Pharmacology

Question 1

Investigation of bovine serum albumin-specific IgE expression in horses

Answer 1

-46 out of 65 healthy horses showed increased BSA-specific IgE at some point post-vaccination compared to baseline
-Horses with severe adverse vaccine reactions (“reactors”) had detectable BSA-specific IgE levels. However, the levels were not consistently higher than in healthy horses. This suggests that while BSA-specific IgE may play a role, it is not the sole determinant of adverse vaccine reactions.
-Plasma from donor horses used for commercial plasma production contained BSA-specific IgE levels comparable to healthy horses.

Index Case Insights:
-The index foal’s reaction illustrates the potential for mast cell degranulation and anaphylaxis when BSA-specific IgE interacts with circulating BSA.
-This aligns with anecdotal reports of fatal hypersensitivity reactions in similar scenarios. Potential for hypersensitivity reactions when commercial plasma with high BSA-specific IgE is administered to foals that have ingested powdered colostrum containing BSA.

Clinical Implications
-Routine vaccinations can elicit BSA-specific IgE, particularly in younger horses.
-Veterinarians should exercise caution when administering plasma to neonates that have consumed powdered colostrum products.

Question 2

Short-term administration of flunixin meglumine or firocoxib does not alter viscoelastic coagulation profiles in healthy horses

Answer 2

Coagulation Profiles:
• Short-term administration of flunixin meglumine (nonselective NSAID) and firocoxib (COX-2–selective NSAID) did not result in clinically significant changes in viscoelastic coagulation parameters in healthy horses.
• Minor statistical differences were observed between the two NSAIDs for clot firmness parameters (A20 and MCF), but these were within reference intervals and not clinically significant.
• Traditional coagulation parameters, such as prothrombin time (PT) and partial thromboplastin time (PTT), were elevated in plasma stored during the flunixin meglumine phase, likely due to artifact.

Differences Between NSAIDs:
• Flunixin meglumine resulted in stronger clot formation (higher A20 and MCF values) compared to firocoxib.
• Both NSAIDs had minimal impact on fibrinogen levels, although a slight increase was noted following flunixin meglumine treatment.

Relevance to COX Pathways:
• The balance between COX-1 (procoagulant) and COX-2 (anticoagulant) activity was maintained during treatment, as evidenced by the lack of hyper- or hypocoagulability.
• The selective inhibition of COX-2 by firocoxib did not disrupt coagulation significantly, which contrasts with thrombotic risks associated with COX-2 inhibitors in humans.

Question 3

Pharmacokinetics of single dose administration of three increasing doses of acetaminophen per os in 1-3-monthold foals

Answer 3

• The study successfully determined the pharmacokinetics of acetaminophen in foals aged 1 to 3 months at doses of 10, 20, and 40 mg/kg.
• Acetaminophen plasma concentrations varied significantly across doses, with a dose-dependent increase in peak plasma concentrations (Cmax) and area under the concentration-time curve (AUC).
• The median terminal half-life of acetaminophen (approximately 2.8 hours) was consistent across all doses, indicating predictable elimination kinetics.

• Peak plasma concentrations (Cmax) were achieved within 4.2 hours of administration for all doses, demonstrating rapid absorption.
• The median Cmax values were 4.4, 6.3, and 14 µg/mL for 10, 20, and 40 mg/kg doses, respectively.
• The AUC values also showed a clear dose-response relationship, increasing with higher doses.
• Despite the higher dose, plasma acetaminophen levels in foals at 20 mg/kg were lower than in adult horses reported in other studies.

• Plasma concentrations achieved with the 10 and 20 mg/kg doses may be insufficient for effective antipyretic or analgesic effects, which typically require levels of 10–20 µg/mL in humans.
• At 40 mg/kg, therapeutic concentrations (>10 µg/mL) were achieved in most foals, making it the most likely effective dose for clinical use.
• The lack of observed adverse effects suggests that these doses are safe for single administration, though repeated dosing requires further study.

Safety Profile:
• No adverse clinical, hematological, or biochemical effects were observed in foals at any dose.
n=6. Age 1-3 months

Single doses of acetaminophen up to 40 mg/kg are safe for 1–3-month-old foals, but only the highest dose is likely to achieve therapeutic plasma concentrations.

Question 4

Pharmacokinetics and clinical efficacy of acetaminophen (paracetamol) in adult horses with mechanically induced lameness

Answer 4

Mechanically induced lameness model, n=9

• Acetaminophen at 30 mg/kg (A30) showed faster onset and greater improvement in lameness scores compared to 20 mg/kg (A20) and placebo (C).
• A30 significantly improved lameness at 2 and 4 hours post-treatment compared to placebo, demonstrating its potential as an effective analgesic.
• A30 was as effective as phenylbutazone (PB) in reducing lameness scores but showed a faster initial response.
• Heart rate was consistently lower in the A30 group compared to other treatments at various time points, indicating effective pain relief and reduced physiological stress.

Pharmacokinetics:
• Both doses of acetaminophen were rapidly absorbed, with peak plasma concentrations achieved within 0.66 hours (A20) and 0.43 hours (A30).
• A30 achieved higher peak concentrations (Cmax) and greater overall exposure (AUC) than A20, consistent with dose-proportional pharmacokinetics.
• The mean elimination half-life was longer for A30 (5.3 hours) than A20 (3.5 hours), likely reflecting slower clearance at the higher dose.
• Therapeutic plasma concentrations (>10 μg/mL) were maintained for longer durations in the A30 group, contributing to its superior clinical efficacy.

• Acetaminophen at 30 mg/kg provides significant pain relief and is suitable as a monotherapy for managing lameness in horses.
• The faster onset of action with A30 makes it advantageous for acute pain management compared to phenylbutazone.
• A therapeutic plasma concentration of 8 μg/mL was exceeded for an average of 5.8 hours (A20) and 7.3 hours (A30), supporting the efficacy of these doses.
• Higher concentrations achieved with A30 align with better clinical outcomes, suggesting the need for dose optimization in future studies.
• A30 reduced heart rate more consistently than other treatments, supporting its analgesic effect.
• Heart rate variability (HRV) data indicated a shift toward vagal tone in the A30 group, correlating with reduced pain and stress.

Question 5

Pharmacokinetics, clinical efficacy, and safety of acetaminophen (paracetamol) in adult horses with naturally occurring chronic lameness

Answer 5

• 30 mg/kg administered orally twice daily for 21 days was safe and resulted in transient improvements in lameness in horses with naturally occurring chronic lameness.
• Plasma pharmacokinetics of APAP demonstrated rapid absorption and no drug accumulation over the 21-day dosing period.

• Tmax (time to maximum concentration) increased from day 7 to day 21, potentially due to variability in gastric emptying.
• Mean plasma protein binding of APAP was approximately 50%, higher than reported in humans.
• No detection of the hepatotoxic metabolite NAPQI, suggesting the administered dose did not overwhelm primary glucuronidation and sulfation pathways.
• Elimination half-life extended slightly by day 21 but remained consistent with previous equine studies.

Lameness Improvements:
• Subjective Evaluations: Whole-body lameness scores showed significant improvement at 2 and 4 hours post-treatment compared to untreated controls. Lamest limb scores also improved significantly, particularly in hindlimb lameness cases.
• Objective Evaluations (BMIS): Improvements in hindlimb push-off lameness were significant at 1, 2, and 8 hours post-treatment. No significant effects were observed for forelimb lameness or hindlimb impact lameness parameters, indicating variability in response depending on lameness type.

Safety Profile:
• No significant changes in liver biopsy scores, clinicopathological markers, or gastric ulcer scores were observed after 21 days of dosing.
• Mild elevations in GGT and calcium were noted but remained within reference ranges and were not clinically significant.
• Total bilirubin levels normalized, likely reflecting the resolution of fasting-related elevations at baseline.

Clinical Implications:
• Acetaminophen at 30 mg/kg twice daily offers a viable option for managing mild chronic lameness in horses.
• Effective as a short-term monotherapy but may not suffice for moderate to severe cases or long-term management.
• Higher doses may be required to achieve comparable pharmacological effects to other species due to increased protein binding and interspecies differences in metabolism.

Question 6

Pharmacokinetics and safety of repeated oral dosing of acetaminophen in adult horses

Answer 6

PK:
• Acetaminophen was rapidly absorbed in horses, with a mean Tmax =1.35 hours after a single dose and 0.99 hours after multiple doses.
• The mean t1/2 increased from 2.78 hours (single dose) to 3.99 hours (multiple doses), suggesting a nonlinear elimination pattern over time.
• No significant drug accumulation occurred with twice-daily dosing over 14 days, as demonstrated by consistent maximum plasma concentrations (Cmax) and lack of significant increases in trough levels.

