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BackgroundOral chemotherapy agents are frequently compounded in veterinary medicine however, the potency of some formulations have been shown to vary from that of Food and Drug Administration (FDA)-approved products.AimsThe objective of this study was to evaluate the potency and stability of three compounded oral chemotherapeutics commonly prescribed to be administered over time.Materials & methodsCompounded chlorambucil 1 mg, cyclophosphamide 5 mg and melphalan 1 mg were obtained and for potency tested upon receipt and 6 weeks later.ResultsPotency ranged from 71 to 104% for chlorambucil and 58 to 109% for melphalan; 1/4 and 2/4 samples were

BackgroundCompounded lomustine is used commonly in veterinary patients. However, the potential variability in these formulations is unknown and concern exists that compounded formulations of drugs may differ in potency from Food and Drug Administration (FDA)-approved products.Hypothesis/objectivesThe initial objective of this study was to evaluate the frequency and severity of neutropenia in dogs treated with compounded or FDA-approved formulations of lomustine. Subsequent analyses aimed to determine the potency of lomustine obtained from several compounding pharmacies.AnimalsThirty-seven dogs treated with FDA-approved or compounded lomustine.MethodsDogs that received compounded or FDA-approved lomustine and had pretreatment and nadir CBCs performed were eligible for inclusion. Variables assessed included lomustine dose, neutrophil counts, and severity of neutropenia. Lomustine 5 mg capsules from 5 compounding sources were tested for potency using high-pressure liquid chromatography (HPLC) with ultraviolet (UV) detection.ResultsTwenty-one dogs received FDA-approved lomustine and 16 dogs were treated with lomustine prescribed from a single compounding pharmacy. All dogs treated with FDA-approved lomustine were neutropenic after treatment; 15 dogs (71%) developed grade 3 or higher neutropenia. Four dogs (25%) given compounded lomustine became neutropenic, with 2 dogs (12.5%) developing grade 3 neutropenia. The potency of lomustine from 5 compounding pharmacies ranged from 50 to 115% of the labeled concentration, with 1 sample within ±10% of the labeled concentration.Conclusions and clinical importanceThese data support broader investigation into the potency and consistency of compounded chemotherapy drugs and highlight the potential need for greater oversight of these products.

The present study characterizes the pharmacokinetic (PK) and pharmacodynamic (PD) relationships of the α2-adrenergic receptor agonists detomidine (DET), medetomidine (MED) and dexmedetomidine (DEX) in parallel groups of horses from in vivo data after single bolus doses. Head height (HH), heart rate (HR), and blood glucose concentrations were measured over 6 h. Compartmental PK and minimal physiologically based PK (mPBPK) models were applied and incorporated into basic and extended indirect response models (IRM). Population PK/PD analysis was conducted using the Monolix software implementing the stochastic approximation expectation maximization algorithm. Marked reductions in HH and HR were found. The drug concentrations required to obtain inhibition at half-maximal effect (IC50 ) were approximately four times larger for DET than MED and DEX for both HH and HR. These effects were not gender dependent. Medetomidine had a greater influence on the increase in glucose concentration than DEX. The developed models demonstrate the use of mechanistic and mPBPK/PD models for the analysis of clinically obtainable in vivo data.

Objective: To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells.Design: In vitro study.Setting: Research laboratory.Animal(s): Immature Sprague-Dawley female rats.Intervention(s): Granulosa cells were cultured in the absence or presence of resveratrol.Main Outcome Measure(s): DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot.Result(s): Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered.Conclusion(s): Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome. [ABSTRACT FROM AUTHOR]

Ketoprofen is an anti-inflammatory drug that is commonly administered to racehorses for the alleviation of musculoskeletal pain and inflammation. This study represents a comprehensive examination of the metabolism (in vivo and in vitro), pharmacokinetics and ex vivo pharmacodynamics, of ketoprofen in horses. The in vitro metabolism as well as specific enzymes responsible for metabolism was determined by incubating liver microsomes and recombinant CYP450 and UGT enzymes with ketoprofen. For the in vivo portion, 15 horses were administered a single intravenous dose of 2.2-mg/kg ketoprofen. Blood and urine samples were collected prior to and up to 120 h post-drug administration. Additional blood samples were collected at select time points and were stimulated with calcium ionophore or lipopolysaccharide, ex vivo, to induce eicosanoid production. Drug, metabolite, and eicosanoid concentrations were determined using LC-MS/MS. Incubation of ketoprofen with equine liver microsomes generated 3-hydroxy ketoprofen, an unidentified hydroxylated metabolite, and ketoprofen glucuronide. Recombinant equine CYP2C23 produced the greatest amount of hydroxylated ketoprofen and recombinant equine UGT1A2 generated ketoprofen glucuronide. Dihydro, 3-hydroxy, and glucuronide metabolites were identified in blood and urine samples. The Vd ss was 0.280, 0.385, and 0.319 L/kg for total ketoprofen, S (+) ketoprofen, and R (-) ketoprofen, respectively. The mean half-life was 6.01 h for total ketoprofen, 2.22 h for S (+) ketoprofen, and 1.72 h for R (-) ketoprofen. Stimulation of ketoprofen-treated blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of TXB 2 , PGE 2 , PGF 2alpha , LTB 4 , and 15(s)-HETE production for up to 120 h post-drug administration., (© 2023 John Wiley & Sons Ltd.)

