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Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry.

The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.

LGD-3303 is a Selective Androgen Receptor Modulator (SARM) that is prohibited in both equine and human sports due to its anabolic properties. The aim of this study was to investigate the equine in vivo metabolite profile of LGD-3303 and identify drug metabolites that can be suitable as new and improved analytical targets for equine doping control. This was performed by an oral administration of 0.05 mg.kg(-1) LGD-3303 to horses, where blood and urine samples were collected up to 96 h after administration. The in vivo samples consisting of plasma, urine and hydrolyzed urine were analyzed utilizing ultra-high performance liquid chromatography hyphenated to a Q Exactive (TM) Orbitrap (TM) high resolution mass spectrometer with a heated electrospray ionization source. A total of eight metabolites of LGD-3303 were tentatively identified, including one carboxylated and several hydroxylated metabolites in combination with glucuronic acid conjugates. A monohydroxylated metabolite is suggested as an analytical target for doping control analysis of plasma and urine after hydrolysis with beta-glucuronidase, due to the high intensity and prolonged detection time in comparison to parent LGD-3303.

Pain treatment of lactating bitches is a clinically relevant, but complicated issue. Published scientific studies regarding the excretion of drugs in canine milk are scarce. When considering the risk of side effects in their offspring, lactating bitches have traditionally received very restricted analgesic and anti-inflammatory therapy. Our aim was to quantify the concentrations of carprofen in milk from lactating bitches and relate those to potential risks for the puppies. A second aim was to evaluate the impact mastitis may have on the concentration of carprofen in milk. A population of 100 bitches was enrolled in the study, among which 88 were bitches treated with carprofen after cesarean section (Group CS), eight were bitches with painful inflammatory conditions (Group I) and four were bitches with mastitis (Group M). The patients enrolled in the study received carprofen 4 mg/kg sc at day 1 followed by 2 mg/kg po every 12 h for the following 2-5 days. Owners were instructed to collect milk once a day for five days. The concentration of carprofen in the milk was quantified with ultra-performance liquid chromatography-tandem mass spectrometry. The data obtained were statistically analyzed as repeated-measures data with a mixed-model approach. Data were used to calculate the theoretical maximum total daily intake of carprofen by the puppies in order to perform a computerized simulation of the plasma concentration of carprofen in the puppies. Follow-up telephone interviews to check the status of the enrolled bitches and their litters occurred at one week and three-six months after treatment with car-profen. The major finding of the study was that the concentration of carprofen in the milk was <700 ng/ mL from bitches undergoing CS or suffering painful conditions other than mastitis. In comparison, administration of 2 mg/kg of carprofen sc or po to adult dogs, results in mean maximal plasma con-centrations of 19480 +/- 5420 ng/mL (mean +/- SD). Moreover, data suggests that inflammation of the mammary gland results in a higher concentration of carprofen in milk (up to 1300 ng/mL). In the computerized simulation, the plasma concentrations of carprofen in puppies in group CS and in group I are one tenth of the concentration in adult dogs receiving carprofen at standard doses. Considering the low excretion into milk, carprofen provides an analgesic alternative to lactating bitches without mastitis.(c) 2022 Published by Elsevier Inc.

Objectives: Asymmetric- and symmetric dimethylarginines (ADMA, SDMA) are elevated in cardiovascular disease (CVD). Preeclampsia is a pregnancy-specific syndrome and is an independent risk factor for subsequent CVD. Aims were to investigate whether ADMA, SDMA levels and L-arginine/ADMA and I.arginine/SDMA ratios during pregnancy and their changes from pregnancy to postpartum are associated to arterial wall layer dimensions and cardiovascular risk factors in women with and without preeclampsia. Study design: Dimethylarginines were analyzed by LC-MS, and the common-carotid-artery (CCA) intima and media thicknesses were estimated using 22-MHz non-invasive ultrasonography in women with preeclampsia (cases = 48) and normal pregnancies (controls = 58) in similar gestational age, with reassessment one-year postpartum. A thick intima, thin media and high intima/media ratio (I/M) indicates a less healthy arterial wall. Results: The median age of cases and controls was 30 years. During pregnancy, women with preeclampsia had higher plasma ADMA, SDMA and lower t-arginine/ADMA and L-arginine/SDMA (all p <0.01) than women with normal pregnancies. Further, ADMA, SDMA, L-arginine/ADMA and L-arginine/SDMA correlated to intima thickness (r(s) = 0.33/0.33/-0.33/-0.35 and p <0.01), UM (r(s) = 0.26/0.28/-0.22/-0.26 and p <0.05) and mean arterial pressure (MAP) (rs = 0.43/0.42/-0.39/-0.40 and p <0.0001). Changes in ADMA, SDMA and t-arginine/SDMA from pregnancy to postpartum correlated to changes in intima thickness (r(s) = 0.22/0.32/-0.21 and p < 0.05/<0.01/<0.05), I/M (r(s) = 0.22/0.31/0.08 and p < 0.05/<0.01/=0.43) and MAP (r(s) = 0.31/0.53/-0.25 and p < 0.01/<0.001/<0.05). No correlations were found for conventional CCA intima-media-thickness. Conclusions: Dimethylarginines were associated to signs of adverse effects on arterial wall layer dimensions and cardiovascular risk factors in women with and without preeclampsia, during pregnancy and to their changes from pregnancy up to one-year postpartum. (C) 2021 The Authors. Published by Elsevier B.V.

Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collected over a time period of 96 h. The in vivo samples were compared with five in vitro incubation models encompassing Cunninghamella elegans, microsomes and S9 fractions of both human and equine origin. The analyses were performed using ultra-high performance liquid chromatography coupled to high resolution Q Exactive(TM) Orbitrap(TM) mass spectrometry (UHPLC-HRMS). A total of 21 metabolites were tentatively identified from the in vivo experiments, of which several novel glucuronides were detected in plasma and urine. In hydrolyzed urine, hydroxylated metabolites dominated. The in vitro models yielded several biotransformation products, including a number of monohydroxylated metabolites matching the in vivo results. The suggested analytical target for equine doping control in plasma is a dihydroxylated metabolite with a net loss of two hydrogens. In urine, the suggested targets are two monohydroxylated metabolites after hydrolysis with beta-glucuronidase, selected both due to prolongation of the detection time and the availability of reference material from the in vitro models.

Background Topical ophthalmic atropine sulfate is an important part of the treatment protocol in equine uveitis. Frequent administration of topical atropine may cause decreased intestinal motility and colic in horses due to systemic exposure. Atropine pharmacokinetics are unknown in horses and this knowledge gap could impede the use of atropine because of the presumed risk of unwanted effects. Additional information could therefore increase safety in atropine treatment.

Results Atropine sulfate (1mg) was administered in two experiments: In part I, atropine sulfate was administered intravenously and topically (manually as eye drops and through a subpalpebral lavage system) to six horses to document atropine disposition. Blood-samples were collected regularly and plasma was analyzed for atropine using UHPLC-MS/MS. Atropine plasma concentration was below lower limit of quantification (0.05 mu g/L) within five hours, after both topical and IV administration. Atropine data were analyzed by means of population compartmental modeling and pharmacokinetic parameters estimated. The typical value was 1.7L/kg for the steady-state volume of distribution. Total plasma clearance was 1.9L/h?kg. The bioavailability after administration of an ophthalmic preparation as an eye drop or topical infusion were 69 and 68%, respectively. The terminal half-life was short (0.8h). In part II, topical ophthalmic atropine sulfate and control treatment was administered to four horses in two dosing regimens to assess the effect on gastro-intestinal motility. Borborygmi-frequency monitored by auscultation was used for estimation of gut motility. A statistically significant decrease in intestinal motility was observed after administration of 1mg topical ophthalmic atropine sulfate every three hours compared to control, but not after administration every six hours. Clinical signs of colic were not observed under any of the treatment protocols.

Conclusions Taking the plasma exposure after topical administration into consideration, data and simulations indicate that eye drops administrated at a one and three hour interval will lead to atropine accumulation in plasma over 24h but that a six hour interval allows total washout of atropine between two topical administrations. If constant corneal and conjunctival atropine exposure is required, a topical constant rate infusion at 5 mu g/kg/24h offers a safe alternative.

Background

Buprenorphine is one of the most used analgesics for postoperative pain in rabbits. The recommended dose in rabbits (0.01–0.05 mg/kg) is the same for intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration, despite lack of pharmacokinetic data. Five male and five female New Zealand White rabbits (mean ± SD body weight 3.1 ± 0.3 kg) were administered 0.05 mg/kg buprenorphine by the IV, IM and SC routes and 0.1 mg/kg by the SC route, in a cross-over design with two-week wash-out periods between treatments. Blood was collected before, and up to 8 h post buprenorphine injection, for determination of serum levels by UPHLC-MS/MS.

Results

The area under the time concentration curve (AUC_0-t) was lower after SC (398 ± 155 ng/mL/min) than IM (696 ± 168 ng/mL/min, p < 0.001) and IV (789 ± 189 ng/mL/min, p < 0.001) administration. The maximum serum concentration was lower after SC (2.2 ± 1.4 ng/mL) than after IM (11 ± 3.2 ng/mL) administration (p < 0.001). The bioavailability was lower after SC (50 ± 19%) than after IM (95 ± 21%) administration (p = 0.006). The elimination half-life was longer after SC (260 ± 120 min) than after IM (148 ± 26 min, p = 0.002) as well as IV (139 ± 33 min) injection (p < 0.001). An increase in the SC dose from 0.05 to 0.1 mg/kg resulted in an increase in the area under the time concentration curve of 50% in female (p = 0.022) and 165% in male rabbits (p < 0.001). The bioavailability did not change in the females (36 ± 14%, p = 0.6), whereas it increased in the males (71 ± 23%, p = 0.008).

Conclusions

The lower bioavailability of 0.05 mg/kg buprenorphine after SC administration could explain the lack of efficacy seen in clinical pain studies in rabbits, using this route. For immediate pain relief, IV or IM administration is therefore be recommended, whereas SC administration may be useful to sustain analgesic serum levels, once efficient pain relief has been achieved. The current data do not support an increase in dose to compensate for the lower SC bioavailability.