Efficacy and Therapeutic Plasma Concentrations:
• Plasma acetaminophen concentrations exceeded the therapeutic threshold for analgesia in humans (10 µg/mL) for only two hours post-administration.
• Previous studies showed that a single 20 mg/kg dose could reduce lameness for up to five hours, suggesting that horses may require lower therapeutic plasma concentrations than humans.

Safety Profile:
• The 14-day dosing regimen was generally well-tolerated, with no clinically significant changes in gastric ulcer scores or hepatic enzyme levels.
• Statistically significant but clinically insignificant changes were noted in albumin, alkaline phosphatase (ALP), total protein, calcium, and creatine kinase levels.
• Sorbitol dehydrogenase (SDH) levels decreased, and total bilirubin increased during the study but were attributed to factors such as grazing or fasting rather than acetaminophen toxicity.

Potential Causes of Observed Changes:
• The variability in absorption (Cmax and Tmax) was attributed to differences in gastric emptying and intestinal absorption among individual horses, particularly under fed conditions.
• The decrease in bioavailability (AUC) and increase in volume of distribution (Vd/F) with multiple doses could reflect changes in protein binding or drug metabolism, though further research is needed to confirm this.

Clinical Implications:
• Efficacy:
-The rapid absorption and short duration above therapeutic concentrations suggest that acetaminophen may require higher or more frequent dosing for sustained analgesic effects in horses.
-Despite the short plasma half-life, its clinical effects on lameness were reported to last longer, potentially indicating local tissue effects or a lower therapeutic requirement in horses.
• Safety:
-Repeated administration of acetaminophen at 20 mg/kg appears safe over two weeks in healthy adult horses, with no evidence of cumulative toxicity.
-Combining acetaminophen with nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the risk of gastrointestinal side effects and warrants careful monitoring.

Question 7

Pharmacokinetics and efficacy of orally administered acetaminophen in adult horses with experimentally induced endotoxemia

Answer 7

PK:
• APA) administered at 30 mg/kg PO in horses with endotoxemia exhibited reduced plasma concentrations compared to healthy horses, likely due to altered drug absorption.
• Cmax in endotoxemic horses was 13.97 µg/mL (30.02 µg/mL in healthy horses)
• Tmax remained similar between endotoxemic and healthy horses, suggesting that delayed gastric emptying was not the primary cause of reduced absorption.
• Reduced AUC in endotoxemic horses points to compromised drug absorption, potentially due to reduced gastric and intestinal perfusion.

Antipyretic Efficacy:
• APAP reduced rectal temperature significantly at 4 and 6 hours post-administration compared to placebo.
• There was no statistical difference in antipyretic efficacy between APAP and flunixin meglumine (FLU), suggesting APAP is a viable alternative for fever management in horses.
• Despite efficacy in reducing fever, APAP showed no significant effect on systemic inflammatory cytokines (IL-1β, IL-10, TNF-α).
Heart Rate Reduction:
• FLU demonstrated superior reduction in heart rate compared to APAP at 4 and 6 hours post-treatment.
• The greater effect of FLU on heart rate was attributed to its more potent COX inhibition and subsequent suppression of thromboxane A2 (TXA2), which has direct chronotropic effects.

Mechanistic Insights:
• Altered Drug Absorption in Endotoxemia:
-Decreased Cmax and AUC in endotoxemic horses likely result from reduced gastrointestinal perfusion and potential venous congestion rather than delayed gastric emptying.
-Findings align with studies in other species where endotoxemia similarly decreased absorption of orally administered drugs.
• COX Inhibition:
-FLU’s ability to reduce heart rate more effectively than APAP highlights its stronger impact on prostanoid-mediated systemic effects during endotoxemia.
• Safety Profile:
-APAP administration did not result in significant changes in hematological or biochemical parameters, indicating good tolerability.
-No adverse effects related to hepatotoxicity or gastrointestinal ulceration were observed, supporting its safety in acute administration for endotoxemic horses.

Question 8

Ex vivo COX-1 and COX-2 inhibition in equine blood by phenylbutazone, flunixin meglumine, meloxicam and firocoxib

Answer 8

COX-1 and COX-2 Selectivity:
• Firocoxib and meloxicam demonstrated significantly less COX-1 inhibition compared to phenylbutazone and flunixin meglumine.
• All four NSAIDs exhibited comparable levels of COX-2 inhibition after multiple doses, indicating similar anti-inflammatory efficacy.
• The high COX-2 selectivity of firocoxib aligns with its reported COX-1/COX-2 inhibitory ratio of 263–643, compared to meloxicam’s lower ratio of 3.8.

Pharmacokinetics:
• Firocoxib had the longest half-life (48.7 hours) and largest volume of distribution among the studied NSAIDs.
• Phenylbutazone and flunixin meglumine displayed shorter half-lives (~6 and ~4.8 hours, respectively), reflecting their need for more frequent dosing.
• Despite short plasma half-lives, phenylbutazone and flunixin maintained COX-1 inhibition beyond expected therapeutic windows, suggesting prolonged physiological effects.
Efficacy of COX-2 Inhibition:
• Firocoxib achieved COX-2 inhibition comparable to traditional NSAIDs, supporting its use as an effective anti-inflammatory agent.
• The addition of a loading dose of firocoxib (0.3 mg/kg) is recommended to achieve therapeutic concentrations more quickly, as it does not reach steady-state until the fifth dose.

Clinical Implications:
NSAID Selection:
• COX-2 selective NSAIDs, such as firocoxib, are preferable for long-term pain management, particularly in patients at risk for gastrointestinal or renal complications.
• Traditional NSAIDs, like flunixin meglumine, may be more effective for immediate, short-term use due to their stronger initial COX-1 and COX-2 inhibition.
Safety Considerations:
• Prolonged COX-1 inhibition by phenylbutazone and flunixin poses a risk of gastrointestinal and renal side effects, especially in dehydrated or systemically compromised horses.
• Firocoxib and meloxicam are safer options for horses with pre-existing renal or gastrointestinal issues, due to reduced COX-1 inhibition.

Question 9

Meloxicam ameliorates the systemic inflammatory response syndrome associated with experimentally induced endotoxemia in adult donkeys

Answer 9

Systemic Inflammatory Response:
• Meloxicam significantly ameliorated SIRS associated with LPS-induced endotoxemia in donkeys.
• Key clinical improvements included reduced tachycardia, faster recovery from leukopenia and neutropenia, and attenuated increases in plasma lactate levels.
• Plasma tumor necrosis factor-alpha (TNFα) and interleukin-1β (IL-1β) concentrations were significantly lower in the meloxicam-treated group compared to controls.
Ex vivo Gene Expression:
• Pro-inflammatory cytokines (TNFα, IL-1β, IL-6, and IL-8) were upregulated in both groups following LPS infusion. NSD between groups
–> Attenuation of TNFα and IL-1β release may reflect post-transcriptional modulation rather than direct suppression of gene expression.
• Anti-inflammatory IL-10 expression peaked earlier in the meloxicam group, suggesting enhanced resolution of inflammation.

Clinical Efficacy:
• Heart Rate: Meloxicam delayed the onset of tachycardia and significantly reduced mean heart rates during the acute phase of endotoxemia.
• Temperature Regulation: While fever was observed in both groups, meloxicam-treated donkeys exhibited a slower rise and earlier stabilization of body temperature.
• Gut Motility: Meloxicam preserved gut motility, which was significantly impaired in the control group.
• Biochemical Changes: Plasma lactate levels normalized faster in meloxicam-treated donkeys.
• Total solids and fibrinogen concentrations were not significantly different between groups, indicating minimal acute phase protein response during the study period

Question 10

Effects of phenylbutazone, firocoxib, and dipyrone on the diuretic response to furosemide in horses

Answer 10

NSAID Impact on Furosemide-Induced Diuresis:
• Pretreatment with phenylbutazone (PB), firocoxib (FX), and dipyrone (DP) reduced furosemide-induced diuresis by approximately 25%, compared to furosemide alone (FU).
• The magnitude of diuretic inhibition was similar across the three NSAIDs, indicating no significant difference in their effects on renal response to furosemide.
Electrolyte Excretion:
• All NSAID treatments attenuated furosemide-induced increases in fractional sodium (FClNa) and chloride (FClCl) clearances during the first hour post-administration.
• Potassium excretion (FClK) increased after furosemide administration but was not significantly affected by NSAID pretreatment.
Interindividual Variability:
• Considerable variability in the magnitude of diuresis was observed among individual mares, both with and without NSAID pretreatment.
• Differences in urine production were less pronounced in horses with lower diuretic response to furosemide.

