Assessing the efficiency of a pharmacokinetic-based algorithm for target-controlled infusion of ketamine in ponies

The objective of this study was to assess a pharmacokinetic algorithm to predict ketamine plasma concentration and drive a target-controlled infusion (TCI) in ponies. Firstly, the algorithm was used to simulate the course of ketamine enantiomers plasma concentrations after the administration of an intravenous bolus in six ponies based on individual pharmacokinetic parameters obtained from a previous experiment. Using the same pharmacokinetic parameters, a TCI of S-ketamine was then performed over 120 min to maintain a concentration of 1 microg/mL in plasma. The actual plasma concentrations of S-ketamine were measured from arterial samples using capillary electrophoresis. The performance of the simulation for the administration of a single bolus was very good. During the TCI, the S-ketamine plasma concentrations were maintained within the limit of acceptance (wobble and divergence <20%) at a median of 79% (IQR, 71-90) of the peak concentration reached after the initial bolus. However, in three ponies the steady concentrations were significantly higher than targeted. It is hypothesized that an inaccurate estimation of the volume of the central compartment is partly responsible for that difference. The algorithm allowed good predictions for the single bolus administration and an appropriate maintenance of constant plasma concentrations.

Effect of ketamine on the limb withdrawal reflex evoked by transcutaneous electrical stimulation in ponies anaesthetised with isoflurane

The purpose of this study was to evaluate the anti-nociceptive activity of ketamine and isoflurane in horses using a limb withdrawal reflex (WR) model. Single and repeated stimulations were applied to the digital nerve of the left forelimb in ponies anaesthetised with isoflurane before, during and after intravenous administration of racemic ketamine. Surface electromyographic activity was recorded from the deltoid muscle. Higher stimulation intensity was required to evoke a reflex during ketamine administration. Furthermore, the amplitudes of response to stimulations were significantly and dose-dependently depressed and a flattening of the stimulus-response curves was observed. The reflex activity recovered partially once the ketamine infusion finished. The results demonstrated that the limb WR can be used to quantify the temporal effect of ketamine on the sensory-motor processing in ponies anaesthetised with isoflurane.

The effects of isoflurane minimum alveolar concentration on withdrawal reflex activity evoked by repeated transcutaneous electrical stimulation in ponies

The aim of this study was to quantify the effects of isoflurane at approximately the minimum alveolar concentration (peri-MAC) on the temporal summation (TS) of reflex activity in ponies. TS was evoked by repeated electrical stimulations applied at 5 Hz for 2 s on the digital nerve of the left forelimb of seven ponies. Surface electromyographic activity was recorded from the deltoid and common digital extensor muscles. TS thresholds and amplitude of response to stimulations of increasing intensities were assessed during anaesthesia at 0.85, 0.95 and 1.05 times the individual MAC, and after anaesthesia in standing animals. Under isoflurane anaesthesia, TS thresholds increased significantly in a concentration-dependent fashion and at each isoflurane MAC, the responses increased significantly for increasing stimulation intensities. A concentration-dependent depression of evoked reflexes with a reduction in the slopes of the stimulus-response function was observed for both muscles. The results demonstrated that with this model it is possible to describe and quantify the effects of anaesthetics on spinal sensory-motor processing in ponies.

Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 microg/ml S-ketamine over 120 min under isoflurane anesthesia was performed in Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses).

Evaluation of administration of isoflurane at approximately the minimum alveolar concentration on depression of a nociceptive withdrawal reflex evoked by transcutaneous electrical stimulation in ponies

OBJECTIVE: To investigate effects of isoflurane at approximately the minimum alveolar concentration (MAC) on the nociceptive withdrawal reflex (NWR) of the forelimb of ponies as a method for quantifying anesthetic potency. ANIMALS: 7 healthy adult Shetland ponies. PROCEDURE: Individual MAC (iMAC) for isoflurane was determined for each pony. Then, effects of isoflurane administered at 0.85, 0.95, and 1.05 iMAC on the NWR were assessed. At each concentration, the NWR threshold was defined electromyographically for the common digital extensor and deltoid muscles by stimulating the digital nerve; additional electrical stimulations (3, 5, 10, 20, 30, and 40 mA) were delivered, and the evoked activity was recorded and analyzed. After the end of anesthesia, the NWR threshold was assessed in standing ponies. RESULTS: Mean +/- SD MAC of isoflurane was 1.0 +/- 0.2%. The NWR thresholds for both muscles increased significantly in a concentration-dependent manner during anesthesia, whereas they decreased in awake ponies. Significantly higher thresholds were found for the deltoid muscle, compared with thresholds for the common digital extensor muscle, in anesthetized ponies. At each iMAC tested, amplitudes of the reflex responses from both muscles increased as stimulus intensities increased from 3 to 40 mA. A concentration-dependent depression of evoked reflexes with reduction in slopes of the stimulus-response functions was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Anesthetic-induced changes in sensory-motor processing in ponies anesthetized with isoflurane at concentrations of approximately 1.0 MAC can be detected by assessment of NWR. This method will permit comparison of effects of inhaled anesthetics or anesthetic combinations on spinal processing in equids.

