Pharmacokinetics and Safety of a Single Subcutaneous or Intramuscular Dose of Ketamine in Healthy Horses

TL;DR

This study examines how ketamine is absorbed and processed in horses when given via injection or under the skin, finding it is safe and effective.

Contribution

The study provides the first pharmacokinetic data for subcutaneous and intramuscular ketamine in horses.

Findings

Subcutaneous ketamine resulted in extremely low plasma concentrations (<5 ng/mL).

Intramuscular ketamine reached peak serum concentrations of 20.9 ng/mL with a terminal half-life of 1.8 hours.

Ketamine metabolites, primarily norketamine, were detected within 5 minutes of intramuscular administration.

Abstract

Pharmacokinetics (PK) of intramuscular (IM) and subcutaneous (SC) ketamine in horses has not been described. This study aimed to evaluate the PK and safety of ketamine and its metabolites after a single SC or IM administration. In Phase 1, two horses received 0.5 or 1 mg/kg of ketamine via SC and IM routes. In Phase 2, eight horses received 0.5 mg/kg IM. Plasma or serum concentrations of ketamine and major metabolites were determined by a validated liquid chromatography‐mass spectrometry method at baseline and selected intervals post‐administration. Subcutaneous administration resulted in extremely low concentrations (< 5 ng/mL). Phase 2 focused only on IM administration. Median peak serum ketamine concentrations after IM administration were 20.9 ng/mL (IQR 15.2–35.9) with a time to peak drug concentration of 1.4 h (IQR = 0.8–1.9 h) and terminal half‐life of 1.8 h (IQR = 1.3–2.6 h). No changes in physical examination or laboratory parameters were observed. Ketamine metabolites were detected within 5 min after IM administration, with norketamine as the predominant metabolite. A single IM administration in healthy horses resulted in rapid absorption and variable inter‐individual concentrations without adverse effects. Future studies should investigate repeated IM dosing and determine therapeutic plasma concentrations in horses.

Figures

Serum ketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum norketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum hydroxynorketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum dehydronorketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Tables

TABLE 1. Accuracy, precision, limit of quantitation (LOQ), and limit of detection (LOD) values for LC–MS/MS analysis of ketamine, norketamine, dehydronorketamine, and hydroxynorketamine in equine plasma and serum. Values represent the average of six replicates.

Analyte	Sample type	Concentration (ng/mL)	Accuracy (% nominal concentration)	Precision (% relative SD)	LOQ (ng/mL)	LOD (ng/mL)	Extraction %
Ketamine	Plasma	1	93	2	0.05	0.025	98
		100	103	5
		500	103	3
	Serum	1	97	6	0.05	0.01
		100	112	3
		500	108	3
Norketamine	Plasma	1	94	3	0.2	0.1	100
		100	110	6
		500	97	3
	Serum	1	94	6	0.05	0.02
		100	102	4
		500	106	3
Dehydronorketamine	Plasma	1	91	3	0.2	0.1	99
		100	108	6
		500	95	4
	Serum	1	92	8	0.05	0.02
		100	102	4
		500	107	2
Hydroxynorketamine	Plasma	1	91	2	0.5	0.4	82
		100	108	7
		500	98	3
	Serum	1	91	8	0.1	0.05
		100	99	2
		500	109	5

TABLE 2. Serum pharmacokinetic parameters for ketamine (0.5 mg/kg) given intramuscularly as a single dose to healthy adult horses (n = 8).

PK parameter	Units	Median (range)
AUC	h × ng/mL	94.8 (76.4–123.6)
Cmax	ng/mL	20.9 (15.2–35.9)
Tmax	h	1.4 (0.8–1.9)
K01	1/h	1.3 (0.6–2.8)
K01_HL	h	0.6 (0.2–1.1)
K10	1/h	0.4 (0.3–0.5)
K10_HL	h	1.8 (1.4–2.6)

TABLE 3. Median and ranges of pharmacokinetic parameters of ketamine metabolites in serum after a single administration of 0.5 mg/kg IM (n = 8).