Clinical Implications:
Renal Function Monitoring:
• The similar inhibitory effects of all three NSAIDs suggest that even COX-2 selective (firocoxib) or atypical NSAIDs (dipyrone) may not provide “renoprotective” benefits compared to nonselective NSAIDs like phenylbutazone.
• Routine monitoring of renal function is advised when using NSAIDs in horses receiving furosemide, especially in cases with underlying dehydration or compromised renal function.
NSAID Selection:
• While dipyrone and firocoxib may offer a reduced risk of gastrointestinal toxicity, their lack of advantage in maintaining diuretic response limits their potential benefit in renal protection.
• Phenylbutazone remains effective but may pose greater risks in horses with pre-existing renal concerns.

Mechanistic Insights:
• Furosemide-induced diuresis depends on prostaglandin E2 (PGE2) production, which enhances solute delivery to the distal nephron and reduces water reabsorption.
• NSAIDs inhibit cyclooxygenase (COX) enzymes, reducing PGE2 production and thereby attenuating the diuretic and natriuretic effects of furosemide.

Question 11

Effects of phenylbutazone alone or in combination with a nutritional therapeutic on gastric ulcers, intestinal permeability, and fecal microbiota in horses

Answer 11

9 days at 4.4mg/kg PBZ:
• increased gastric ulcer scores (1), intestinal permeability, and altered fecal microbiota.
• Increased permeability, as measured by circulating 16S rDNA, was transient, peaking on Day 54 (five days into treatment) and resolving by Day 59.
• Significant loss of beneficial genera, such as Pseudobutyrivibrio, was observed, potentially contributing to intestinal inflammation.
Nutritional Therapeutic Mitigation:
• Co-administration of a nutritional therapeutic attenuated phenylbutazone-induced increases in intestinal permeability and gastric ulcer scores.
• 6 of the 10 horses developed glandular ulceration with a mean increase of 1.1 grades. Only 1 horse in the nutritional therapeutic group developed phenylbutazone-induced glandular ulceration. The difference in glandular ulcer scores was significant in the
phenylbutazone-treated horses (P = .02) but not in the control and phenylbutazone plus nutritional therapeutic groups
• Horses receiving the nutritional therapeutic exhibited stabilization in fecal microbiota, maintaining beneficial bacteria like Pseudobutyrivibrio.

Phenylbutazone-induced GI effects likely resulted from:
• Topical effects causing intestinal epithelial cell death.
• Reduced prostaglandin (PGE2) production leading to diminished mucus secretion and mucosal protection.
• Disruption of tight junction proteins (e.g., ZO-1) via oxidative stress from reactive oxygen species (ROS), increasing permeability.
• Altered antimicrobial peptide secretion by Paneth cells.
• Loss of beneficial bacteria, such as Pseudobutyrivibrio, may exacerbate intestinal inflammation due to their role in butyrate production, a short-chain fatty acid crucial for maintaining epithelial barrier integrity.

Clinical Implications:
• Healthy adult horses showed only transient increases in GI permeability, but more severe effects could occur in compromised or young horses.
• Clinical signs of GI injury were absent, indicating potential subclinical injury in this population.
• NSAIDs like phenylbutazone pose risks for right dorsal colitis and other GI complications, emphasizing the need for protective strategies.
• The nutritional therapeutic showed potential in mitigating NSAID-induced GI effects, possibly through its components (e.g., omega-3 fatty acids, antioxidants, glutamine, prebiotics).

Question 12

Pharmacokinetics and anti-inflammatory effects of flunixin meglumine as a sole agent and in combination with phenylbutazone in exercised Thoroughbred horses

Answer 12

• Administration of phenylbutazone (PBZ) 24 hours after flunixin meglumine (FM) significantly increased FM clearance compared to FM administered alone.
• No significant differences were noted in the elimination half-life or other pharmacokinetic parameters, except clearance.

Anti-inflammatory Effects:
• FM alone effectively inhibited both constitutively expressed (COX-1) and inducible (COX-2) cyclooxygenase enzymes, as shown by the suppression of thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and prostaglandin F2 alpha (PGF2α).
• Co-administration of PBZ prolonged the suppression of TXB2 and PGE2, indicating additive or synergistic inhibition of COX enzymes.
• Leukotriene B4 (LTB4) and 5-HETE concentrations increased transiently post-FM administration

Metabolism Interaction:
• The slight increase in FM concentrations shortly after PBZ administration may result from competitive displacement from plasma protein binding sites due to PBZ’s high binding affinity.

Clinical Implications:
• The prolonged anti-inflammatory effects observed with combined FM and PBZ administration suggest potential benefits in managing severe inflammatory conditions.
• However, the enhanced COX inhibition may increase the risk of adverse effects, such as gastrointestinal or renal complications, particularly with prolonged use.

Additive Effects:
• The suppression of COX-1 (TXB2) and COX-2 (PGE2) by both FM and PBZ suggests an additive effect when the drugs are used in combination.
• Prolonged inhibition of COX-2–related mediators, such as PGE2, highlights the cumulative pharmacodynamic impact of sequential NSAID administration.
Arachidonic Acid Pathway:
• NSAID-induced COX inhibition leads to arachidonic acid being redirected to the lipoxygenase pathway, explaining the transient increase in LTB4 and 5-HETE concentrations.

Question 13

Localisation of cannabinoid and cannabinoid-related receptors in the equine dorsal root ganglia

Answer 13

Expression of Cannabinoid and Related Receptors:
• CB1R: Observed in all sensory neurons and satellite glial cells (SGCs)
• CB2R: Expressed in 80% of neurons and SGCs
• PPARα: Detected in sensory neurons, SGCs, and vascular endothelial cells, aligning with its known roles in anti-inflammatory and neuroprotective effects.
• TRPA1: Found in sensory neurons and SGCs, with higher expression in unmyelinated nerve fibers, supporting its involvement in nociception and pain signaling.
• 5-HT1aR: Localized in sensory neurons, SGCs, and Schwann cells, indicating its participation in serotonergic modulation of nociception.
The distribution of these receptors in equine dorsal root ganglia (DRG) aligns partially with observations in humans, rodents, and dogs, though notable differences exist in receptor localization (e.g., CB1R and CB2R in SGCs).

Mechanistic Insights:
• CB1R influences nociception by modulating neurotransmitter release at sensory neuron synapses.
• CB2R, through its expression in both neurons and SGCs, is implicated in peripheral pain modulation and anti-inflammatory pathways. Activation of CB2R has demonstrated efficacy in reducing nociceptive signaling in human and rodent studies, suggesting similar therapeutic potential in equine medicine.
• PPARα: Acts as a transcription factor with rapid non-genomic effects, contributing to the anti-inflammatory and neuroprotective properties of cannabinoids. Its vascular localization suggests a role in regulating blood flow and homeostasis within the DRG.
• TRPA1: Known for mediating somatic and visceral pain responses, particularly to mechanical, chemical, and thermal stimuli.
• 5-HT1aR: Plays a critical role in serotonergic analgesia by inhibiting glutamate release and reducing pain signal transmission.

Question 14

Pharmacokinetics and oral bioavailability of cannabidiol in horses

Answer 14

• Cannabidiol (CBD) displayed a tri-compartmental distribution profile, indicative of widespread tissue penetration.
• The volume of distribution (Vss) was large (36 L/kg), suggesting significant sequestration in tissues like fat, central nervous system, and joints.
• Clearance (Cl) was high at 1.46 L/h/kg, pointing to efficient hepatic elimination.
• The oral bioavailability of CBD was low (~14%), consistent across both sesame oil and micellar formulations.
• Micellar formulations provided faster absorption and higher peak plasma concentrations (Cmax) compared to oil formulations, but bioavailability remained similar.

Absorption and Bioavailability:
• Low oral bioavailability is attributed to CBD’s high lipophilicity, limited intestinal absorption, and extensive hepatic first-pass metabolism.
Half-life and Steady-State:
• The terminal half-life (t1/2) was approximately 24-34 hours, longer than prior studies, reflecting slow drug release from peripheral compartments.
• Simulations indicated steady-state concentrations were reached after 6-7 days with twice-daily oral administration.

Therapeutic Applications:
• Both micellar and oil-based formulations can be used for oral CBD administration, with the choice depending on desired pharmacokinetic profiles:
-Micellar formulations: Faster peak levels, suitable for acute conditions.
-Oil formulations: Sustained levels, possibly better for chronic conditions.

Mechanistic Insights:
• First-Pass Effect: Extensive first-pass metabolism by the liver contributes significantly to CBD’s low systemic availability after oral administration. The presence of major metabolites (e.g., 7-COOH CBD) in other studies supports this finding.
• Lipophilic Drug Challenges: The high lipophilicity (log P ~6.3) and low water solubility of CBD necessitate lipid-based or micellar delivery systems for improved absorption.

