethasone plasma exposure characterised by low peak in plasma
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dexamethasone concentration above the lower limit of quantification (LOQ) characterised by a
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relatively mildly increased plasma dexamethasone concentration of several days duration compared to
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the DEX IV study (Figs. 2 and 3). The use of an intravenous solution in DEX IV gave a pattern of
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high immediate concentrations but more rapid decrease below LOQ with lower variation between the
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horses compared to the DEX IM study. The plasma glucose time courses corresponded well to plasma
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dexamethasone time-courses in both studies (Fig. 4). The pharmacodynamic parameters were in
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general estimated with acceptable precision (Table 1). The potency (EC50)-value for glucose response
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presented here is also ten-fold higher than the potency value for cortisol response in the horse [11,
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12]. The variation in parameter estimates in this study is similar compared to parameter variation in
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other studies. For instance the potency (EC50)-value varies four to ten-fold in this study. This is
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consistent with the ten-fold variation in different potency-values previously reported for both horses
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and dogs and the kout-parameter in this study showed lower between animal variation compared with
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that for cortisol response in horses or the response to nimesulide in dogs [12, 23, 24]. The R0 was
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4.20 and 5.42 in the DEX IM and the DEX IV study, respectively. Both values are consistent with
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previously reported pre-race or pre-experimental baseline plasma glucose concentrations [13, 25-27].
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It is also within the range of the internal laboratory reference value for plasma glucose (3.6-6.5
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mmol/L) where the samples were analysed.
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In this study, the glucose response was predicted by means of a turnover model with stimulatory
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function for production of the response. Glucocorticoids exert their plasma glucose response by
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increasing the rate of gluconeogenesis and decreasing the tissue uptake of glucose. Decreased uptake
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is by means of decreased tissue insulin sensitivity [28, 29]. Glucose homeostasis is strongly associated
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ACCEPTED MANUSCRIPT with other hormones e.g. insulin and glucagon. Plasma insulin is elevated in glucocorticoid treated
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horses [13]. The most elegant studies modelling glucose response to drug exposure has included a
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moderator of glucose response, e.g. insulin concentrations [30, 31]. In those studies, the moderator
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has allowed separation of different mechanisms in plasma glucose regulation resulting in more precise
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parameter estimates. The model in this study did not include any moderator of glucose dynamics other
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than dexamethasone exposure. As a consequence, the potency (EC50)-values presented here is of
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lower value for glucose regulation. However, from an anti-doping perspective the data are useful
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since the focus is on the drug response. The model predicted response-time courses are acceptable
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even though the model constantly underestimated the peaks of the response. Conclusively, the results
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presented in this study should mainly function as input to future research and anti-doping procedures
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although the weaknesses might be considered when interpreting the results and choosing an
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uncertainty factor in the screening limit decision process.
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Both endogenous and exogenous glucocorticoids increase endurance in rats and humans [32-36]. It is
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known that carbohydrate supplementation during exercise prevents reduction in performance capacity
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in humans and that glucocorticoids have a gluconeogenetic effect, for instance by upregulation of
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gluconeogenetic enzymes [37-43] . During prolonged exercise (e.g. endurance sports) plasma glucose
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concentrations decrease and fatigue develops [26, 27, 44]. If gluconeogenesis is inhibited these
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changes develop quicker [45]. Supplementary carbohydrates during exercise maintain blood glucose
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concentrations, attenuate decrease in both maximum muscle contraction force and technical skills and
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delay the development of fatigue [37-41]. The gluconeogenetic formation of glucose from lactate has
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also been described as one of the main pathways for lactate disposal [46]. There is no currently
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available evidence that pre-exercise blood glucose elevation increases performance in horses. On the
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contrary, there is conflicting evidence that pre-exercise carbohydrate feeding that increases blood
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glucose concentrations may result in more severe hypoglycaemia during exercise [25, 47-49].