PK parameter	Units	NK	OHNK	DHNK
Tmax	h	1.5 (0.3–2.0)	0.3 (0.1–1.0)	1.5 (0.7–2.0)
Cmax	ng/mL	46.8 (35.6–71.2)	16.3 (8.6–55.1)	20.7 (14.7–37.5)
AUClast	h × ng/mL	200.8 (160.6–244.5)	31.6 (17.9–97.8)	82.1 (62.4–103.4)

TABLE 4. Muscle enzyme activities measured before and after drug administration. Values are reported as mean ± range. No statistically significant differences were observed between pre‐ and post‐administration values.

	Time point	Median (U/L)	Range (U/L)	p
CK	Baseline	373	208–945	0.054
	360 min	389.5	309–934
AST	Baseline	326	256–503	0.057
	360 min	328	268–520

Full text

Introduction

1

Pain management in horses remains challenging for veterinarians. Non‐steroidal anti‐inflammatory drugs (NSAIDs), opioids, and alpha‐2 adrenergic agonists are commonly used; however, their effectiveness is often limited by adverse effects, short duration of action, and relatively high cost (Sellon et al. 2009; Flood and Stewart 2022). Prolonged NSAID use is associated with complications such as right dorsal colitis and renal papillary necrosis (Flood and Stewart 2022). When administered alone, opioids may induce excitatory responses and contribute to gastrointestinal ileus (Elfenbein et al. 2014). Ketamine is an N‐methyl‐D‐aspartate (NMDA) receptor antagonist primarily used as a dissociative anesthetic in veterinary medicine (Muir 2010). At subanesthetic doses, it provides analgesic effects in horses and other species (Guedes et al. 2012). While NMDA receptor antagonism underlies some of its analgesic mechanism, ketamine also interacts with other receptors including nicotinic, muscarinic, opioid, monoaminergic receptors, and voltage‐sensitive sodium channels (Bell and Kalso 2018). Additionally, ketamine has anti‐inflammatory properties by modulating the production of proinflammatory mediators (Dale et al. 2012). In rodents, ketamine metabolites such as norketamine, hydroxynorketamine, and dehydronorketamine play key roles in both analgesic and antidepressant effects (Gomes et al. 2024; Sałat et al. 2015), potentially through synergy with endogenous opioids (Gomes et al. 2024).

In horses, intravenous (IV) continuous rate infusion (CRI) of ketamine at subanesthetic doses is safe and provides analgesic effects (Casoni et al. 2015; Fielding et al. 2006). Its use, however, is limited to supervised settings requiring careful patient monitoring (Elfenbein et al. 2011). Intramuscular (IM), subcutaneous (SC), and oral administration of ketamine provide analgesia with minimal adverse effects in humans and dogs (Blonk et al. 2010; Potvin et al. 2023; Kaka et al. 2016). Empirical IM and SC use of ketamine in horses at subanesthetic doses has been reported, but pharmacokinetic data supporting these routes of administration is lacking. The hypothesis of this study is that SC and IM administration of ketamine to healthy adult horses will achieve blood (serum and plasma) drug concentrations above 70 ng/mL without adverse effects, and that ketamine metabolites will also be detectable in blood. A target concentration of 70 ng/mL was selected because plasma ketamine concentrations of 70–160 ng/mL are associated with analgesic effects without adverse effect in humans (Zanos et al. 2018). The objectives of this study are to describe the pharmacokinetics (PK) of ketamine and its metabolites following a single dose of SC or IM administration, and to evaluate safety of these procedures based on physical examination data (heart rate, respiratory rate, rectal temperature, gastrointestinal sounds, and injection site reactions), changes in muscle enzyme activities, and serum amyloid A concentrations.