Question 15

The pharmacokinetics of a fentanyl matrix patch applied at three different anatomical locations in horses

Answer 15

• Fentanyl matrix patches provided measurable plasma concentrations across all anatomical locations: inguinal abdomen (TXA), dorsal metacarpus (TXM), and ventral tail base (TXT).
• The maximum plasma concentration (Cmax) ranged from 1.55 to 2.07 ng/mL, comparable to prior studies using reservoir patches.
• Tmax was location-dependent, with TXA and TXT showing shorter times (10 hours) compared to TXM (14.3 hours).
• The area under the curve (AUC) values were smaller than those reported in studies with reservoir patches, likely due to differences in patch formulations and methodologies.

Site-Specific Absorption:
• TXA and TXT demonstrated higher plasma concentrations and greater AUC values than TXM, aligning with previous findings showing superior absorption in areas with thinner, more vascularized skin.
• Variability in absorption across locations may reflect differences in skin thickness, vascularity, and local drug penetration.

Comparison with Reservoir Patches:
• Matrix patches had lower AUC values than reservoir patches, likely due to:
-Differences in patch design and drug delivery mechanisms.
-Variability in application methodology, including preparation of the application site (e.g., shaving, cleaning).
-Potential residual drug in patches after removal, which was not quantified in this study.

Analgesic Potential:
• Plasma fentanyl levels achieved in this study (1.55–2.07 ng/mL) are consistent with concentrations reported to provide analgesia in other species.
• However, additional studies are needed to determine whether these levels provide effective pain relief in horses without adjunct NSAIDs or other therapies.
• Pain types (e.g., visceral vs. somatic) may respond differently to fentanyl, requiring tailored clinical applications.

Safety and Tolerability:
• No significant effects on heart rate, respiratory rate, or rectal temperature were observed during the study, indicating the patches were well-tolerated.
• No local adverse effects, such as erythema or irritation, were noted at any application sites, contrasting with previous studies reporting mild irritation with reservoir patches.

Question 16

Plasma disposition of gabapentin after escalating intragastric doses in adult horses

Answer 16

PK:
• Gabapentin plasma concentrations increased with escalating doses from 10 to 160 mg/kg, but not in a proportional manner.
• AUC and Cmax increased less than dose-proportionally, indicating saturation of intestinal absorption pathways.
• Terminal half-life varied across doses, with a range of 2 to 15.7 hours, longer than previously reported in lower-dose studies (5–20 mg/kg).
Dose Proportionality:
• Plasma gabapentin concentrations did not increase linearly with higher doses, a phenomenon attributed to the saturation of intestinal transporters, similar to findings in humans and dogs.
• At doses ≥120 mg/kg, further increases in gabapentin exposure were minimal, suggesting limited additional benefit at higher doses.
Efficacy Insights:
• Simulated plasma concentrations indicated that dosing regimens of 10 mg/kg every 8 hours, 20 mg/kg every 12 hours, or 80 mg/kg every 24 hours could maintain plasma levels above the estimated effective concentration (EC50) for neuropathic pain in other species.
• Despite these pharmacokinetic insights, the lack of validated neuropathic pain models in horses limits the ability to correlate plasma levels with clinical efficacy.

Safety:
• Gabapentin was well-tolerated at all doses, with mild sedation observed in one horse at higher doses (120 and 160 mg/kg). No significant changes in physical examination parameters were noted.
• Long-term safety of gabapentin, particularly concerning hepatic and renal function, remains unverified.

Question 17

Pharmacokinetics and pharmacodynamics of repeat dosing of gabapentin in adult horses

Answer 17

PK:
• Gabapentin administered at 40 mg/kg and 120 mg/kg every 12 hours resulted in predictable plasma drug accumulation over 14 days.
• Higher doses (120 mg/kg) produced significantly greater plasma concentrations and AUC compared to 40 mg/kg, suggesting dose-dependent absorption and accumulation.
• Median trough concentrations after repeated administration were 6.4 µg/mL (40 mg/kg) and 17 µg/mL (120 mg/kg), with the latter achieving theoretical effective analgesic concentrations (EC50 = 16.7 µg/mL).
• shows dose-dependent accumulation, consistent with saturation of renal elimination or absorption mechanisms.

Safety:
• No clinical signs of sedation or ataxia were observed in any horses at either dose during the study.
• Biochemistry profiles remained within normal limits, suggesting no adverse effects on liver or kidney function.
• healthy adult mares for 14 days

Efficacy Potential:
• The 120 mg/kg dose is more likely to achieve plasma concentrations associated with analgesia in humans and rats, making it suitable for treating equine pain.
• The 40 mg/kg dose, while safe, may require adjustment in dosing intervals to maintain therapeutic levels in some horses.
• Variability in plasma concentrations suggests the need for individualized dosing regimens based on the horse’s pharmacokinetic profile.

Question 18

Effect of the p38 MAPK inhibitor doramapimod on the systemic inflammatory response to intravenous lipopolysaccharide in horses

Answer 18

• Doramapimod significantly reduced the systemic inflammatory response to LPS infusion in horses.
• The drug decreased heart rate, rectal temperature, blood pressure changes, leukocyte depletion, and cytokine production (TNF-α and IL-1β) following LPS administration.
• Peak cytokine levels were reduced, and the total inflammatory burden, as measured by the AUC for TNF-α and IL-1β, was significantly lower in the doramapimod-treated group.
Mechanism of Action:
• Doramapimod inhibits the p38 MAPK pathway, a key regulator of inflammatory cytokine transcription and post-transcriptional stabilization.
• This mechanism directly targets the upstream activation of pro-inflammatory mediators, offering broader anti-inflammatory potential compared to traditional NSAIDs, which act further downstream.
Safety:
• Doramapimod was well tolerated in healthy horses, with no observed adverse effects on clinical parameters or hematological and biochemical profiles.
• The drug’s safety profile supports its potential for short-term use in systemic inflammatory conditions, such as SIRS in equine patients.
• Short-term Use: Doramapimod’s effectiveness in reducing inflammation within hours of administration suggests its utility for acute inflammatory crises. The transient nature of cytokine suppression aligns with the critical time frame for intervention in acute SIRS cases.

• Doramapimod could complement existing treatments such as NSAIDs or polymyxin B in managing endotoxemia or other inflammatory conditions.

Question 19

Safety and efficacy of subcutaneous alpha-tocopherol in healthy adult horses

Answer 19

• Subcutaneous administration of the 500 IU/mL alpha-tocopherol formulation significantly increased serum and cerebrospinal fluid (CSF) concentrations compared to the 600 IU/mL formulation and oral administration.
• Serum alpha-tocopherol levels exceeded normal reference ranges within 24–48 hours of subcutaneous administration in Phase 2, suggesting effective systemic absorption.
• All horses developed notable tissue swelling at the injection site, regardless of the product concentration. One horse experienced persistent swelling that required surgical drainage, and histology confirmed sterile granulomatous inflammation.
• The extent of tissue reaction limits the clinical applicability of the subcutaneous formulation, despite its efficacy in raising vitamin E levels.
• Muscle concentrations were highly variable and showed no statistically significant increase post-administration, indicating preferential distribution to other tissues.

• Oral supplementation achieved a modest increase in serum alpha-tocopherol levels, but CSF concentrations remained suboptimal.
• Subcutaneous administration resulted in faster and more substantial increases in both serum and CSF alpha-tocopherol levels, making it potentially beneficial for urgent correction of deficiency.

• Subcutaneous alpha-tocopherol could serve as a rapid intervention for severe vitamin E deficiency, particularly in horses with conditions affecting oral absorption.
• Its application may be considered for equine neurological diseases where increasing CSF concentrations quickly is critical.
• The 500 IU/mL formulation is preferable to the 600 IU/mL formulation due to its higher efficacy and potentially reduced local irritation.
• Oral administration remains the safest option for long-term supplementation but may not suffice for rapid correction in acute cases.

Question 20

Antimicrobial susceptibility of bacterial isolates from ambulatory and referral practices

Answer 20

• Resistance rates were significantly higher in isolates from referral hospitals compared to ambulatory practices.
• Penicillin-Gentamicin (P-G) sensitivity: 91% of ambulatory isolates were sensitive, compared to only 64% of referral hospital isolates.
• Trimethoprim-Sulfamethoxazole (TMPS) sensitivity: 82% of ambulatory isolates were sensitive, compared to 56% of referral hospital isolates.

• No second-line antimicrobial showed consistently high efficacy for isolates resistant to first-line antimicrobials.
• Referral hospital isolates had the lowest sensitivity rates to second-line antimicrobials:
-Tetracycline: 8%
-Ceftiofur: 29%
-Enrofloxacin: 40%

The higher resistance rates in referral hospitals are likely due to:
• Greater antimicrobial selection pressure.
• Increased transmission of resistant strains in hospital settings.
• Higher stress levels in hospitalized horses, leading to increased shedding of resistant bacteria.