Given the potential for long-term inhibition of bone remodeling/healing and detrimental effects to horses in training, bisphosphonates are tightly regulated in horseracing. Hair has proven to be an effective matrix for detection of drug administration to horses and has been particularly effective in detecting drugs for a long period of time post administration. Thus, hair may prove to be a useful matrix for detection of administration of this class of drugs. The objective of the current study was to develop an assay and assess the usefulness of hair as a matrix for long-term detection of clodronate to horses. Seven horses received a single intramuscular administration of 1.8 mg/kg clodronate. Hair samples were collected prior to and up to 6 months post administration. A liquid chromatography-tandem mass spectrometry method was developed and concentrations of clodronate measured in hair samples. The drug was first detected on day 7 in 4/7 horses, and on days 14, 28 and 35 in the remaining three horses. In 4/7 horses, clodronate was still detectable 6 months post administration. Results of this study demonstrate that, although there was significant inter-individual variability in detection times (63 to 180 days) and several intermediate times where the drug could not be detected but was subsequently detected in later timepoints, clodronate administration was detectable in hair for a prolonged period in most of the horses (4/7) studied., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Different structurally related phenylpiperidine opioids exhibit different isoflurane-sparing effects in cats. Because minimum alveolar concentration (MAC) in cats is affected only by very high plasma concentrations of some phenylpiperidine opioids, we hypothesized these effects are caused by actions on nonopioid receptors. Using a prospective, randomized, crossover design, six cats were anesthetized with isoflurane, intubated, ventilated, and instrumented. Isoflurane MAC was measured in triplicate using a tail-clamp and bracketing technique. A computer-controlled intravenous infusion using prior pharmacokinetic models targeted plasma concentrations of 60 ng/ml fentanyl, 10 ng/ml sufentanil, or 500 ng/ml alfentanil, and isoflurane MAC was measured in duplicate. Next, naltrexone 0.6 mg/kg was administered to cats hourly during the opioid infusion, and isoflurane MAC was measured in duplicate. Blood was collected during MAC determinations to measure opioid concentrations. Responses were analyzed using repeated measures ANOVA with significance at p < .05. Alfentanil and sufentanil decreased isoflurane MAC by 16.4% and 6.4%, respectively, and these effects were completely reversed by naltrexone. Fentanyl had no significant effect on isoflurane MAC. Alfentanil and sufentanil modestly reduce isoflurane MAC via agonist effects on opioid receptors. However, these effects are too small to justify clinical use of phenylpiperidine opioids as single agents to reduce MAC in cats., (© 2020 John Wiley & Sons Ltd.)

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3βHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3β-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3β-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Background: Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids., Objectives: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS., Study Design: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation., Methods: Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation., Results: Estrone sulphate concentrations peaked by week 26 at almost 60 μg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed., Main Limitations: Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards., Conclusions: Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation., (© 2019 EVJ Ltd.)

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1β and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The authors apologize for errors in Figure 6 of their article published in the October 2017 issue of Reproduction (vol 154 iss 4 pp 445–454). The authors explain that the addition of data (Figure 6) on steroid concentrations in the chorioallantois to their manuscript on fetal adrenal and fetal gonadal steroids during development of the equine fetus was made in response to reviewer comments. However, in compiling, summarizing and graphing the data, the wrong units were used in the final figure. The manuscript as published represents the data in Figure 6 as “ng/g”, when in fact they are “nmol/g”. The authors very much regret having made the mistake and sincerely apologize for any confusion this might have caused., (© 2019 Society for Reproduction and Fertility)

In vivo and in vitro evidence indicates that the bioactive, 5α-reduced progesterone metabolite, 5α-dihydroprogesterone (DHP) is synthesized in the placenta, supporting equine pregnancy, but its appearance in early pregnancy argues for other sites of synthesis also. It remains unknown if DHP circulates at relevant concentrations in cyclic mares and, if so, does synthesis involve the non-pregnant uterus? Jugular blood was drawn daily from cyclic mares ( n  = 5). Additionally, ovariectomized mares (OVX) and geldings were administered progesterone (300 mg) intramuscularly. Blood was drawn before and after treatment. Incubations of whole equine blood and hepatic microsomes with progesterone were also investigated for evidence of DHP synthesis. Sample analysis for progesterone, DHP and other steroids employed validated liquid chromatography-tandem mass spectrometry methods. Progesterone and DHP appeared a day (d) after ovulation in cyclic mares, was increased significantly by d3, peaking from d5 to 10 and decreased from d13 to 17. DHP was 55.5 ± 3.2% of progesterone concentrations throughout the cycle and was highly correlated with it. DHP was detected immediately after progesterone administration to OVX mares and geldings, maintaining a relatively constant ratio with progesterone (47.2 ± 2.9 and 51.2 ± 2.7%, respectively). DHP was barely detectable in whole blood and hepatic microsome incubations. We conclude that DHP is a physiologically relevant progestogen in cyclic, non-pregnant mares, likely stimulating the uterus, and that it is synthesized peripherally from luteal progesterone but not in the liver or blood. The presence of DHP in pregnant perissodactyla as well as proboscidean species suggests horses may be a valuable model for reproductive endocrinology in other exotic taxa., (© 2018 Society for Endocrinology.)

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r 2  = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3βHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3βHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable ( P  < 0.05) and transcript was decreased. Trilostane inhibited 3βHSD significantly more in testis than placenta, suggesting possible existence of different 3βHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3βHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses., (© 2018 Society for Reproduction and Fertility.)

Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher but decreased progressively with GA. Tissue steroid concentrations generally paralleled those in serum with time. Adrenal and gonadal tissue pregnenolone concentrations were similar and 100-fold higher than those in allantochorion. DHEA was far higher in gonads than adrenals and progesterone was higher in adrenals than gonads. Androstenedione decreased with GA in adrenals but not in gonads. Transcript analysis generally supported these data. CYP17A1 was higher in fetal gonads than adrenals or allantochorion, and HSD3B1 was higher in fetal adrenals and allantochorion than gonads. CYP11A1 transcript was also significantly higher in adrenals and gonads than allantochorion and CYP19 and SRD5A1 transcripts were higher in allantochorion than either fetal adrenals or gonads. Given these data, and their much greater size, the fetal gonads are the source of DHEA and likely contribute more than fetal adrenal glands to circulating fetal pregnenolone concentrations. Low CYP11A1 but high HSD3B1 and SRD5A1 transcript abundance in allantochorion, and low tissue pregnenolone, suggests that endogenous placental pregnenolone synthesis is low and likely contributes little to equine placental 5α-reduced pregnane secretion., (© 2017 Society for Reproduction and Fertility.)