Material and Methods

2

Experimental Animals

2.1

All experimental protocols were approved by the Washington State University Institutional Care and Use Committee ASAF#7217. In Phase 1, a pilot study was conducted using two healthy adult Thoroughbred mares, aged 8 and 22 years, with body weights of 520 and 560 kg, respectively. In Phase 2, 7 healthy Quarter Horse mares and 1 healthy Morgan‐cross mare were used. Horses ranged in age from 14 to 18 years, with body weights between 554 and 604 kg. Horses were housed individually in 4 × 4‐m indoor stalls and were only restrained for catheter placement and blood collection. Grass hay was offered ad libitum and water was available at all times.

Phase 1

2.2

A pilot study was performed to determine the optimal dose and route of ketamine administration. Horses were brought indoors in the early morning and remained housed in individual stalls for the duration of the sampling period. On day 1, Horse 1 received a single SC dose of 0.5 mg/kg of ketamine hydrochloride (Ketaset, ZOETIS) and Horse 2 received a single IM dose of 1.0 mg/kg. After an 8‐day washout period, ketamine was administered once to Horse 1 at 0.5 mg/kg IM and once to Horse 2 at 1.0 mg/kg SC. An 8‐day washout period was selected because it greatly exceeds 10 times the reported elimination half‐life of ketamine in horses (estimated at approximately 45 min) and provided practical flexibility for sampling (Fielding et al. 2006). For both horses, SC injections were made in the pectoral region and IM injections were made into the mid‐cervical trapezius muscle on the left side of the neck.

A 14‐G, 13.3 cm, polyurethane IV catheter (MILA International, INC, Kentucky, USA) was placed sterilely in the left jugular vein and blood samples were collected for the analysis of ketamine and its metabolites by aspiration with a syringe prior to drug administration (time 0) and at 5,15, 20, 30, 40, 60, 120, 180, and 360 min after drug administration. For blood collection, 8 mL of blood was initially aspirated and discarded. A second sample of 10 mL was then collected for analysis. Samples were collected into sterile vacutainer tubes with EDTA for plasma separation, centrifuged, and stored at −20°C until plasma ketamine and metabolite concentrations were analyzed.

Physical examination data (heart rate, respiratory rate, rectal temperature, gastrointestinal sounds, and injection site reactions) from each horse was recorded at baseline and 360 min post‐administration. Gastrointestinal auscultation scores were assessed as previously described (Sellon et al. 2009). Local injection site reactions were evaluated at 24 h post‐administration through palpation and subjective assessment of skin lesions and neck flexion.

Phase 2

2.3

Based on preliminary data from Phase 1, a dose of 0.5 mg/kg IM was selected for further investigation. In Phase 2, eight healthy adult female horses received a single dose of ketamine hydrochloride (Ketamine III, DECHRA) at 0.5 mg/kg IM on the left side of the neck. Horses were restrained and managed as previously described for Phase 1.

Blood samples for determination of serum ketamine and metabolite concentrations were collected from the jugular vein catheter by aspiration with a syringe prior to drug administration as previously described for Phase 1. Samples were transferred into sterile vacutainer tubes without anticoagulant for serum separation. After centrifugation at 19,479× g (13,5000 rpm) for 15 min at 23°C, serum samples were separated and stored at −20°C until analysis.

Serum samples were obtained at baseline and 360 min post‐injection for determination of serum creatine kinase (CK) and aspartate aminotransferase (AST) activities using a clinical chemistry system analyzer (SIEMENS Dimension). Analysis was performed within 4 h of collection. Serum amyloid A (SAA) concentrations were measured immediately after blood collection using a stall‐side kit (Equine SAA test, VMRD+, Pullman, WA, USA).

Physical examination data (heart rate, respiratory rate, rectal temperature, gastrointestinal sounds and injection site reactions) were assessed at baseline and at 30‐, 60‐, 120‐, 180‐, and 360‐min post‐administration. Local injection site reactions were evaluated as previously described in Phase 1.

Drug and Metabolite Concentration Determination

2.3.1

Plasma and serum calibrators were prepared by dilution of the working standard solutions for ketamine, norketamine, dehydronorketamine, and hydroxynorketamine (Cerilliant, Round Rock, TX) with drug‐free equine plasma or serum to concentrations ranging from 0.05 to 1000 ng/mL. Only norketamine was a racemic mixture. Calibration curves and negative control samples were prepared fresh for each quantitative assay. Quality control samples (equine plasma or serum fortified with analyte at three concentrations within the standard curve) were included with each sample set as a check of accuracy.