Question 21

Effects of corn oil on ponazuril serum and CSF concentrations in horses

Answer 21

Co-administration of ponazuril with corn oil (PONOIL) resulted in significantly higher serum and cerebrospinal fluid (CSF) concentrations compared to ponazuril alone (PON).
• Steady-state serum concentrations for PONOIL (6.2 ± 0.9 mg/L) were higher than PON (4.5 ± 1.0 mg/L; P = .004).
• Steady-state CSF concentrations for PONOIL (0.213 ± 0.04 mg/L) exceeded those for PON (0.162 ± 0.04 mg/L; P = .03).

• CSF ponazuril concentrations were not significantly higher on Day 7, despite higher serum concentrations, suggesting delayed equilibration between serum and CSF.
• Higher serum concentrations on Days 14 and 21 directly correlated with increased CSF levels, indicating that serum concentrations drive CSF accumulation.
• Substantial inter-horse variability in both serum and CSF concentrations was observed, especially in the PON group.
• Some horses achieved CSF concentrations close to or below the effective inhibitory concentration for Sarcocystis neurona, highlighting variability in pharmacokinetics.

Enhanced Absorption:
• Corn oil enhances ponazuril absorption due to the drug’s high lipophilicity, improving bioavailability and systemic concentrations.
• Higher serum concentrations translate into greater CSF penetration, driven by passive diffusion across the blood-brain barrier (BBB).
Delayed CSF Equilibration:
• The time to achieve maximal CSF concentrations may reflect slower BBB crossing and compartmental distribution dynamics of ponazuril.

Question 22

Evaluation of safety, humoral immune response and faecal shedding in horses inoculated with a modified-live bovine coronavirus vaccination

Answer 22

• The modified-live bovine coronavirus (BCoV) vaccine was safe to administer via oral, intranasal, and intrarectal routes.
• No clinical signs of enteric coronavirus infection (e.g., lethargy, fever, diarrhea, or colic) were observed in vaccinated horses.
• No evidence of vaccine virus shedding in feces supports the hypothesis that the vaccine virus did not replicate in the equine intestinal tract.

• Viral shedding was detected in nasal secretions of two intranasally vaccinated horses within 24 hours post-vaccination, attributed to residual vaccine virus rather than active replication.
• Absence of viral shedding in feces across all groups indicates minimal risk of environmental contamination.
• Seroconversion occurred in only 27% of vaccinated horses, with one seroconversion from each vaccination route.
-The limited humoral response may result from inadequate replication of the vaccine virus in equine cells, the low antigenic mass in the vaccine, or pre-existing cross-reactive antibodies to equine coronavirus (ECoV).

Immune Activation:
• The study aimed to bypass gastric degradation by testing alternative administration routes (e.g., intrarectal), hypothesizing improved mucosal immunity. However, the serological response remained limited.
• Prior evidence from foal studies indicates that intrarectal vaccination may enhance cell-mediated immunity, but this was not assessed here.
• BCoV’s close antigenic relationship to ECoV makes it a suitable model for vaccine testing, but the limited replication of the modified-live BCoV strain in equine tissues may have reduced its immunogenicity.

Question 23

Effects of administration of ascorbic acid and low- dose hydrocortisone after infusion of sublethal doses of lipopolysaccharide to horses

Answer 23

Clinical and Cytokine Effects:
• Administration of ascorbic acid (AA), hydrocortisone (HC), or their combination did not significantly impact clinical signs, plasma serum amyloid A (SAA) concentrations, or pro-inflammatory cytokine expression compared to placebo.
• A notable finding was that AA administration resulted in higher segmented neutrophil counts at 6 and 12 hours post-LPS infusion, suggesting a protective effect against neutrophil depletion caused by oxidative stress.
Cortisol Response and CIRCI:
• LPS infusion increased serum cortisol levels, consistent with stress responses, but the cortisol response to ACTH stimulation indicated CIRCI in all tested horses.
• AA did not improve cortisol responses, suggesting its limited role in mitigating CIRCI in this experimental model- but was not tested in the HC group due to cross reaction with assays and exogenous HC.
• CIRCI is relevant in septic horses and requires further investigation regarding the role of HC as adjunctive therapy.
Plasma Ascorbic Acid Levels:
• Plasma AA levels decreased in 83% of horses post-LPS infusion, indicative of consumption by inflammatory processes.
• Horses administered exogenous AA showed elevated plasma AA levels at all time points, reinforcing its role in replenishing depleted stores during inflammation.

Neutrophil Protection:
• AA likely mitigates oxidative stress-induced neutrophil destruction through its antioxidant properties, reducing reactive oxygen species (ROS) and protecting cellular integrity.
Synergistic Effects:
• HC upregulates the sodium-dependent vitamin C transporter-2 (SVCT2), facilitating cellular AA uptake. This synergy may enhance AA’s antioxidative and anti-inflammatory effects, though no such additive effects were observed in this study.

Question 24

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses

Answer 24

• Administration of PB-MS did not significantly alter clinical signs (e.g., rectal temperature, pain score) or inflammatory markers (e.g., serum amyloid A (SAA), TNF-α, IL-6) compared to placebo controls.
• A notable reduction in IL-1β gene expression was observed in the PB-MSC group at specific time points (T0 and T1 hours), but this was deemed unlikely to be clinically meaningful given the short timeframe.

• Plasma reactive oxygen metabolite (dROM) concentrations and intracellular reactive oxygen species (ROS) levels were not significantly different between treatment groups.
• Intracellular ROS increased over time in the placebo group, suggesting a potential antioxidant effect of PB-MSC. However, no significant differences in plasma antioxidant capacity (PAC) were observed.
• No significant differences in leukocyte or neutrophil counts were detected between PB-MSC and placebo groups, although PB-MSC treatment was associated with lower lymphocyte counts overall.

PB-MSC Modulation of Inflammation:
• PB-MSC are known to regulate inflammation by interacting with immune cells and secreting anti-inflammatory mediators. However, the lack of robust effects in this study may reflect insufficient PB-MSC activation or an inadequate dose relative to the inflammatory burden induced by LPS.
• PB-MSC were administered 30 minutes after LPS infusion to simulate clinical endotoxemia. The timing may have limited their efficacy, as prolonged inflammation could be necessary to fully activate the immunosuppressive properties of PB-MSC.
• PB-MSC may have become trapped in organ microvasculature (e.g., lungs), reducing their systemic availability and impact on inflammation.

Question 25

Pharmacokinetics and tolerability of multiple-day oral dosing of mycophenolate mofetil in healthy horses

Answer 25

• Mycophenolate mofetil (MMF) was converted to mycophenolic acid (MPA) and further metabolized into MPG, MPAG, and AcMPAG.
• Minimal accumulation of MPA and metabolites occurred over the 7-day dosing period.
• Prolonged MPA half-life (T½) on Day 7 compared to Day 1 suggests potential alterations in metabolism or excretion over time.
• Enterohepatic recirculation contributed to MPA plasma concentration variability, with a secondary peak observed between 3–5 hours post-administration.
• Adverse effects were noted, including hyporexia, decreased gastrointestinal motility, and reduced fecal output by Day 7.
• All horses developed myelosuppression (neutropenia and lymphopenia) and hyperbilirubinemia, indicative of hematologic and hepatic toxicity.
• Salmonellosis was detected in 2 horses post-treatment, likely due to immunosuppression.

• Hyperbilirubinemia may result from drug-induced liver injury or fasting-associated bilirubin increases.
• Hypertriglyceridemia reflected lipid mobilization due to negative energy balance from hyporexia.

Accumulation and Therapeutic Range:
• Weak metabolite accumulation (AI < 2) was observed, consistent with limited systemic buildup.
• High MPA fluctuation percentage suggests challenges in maintaining therapeutic plasma levels without exceeding toxicity thresholds.

Immunosuppressive Mechanism:
• MMF’s inhibition of purine synthesis suppresses lymphocyte proliferation, explaining observed lymphopenia.
• AcMPAG may contribute to gastrointestinal toxicity via cytokine release from mononuclear leukocytes.
Enterohepatic Recirculation:
• Reabsorption of MPA through the enterohepatic cycle amplifies exposure, potentially exacerbating toxicity in prolonged treatment.

Question 26

The effects of orally administered trazodone on ambulation and recumbency in healthy horses

Answer 26

• Trazodone at 7.5 mg/kg significantly reduced step frequency in healthy horses by approximately 44%, supporting its potential use in conditions requiring decreased ambulation, such as acute laminitis.
• The 2.5 mg/kg dose also decreased step frequency but showed inconsistent results due to significant intra-horse variability.
• Trazodone did not increase time spent in recumbency, suggesting it may not be effective for encouraging recumbency as part of laminitis management.
• Horses treated with 7.5 mg/kg exhibited consistent sedation, including eyelid droop, mild ataxia, and lack of response to stimuli, beginning within 30 minutes and lasting up to 8 hours post-administration.
• Trazodone was well-tolerated overall, with no significant adverse effects on heart rate (HR), QTc interval, or fecal output.
• One horse experienced transient sinus tachycardia, muscle fasciculations, and sweating after the second 7.5 mg/kg dose, resolving without intervention and not recurring with subsequent doses.