Effects of an alveolar recruitment maneuver on cardiovascular and respiratory parameters during total intravenous anesthesia in ponies

OBJECTIVE: To evaluate pulmonary and cardiovascular effects of a recruitment maneuver (RM) combined with positive end-expiratory pressure (PEEP) during total intravenous anesthesia in ponies. ANIMALS: 6 healthy adult Shetland ponies. PROCEDURE: After premedication with detomidine (10 microg/kg, IV), anesthesia was induced with climazolam (0.06 mg/kg, IV) and ketamine (2.2 mg/kg, IV) and maintained with a constant rate infusion of detomidine (0.024 mg/kg/h), climazolam (0.036 mg/kg/h), and ketamine (2.4 mg/kg/h). The RM was preceded by an incremental PEEP titration and followed by a decremental PEEP titration, both at a constant airway pressure difference (deltaP) of 20 cm H2O. The RM consisted of a stepwise increase in deltaP by 25, 30, and 35 cm H2O obtained by increasing peak inspiratory pressure (PIP) to 45, 50, and 55 cm H2O, while maintaining PEEP at 20 cm H2O. Hemodynamic and pulmonary variables were analyzed at every step of the PEEP titration-RM. RESULTS: During the PEEP titration-RM, there was a significant increase in PaO 2 (+12%), dynamic compliance (+ 62%), and heart rate (+17%) and a decrease in shunt (-19%) and mean arterial blood pressure (-21%) was recorded. Cardiac output remained stable. CONCLUSIONS AND CLINICAL RELEVANCE: Although baseline oxygenation was high, Pa(O2) and dynamic compliance further increased during the RM. Despite the use of high PIP and PEEP and a high tidal volume, limited cardiovascular compromise was detected. A PEEP titration-RM may be used to improve oxygenation in anesthetized ponies. During stable hemodynamic conditions, PEEP titration-RM can be performed with acceptable adverse cardiovascular effects.

Stereoselective pharmacokinetics of ketamine and norketamine after racemic ketamine or S-ketamine administration in Shetland ponies sedated with xylazine

The pharmacokinetics of ketamine and norketamine enantiomers after administration of intravenous (IV) racemic ketamine (R-/S-ketamine; 2.2mg/kg) or S-ketamine (1.1mg/kg) to five ponies sedated with IV xylazine (1.1mg/kg) were compared. The time intervals to assume sternal and standing positions were recorded. Arterial blood samples were collected before and 1, 2, 4, 6, 8 and 13min after ketamine administration. Arterial blood gases were evaluated 5min after ketamine injection. Plasma concentrations of ketamine and norketamine enantiomers were determined by capillary electrophoresis and were evaluated by non-linear least square regression analysis applying a monocompartmental model. The first-order elimination rate constant was significantly higher and elimination half-life and mean residence time were lower for S-ketamine after S-ketamine compared to R-/S-ketamine administration. The maximum concentration of S-norketamine was higher after S-ketamine administration. Time to standing position was significantly diminished after S-ketamine compared to R-/S-ketamine. Blood gases showed low-degree hypoxaemia and hypercarbia.

Stereoselective pharmacokinetics of ketamine and norketamine after racemic ketamine or S-ketamine administration during isoflurane anaesthesia in Shetland ponies

BACKGROUND: The arterial pharmacokinetics of ketamine and norketamine enantiomers after racemic ketamine or S-ketamine i.v. administration were evaluated in seven gelding ponies in a crossover study (2-month interval). METHODS: Anaesthesia was induced with isoflurane in oxygen via a face-mask and then maintained at each pony's individual MAC. Racemic ketamine (2.2 mg kg(-1)) or S-ketamine (1.1 mg kg(-1)) was injected in the right jugular vein. Blood samples were collected from the right carotid artery before and at 1, 2, 4, 8, 16, 32, 64, and 128 min after ketamine administration. Ketamine and norketamine enantiomer plasma concentrations were determined by capillary electrophoresis. Individual R-ketamine and S-ketamine concentration vs time curves were analysed by non-linear least square regression two-compartment model analysis using PCNonlin. Plasma disposition curves for R-norketamine and S-norketamine were described by estimating AUC, C(max), and T(max). Pulse rate (PR), respiratory rate (R(f)), tidal volume (V(T)), minute volume ventilation (V(E)), end-tidal partial pressure of carbon dioxide (PE'(CO(2))), and mean arterial blood pressure (MAP) were also evaluated. RESULTS: The pharmacokinetic parameters of S- and R-ketamine administered in the racemic mixture or S-ketamine administered separately did not differ significantly. Statistically significant higher AUC and C(max) were found for S-norketamine compared with R-norketamine in the racemic group. Overall, R(f), V(E), PE'(CO(2)), and MAP were significantly higher in the racemic group, whereas PR was higher in the S-ketamine group. CONCLUSIONS: Norketamine enantiomers showed different pharmacokinetic profiles after single i.v. administration of racemic ketamine in ponies anaesthetised with isoflurane in oxygen (1 MAC). Cardiopulmonary variables require further investigation.