Prior to analysis, 0.1 mL of plasma or serum was diluted with 100 μL of water containing d4‐ketamine (Cerilliant, Round Rock, TX) internal standard at 0.01 ng/μL. The samples were then vortexed briefly to mix, and subsequently, 3 mL of methyl‐tert‐butyl ether was added to each plasma or serum sample, and the samples were mixed by rotation for 20 min at 40 rpm. After rotation, samples were centrifuged at 3300 rpm (2260 g) for 5 min at 4°C, and the top organic layer was transferred to a 12 × 75 mm glass tube. The samples were dried under nitrogen, dissolved in 120 μL of 5% acetonitrile (ACN) in water with 0.2% formic acid, and 25 μL was injected into the liquid chromatography–tandem mass spectrometry (LC–MS/MS) system.

Quantitative analysis was performed on a TSQ Altis triple quadrupole mass spectrometer coupled with a Vanquish liquid chromatography system (Thermo Scientific, San Jose, CA). Chromatography employed an Eclipse XDB‐Phenyl 15 cm × 2.1 mm 5 μm column (Agilent, Palo Alto, CA) and a linear gradient of ACN in water with 0.2% formic acid at a flow rate of 0.4 mL/min. The initial ACN concentration was held at 0% for 0.25 min, increased to 50% over 6.35 min, and flushed for 0.2 min at 90% ACN, before re‐equilibrating for 3.2 min at initial conditions.

Detection and quantification were conducted using selective reaction monitoring (SRM) of the initial precursor ion for ketamine (mass to charge ratio (m/z) 238.1), norketamine (m/z 224.1), dehydronorketamine (m/z 222.1), hydroxynorketamine (m/z 240.2), and the internal standard d4‐ketamine (m/z 242.1). The response for the product ions for ketamine (m/z 124.9, 206.9), norketamine (m/z 125.1, 207.1), dehydronorketamine (m/z 141.0, 142.0), hydroxynorketamine (m/z 124.9, 151.1), and the internal standard d4‐ketamine (m/z 128.9) were plotted, and peaks at the proper retention time were integrated using Quanbrowser software (Thermo Scientific).

Precision and accuracy of the assay were determined by assaying quality control samples in replicates (n = 6). Accuracy was reported as percent nominal concentration and precision was reported as percent relative standard deviation (Table 1). The limit of quantitation and limit of detection for all analytes are also shown in Table 1.

Data Analysis

2.4

Pharmacokinetic Analysis

2.4.1

Pharmacokinetic parameters of ketamine in serum were estimated using compartmental analysis. Pharmacokinetic parameters of ketamine metabolites in serum were estimated using non‐compartmental analysis.

For compartmental analysis, a standard two‐stage approach was used to select the simplest model that best fits the observed concentration data. Serum drug concentrations were plotted on linear and semi‐logarithmic graphs for analysis and to allow visual assessment of the best model for PK analysis. The compartment structure was defined, and standard procedures and diagnostic tools (standard errors of the estimates, correlation matrix, and residual plots, F‐test, Akaike's information criterion, and Schwarz criterion) were used to select the best model. Compartmental analysis was finally performed by using a uniform weighting scheme.

A 1‐compartment model, first‐order absorption, no lag time was used with corresponding equations of C(T) = D × K01/V/(K01−K10) × exp(−K10 × T) − exp(−K01 × T) where C is concentration, D is dose, K01 is the absorption rate post‐administration, assuming first‐order absorption, K10 is the elimination rate post‐administration and T is time.

Estimated pharmacokinetic parameters include area under the plasma concentration‐time curve from 0 h to infinity after dosing (AUC0–∞), maximum concentration (Cmax), time to maximum concentration (Tmax), absorption rate constant (K01), half‐life of absorption (K01_HL), terminal elimination rate constant (K10), and half‐life of the terminal elimination portion of the curve after IM administration (K10_HL). All PK analyses were estimated using Phoenix WinNonlin v10.2.