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However the doping of sport horses could aim at both increasing performance (so called positive
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doping) and decreasing the performance (so called negative doping) [50]. The effects on performance
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described above and the correlation between increased blood-glucose levels and anti-rheumatic
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ACCEPTED MANUSCRIPT response in man [18] strongly suggest that plasma glucose concentrations are relevant for the anti-
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doping control in sport-horses. The pharmacodynamics parameters presented in this study increase the
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evidence-base for dexamethasone screening limits. Unfortunately, IFHA has not yet published an
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international SL for dexamethasone in plasma but there is an international SL for dexamethasone in
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urine (0.2 µg/L) [51]. In urine, dexamethasone concentration is approximately ten-fold higher
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compared to the concurrent concentration in plasma [11]. Since the plasma/urine ratio is not a robust
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factor [5] a conservative attitude when transforming irrelevant plasma concentrations to irrelevant
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urine concentrations may be appropriate. Toutain & Lassourd [5] established that the effective plasma
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concentration can be calculated by dividing the standard dose (per dosing interval) with the clearance
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(per dosing interval). Using data collected from an experimental study by Soma et al. [52], the dose
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50 µg/kg and the clearance 0.5 L · h ⁄ kg result in the effective plasma concentration 4 µg/L in horses.
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Applying an uncertainty factor 500 to the effective plasma concentration 4 µg/L creates an irrelevant
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plasma concentration (IPC) that translates into the SL 0.008 µg/L. This uncertainty factor consist of a
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factor 50 that transforms an effective concentration to an irrelevant concentration and a factor 10 that
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takes inter-individual variability into account [5]. The value 0.008 µg/L is below the EC50-value for
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glucose response presented in this study and lower than the median IC50-values for inhibiting cortisol
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secretion into plasma presented elsewhere [11, 12]. The use of the uncertainty factor 50 (that
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according to Toutain & Lassourd [5]convert a concentration close to a IC50-value to an irrelevant
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concentration) on the lowest IC50-value presented in this study produce the irrelevant concentration
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0.006µg/L which is consistent with 0.008 µg/L. Applying the ten-fold ratio between plasma and urine,
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the irrelevant plasma concentrations suggested in this study result in the irrelevant urine concentration
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0.08 µg/L. That can be compared with 0.2 µg/L given by IFHA. Obviously, 0.008 µg/L plasma and
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0.08 µg/L urine are concentrations without biologically relevant effect that does not alter the
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performance of horses and lower concentrations can be considered as traces of legitimate medication.
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Acknowledgment
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This work was supported by the Swedish-Norwegian Foundation for Equine Research and the Nordic
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Equine Medication and Anti-Doping Committee. Preliminary results were presented as an abstract, an
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oral presentation and in conference proceedings of the 20th International Conference of Racing
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Analysts and Veterinarians (ICRAV), Mauritius, 20th-28th September 2014.
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References 1. Fédération Equestre Internationale. 2018. Treatment and Prohibited Substances. http://inside.fei.org/fei/your-role/veterinarians/cleansport. Accessed 2018 04 16. 2. International Association of Horseracing Authorites. 2018. International Agreement on Breeding, Racing and Wagering. Available from: http://www.horseracingintfed.com/resources/2016Agreement.pdf. Accessed 2018 04 16. 3. Barragry, T., Doping and drug detection times in horses: New data for therapeutic agents. Irish Veterinary Journal, 2006. 59(7): p. 394-398. 4. Webbon, P., Medication: a way forward from zero tolerance to irrelevant plasma concentrations. Equine Vet J, 2002. 34(3): p. 220-1. 5. Toutain, P.L. and V. Lassourd, Pharmacokinetic/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse. Equine Veterinary Journal, 2002. 34(3): p. 242-9. 6. Toutain, P.L., Veterinary medicines and competition animals: the question of medication versus doping control. Handbook of experimental pharmacology, 2010(199): p. 315-39. 7. European Horseracing Science and Liason Committée. 2018, The Science behind this work. https://www.ehslc.com/detection-times/the-science-behind-this-work. Accessed 2018 04 16. 8. International Association of Horseracing Authorites. 2018. Screening limits in Plasma. http://www.horseracingintfed.com/default.asp?section=IABRW&area=6. Accessed 2018 04 16. 9. Gabrielsson, J. and A.R. Green, Quantitative pharmacology or pharmacokinetic pharmacodynamic integration should be a vital component in integrative pharmacology. J Pharmacol Exp Ther, 2009. 331(3): p. 767-74. 10. Wright, D.F., H.R. Winter, and S.B. Duffull, Understanding the time course of pharmacological effect: a PKPD approach. Br J Clin Pharmacol, 2011. 71(6): p. 815-23.
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16. 17. 18. 19.