Effects of a low dose infusion of racemic and S-ketamine on the nociceptive withdrawal reflex in standing ponies

OBJECTIVE: To investigate the effect of plasma concentrations obtained by a low dose constant rate infusion (CRI) of racemic ketamine or S-ketamine on the nociceptive withdrawal reflex (NWR) in standing ponies. STUDY DESIGN: Prospective, blinded, cross-over study. ANIMALS: Six healthy 5-year-old Shetland ponies. METHODS: Ponies received either 0.6 mg kg(-1) racemic ketamine (group RS) or 0.3 mg kg(-1) S-ketamine (group S) intravenously (IV), followed by a CRI of 20 microg kg(-1)minute(-1) racemic ketamine (group RS) or 10 microg kg(-1)minute(-1) S-ketamine (group S) for 59 minutes. The NWR was evoked by transcutaneous electrical stimulation of a peripheral nerve before drug administration, 15 and 45 minutes after the start of the bolus injection and 15 minutes after the end of the CRI. Electromyographic responses were recorded and analysed. Arterial blood was collected before stimulation and plasma concentrations of ketamine and norketamine were measured enantioselectively using capillary electrophoresis. Ponies were video recorded and monitored to assess drug effects on behaviour, heart rate (HR), mean arterial blood pressure (MAP) and respiratory rate. RESULTS: The NWR was significantly depressed in group RS at plasma concentrations between 20 and 25 ng mL(-1) of each enantiomer. In group S, no significant NWR depression could be observed; plasma concentrations of S-ketamine (9-15 ng mL(-1)) were lower, compared to S-ketamine concentrations in group RS, although this difference was not statistically significant. Minor changes in behaviour, HR and MAP only occurred within the first 5-10 minutes after bolus drug administration in both groups. CONCLUSION: Antinociceptive activity in standing ponies, demonstrated as a depression of the NWR, could only be detected after treatment with racemic ketamine. S-ketamine may have lacked this effect as a result of lower plasma concentrations, a more rapid metabolism or a lower potency of S-ketamine in Equidae so further investigation is necessary.

Stereoselective pharmacokinetics of ketamine and norketamine after constant rate infusion of a subanesthetic dose of racemic ketamine or S-ketamine in Shetland ponies

OBJECTIVE: To evaluate pharmacokinetics of ketamine and norketamine enantiomers after constant rate infusion (CRI) of a subanesthetic dose of racemic ketamine or S-ketamine in ponies. ANIMALS: Five 6-year-old Shetland pony geldings that weighed between 101 and 152 kg. PROCEDURES: In a crossover study, each pony received a CRI of racemic ketamine (loading dose, 0.6 mg/kg; CRI, 0.02 mg/kg/min) and S-ketamine (loading dose, 0.3 mg/kg; CRI, 0.01 mg/kg/min), with a 1-month interval between treatments. Arterial blood samples were collected before and at 5, 15, 30, 45, and 60 minutes during drug administration and at 5, 10, 30, and 60 minutes after discontinuing the CRI. Plasma ketamine and norketamine enantiomers were quantified by use of capillary electrophoresis. Individual R-ketamine and S-ketamine concentration-versus-time curves were analyzed by use of a monocompartmental model. Plasma disposition curves for R-norketamine and S-norketamine were described by estimating the area under the concentration-versus-time curve (AUC), maximum concentration (Cmax), and time until Cmax. RESULTS: Plasma concentrations of S-ketamine decreased and biodegradation products increased more rapidly after S-ketamine CRI, compared with results after racemic ketamine CRI. The R-norketamine was eliminated faster than was the S-norketamine. Significant differences between treatments were found for the AUC of S-ketamine and within the racemic ketamine CRI for the AUC and Cmax of norketamine isomers. CONCLUSIONS AND CLINICAL RELEVANCE: CRI of S-ketamine may be preferable over CRI of racemic ketamine in standing equids because the S-enantiomer was eliminated faster when infused alone instead of as part of a racemic mixture.