Safety parameters were assessed for normality using the Shapiro–Wilk test, and the Wilcoxon signed‐rank test was used to evaluate non‐parametric data when comparing safety parameters (CK, AST, and SAA) before and after IM administration of ketamine. The analysis was performed with R studio software 4.2.2 (R study by Posit Software, PBC, Version 2023).

Results

3

In Phase 1, a single SC dose of 0.5 mg/kg resulted in a Cmax of 4.35 ng/mL at a Tmax of 20 min, while a 1 mg/kg dose resulted in a Cmax of 2.78 ng/mL at a Tmax of 60 min. Other PK parameters were not calculated. Mild local reactions at 24 h post‐administration were observed at the SC injection site characterized by localized swelling, mild inflammation, local sweating, and edema. Intramuscular administration of a single dose of 0.5 mg/kg resulted in a Cmax of 28.0 ng/mL at 5 min after ketamine administration, while a single dose of 1 mg/kg resulted in a Cmax of 8.72 ng/mL at 30 min after ketamine administration (Supporting Information Items S1–S5). No adverse reactions at the IM injection sites were observed.

In Phase 2, ketamine concentrations were detectable in serum as early as 5 min after IM injection in seven of eight horses, but there were marked differences in absorption patterns between horses. Horse 6 and 8 showed a rapid increase in serum ketamine concentrations, peaking and declining faster than in the other horses (Figure 1). Horse 5 exhibited a rapid increase in serum ketamine concentration followed by sustained high concentrations over time. Median peak serum ketamine concentration after IM administration was 20.9 ng/mL (IQR = 15.2–35.9 ng/mL) with time to peak drug concentration of 1.4 h (IQR = 0.8–1.9 h) and terminal half‐life of 1.8 h (IQR = 1.3–2.6 h). Other PK parameters are shown in Table 2. Ketamine metabolites were detectable 5 min after drug administration (Figures 2, 3, 4 and Table 3). Norketamine was the predominant metabolite.

Serum ketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum norketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum hydroxynorketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

Serum dehydronorketamine concentrations (ng/mL) in healthy adult horses (n = 8) after a single intramuscular administration of ketamine hydrochloride at 0.5 mg/kg.

All vital parameters remained within the normal limits and no local site reactions were observed. Due to the non‐normal distribution of data, the Wilcoxon signed‐rank test was used to assess differences between paired samples. No significant differences were observed over time in CK and AST activities (Table 4). Serum amyloid A concentrations were below the detection threshold (< 20 μg/mL) prior to and at 360 min after ketamine administration.

Discussion

4

The use of subanesthetic IV doses of ketamine for pain management in horses has been previously described (Flood and Stewart 2022; Elfenbein et al. 2014; Bell and Kalso 2018; Kaka et al. 2016). Alternative routes of administration, such as SC or IM, have been used empirically despite a lack of supporting pharmacokinetic data. The primary objective of this study was to describe the PK of ketamine and its metabolites following a single SC or IM administration in healthy horses. To the authors' knowledge, this is the first study to report PK data of ketamine and its metabolites after a single SC or IM administration of ketamine hydrochloride of 0.5 or 1 mg/kg in healthy adult horses.

In Phase 1, SC ketamine administration resulted in markedly lower plasma ketamine concentrations than were observed after IM administration. Mild local site reactions were observed after SC injection. The observation that plasma ketamine concentrations were higher after administration of the lower dose (0.5 mg/kg) compared to the higher dose (1 mg/kg), regardless of the route of administration in these two horses was unexpected. Given that only two horses were included in this phase, individual factors such as local blood flow, tissue composition and fat‐to‐muscle ratio may have influenced the rate and extent of absorption. Differences in absorption observed between the two horses in Phase 1 were consistent with the variability noted in Phase 2, likely reflecting physiological differences in absorption and metabolism. These findings are illustrated in Supporting Information Items S1–S5, which show higher concentrations of metabolites compared to the parent compound. These results highlight the variability in ketamine disposition among individuals and emphasize the influence of both absorption and metabolic factors.