Romifidine is an alpha-2 adrenergic agonist used for sedation and analgesia in horses. As it is a prohibited substance, its purported use at low doses in performance horses necessitates further study. The primary goal of the study reported here was to describe the serum concentrations and pharmacokinetics of romifidine following low-dose administration immediately prior to exercise, utilizing a highly sensitive liquid chromatography-tandem mass spectrometry assay that is currently employed in many drug testing laboratories. An additional objective was to describe changes in heart rate and rhythm following intravenous administration of romifidine followed by exercise. Eight adult Quarter Horses received a single intravenous dose of 5 mg (0.01 mg/kg) romifidine followed by 1 h of exercise. Blood samples were collected and drug concentrations measured at time 0 and at various times up to 72 h. Mean ± SD systemic clearance, steady-state volume of distribution and terminal elimination half-life were 34.1 ± 6.06 mL/min/kg and 4.89 ± 1.31 L/kg and 3.09 ± 1.18 h, respectively. Romifidine serum concentrations fell below the LOQ (0.01 ng/mL) and the LOD (0.005 ng/mL) by 24 h postadministration. Heart rate and rhythm appeared unaffected when a low dose of romifidine was administered immediately prior to exercise., (© 2017 John Wiley & Sons Ltd.)

OBJECTIVE To measure concentrations of trazodone and its major metabolite in plasma and urine after administration to healthy horses and concurrently assess selected physiologic and behavioral effects of the drug. ANIMALS 11 Thoroughbred horses enrolled in a fitness training program. PROCEDURES In a pilot investigation, 4 horses received trazodone IV (n = 2) or orally (2) to select a dose for the full study; 1 horse received a vehicle control treatment IV. For the full study, trazodone was initially administered IV (1.5 mg/kg) to 6 horses and subsequently given orally (4 mg/kg), with a 5-week washout period between treatments. Blood and urine samples were collected prior to drug administration and at multiple time points up to 48 hours afterward. Samples were analyzed for trazodone and metabolite concentrations, and pharmacokinetic parameters were determined; plasma drug concentrations following IV administration best fit a 3-compartment model. Behavioral and physiologic effects were assessed. RESULTS After IV administration, total clearance of trazodone was 6.85 ± 2.80 mL/min/kg, volume of distribution at steady state was 1.06 ± 0.07 L/kg, and elimination half-life was 8.58 ± 1.88 hours. Terminal phase half-life was 7.11 ± 1.70 hours after oral administration. Horses had signs of aggression and excitation, tremors, and ataxia at the highest IV dose (2 mg/kg) in the pilot investigation. After IV drug administration in the full study (1.5 mg/kg), horses were ataxic and had tremors; sedation was evident after oral administration. CONCLUSIONS AND CLINICAL RELEVANCE Administration of trazodone to horses elicited a wide range of effects. Additional study is warranted before clinical use of trazodone in horses can be recommended.

Although developed as a therapeutic medication, meldonium has found widespread use in human sports and was recently added to the World Anti-Doping Agency's list of prohibited substances. Its reported abuse potential in human sports has led to concern by regulatory authorities about the possible misuse of meldonium in equine athletics. The potential abuse in equine athletes along with the limited data available regarding the pharmacokinetics and pharmacodynamics of meldonium in horses necessitates further study. Eight exercised adult thoroughbred horses received a single oral dose of 3.5, 7.1, 14.3 or 21.4 mg/kg of meldonium. Blood and urine samples were collected and analyzed using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined using non-compartmental analysis. Maximum serum concentrations ranged from 440.2 to 1147 ng/mL and the elimination half-life from 422 to 647.8 h. Serum concentrations were below the limit of quantitation by days 4, 7, 12 and 12 for doses of 3.5, 7.1, 14.3 and 21.4 mg/kg, respectively. Urine concentrations were below the limit of detection by day 44 following administration of 3.5 mg/kg and day 51 for all other dose groups. No adverse effects were observed following meldonium administration. While the group numbers were small, changes in heart rate were observed in the 3.5 mg/kg dose group (n = 1). Glucose concentrations changed significantly in all dose groups studied (n = 2 per dose group). Similar to that reported for humans, the detection time of meldonium in biological samples collected from horses is prolonged, which should allow for satisfactory regulation in performance horses. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The use of corticosteroids, such as betamethasone, in performance horses is tightly regulated. The objective of the current study was to describe the plasma pharmacokinetics of betamethasone as well as time-related urine and synovial fluid concentrations following intra-articular administration to horses. Twelve racing-fit adult Thoroughbred horses received a single intra-articular administration (9 mg) of a betamethasone sodium phosphate and betamethasone acetate injectable suspension into the right antebrachiocarpal joint. Blood, urine, and synovial fluid samples were collected prior to and at various times up to 21 days post drug administration. All samples were analyzed using tandem liquid chromatography-mass spectrometry. Plasma data were analyzed using compartmental pharmacokinetic modeling. Maximum measured plasma betamethasone concentrations were 3.97 ± 0.23 ng/mL at 1.45 ± 0.20 h. The plasma elimination half-life was 7.48 ± 0.39 h. Betamethasone concentrations were below the limit of detection in all horses by 96 h and 7 days in plasma and urine, respectively. Betamethasone fell below the limit of detection in the right antebrachiocarpal joint between 14 and 21 days. Results of this study provide information that can be used to regulate the use of intra-articular betamethasone in the horse. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