20. 21. 22.
23.
24.
25.
26.
27.
28. 29. 30. 31.
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SC
14.
M AN U
13.
TE D
12.
Ekstrand, C., et al., Plasma concentration-dependent suppression of endogenous hydrocortisone in the horse after intramuscular administration of dexamethasone-21isonicotinate. Journal of Veterinary Pharmacology and Therapeutics, 2015. 38(3): p. 235-42. Ekstrand, C., et al., A quantitative approach to analysing cortisol response in the horse. J Vet Pharmacol Ther, 2016. 39(3): p. 255-263. Tiley, H.A., R.J. Geor, and L.J. McCutcheon, Effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses. American journal of veterinary research, 2007. 68(7): p. 753-9. Soma, L.R., et al., Pharmacokinetics of dexamethasone with pharmacokinetic/pharmacodynamic model of the effect of dexamethasone on endogenous hydrocortisone and cortisone in the horse. Journal of Veterinary Pharmacology and Therapeutics, 2005. 28(1): p. 71-80. Abraham, G., et al., Serum thyroid hormone, insulin, glucose, triglycerides and protein concentrations in normal horses: association with topical dexamethasone usage. Veterinary journal, 2011. 188(3): p. 307-12. Cartmill, J.A., et al., Leptin secretion in horses: effects of dexamethasone, gender, and testosterone. Domest Anim Endocrinol, 2006. 31(2): p. 197-210. Ringler, I. and W.E. Dulin, Potency relationships of various C-21 steroids as measured by two methods of liver glycogen deposition. Proc Soc Exp Biol Med, 1962. 110: p. 869-72. Ringler, I., et al., Biological potencies of chemically modified adrenocorticosteroids in rat and man. Metabolism, 1964. 13: p. 37-44. Andersson, L. and T. Olsson, The effect of two glucocorticoids on plasma glucose and milk production in healthy cows and the therapeutic effect in ketosis. Nordisk Veterinaermedicin, 1984. 36(1-2): p. 13-8. Wierda, A., et al., Effects of two glucocorticoids on milk yield and biochemical measurements in healthy and ketotic cows. Vet Rec, 1987. 120(13): p. 297-9. Shpigel, N.Y., et al., Use of corticosteroids alone or combined with glucose to treat ketosis in dairy cows. J Am Vet Med Assoc, 1996. 208(10): p. 1702-4. Braun, R.K., E.N. Bergman, and T.F. Albert, Effects of various synthetic glucocorticoids on milk production and blood glucose and ketone body concentrations in normal and ketotic cows. Journal of the American Veterinary Medical Association, 1970. 157(7): p. 941-6. Toutain, P.L., et al., Plasma concentrations and therapeutic efficacy of phenylbutazone and flunixin meglumine in the horse: pharmacokinetic/pharmacodynamic modelling. J Vet Pharmacol Ther, 1994. 17(6): p. 459-69. Toutain, P.L., et al., A pharmacokinetic/pharmacodynamic approach vs. a dose titration for the determination of a dosage regimen: the case of nimesulide, a Cox-2 selective nonsteroidal anti-inflammatory drug in the dog. J Vet Pharmacol Ther, 2001. 24(1): p. 43-55. Stull, C.L. and A.V. Rodiek, Stress and glycemic responses to postprandial interval and feed components in exercising horses. Journal of Equine Veterinary Science, 1995. 15(9): p. 382386. Essen-Gustavsson, B., K. Karlstrom, and A. Lindholm, Fibre types, enzyme activities and substrate utilisation in skeletal muscles of horses competing in endurance rides. Equine Vet J, 1984. 16(3): p. 197-202. Larsson, J., et al., Physiological parameters of endurance horses pre- compared to post-race, correlated with performance: a two race study from scandinavia. ISRN veterinary science, 2013. 2013: p. 684353. Baxter, J.D. and P.H. Forsham, Tissue effects of glucocorticoids. Am J Med, 1972. 53(5): p. 573-89. Pagano, G., et al., An in vivo and in vitro study of the mechanism of prednisone-induced insulin resistance in healthy subjects. J Clin Invest, 1983. 72(5): p. 1814-20. Cao, Y., W. Gao, and W.J. Jusko, Pharmacokinetic/pharmacodynamic modeling of GLP-1 in healthy rats. Pharmaceutical research, 2012. 29(4): p. 1078-86. Jin, J.Y. and W.J. Jusko, Pharmacodynamics of Glucose Regulation by Methylprednisolone. II. Normal Rats. Biopharmaceutics & Drug Disposition, 2009. 30(1): p. 35-48.