Background: Dexamethasone is used for the intra-articular route of administration in management of aseptic arthritis in horses. Despite its widespread use there is very little quantitative data of the disposition and response to dexamethasone. The aim of this study was to investigate and describe the synovial fluid and plasma dexamethasone concentration over time and to explore the relation between synovial fluid concentration and response using clinical endpoints as response biomarkers after IA injection of dexamethasone disodium salt solution in an equine model of synovitis.

Results: Inflammation was induced in the radiocarpal joint of six horses by injection of 2ng lipopolysaccharide (LPS). Two hours later either saline or dexamethasone was injected in the same joint in a two treatment cross over design. Each horse was treated once with one of the six doses dexamethasone used (0.01, 0.03, 0.1, 0.3, 1 or 3mg) and once with saline. Dexamethasone was quantified by means of UHPLC-MS/MS. Dexamethasone disposition was characterised by means of a non-linear mixed effects model. Lameness was evaluated both objectively with an inertial sensor based system and subjectively scored using a numerical scale (0-5). Joint circumference, skin temperature over the joint and rectal temperature were also recorded. The LPS-challenge induced lameness in all horses with high inter-individual variability. Dexamethasone significantly decreased lameness compared with saline. Other variables were not statistically significant different between treatments. Objective lameness scoring was the most sensitive method used in this study to evaluate the lameness response. A pharmacokinetic/pharmacodynamic model was successfully fitted to experimental dexamethasone and lameness data. The model allowed characterization of the dexamethasone synovial fluid concentration-time course, the systemic exposure to dexamethasone after intra-articular administration and the concentration-response relation in an experimental model of synovitis.

Conclusions: The quantitative data improve the understanding of the pharmacology of dexamethasone and might serve as input for future experiments and possibly contribute to maintain integrity of equine sports.

Objective: Endothelial function, including the nitric oxide (NO)-pathway, has previously been extensively investigated in heart failure (HF). In contrast, studies are lacking on the NO pathway after heart transplantation (HT). We therefore investigated substances in the NO pathway prior to and after HT in relation to hemodynamic parameters.

Design: 12 patients (median age 50.0 yrs, 2 females), heart transplanted between June 2012 and February 2014, evaluated at our hemodynamic lab, at rest, prior to HT, as well as four weeks and six months after HT were included. All patients had normal left ventricular function post-operatively and none had post-operative pulmonary hypertension or acute cellular rejection requiring therapy at the evaluations. Plasma concentrations of ADMA, SDMA, L-Arginine, L-Ornithine and L-Citrulline were analyzed at each evaluation.

Results: In comparison to controls, the plasma L-Arginine concentration was low and ADMA high in HF patients, resulting in low L-Arginine/ADMA-ratio pre-HT. Already four weeks after HT L-Arginine was normalized whereas ADMA remained high. Consequently the L-Arginine/ADMA-ratio improved, but did not normalize. The biomarkers remained unchanged at the six-month evaluation and the L-Arginine/ADMA-ratio correlated inversely to pulmonary vascular resistance (PVR) six months post-HT.

Conclusions: Plasma L-Arginine concentrations normalize after HT. However, as ADMA is unchanged, the L-Arginine/ADMA-ratio remained low and correlated inversely to PVR. Together these findings suggest that (i) the L-Arginine/ADMA-ratio may be an indicator of pulmonary vascular tone after HT, and that (ii) NO-dependent endothelial function is partly restored after HT. Considering the good postoperative outcome, the biomarker levels may be considered “normal” after HT.

BackgroundCetirizine is an antihistamine used in dogs, but plasma concentrations in relation to effect after oral administration are not well studied. This study investigated cetirizine exposure and the plasma cetirizine concentration-antihistamine response relation in the dog following oral administration of cetirizine.ResultsEight Beagle dogs were included in a cross-over study consisting of two treatments. In treatment one, cetirizine 2-4mg/kg was administered per os once daily for 3days. The other treatment served as a control. Wheal diameter induced by intra-dermal histamine injections served as response-biomarker. Cetirizine plasma concentration was quantified by UHPLC-MS/MS. Median (range) cetirizine plasma terminal half-life was 10h (7.9-16.5). Cetirizine significantly inhibited wheal formation compared with the premedication baseline. Maximum inhibition of wheal formation after treatment with cetirizine per os was 100% compared with premedication wheal diameter. The median (range) IC50-value for reduction in wheal area was 0.33 mu g/mL (0.07-0.45). The median (range) value for the sigmoidicity factor was 1.8 (0.8-3.5). A behavioral study was also conducted and revealed no adverse effects, such as sedation.ConclusionThe results indicate that a once-daily dosing regimen of 2-4mg/kg cetirizine per os clearly provides a sufficient antihistamine effect. Based on this experimental protocol, cetirizine may be an option to treat histamine-mediated inflammation in the dog based on this experimental protocol but additional clinical studies are required.