There is limited data describing xylazine serum concentrations in the horse and no reports of concentrations beyond 24 hours. The primary goal of the study reported here was to update the pharmacokinetics of xylazine following intravenous (IV) administration in order to assess the applicability of current regulatory recommendations. Pharmacodynamic parameters were determined using PK-PD modeling. Sixteen exercised adult Thoroughbred horses received a single IV dose of 200 mg of xylazine. Blood and urine samples were collected at time 0 and at various times for up to 96 hours and analyzed using liquid chromatography tandem mass spectrometry. Xylazine serum concentrations were best fit by a 3-compartment model. Mean ± SEM systemic clearance, volume of distribution at steady state, beta half-life and gamma half-life were 12.7 ± 0.735 mL/min/kg, 0.660 ± 0.053 L/kg, 2.79 ± 0.105 hours and 26.0 ± 1.9, respectively. Immediately following administration, horses appeared sedate as noted by a decrease in chin-to-ground distance, decreased locomotion and decreased heart rate (HR). Sedation lasted approximately 45 minutes. Glucose concentrations were elevated for 1-hour post administration. The EC50 (IC50) was 636.1, 702.2, 314.1 and 325.7 ng/mL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. The Emax (Imax) was 27.3 beats per minute, 47.5%, 42.4 cm and 0.28 mg/dL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. Pharmacokinetic parameters differ from previous reports and a prolonged detection time suggests that an extended withdrawal time, beyond current regulatory recommendations, is warranted to avoid inadvertent positive regulatory findings in performance horses. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Equine practitioners should follow these recommendations when using compounded medications: (1) the decision must be veterinary driven, based on a valid veterinarian-client-patient relationship and on evidence-based medicine; (2) compliance with the Animal Medicinal Drug Use Clarification Act of 1994; and (3) use limited to (a) horses for which no other method or route of drug delivery is practical; (b) those drugs for which safety, efficacy, and stability have been demonstrated; or (c) disease conditions for which a quantifiable response to therapy or drug concentration can be monitored., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The use of anti-ulcer medications, such as cimetidine, ranitidine, and omeprazole, is common in performance horses. The use of these drugs is regulated in performance horses, and as such a withdrawal time is necessary prior to competition to avoid a medication violation. To the authors' knowledge, there are no reports in the literature describing repeated oral administrations of these drugs in the horse to determine a regulatory threshold and related withdrawal time recommendations. Therefore, the objective of the current study was to describe the disposition and elimination pharmacokinetics of these anti-ulcer medications following oral administration to provide data upon which appropriate regulatory recommendations can be established. Nine exercised Thoroughbred horses were administered 20 mg/kg BID of cimetidine or 8 mg/kg BID of ranitidine, both for seven doses or 2.28 g of omeprazole SID for four doses. Blood samples were collected, serum drug concentrations were determined, and elimination pharmacokinetic parameters were calculated. The serum elimination half-life was 7.05 ± 1.02, 7.43 ± 0.851 and 3.94 ± 1.04 h for cimetidine, ranitidine, and omeprazole, respectively. Serum cimetidine and ranitidine concentrations were above the LOQ and omeprazole and omeprazole sulfide below the LOQ in all horses studied upon termination of sample collection., (© 2016 John Wiley & Sons Ltd.)

Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography-mass spectrometry. The biopotency of dominant metabolites was assessed using luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0-100nM) of progesterone, 20αDHP and 3α,20βDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20βDHP and 20αDHP (800-400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4-2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20βDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20βDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta., (© 2016 Society for Reproduction and Fertility.)

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies., (© 2016 Society for Endocrinology.)

Cetirizine is an antihistamine used in performance horses for the treatment of hypersensitivity reactions and as such a withdrawal time is necessary prior to competition. The objective of the current study was to describe the disposition and elimination of cetirizine following oral administration in order to provide additional serum concentration data upon which appropriate regulatory recommendations can be established. Nine exercised thoroughbred horses were administered 0.4 mg/kg of cetirizine orally BID for a total of five doses. Blood samples were collected immediately prior to drug administration and at various times postadministration. Serum cetirizine concentrations were determined and selected pharmacokinetic parameters determined. The serum elimination half-life was 5.83 ± 0.841 h. Average serum cetirizine concentrations were still above the LOQ of the assay (0.05 ng/mL) at 48 h (final sample collected) postadministration of the final dose., (© 2016 John Wiley & Sons Ltd.)

Methocarbamol (MCBL) is commonly used in performance horses for the treatment of skeletal muscle disorders. Current regulatory recommendations for show horses and racehorses are based on a single oral dose of 5 g, although doses in excess of this are often administered. The goal of the current study was to characterize the disposition of MCBL following higher dose administration and administration in combination with another commonly used drug in performance horses, phenylbutazone (PBZ). Exercised Thoroughbred horses were administered various doses of MCBL as a sole agent and MCBL in combination with PBZ. Blood samples were collected at various times, concentrations of MCBL and PBZ measured using LC-MS/MS and pharmacokinetic parameters calculated using compartmental analysis. Following administration of 15 g of MCBL, either as part of a single- or multiple-dose regimen, a number of horses exceeded the Association of Racing Commissioners International and the United States Equestrian Federation's recommended regulatory threshold at the recommended withdrawal time. There was not a significant difference between horses that received only MCBL and those that received MCBL and PBZ. Results of the current study support an extended withdrawal guideline when doses in excess of 5 g are administered., (© 2016 John Wiley & Sons Ltd.)