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Gorostiaga, E.M., S.M. Czerwinski, and R.C. Hickson, Acute glucocorticoid effects on glycogen utilization, O2 uptake, and endurance. J Appl Physiol (1985), 1988. 64(3): p. 1098106. Sellers, T.L., et al., Effect of the exercise-induced increase in glucocorticoids on endurance in the rat. J Appl Physiol (1985), 1988. 65(1): p. 173-8. Le Panse, B., et al., Short-term glucocorticoid intake improves exercise endurance in healthy recreationally trained women. Eur J Appl Physiol, 2009. 107(4): p. 437-43. Arlettaz, A., et al., Effects of short-term prednisolone intake during submaximal exercise. Medicine and Science in Sports and Exercise, 2007. 39(9): p. 1672-8. Viru, M., et al., Glucocorticoids in metabolic control during exercise: glycogen metabolism. J Sports Med Phys Fitness, 1994. 34(4): p. 377-82. Coyle, E.F., et al., Carbohydrate feeding during prolonged strenuous exercise can delay fatigue. J Appl Physiol Respir Environ Exerc Physiol, 1983. 55(1 Pt 1): p. 230-5. Farris, J.W., et al., Effect of tryptophan and of glucose on exercise capacity of horses. J Appl Physiol (1985), 1998. 85(3): p. 807-16. Harper, L.D., et al., Physiological and performance effects of carbohydrate gels consumed prior to the extra-time period of prolonged simulated soccer match-play. J Sci Med Sport, 2015. Nybo, L., CNS fatigue and prolonged exercise: effect of glucose supplementation. Med Sci Sports Exerc, 2003. 35(4): p. 589-94. Patterson, S.D. and S.C. Gray, Carbohydrate-gel supplementation and endurance performance during intermittent high-intensity shuttle running. Int J Sport Nutr Exerc Metab, 2007. 17(5): p. 445-55. Frijters, R., et al., Prednisolone-induced differential gene expression in mouse liver carrying wild type or a dimerization-defective glucocorticoid receptor. BMC Genomics, 2010. 11: p. 359. Yoon, J.C., et al., Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature, 2001. 413(6852): p. 131-8. Lucke, J.N. and G.M. Hall, Biochemical changes in horses during a 50-mile endurance ride. Vet Rec, 1978. 102(16): p. 356-8. John-Alder, H.B., R.M. McAllister, and R.L. Terjung, Reduced running endurance in gluconeogenesis-inhibited rats. Am J Physiol, 1986. 251(1 Pt 2): p. R137-42. Jitrapakdee, S., Transcription factors and coactivators controlling nutrient and hormonal regulation of hepatic gluconeogenesis. Int J Biochem Cell Biol, 2012. 44(1): p. 33-45. Lawrence, L., et al., Feeding staus affects glucose-metabolism in exercising horses. Journal of Nutrition, 1993. 123(12): p. 2152-2157. Bullimore, S.R., et al., Carbohydrate supplementation of horses during endurance exercise: Comparison of fructose and glucose. Journal of Nutrition, 2000. 130(7): p. 1760-1765. Vervuert, I., M. Coenen, and A. Bichmann, Comparison of the effects of fructose and glucose supplementation on metabolic responses in resting and exercising horses. Journal of Veterinary Medicine Series a-Physiology Pathology Clinical Medicine, 2004. 51(4): p. 171177. Wong, J.K. and T.S. Wan, Doping control analyses in horseracing: a clinician's guide. Vet J, 2014. 200(1): p. 8-16. International Association of Horseracing Authorites. 2018. Screening limits in Urine. http://www.ifhaonline.org/default.asp?section=IABRW&area=1. Accessed 2018 04 16. Soma, L.R., et al., Pharmacokinetics of dexamethasone following intra-articular, intravenous, intramuscular, and oral administration in horses and its effects on endogenous hydrocortisone. Journal of Veterinary Pharmacology and Therapeutics, 2013. 36(2): p. 18191.
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Table 1. Pharmacodynamic parameters and their precision (relative standard deviation).