Pharmacokinetics:
• linear pharmacokinetics, achieving steady-state concentrations by the fourth dose with an accumulation ratio of approximately 1.45 at 7.5 mg/kg.
• The active metabolite m-chlorophenylpiperazine (m-CPP) was present but at low concentrations, suggesting minimal contribution to trazodone’s effects.

Safety Monitoring:
• While generally safe, close monitoring is advised during initial administration, particularly in horses receiving higher doses, to detect any idiosyncratic reactions like tachycardia or excitability.

Accumulation Ratio is defined as the ratio of the concentration of a compound at steady state to the concentration after a single dose,

Question 27

Effects of intravenous administration of ascorbic acid on oxidative status in healthy adult horses

Answer 27

These are effects in healthy horses, needs assessing in sepsis. Single dose only

• High-dose IV administration of AA (100 mg/kg) significantly reduced plasma derivatives of reactive oxygen metabolites (dROM) at 2 hours post-administration, indicating a decrease in circulating ROS.
• By 6 hours post-administration, dROM levels returned to baseline, suggesting a transient effect.
• Lower doses (25 mg/kg and 50 mg/kg) did not produce a significant impact on dROM, possibly due to insufficient plasma concentrations.
• Neither basal nor stimulant-induced intraerythrocytic ROS concentrations were affected by AA administration at any dose, which might reflect insensitivity of the assay or minimal oxidative stress in the study’s healthy horse population.
• Stimulant-induced neutrophil ROS production was not significantly reduced, potentially due to the study’s design, which measured ROS production after plasma AA levels had declined.
• Plasma antioxidant capacity did not increase significantly at any dose or time point, suggesting that AA supplementation did not enhance the overall antioxidant status in healthy horses. It is possible that plasma PAC measurements failed to capture the transient increases in antioxidant activity immediately post-AA infusion.
• Plasma AA concentrations increased dose-dependently and remained elevated for 6 hours post-administration, confirming effective systemic absorption.

ROS Scavenging:
• High-dose AA may reduce extracellular ROS, as evidenced by decreased dROM levels, but its effects on intracellular ROS appear limited, possibly due to barriers in AA transport into cells.
• The lack of significant changes in PAC and neutrophil ROS production could indicate that endogenous antioxidant mechanisms were sufficient to manage the low oxidative stress in healthy horses.

Question 28

Effect of sirolimus on insulin dynamics in horses

Answer 28

• A significant reduction in insulin levels was observed 24 hours post-sirolimus administration compared to placebo.
• Sirolimus decreased serum insulin concentrations during the oral glucose test (OGT) by 37% at 60 minutes and 28% at 120 minutes post-dosing.
• There was no significant effect on blood glucose concentrations at the same timepoints, suggesting targeted suppression of insulin secretion.

• The lack of an effect at 4 hours post-administration, despite high sirolimus concentrations, implies that the drug’s effects are not solely dependent on blood concentration.
• Insulin suppression appeared transient, with no significant differences observed at 72 or 144 hours post-administration.

Effects in Induced Insulin Dysregulation (ID):
• In horses with dexamethasone-induced ID, sirolimus treatment restored insulin responses to near baseline levels after 7 days, suggesting therapeutic potential for managing hyperinsulinemia-associated laminitis (HAL).

mTOR Pathway Modulation:
• Sirolimus inhibits the mechanistic target of rapamycin (mTOR) pathway, which plays a role in insulin secretion and β-cell function.
• Its delayed effect on insulin suppression may relate to the indirect inhibition of mTOR complex 2 (mTORC2), which affects insulin production via cytoskeletal regulation and β-cell granule formation.
• The transient nature of the response may reflect the timing of mTORC2 inhibition, which typically requires longer drug exposure.

Management of HAL:
• Sirolimus may be a promising therapeutic for reducing hyperinsulinemia in horses with ID, particularly in preventing HAL, given its ability to modulate insulin responses without affecting glucose levels.
• Daily dosing might be necessary to maintain therapeutic effects due to its transient impact.
• While 0.06 mg/kg was effective, further studies are needed to optimize dosing intervals and establish safe long-term protocols.
• Oral sirolimus showed potential for efficacy despite low bioavailability, which could enhance its practicality in clinical use.

Safety Profile:
• Sirolimus was well-tolerated in short-term administration, with transient pigmenturia and hemolysis attributed to the propylene glycol vehicle rather than the drug itself.
• Long-term safety remains uncertain, particularly regarding its immunosuppressive properties, which could pose risks in horses.

Question 29

Monitoring trends in antimicrobial susceptibility patterns of bacterial isolates from horses with ulcerative keratitis

Answer 29

• The predominant bacterial genera isolated were Streptococcus spp., Staphylococcus spp., and Pseudomonas aeruginosa.
• Over time, there has been a notable increase in the proportion of Staphylococcus spp. and a decrease in Streptococcus spp., suggesting a shift in the microbial population associated with equine ulcerative keratitis.
• This shift aligns with global trends in opportunistic infections in both veterinary and human ocular settings.

Antimicrobial Susceptibility:
• Chloramphenicol and ciprofloxacin exhibited high levels of in vitro efficacy across isolates.
• Streptococcus spp. showed high susceptibility to chloramphenicol, cephalothin, and erythromycin, but lower susceptibility to aminoglycosides such as gentamicin and tobramycin.
• Pseudomonas aeruginosa was most susceptible to ciprofloxacin, but less so to other antibiotics like gentamicin and tobramycin.
• Combination therapies targeting both gram-positive and gram-negative organisms yielded higher susceptibility rates compared to monotherapies.

Therapeutic Implications:
Empirical Treatment Recommendations:
• For broad-spectrum initial therapy, combining antibiotics like gentamicin with cefazolin or ciprofloxacin with chloramphenicol is recommended until culture results are available.
• Aminoglycosides alone are insufficient against Streptococcus spp., emphasizing the need for combination therapy in empirical treatment.

• Over the last two decades, antimicrobial resistance patterns in this population have shown minimal change, contrasting with global concerns of rising resistance.
• Methicillin-resistant Staphylococcus spp. prevalence remains consistent (~33%), in line with human medical trends.

Question 30

Pharmacokinetics of multiple doses of chloramphenicol in fed adult horses

Answer 30

• Chloramphenicol, when administered orally to adult horses, shows low serum concentrations at steady state.
• Rapid elimination half-life (average 3.22 hours) and quick time to peak serum concentration (~0.5 hours) were observed.

• Chloramphenicol’s efficacy depends on maintaining serum concentrations above the Minimum Inhibitory Concentration (MIC) for at least 50% of the dosing interval.
• Current CLSI susceptibility breakpoints (4 µg/mL for streptococci and 8 µg/mL for other bacteria) were not consistently met:
• None of the horses achieved serum concentrations ≥8 µg/mL for at least 50% of the dosing interval.
• Only one horse achieved concentrations ≥4 µg/mL for ≥4 hours, and three achieved ≥4 µg/mL for ≥3 hours.
• Average serum concentration was 5.0 µg/mL, but this varied widely between individuals, complicating a universal dosing recommendation.

Accumulation and Metabolism:
• Observed accumulation of chloramphenicol over the study period suggests reduced metabolism or elimination.
• This may be due to chloramphenicol’s inhibitory effect on cytochrome P450 enzymes, consistent with findings in human and equine studies.
• Tissue concentrations were not assessed- may accumulatye

• Clinicians are advised to determine the MIC for specific bacterial targets and adjust dosing accordingly.

Question 31

Fluoroquinolone exposure in utero did not affect articular cartilage of resulting foals

Answer 31

• In utero exposure to enrofloxacin did not result in detectable cartilage changes or significant joint abnormalities in neonatal foals by 30 days of age. At either therapeutic (7.5) or supratherapeutic(15mg/kg) doses when treated from 280d gestation for 14 days.
• Radiographs and MRI identified osteochondral changes in both treated and untreated foals, but there were no significant differences in prevalence or severity of lesions across groups.
• Severe cartilage erosion and joint effusion were observed in foals treated with enrofloxacin post-natally, suggesting potential toxicity when administered after birth.