Determination of the minimum alveolar concentration of isoflurane in Shetland ponies using constant current or constant voltage electrical stimulation

OBJECTIVE: To determine the minimum alveolar concentration (MAC) of isoflurane in Shetland ponies using a sequence of three different supramaximal noxious stimulations at each tested concentration of isoflurane rather than a single stimulation. STUDY DESIGN: Prospective, experimental trial. ANIMALS: Seven 4-year-old, gelding Shetland ponies. METHODS: The MAC of isoflurane was determined for each pony. Three different modes of electrical stimulation were applied consecutively (2 minute intervals): two using constant voltage (90 V) on the gingiva via needle- (CVneedle) or surface-electrodes (CVsurface) and one using constant current (CC; 40 mA) via surface electrodes applied to the skin over the digital nerve. The ability to clearly interpret the responses as positive, the latency of the evoked responses and the inter-electrode resistance were recorded for each stimulus. RESULTS: Individual isoflurane MAC (%) values ranged from 0.60 to 1.17 with a mean (+/-SD) of 0.97 (+/-0.17). The responses were more clearly interpreted with CC, but did not reach statistical significance. The CVsurface mode produced responses with a longer delay. The CVneedle mode was accompanied by variable inter-electrode resistances resulting in uncontrolled stimulus intensity. At 0.9 MAC, the third stimulation induced more positive responses than the first stimulation, independent of the mode of stimulation used. CONCLUSIONS: The MAC of isoflurane in the Shetland ponies was lower than expected with considerable variability among individuals. Constant current surface electrode stimulations were the most repeatable. A summation over the sequence of three supramaximal stimulations was observed around 0.9 MAC. CLINICAL RELEVANCE: The possibility that Shetland ponies require less isoflurane than horses needs further investigation. Constant current surface-electrode stimulations were the most repeatable. Repetitive supramaximal stimuli may have evoked movements at isoflurane concentrations that provide immobility when single supramaximal stimulation was applied.

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.

Selection signatures in Shetland ponies.

Shetland ponies were selected for numerous traits including small stature, strength, hardiness and longevity. Despite the different selection criteria, Shetland ponies are well known for their small stature. We performed a selection signature analysis including genome-wide SNPs of 75 Shetland ponies and 76 large-sized horses. Based upon this dataset, we identified a selection signature on equine chromosome (ECA) 1 between 103.8 Mb and 108.5 Mb. A total of 33 annotated genes are located within this interval including the IGF1R gene at 104.2 Mb and the ADAMTS17 gene at 105.4 Mb. These two genes are well known to have a major impact on body height in numerous species including humans. Homozygosity mapping in the Shetland ponies identified a region with increased homozygosity between 107.4 Mb and 108.5 Mb. None of the annotated genes in this region have so far been associated with height. Thus, we cannot exclude the possibility that the identified selection signature on ECA1 is associated with some trait other than height, for which Shetland ponies were selected.

Seroprevalence of Leptospira spp. in clinically healthy horses in Switzerland

A retrospective, cross-sectional study was conducted to determine the leptospiral seroprevalence in clinically healthy horses in Switzerland. A representative sample of 615 horse sera was examined by microscopic agglutination test for the presence of antibodies against 15 Leptospira spp. serovars. In total, 58.5 % (n = 360) of the horses were positive for one or more of the antigens analysed, with 20.3 % of them showing titres >= 400. The most prevalent serovar was Pyrogenes (22.6 %), followed by serovars Canicola (22.1 %) and Australis (19.2 %). Older horses, mares, ponies and animals spending increased time on pasture exhibited significantly higher prevalence rates (p < 0.05). Moreover, the prevalence was higher in summer and autumn (p = 0.003). The high seroprevalence in healthy horses indicates that they are often exposed to or infected with Leptospira spp. without developing signs of disease. Therefore, other laboratory and clinical data should always be taken into consideration when interpreting serological test results for Leptospira spp.

Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis

A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calibrator concentration are < 8 and < 14%, respectively. The LOD for all enantiomers is 0.01 microg/mL. After i.v. bolus administration of 2.2 mg/kg racemic ketamine, the assay is demonstrated to provide reliable data for plasma samples of ponies under isoflurane anesthesia, of ponies premedicated with xylazine, and of one horse that received romifidine, L-methadone, guaifenisine, and isoflurane. In animals not premedicated with xylazine, the ketamine N-demethylation is demonstrated to be enantioselective. The concentrations of the two ketamine enantiomers in plasma are equal whereas S-norketamine is found in a larger amount than R-norketamine. In the group receiving xylazine, data obtained do not reveal this stereoselectivity.