Guaifenesin is an expectorant commonly used in performance horses to aid in the clearance of mucus from the airways. Guaifenesin is also a centrally acting skeletal muscle relaxant and as such is a prohibited drug with withdrawal necessary prior to competition. To the authors' knowledge, there are no reports in the literature describing single or multiple oral administrations of guaifenesin in the horse to determine a regulatory threshold and related withdrawal time. Therefore, the objective of the current study was to describe the pharmacokinetics of guaifenesin following oral administration in order to provide data upon which appropriate regulatory recommendations can be established. Nine exercised Thoroughbred horses were administered 2 g of guaifenesin orally BID for a total of five doses. Blood samples were collected immediately prior to drug administration and at various times postadministration. Serum guaifenesin concentrations were determined and pharmacokinetic parameters calculated. Guaifenesin was rapidly absorbed (Tmax of 15 min) following oral administration. The Cmax was 681.3 ± 323.8 ng/mL and 1080 ± 732.8 following the first and last dose, respectively. The serum elimination half-life was 2.62 ± 1.24 h. Average serum guaifenesin concentrations remained above the LOQ of the assay (0.5 ng/mL) by 48 h postadministration of the final dose in 3 of 9 horses., (© 2016 John Wiley & Sons Ltd.)

The aim of this study was to determine the pharmacokinetics of amikacin and penicillin G sodium when administered in combination as an intravenous regional limb perfusion (IVRLP) to horses. Seven healthy adult horses underwent an IVRLP in the cephalic vein with 2 g of amikacin sulfate and 10 mill IU of penicillin G sodium diluted to 60 mL in 0.9% saline. A pneumatic tourniquet set at 450 mmHg was left in place for 30 min. Synovial fluid was collected from the metacarpophalangeal joint 35 min and 2, 6, 12, and 24 h after infusion of the antimicrobials. Concentrations of amikacin and penicillin in synovial fluid were quantitated by liquid chromatography tandem-mass spectrometry analysis. Therapeutic concentrations of amikacin and penicillin for equine-susceptible pathogens were achieved in the synovial fluid. Maximum synovial concentrations (Cmax) (mean ± SE) for amikacin and penicillin were 132 ± 33 μg/mL and 8474 ± 5710 ng/mL, respectively. Only 3 horses had detectable levels of penicillin at 6 h and 1 at the 12 h sample. The combination of amikacin with penicillin G sodium via IVDLP resulted in reported therapeutic concentrations of both antibiotics in the synovial fluid. The Cmax:MIC (minimum inhibitory concentration) ratio for amikacin was 8:1 and Time > MIC for penicillin was 6 h. At 24 h, the mean concentration of amikacin was still above 4 μg/mL. Terminal elimination rate constants (T1/2 lambdaz) were 13.6 h and 2.8 h for amikacin and penicillin, respectively. The use of IVDLP with penicillin may therefore not be practical as rapid clearance of penicillin from the synovial fluid requires frequent perfusions to maintain acceptable therapeutic concentrations.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development., (© 2016 Society for Reproduction and Fertility.)

The aim of the study was to describe the pharmacokinetics and selected pharmacodynamics of intravenous dexmedetomidine in horses. Eight adult horses received 5 μg/kg dexmedetomidine IV. Blood samples were collected before and for 10 h after drug administration to determine dexmedetomidine plasma concentrations. Pharmacokinetic parameters were calculated using noncompartmental analysis. Data from one outlier were excluded from the statistical summary. Behavioral and physiological responses were recorded before and for 6 h after dexmedetomidine administration. Dexmedetomidine concentrations decreased rapidly (elimination half-life of 8.03 ± 0.84 min). Time of last detection varied from 30 to 60 min. Bradycardia was noted at 4 and 10 min after drug administration (26 ± 8 and 29 ± 8 beats/min respectively). Head height decreased by 70% at 4 and 10 min and gradually returned to baseline. Ability to ambulate was decreased for 60 min following drug administration, and mechanical nociceptive threshold was increased during 30 min. Blood glucose peaked at 30 min (134 ± 24 mg/dL) and borborygmi were decreased for the first hour after dexmedetomidine administration. Dexmedetomidine was quickly eliminated as indicated by the rapid decrease in plasma concentrations. Physiological, behavioral, and analgesic effects observed after dexmedetomidine administration were of short duration., (© 2014 John Wiley & Sons Ltd.)

In order to ensure the welfare of performance horses and riders as well as the integrity of the sport, the use of both therapeutic and illegal agents in horse racing is tightly regulated. While Dehydroepiandrosterone (DHEA) is not specifically banned from administration to racehorses in the United States and no screening limit or threshold concentration exists, the metabolic conversion of DHEA to testosterone make its presence in nutritional supplements a regulatory concern. The recommended regulatory threshold for total testosterone in urine is 55 and 20 ng/mL for mares and geldings, respectively. In plasma, screening and confirmation limits for free testosterone (mares and geldings), of no greater than 0.1 and 0.025 ng/mL, respectively are recommended. DHEA was administered orally, as part of a nutritional supplement, to 8 exercised female thoroughbred horses and plasma and urine samples collected at pre-determined times post administration. Using liquid chromatography-mass spectrometry (LC-MS), plasma and urine samples were analyzed for DHEA, DHEA-sulfate, testosterone, testosterone-sulfate, pregnenolone, androstenedione, and androstenediol. DHEA was rapidly absorbed with maximal plasma concentrations reaching 52.0 ± 43.8 ng/mL and 32.1 ± 12.9 ng/mL for DHEA and DHEA sulfate, respectively. Free testosterone was not detected in plasma or urine samples at any time. Maximum sulfate conjugated testosterone plasma concentrations were 0.98 ± 1.09 ng/mL. Plasma testosterone-sulfate concentrations did not fall below 0.1 ng/mL and urine testosterone-sulfate below 55 ng/mL until 24-36 h post DHEA administration. Urine testosterone sulfate concentrations remained slightly above baseline levels at 48 h for most of the horses studied., (Copyright © 2014 John Wiley & Sons, Ltd.)