• Results align with earlier findings that enrofloxacin crosses the placenta without inducing gross or histological lesions.
• Toxicity observed in post-natally treated foals mirrors previous studies indicating potential adverse effects at neonatal stages, even at therapeutic doses.
• no long term follow up

Question 32

Administration of enrofloxacin during late pregnancy failed to induce lesions in the resulting newborn foals

Answer 32

• Administration of enrofloxacin to late pregnant mares (280d 14d) did not cause observable chondrotoxic lesions in fetuses by 5 weeks of age.
• Cellular changes seen in foals’ cartilage across all groups were consistent with normal developmental processes, not enrofloxacin exposure.
• Severe cartilage erosion and joint effusion were observed only in post-natally treated foals, emphasizing potential age-related susceptibility.

• Hindlimb tendons demonstrated higher elastic modulus and tensile strength compared to forelimbs, a previously unreported finding in adult horses.
• Plasma magnesium levels were normal in all mares, and no changes occurred during the study.
• Magnesium deficiency is known to mimic fluoroquinolone-induced lesions; thus, its status should be monitored in clinical applications.

• Fetal fluoroquinolone exposure may require long-term evaluation as adverse effects could manifest later in development.

• Enrofloxacin reaches therapeutic levels in fetal fluids, indicating its efficacy for severe placental infections.

Question 33

Pharmacokinetics of intravenous and oral administration of enrofloxacin to the late-term pregnant and non-pregnant mares

Answer 33

• Differences in pharmacokinetics of enrofloxacin were observed between pregnant and non-pregnant mares.
• higher enrofloxacin AUC values in pregnant mares after oral administration.
• The recommended dosages (5 mg/kg IV and 7.5 mg/kg PO) are appropriate for treating susceptible pathogens (MIC < 0.25 µg/mL) in both groups.

Oral Administration Observations:
• Enrofloxacin was well-absorbed, with bioavailability comparable to previous studies.
• Oral bioavailability did not differ significantly between pregnant and non-pregnant mares.
• Ciprofloxacin Cmax was lower, and Tmax was longer in pregnant mares, possibly due to delayed gastric emptying during pregnancy.

Intravenous Administration Observations:
• Slight tendencies for increased AUC and decreased systemic clearance in pregnant mares compared to non-pregnant mares.
• Ciprofloxacin concentrations at 24 hours post-dose were higher in pregnant mares, suggesting altered metabolism or elimination.

Clinical Implications:
• The efficacy of enrofloxacin for pathogens with MIC > 0.25 µg/mL may require dosage adjustments, particularly in non-pregnant mares.
• For bacteria with intermediate susceptibility (MIC = 0.25–1 µg/mL), achieving effective AUC/MIC ratios may necessitate increased dosages.

Question 34

Gentamicin plasma concentrations in hospitalised horses and retrospective minimal inhibitory concentrations of gram-negative equine pathogens

Answer 34

• Gentamicin administered at 10 mg/kg IV every 24 hours achieved the target peak plasma concentration (≥20 µg/mL) in 90% of horses.
• The majority (97%) of horses met the target trough concentration (<2 µg/mL) 20 hours post-administration, minimizing the risk of nephrotoxicity.
• Despite achieving target concentrations, the plasma concentration-to-MIC ratio (≥10:1) was only met for 24% of gram-negative bacteria.

• Peak concentrations (C1h) were within an acceptable range for most horses (75% between 23.5 and 36.2 µg/mL).
• Azotemia was associated with significantly higher C1h due to decreased clearance from reduced glomerular filtration rates.
• Horses with systemic inflammatory response syndrome (SIRS) did not show significant differences in peak or trough concentrations compared to non-SIRS horses.

Minimum Inhibitory Concentrations (MICs) for Local Gram-Negative Pathogens:
• 28% of bacterial isolates were susceptible to gentamicin, with MICs ≤2 µg/mL.
• Resistance was prevalent, with 70% of isolates showing MICs ≥16 µg/mL.
• Only 2% of intermediate isolates would benefit from increasing the gentamicin plasma target to ≥40 µg/mL.

• Aminoglycosides are most effective when the plasma peak concentration:MIC ratio is between 8:1 and 10:1.
• An MIC ≤ 2 µg/mL for gentamicin, are considered susceptible.
• A plasma concentration of 16–20 µg/mL (8–10 × MIC) for gentamicin should therefore be sufficient, but targeting plasma concentrations of 36–40 µg/mL could provide additional effects against strains, with an MIC of >2–4 µg/mL.
• Current trough recommendations from human medicine that have been adopted for horses are plasma concentrations <2 µg/mL. This should be maintained for 4 hours before administering the next dose

Question 35

Acute phase protein concentrations following serial procaine penicillin G injections in horses

Answer 35

5 days BID IM PPG
• Increased CK days 1-6
• AST days 2-7 and 10
• fibrinogen days 6-8 and 10
• SAA day 6.
• No change in HAP

• SAA concentrations showed dramatic increases, reaching up to 317 times baseline in some horses. Significant individual variability in SAA response was noted, with no clinical differences observed between horses that developed an APR and those that did not. SAA peaked later than expected (Day 6), potentially due to cumulative effects from repeated injections. Was only cumulatively > baseline on this day, but was significantly greater in some individuals
• Fibrinogen (FIB): FIB concentrations increased less dramatically than SAA, consistent with its classification as a moderate APP. Peak FIB levels coincided with SAA peaks, despite expectations of an earlier SAA peak.

• Serial IM PPG injections may confound the interpretation of APP concentrations in clinical settings, potentially leading to a misdiagnosis of ineffective treatment if APP levels are elevated.

Question 36

Adverse effects of polymyxin B administration to healthy horses

Answer 36

PolyB administration caused mild to moderate, transient ataxia in healthy horses.
• Ataxia severity correlated with the number of PolyB doses and co-administration of gentamicin.
• Neurotoxicosis included signs such as muscular weakness and proprioceptive ataxia, which are consistent with neuromuscular and sensory system abnormalities.

• Ataxia occurred in all horses during PolyB administration, often peaking within 3–5 days of treatment initiation. Symptoms resolved within a few days after cessation of treatment
• Co-administration of gentamicin exacerbated ataxia severity, potentially through combined neurotoxic mechanisms. Gentamicin’s presynaptic inhibition of acetylcholine release may amplify PolyB’s neurotoxic effects.
• Acute kidney injury was not observed in any horses despite known nephrotoxic risks associated with PolyB in humans. Three horses receiving PolyB with gentamicin showed elevated urinary GGT/creatinine ratios, indicating mild tubular cell damage. Synergistic nephrotoxicity between PolyB and gentamicin is likely, given their accumulation in renal tubular cells.

Neurotoxicity mechanisms include:
• Neuromuscular blockade through reduced acetylcholine sensitivity.
• Mitochondrial dysfunction causing oxidative stress and cellular damage.
• Binding to lipid-rich tissues, although CSF PolyB concentrations were low, suggesting minimal central nervous system penetration.

PolyB is a crtically important drug for human MDR. This study was healthy horses only.

Question 37

Pharmacokinetics of amikacin after intravenous, intra-articular, and combined intravenous and intra-articular administration in healthy neonatal foals

Answer 37

• Intravenous (IV) administration of amikacin at 25 mg/kg once daily achieves therapeutic plasma concentrations (≥53 µg/mL at 30 minutes) in foals aged 7–23 days. The mean was 54 for 7do, some had <53
• Combined IV and intra-articular (IA) administration met therapeutic plasma levels in older foals (14–23 days) but not in younger foals (7–9 days).
• Intra-articular-only administration at 8.3 mg/kg failed to achieve therapeutic plasma concentrations for systemic infections in all age groups.

Pharmacokinetic Observations:
• Younger foals (7–9 days old) exhibited lower peak plasma concentrations compared to older foals due to their larger volume of distribution.
• Plasma amikacin concentrations after IA administration peaked later and were lower than those achieved with IV administration.
• Bioavailability of amikacin from the IA route was nearly 100%, confirming rapid systemic absorption.
• Plasma trough concentrations were below the limit of quantification (2.5 µg/mL) at 24 hours for all protocols, indicating minimal risk of nephrotoxicity.

Implications for Clinical Use:
•IV-Only Protocol: Effective for older foals but may require increased doses for some younger foals to achieve therapeutic plasma concentrations.
• Combined IV + IA Protocol: Effective for localized and systemic infections in older foals but less so for younger foals due to delayed systemic absorption of the IA dose. Concurrent IV and IA administration provides therapeutic synovial fluid concentrations.
• IA-Only Protocol: Provides effective synovial fluid concentrations but inadequate systemic coverage, limiting its use to localized infections. Should not be used as a standalone treatment for septicemia.

Question 38

Gentamicin-induced sensorineural auditory loss in healthy adult horses

Answer 38

• Gentamicin at the standard intravenous (IV) dose of 6.6 mg/kg for 7 days caused sensorineural auditory loss in 70% (7/10) of the healthy horses.
• Auditory loss was partial in 4 horses and complete in 3 (unilateral in 2, bilateral in 1).
• Four of the affected horses recovered auditory function within 30 days post-treatment, while three cases remained partially or completely deaf.
• Vestibular toxicity was not observed in any of the horses, and nephrotoxicity, as measured by serum creatinine, was within normal limits.