In Phase 2, IM injection of ketamine resulted in relatively rapid absorption with a Tmax of 1.4 h, suggesting that IM administration of this drug might be beneficial in horses in which rapid onset of analgesia is desirable. The concentration of ketamine in plasma declined relatively quickly, as reflected in a short terminal half‐life. This behavior may contribute to the relatively brief duration of pharmacological effects typically associated with ketamine in clinical settings as described with other routes of administration (Kronenberg 2002).

Effective analgesic plasma concentrations of ketamine have not been established in horses. However, in humans, plasma concentrations between 70 and 160 ng/mL have been associated with analgesic effects (Zanos et al. 2018). Based on this, the lower threshold of 70 ng/mL was used as a reference point in this study and in our study hypothesis to evaluate the potential for analgesic efficacy. Although these serum concentrations were not achieved in the horses in this study, the absence of a therapeutic effect in horses cannot be ruled out. Serum concentrations may not accurately reflect the drug and metabolite concentrations present at active sites in the CNS, with the possibility that lower plasma drug concentrations may be consistent with anecdotally observed analgesic effects in horses.

Ketamine metabolites are synthesized in the liver, primarily through nitrogen demethylation by cytochrome P450 enzymes, with the principal enzyme activity of CYP3A4 and a minor contribution of CYP2B6 and CYP2CA (Zanos et al. 2018; Rao et al. 2016; Hijazi and Boulieu 2002). The metabolite profiles demonstrated rapid conversion of ketamine, with major metabolites detectable as early as 5 min post‐administration. Norketamine was the predominant metabolite, as indicated by the highest Cmax and AUC values compared with HNK and DHNK, which were present at lower concentrations, consistent with previous studies identifying NK as the primary metabolite (Zanos et al. 2018; Pypendop and Ilkiw 2005). Ketamine metabolites remain in circulation longer than the parent compound, presumably due to slower renal clearance and greater polarity (Zanos et al. 2018). These findings reinforce the importance of considering both parent drug and metabolite dynamics when evaluating ketamine's therapeutic potential in equine patients.

No adverse reactions or significant changes in physical examination parameters were observed after IM administration of ketamine. Serum amyloid A concentration and muscle enzyme activities (CK and AST) were measured to assess the degree of muscle damage associated with IM administration. In horses, repeated IM injections have been associated with local tissue irritation and muscle injury, as evidenced by increased concentrations of SAA and increased CK and AST activities (Gordon et al. 2023). Muscle enzyme activities, specifically CK and AST, did not change significantly within the 6‐h period after IM injection. Prior to administration, five horses exhibited mildly increased baseline CK activities, and two had mildly increased baseline AST activities. The increased baseline values likely reflect individual variability or subclinical breed‐related muscle physiology. It is also possible that these values, while outside standard reference intervals, represent normal baseline activities for these individual horses. While there were no significant increases within the monitoring time (360 min) it is possible that some changes in muscle enzyme levels could have occurred beyond this timeframe, but they are unlikely to be clinically relevant.

An important limitation of this study is the small number of horses used in Phase 1. Considering the observed variability in drug absorption, metabolism and elimination, it is possible that the initial data did not reflect the benefits of using a higher dose or SC route of administration. The study population consisted of Thoroughbred horses in Phase 1 and Quarter Horses in Phase 2. This difference reflects the availability of horses at the time of the scheduled experiments. The authors are unaware of any documented differences in ketamine or other drug metabolism between Thoroughbreds and Quarter Horses, breeds that are closely related genetically. The findings in Thoroughbreds and Quarter Horses may not, however, be broadly applicable to other breeds that may be more genetically diverse. Sex‐dependent variations in PK may also complicate extrapolation of these results. Ketamine metabolism can differ significantly between sexes in both animals and humans (Saland et al. 2018; Highland et al. 2022). Because this study population consisted exclusively of mares (females), the applicability of the findings to geldings and stallions (males) should be assessed in future studies.