Reason for Performing Study: The use of firocoxib in horses and its ability to affect performance and potential to allow a horse to compete when it otherwise should not, necessitates establishing appropriate withdrawal time guidelines prior to performance., Objectives: To describe plasma concentrations and characterise the pharmacokinetics of 3 firocoxib formulations following multiple administrations of the label dose, with respect to recommended plasma thresholds for performance horses., Study Design: Balanced 3-way crossover prospective study., Methods: Nine healthy mature horses were administered firocoxib injectable solution (0.09 mg/kg bwt i.v. s.i.d. for 5 days), firocoxib paste (0.1 mg/kg bwt per os s.i.d. for 14 days) and firocoxib tablets (57 mg s.i.d. for 14 days). Blood samples were collected at Time 0 and at various times post drug administration until plasma concentrations were below the limit of detection of the assay. Plasma samples were analysed using liquid chromatography-mass spectrometry and data analysed using noncompartmental analysis., Results: The mean plasma half-life was 1.64 ± 0.737, 1.70 ± 0.800 and 1.73 ± 0.767 days for injectable, paste and tablet formulations, respectively. Plasma concentrations fell below the Racing Medication and Testing Consortium's recommended threshold for racehorses (20 ng/ml) by 7 days post administration of the final dose for all formulations. Plasma concentrations never exceeded the threshold concentration (240 ng/ml) for horse competing in US Equestrian Federation events for any of the formulations., Conclusions: This study extends current knowledge regarding the pharmacokinetics of firocoxib and provides information that can be used to establish appropriate withdrawal time guidelines following multiple administrations, with respect to already established plasma regulatory threshold concentrations., (© 2013 EVJ Ltd.)

This study reports the pharmacokinetics of buprenorphine, following i.v. and buccal administration, and the relationship between buprenorphine concentration and its effect on thermal threshold. Buprenorphine (20 μg/kg) was administered intravenously or buccally to six cats. Thermal threshold was determined, and arterial blood sampled prior to, and at various times up to 24 h following drug administration. Plasma buprenorphine concentration was determined using liquid chromatography/mass spectrometry. Compartment models were fitted to the time-concentration data. Pharmacokinetic/pharmacodynamic models were fitted to the concentration-thermal threshold data. Thermal threshold was significantly higher than baseline 44 min after buccal administration, and 7, 24, and 104 min after i.v. administration. A two- and three-compartment model best fitted the data following buccal and i.v. administration, respectively. Following i.v. administration, mean ± SD volume of distribution at steady-state (L/kg), clearance (mL·min/kg), and terminal half-life (h) were 11.6 ± 8.5, 23.8 ± 3.5, and 9.8 ± 3.5. Following buccal administration, absorption half-life was 23.7 ± 9.1 min, and terminal half-life was 8.9 ± 4.9 h. An effect-compartment model with a simple effect maximum model best predicted the time-course of the effect of buprenorphine on thermal threshold. Median (range) ke0 and EC50 were 0.003 (0.002-0.018)/min and 0.599 (0.073-1.628) ng/mL (i.v.), and 0.017 (0.002-0.023)/min and 0.429 (0.144-0.556) ng/mL (buccal)., (© 2013 John Wiley & Sons Ltd.)

The aim of this study was to examine the effect of the sampling site on the drug concentration-time profile, following intravenous or buccal (often called 'oral transmucosal') drug administration. Buprenorphine (20 μg/kg) was administered IV or buccally to six cats. Blood samples were collected from the carotid artery and the jugular and medial saphenous veins for 24 h following buprenorphine administration. Buprenorphine concentration-time data were examined using noncompartmental analysis. Pharmacokinetic parameters were compared using the Wilcoxon signed rank test, applying the Bonferroni correction. Significance was set at P < 0.05. Following IV administration, no difference among the sampling sites was found. Following buccal administration, maximum concentration [jugular: 6.3 (2.9-9.8), carotid: 3.4 (1.9-4.9), medial saphenous: 2.5 (1.7-4.1) ng/mL], area under the curve [jugular: 395 (335-747), carotid: 278 (214-693), medial saphenous: 255 (188-608) ng·min/mL], and bioavailability [jugular: 47 (34-67), carotid: 32 (20-52), medial saphenous: 23 (16-55)%] were higher in the jugular vein than in the carotid artery and medial saphenous vein. Jugular venous blood sampling is not an acceptable substitute for arterial blood sampling following buccal drug administration.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Statins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of the cellular production of cholesterol and other products of the mevalonate pathway. Statins exert hepatic and extrahepatic effects, modulating the function of various tissues and organs, including ovaries. Previously, we have demonstrated that simvastatin inhibited cellular proliferation and reduced androgen production by ovarian theca-interstitial cells. The above actions are of translational relevance to the most common endocrine disorder among women in reproductive age: polycystic ovary syndrome. However, different statins may have distinctly different profiles of effects on cholesterol and androgens. The present study was designed to compare the effects of several statins on growth and steroidogenesis of rat theca-interstitial cells. The cells were incubated in the absence (control) or in the presence of simvastatin, lovastatin, atorvastatin, or pravastatin. Assessment of effects of statins on cell growth was carried out by evaluation of DNA synthesis and by estimation of the number of viable cells. Effects on steroidogenesis were evaluated by quantification of steroid production and expression of mRNA for the key enzyme regulating androgen production: Cyp17a1. Among tested statins, simvastatin exerted the greatest inhibitory effects on all tested parameters. The rank order of the effects of the tested statins is as follows: simvastatin > lovastatin > atorvastatin ≥ pravastatin. While the lipophilicity is likely to play a major role in determining the ability of statins to act on nonhepatic cells, other factors unique to individual cell types are also likely to be relevant.