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DEX IV
Kout (1/h)
R0 (mmol/L)
Horse
EC50 (µg/L)
Kout (1/h)
R0 (mmol/L)
1 2 3 4 5 6 Median Range
0.31 ± 97.4 0.74 ± 43.4 3.26 ± 113 0.73 ± 67.3 0.51 ± 47.1 0.34 ± 68.1 0.62 0.31 – 3.26
0.11 ± 87.6 0.18 ± 77.9 0.27 ± 641 0.21 ± 120 0.07 ± 51.4 0.08 ± 56.4 0.15 0.07 – 0.27
4.88 ± 11.2 4.20 ± 5.16 4.34 ± 5.23 4.19 ± 8.09 4.25 ± 5.02 4.31 ± 8.14 4.23 4.19 – 4.88
A B C D E F Median Range
2.11 ± 47.9 0.66 ± 45.2 2.36 ± 91.7 0.62 ± 43.6 0.72 ± 34.6 0.57 ± 44.6 0.69 0.57 – 2.36
0.48 ± 36.3 0.17 ± 21.6 0.28 ± 56.1 0.24 ± 18.7 0.26 ± 16.7 0.20 ± 19.4 0.25 0.17 – 0.48
5.37 ± 1.6 5.22 ± 1.5 5.96 ± 2.0 5.32 ± 1.7 5.46 ± 1.4 5.57 ± 1.5 5.42 5.22 – 5.96
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DEX IM Horse
EC50, kout and R0 are the plasma dexamethasone concentration at 50 % of the response, the fractional turnover rate and the unaffected baseline of response, respectively.
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Fig. 1. Schematic illustration of the dexamethasone disposition models for the DEX IM study (1A), the DEX IV study (2A) and the glucose turnover models from the DEX IM study (1B) and DEX IV study (2B) describing the dexamethasone-induced changes in glucose response in horses. In the upper plot, DEX IM, Cp, ka, and k denote the dose (administered intramuscularly), the dexamethasone plasma concentration, the absorption- and the elimination rate constants of dexamethasone in plasma respectively. In the lower plot, DEX IV represents the bolus + intravenous infusion regimen. Vc, Vt, Cl and Cld represent the central and peripheral volume of distribution, clearance and inter-compartmental distribution parameter, respectively. The plasma exposure (Equations 1, 2 and 3) then served to ‘drive’ the drug-mechanism function (Equation 4) acting on the turnover rate of glucose in respectively the DEX IM study (2A) and the DEX IV study (2B). S(C), kin, kout and R represent the stimulatory drug mechanism function, the turnover rate, the fractional turnover rate and the glucose response, respectively. One transit compartment (shaded) was used to capture the delayed onset of action on glucose response in the DEX IV study (2B).
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Fig. 2. Mean observed (filled circles) and mean predicted (lines) plasma concentrations of dexamethasone (upper plot, A) and glucose (lower plot, B). The plots only include data from ten days and not the entire sampling period (28 days). The concentration-time courses are produced after administration of 30 µg/kg dexamethasone-21-isonicotinate intramuscularly. * = statistically different compared with day 0.
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Fig. 3. Mean observed (symbols) and mean predicted (lines) plasma concentrations of dexamethasone (upper plot, A) and glucose (lower plot, B) showing that only the highest dose dexamethasone increased glucose concentrations in plasma. Dexamethasone 21-phosphate disodium salt was administered as an intra-venous (IV) bolus dose followed by three hours constant rate infusion (bolus + infusion) at four different dose-levels: saline control (empty circles), 0.1+0.07 µg/kg (squares), 1+0.7 µg/kg (triangles) and 10+7 µg/kg (filled circles). * = significant difference between treatment with the highest dose dexamethasone and treatment with saline.
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Fig. 4. Predicted plasma dexamethasone-time (upper plots) and glucose-time (lower plots) courses from six horses in the DEX IM (left plots) study and six horses in the DEX IV (right plots) study, respectively. Note that only predicted concentration-time profiles based on data from the 17µg/kg study occasion of the DEX IV study are included in the figure.
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ACCEPTED MANUSCRIPT Administration of dexamethasone increased plasma glucose concentration The potency-value for glucose response to dexamethasone ranged 0.47-2.45 µg/L
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Pharmacodynamic parameters strengthen medication-control in equine sports