In Phase 1 of this study, ketamine and its metabolites were measured in plasma samples and in Phase 2 serum samples were used. The assays used were fully validated for both sample types (H. Knych, personal communication, 2024; Table 1). It remains possible that the different sample types may have introduced some degree of variability in the results, but it is unlikely that the variability would be significant given the stringent validation protocols used.

The lack of clearly defined concentrations of ketamine demonstrated to provide analgesic effects in horses makes it difficult to determine whether the detected plasma or serum concentrations of ketamine and its metabolites might be clinically effective. It is possible that analgesic thresholds may differ substantially from those established in humans and other animal models. Pharmacokinetics of ketamine may be affected by the use of adjunctive medications. This is important to consider because ketamine is typically administered as part of a multimodal analgesic protocol in clinical settings. As such, the pharmacokinetic data presented may not fully reflect common clinical use, where drug–drug interactions occur, particularly with agents that induce or inhibit cytochrome P450 enzymes and could alter ketamine metabolism and disposition.

Two different commercially available formulations of ketamine hydrochloride were used in this study during Phase 1 and Phase 2 due to supply issues. Although both products were FDA‐approved, and contained the same active ingredient, minor differences in excipients or formulation could affect ketamine PK and its metabolites' transformation.

Despite these limitations, the study provides valuable information as the first to characterize the PK of IM ketamine in horses. The rapid appearance of ketamine and its metabolites after IM administration supports its potential use as a rescue analgesic or as part of a multimodal analgesic strategy, particularly in situations where IV administration is impractical.

Conclusions

5

Subcutaneous administration of ketamine resulted in low plasma concentrations of both the parent drug and all metabolites. Low plasma concentrations after SC administration suggest that this route of administration is unlikely to achieve meaningful systemic exposure and is unlikely to provide significant analgesic effects in horses. In the absence of pharmacodynamic testing, however, this cannot be stated with certainty. A single IM dose of 0.5 mg/kg ketamine produced detectable concentrations of ketamine and its metabolites within minutes, without adverse effects. The lack of information regarding correlations between serum concentrations of ketamine or its metabolites with analgesic effects in horses currently limits the ability to make definitive clinical dosing recommendations. Pharmacokinetic profiles demonstrated considerable inter‐individual variability in parent drug and metabolite concentrations after IM administration, which may affect the predictability and consistency of clinical responses. Future research should focus on evaluating repeated IM dosing protocols and identifying therapeutic target concentrations of ketamine and metabolites to guide its clinical use in equine patients.

Author Contributions

All authors were involved in conception and design of this project. A.R., D.C.S., M.G.S., E.P., and Z.M.P. conducted the investigation. A.R., D.C.S., M.G.S., E.P., and N.F.V. analyzed and interpreted the data. A.R. prepared the original draft. All authors were involved in review and editing.

Funding

Funding for this study was generously provided by the McEachern Endowment of Washington State University College of Veterinary Medicine.

Ethics Statement

This research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Washington State University (approval number ASAF 7217).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Item S1: Plasma ketamine concentrations (ng/mL) of healthy adult horses (n = 2) over time following a single intramuscular or subcutaneous administration at 0.5 or 1.0 mg/kg.

Item S2: Plasma concentrations of ketamine and its metabolites after a single subcutaneous (SC) administration of ketamine hydrochloride at 0.5 mg/kg in Horse 1.

Item S3: Plasma concentrations of ketamine and its metabolites after a single intramuscular (IM) administration of ketamine hydrochloride at 0.5 mg/kg in Horse 1.

Item S4: Plasma concentrations of ketamine and its metabolites after a single subcutaneous (SC) administration of ketamine hydrochloride at 1 mg/kg in Horse 2 (n = 1).

Item S5: Plasma concentrations of ketamine and its metabolites after a single intramuscular (IM) administration of ketamine hydrochloride at 1 mg/kg in Horse 2 (n = 1).