Background: Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis., Methods: Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 μM) and/or resveratrol (3-10 μM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test., Results: Resveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin., Conclusions: The present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

The aim of this study was to compare the pharmacokinetics of fentanyl, alfentanil, and sufentanil in isoflurane-anesthetized cats. Six adult cats were used. Anesthesia was induced and maintained with isoflurane in oxygen. End-tidal isoflurane concentration was set at 2% and adjusted as required due to spontaneous movement. Fentanyl (10 μg/kg), alfentanil (100 μg/kg), or sufentanil (1 μg/kg) was administered intravenously as a bolus, on separate days. Blood samples were collected immediately before and for 8 h following drug administration. Plasma drug concentration was determined using liquid chromatography/mass spectrometry. Compartment models were fitted to concentration-time data. A 3-compartment model best fitted the concentration-time data for all drugs, except for 1 cat in the sufentanil group (excluded from analysis). The volume of the central compartment and the volume of distribution at steady-state (L/kg) [mean ± SEM (range)], the clearance (mL/min/kg) [harmonic mean ± pseudo-SD (range)], and the terminal half-life (min) [median (range)] were 0.25 ± 0.04 (0.09-0.34), 2.18 ± 0.16 (1.79-2.83), 18.6 ± 5.0 (15-29.8), and 151 (115-211) for fentanyl; 0.10 ± 0.01 (0.07-0.14), 0.89 ± 0.16 (0.68-1.83), 11.6 ± 2.6 (9.2-15.8), and 144 (118-501) for alfentanil; and 0.06 ± 0.01 (0.04-0.10), 0.77 ± 0.07 (0.63-0.99), 17.6 ± 4.3 (13.9-24.3), and 54 (46-76) for sufentanil. Differences in clearance and volume of distribution result in similar terminal half-lives for fentanyl and alfentanil, longer than for sufentanil., (© 2013 John Wiley & Sons Ltd.)

Objective: To describe the effects of alpha2 -adrenergic receptor antagonists on the pharmacodynamics of sublingual (SL) detomidine in the horse., Study Design: Randomized crossover design., Animals: Nine healthy adult horses with an average age of 7.6 ± 6.5 years., Methods: Four treatment groups were studied: 1) 0.04 mg kg(-1) detomidine SL; 2) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 0.075 mg kg(-1) yohimbine intravenously (IV); 3) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 4 mg kg(-1) tolazoline IV; and 4) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 0.12 mg kg(-1) atipamezole IV. Each horse received all treatments with a minimum of 1 week between treatments. Blood samples were obtained and plasma analyzed for yohimbine, atipamezole and tolazoline concentrations by liquid chromatography-mass spectrometry. Behavioral effects, heart rate and rhythm, glucose, packed cell volume (PCV) and plasma proteins were monitored., Results: Chin-to-ground distance increased following administration of the antagonists, however, this effect was transient, with a return to pre-reversal values as early as 1 hour. Detomidine induced bradycardia and increased incidence of atrioventricular blocks were either transiently or incompletely antagonized by all antagonists. PCV and glucose concentrations increased with tolazoline administration, and atipamezole subjectively increased urination frequency but not volume., Conclusions and Clinical Relevance: At the doses administered in this study, the alpha2 -adrenergic antagonistic effects of tolazoline, yohimbine and atipamezole on cardiac and behavioral effects elicited by SL administration of detomidine are transient and incomplete., (© 2013 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia.)

Objective: To assess tear and plasma concentrations of doxycycline following oral administration to northern elephant seals (Mirounga angustirostris)., Design: Pharmacokinetic study., Animals: 18 juvenile northern elephant seals without signs of ocular disease., Procedures: Study seals were receiving no medications other than a multivitamin and were free from signs of ocular disease as assessed by an ophthalmic examination. Doxycycline (10 or 20 mg/kg [4.5 or 9.1 mg/lb]) was administered orally every 24 hours for 4 days. Tear and plasma samples were collected at fixed time points, and doxycycline concentration was assessed by means of liquid chromatography-tandem mass spectrometry. Concentration-time data were calculated via noncompartmental analysis., Results: Following administration of doxycycline (10 mg/kg/d, PO), maximum plasma doxycycline concentration was 2.2 μg/mL at 6.1 hours on day 1 and was 1.5 μg/mL at 4.0 hours on day 4. Administration of doxycycline (20 mg/kg/d, PO) produced a maximum plasma doxycycline concentration of 2.4 μg/mL at 2.3 hours on day 1 and 1.9 μg/mL at 5.8 hours on day 4. Doxycycline elimination half-life on day 4 in animals receiving doxycycline at a dosage of 10 or 20 mg/kg/d was 6.7 or 5.6 hours, respectively. Mean plasma-to-tear doxycycline concentration ratios over all days were not significantly different between the low-dose (9.85) and high-dose (9.83) groups. For both groups, doxycycline was detectable in tears for at least 6 days following cessation of dosing., Conclusions and Clinical Relevance: Oral administration of doxycycline at the doses tested in the present study resulted in concentrations in the plasma and tears of northern elephant seals likely to be clinically effective for treatment of selected cases of systemic infectious disease, bacterial ulcerative keratitis, and ocular surface inflammation. This route of administration should be considered for treatment of corneal disease in northern elephant seals and possibly other related pinniped species.

Objective: To determine the effects of domperidone on in vivo and in vitro measures of gastrointestinal tract motility and contractility in healthy horses., Sample: 18 adult horses and tissue samples from an additional 26 adult horses., Procedures: Domperidone or placebo paste was administered to healthy horses in a 2-period crossover study. Gastric emptying was evaluated after oral administration of domperidone paste (1.1 or 5.0 mg/kg) or placebo paste by means of the acetaminophen absorption test in 12 horses. Frequency of defecation, weight of feces produced, fecal moisture, and stomach-to-anus transit time of microspheres were evaluated after administration of domperidone paste (1.1 mg/kg) or placebo paste in 6 horses. The effect of domperidone on smooth muscle contractile activity in samples of duodenum, jejunum, ileum, or colon obtained from 26 horses immediately after euthanasia (for nonsystemic medical problems) was investigated., Results: Oral administration of 5.0 mg of domperidone/kg increased peak plasma acetaminophen concentration and area under the curve, indicating increased gastric emptying. Administration of 1.1 mg of domperidone/kg had no effect on gastric emptying, transit time, defecation frequency, or amount and moisture of excreted feces. Contractile activities of circular and longitudinal muscle strips from the duodenum, jejunum, ileum, or colon were not altered by domperidone. Dopamine increased contractile activity of longitudinal muscle strips but not that of circular muscle strips from the midjejunum. Domperidone decreased the dopamine-induced contractile activity of midjejunal longitudinal muscle strips., Conclusions and Clinical Relevance: The potential beneficial effects of domperidone in horses with ileus need to be evaluated in horses with decreased gastric emptying or adynamic ileus.

Objective: To determine the response to neostigmine of the contractile activity of the jejunum and pelvic flexure and the effects of a continuous rate infusion (CRI) of neostigmine in horses., Animals: 7 adult horses and tissue from 12 adult horses., Procedures: A CRI of neostigmine (0.008 mg/kg/h) or placebo was administered to 6 horses in a crossover study design. Gastric emptying was evaluated by the acetaminophen test. The frequency of defecation and urination and the consistency and weight of feces were recorded throughout the experiment. The effect of neostigmine on smooth muscle contractile activity was evaluated in tissues from the jejunum and pelvic flexure. The effect of neostigmine and acetylcholine after incubation with muscarinic receptor antagonists (atropine and DAU 5884) and an acetylcholinesterase inhibitor (edrophonium) was also investigated in vitro., Results: No difference was observed between neostigmine and placebo for time to reach peak plasma acetaminophen concentration and absorption rate constant. A CRI of neostigmine increased fecal production and frequency of urination. Neostigmine induced a dose-dependent increase of contractile amplitude in jejunum and pelvic flexure muscle strips. Incubation of muscle strips with atropine and DAU 5884 inhibited the response to acetylcholine and neostigmine. Incubation of smooth muscle strips from the jejunum with edrophonium increased the response to acetylcholine and had no effect on the response to neostigmine in vitro., Conclusions and Clinical Relevance: A CRI of neostigmine increased fecal production and urination frequency in horses. A CRI of neostigmine did not decrease gastric emptying. Neostigmine stimulated contractile activity of jejunum and pelvic flexure smooth muscle strips in vitro.

Objective: To determine the pharmacokinetics, pharmacodynamics, and safety of zoledronic acid in horses., Animals: 8 healthy horses., Procedures: A single dose of zoledronic acid (0.057 mg/kg, IV) was administered during a 30-minute period. Venous blood was collected at several time points. Zoledronic acid concentration in plasma was measured by liquid chromatography-tandem mass spectrometry, and pertinent pharmacokinetic parameters were determined. Plasma was analyzed for total calcium, BUN, and creatinine concentrations and a marker for bone resorption (C-terminal telopeptides of type I collagen)., Results: Zoledronic acid was safely administered IV during a 30-minute period, and no adverse effects were observed. Plasma concentrations of zoledronic acid were consistent with a 2-compartment mammillary model. Plasma concentrations of zoledronic acid were detected for up to 8 hours after administration. Mean total calcium concentrations in plasma were less than the reference range 7 days after zoledronic acid administration. A marker for bone remodeling decreased in concentration after zoledronic acid administration and remained low for the 1-year duration of the study. No changes in BUN and creatinine concentrations were observed after zoledronic acid administration., Conclusions and Clinical Relevance: Zoledronic acid was safely administered in healthy horses. Zoledronic acid is reported as the strongest bisphosphonate presently available, and studies evaluating potential benefits of zoledronic acid in horses with orthopedic conditions are warranted.

Objective: To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model., Design: In vivo and in vitro studies., Setting: Research laboratory., Animal(s): Immature Sprague-Dawley female rats., Intervention(s): In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 μg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole., Main Outcome Measure(s): Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction., Result(s): In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures., Conclusion(s): These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To test the hypothesis that application of a rope restraint device would result in behavioral, electroencephalographic, and humoral changes consistent with sleep and analgesia in neonatal foals., Animals: 8 healthy neonatal foals., Procedures: Following acclimatization to experimental conditions, each foal underwent a series of assessments before and during or at the end of a period of restraint via application of a restraint device (soft linen rope). Assessments included measurements of heart and respiratory rates, rectal temperature, and circulating β-endorphin and steroid hormone concentrations and evaluations of mentation and body position (behavior), electroencephalographic patterns, and pain tolerance., Results: All foals were lively with apparently normal behavior prior to restraint. During application of the restraint device, foals assumed lateral recumbency with relaxed, somnolent behavior. Heart and respiratory rates and rectal temperature uniformly decreased as a result of the procedure. Electroencephalographic recordings (completed for 3 foals only) revealed patterns consistent with slow wave sleep. Plasma ACTH, dehydroepiandrosterone sulfate, and androstenedione concentrations significantly increased during restraint, compared with prerestraint values. The foals' tolerance to noxious stimuli significantly increased during restraint; however, this was independent of the concentration of circulating β-endorphin., Conclusions and Clinical Relevance: In neonatal foals, the evaluated form of restraint resulted in a decrease in heart and respiratory rates and rectal temperature. Squeeze-induced somnolence may resemble the effects of compression of the fetus in the birth canal and lead to inhibition of voluntary activity. Use of this technique to safely restrain neonatal foals during minor procedures warrants further evaluation.

A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025-10 ng mL(-1)) and quantitation (range 0.125-25 ng mL(-1)) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture., (Copyright © 2012 Elsevier B.V. All rights reserved.)