Mechanisms and Pathophysiology:
• Sensorineural auditory loss was attributed to damage to hair cells in the cochlea.
• Evidence of sensorineural rather than conductive auditory loss was supported by absent responses in bone conduction brainstem auditory evoked responses (BAER).
• Cochlear damage is consistent with known aminoglycoside-induced ototoxicity mechanisms involving oxidative stress and apoptosis.
• No significant correlation was found between peak gentamicin concentrations (>28 µg/mL) and the development or recovery of auditory loss.

• High-dose gentamicin protocols, as proposed for achieving higher plasma concentrations for bacterial pathogens, may increase the risk of ototoxicity and require further evaluation.

Question 39

Pharmacokinetic and pharmacodynamic effects of 2 registered omeprazole preparations and varying dose rates in horses

Answer 39

• Both the reference omeprazole paste (REF) and the novel in-feed granule preparation (NOV) effectively increased gastric pH and reduced gastric squamous ulcer scores.
• Plasma omeprazole concentrations were higher for NOV due to the larger administered dose, but pharmacodynamic (PD) effects, including gastric pH changes, were comparable between the two products.
• No accumulation of omeprazole was observed after repeated administration of either product, confirming its rapid elimination and lack of dose-dependent retention.

• NOV demonstrated greater bioavailability compared to REF, attributed to its larger dose and possibly its formulation.
• Feeding during NOV administration did not reduce absorption, contrary to previous concerns about food interference with bioavailability.
• Both products maintained gastric pH above 4 for a significant portion of the day, essential for ulcer healing.
• Repeated administration over six days resulted in lower gastric squamous ulcer scores for both products, with similar efficacy despite dose differences.
• The lack of a dose-response relationship for NOV suggests that the maximum pharmacodynamic effect was achieved at the tested doses.

Pharmacokinetics (PK):
• Omeprazole was rapidly absorbed, with Tmax occurring within 60 minutes for both products.
• Elimination was rapid (mean half-life ~60 minutes), and there was no evidence of drug accumulation after repeated dosing.
• The enteric coating in both formulations successfully protected the drug from degradation in the stomach, ensuring effective absorption.

Clinical Implications:
• NOV offers a practical alternative to REF, particularly for horses that are refractory to oral paste administration.
• Both formulations are suitable for once-daily administration, providing effective gastric ulcer management without the risk of drug accumulation.
• Administration of NOV with small amounts of feed is feasible and does not compromise efficacy.

Question 40

Evaluation of the effects of mediumterm (57day) omeprazole administration and discontinuation on serum gastrin and chromogranin A concentrations in horses

Answer 40

• Omeprazole treatment for 57 days significantly increased serum gastrin concentrations (2.5-fold within 7 days of initiation).
• Gastrin concentrations returned to baseline within 2–4 days after discontinuation.
• Serum chromogranin A (CgA) concentrations did not show a significant change during treatment or after discontinuation.
• This suggests minimal trophic effects on enterochromaffin-like (ECL) cells during the study period.
• This could indicate a lower predisposition to rebound gastric hyperacidity (RGH) compared to humans.

• Findings do not support the need for tapering omeprazole treatment after medium-term administration in horses.
• Management strategies post-discontinuation should focus on reducing ESGD risk through dietary modifications and reduced exercise stress.
• Rapid normalization of serum gastrin concentrations supports the safety of discontinuing omeprazole abruptly, particularly after medium-term (57d) use.

Comparison with Previous Studies:
• Findings are consistent with studies showing rapid gastrin normalization in humans post-PPI discontinuation, but the shorter duration in horses is notable.
• Previous equine studies demonstrated a doubling of serum gastrin within 14 days of omeprazole use, aligning with the 7-day increase observed here.
• Unlike findings in humans and other species, horses may have a diminished response in ECL cell activity during PPI therapy.

Question 41

Use of antimicrobials licensed for systemic administration in UK equine practice

Answer 41

• Systemic antimicrobials were prescribed to 19.5% of equids in the study population.
• Category B antimicrobials (highest-priority critically important antimicrobials, HPCIA) were used in 1.9% of equids and 8.9% of antimicrobial courses.
• Potentiated sulphonamides were the most commonly prescribed class, followed by tetracyclines and natural penicillins.
• Despite stewardship guidelines, HPCIA use was common, with 71.7% of these courses prescribed as first-line therapy.
• Bacteriological culture was conducted in only 19.1% of HPCIA courses, highlighting a gap in evidence-based prescribing.

Indications for AMU:
• Integumentary disorders (40.5%) were the most frequent indication for systemic AMU, followed by prophylactic use (29.0%) and musculoskeletal conditions (22.8%).
• HPCIA prescriptions were most often associated with urogenital disorders (31.1%), integumentary disorders (25.2%), and respiratory conditions (15.9%).

Drivers of AMU Variability:
• Significant variability in AMU was observed across practices, likely influenced by differences in caseload complexity, clinician habits, and practice guidelines.
• Equids in their first year of life had higher odds of systemic and HPCIA AMU, reflecting greater susceptibility to infections.

Question 42

Antimicrobial prescribing and resistance surveillance in equine practice

Answer 42

• The study shows a reduction in the use of antimicrobials compared to previous reports, particularly high-priority critically important antimicrobials (HPCIAs).
• Potentiated sulphonamides were the most frequently reported antimicrobials, used by 86% of respondents.
• Despite reduced use, HPCIAs like ceftiofur (7.4%) and enrofloxacin (1.9%) were still reported as commonly used antimicrobials.
• The most common indication for HPCIA use was neonatal sepsis, particularly in foals.

• Over 40% of respondents encountered multi-drug resistant (MDR) infections in the last 12 months, emphasizing the prevalence of antimicrobial resistance (AMR) in equine practice.
• Resistance was commonly seen in surgical site infections and infections involving wounds, uterine infections, and respiratory systems.

Adherence to Guidelines:
• There was a notable improvement in antimicrobial stewardship practices compared to past studies, with 54.4% of respondents reporting the presence of antimicrobial use policies in their practice.
• The BEVA Protect Me toolkit was frequently cited as a guiding resource for antimicrobial use.
Inconsistent Testing:
• Bacteriology culture and sensitivity testing were underutilized, with many prescribing antimicrobials empirically.
• Only 47.3% of respondents reported always or frequently performing bacterial culture when infections were suspected.
Surgical Prophylaxis:
• Many respondents used prophylactic antimicrobials in clean surgeries despite evidence suggesting they are unnecessary in most cases.
• Extended durations of post-operative prophylactic antimicrobial therapy were common, often exceeding recommended guidelines.

Question 43

Antibiotic usage in 14 equine practices over a 10-year period (2012-2021)

Answer 43

Analysis of antibiotic usage over a 10-year period showed varied trends in both mass-based metrics and defined daily dose (DDD) metrics.
• Potentiated sulphonamides remained the most frequently used antibiotic class across the study period, making up the majority of antibiotic usage by weight.

Comparison of Metrics:
• Mass-based metrics (e.g., mg/kg) were highlighted as crude and potentially misleading due to differences in dose rates among antibiotics.
• DDD metrics (e.g., DDDvet/animal/year and DDD/1000) provided a more standardized approach, addressing some limitations of mass-based methods.

High-Priority Critically Important Antibiotics (HPCIAs):
• Usage of HPCIAs, including classes like cephalosporins, macrolides, and fluoroquinolones, declined significantly over the study period.
• A shift in practice toward reduced HPCIA reliance aligns with stewardship goals.

Recommendations:
• Adoption of standardized metrics, such as DDDvet, across the equine industry is suggested for consistent monitoring and comparison.
• Enhanced accuracy in reporting systems and uniform methodologies could improve data reliability.
• Continued emphasis on antimicrobial stewardship, including reducing unnecessary HPCIA usage.

Question 44

Antimicrobial resistance in clinical bacterial isolates from horses in the UK

Answer 44

• Multidrug resistance (MDR) was particularly high in isolates from surgical site infections (SSIs), catheter-related infections (CRIs), and orthopedic infections.
• Enterococcus spp. showed no readily available treatment options for 30.2% of isolates, posing significant therapeutic challenges.
• Resistance to high-priority critically important antimicrobials (HPCIAs), such as cephalosporins and fluoroquinolones, was observed across various bacterial species, with E. coli and Citrobacter spp. exhibiting resistance rates of >20% for fluoroquinolones.

• MDR and resistance rates varied significantly across sample sites, with SSIs and orthopedic infections consistently showing higher resistance levels than respiratory or urogenital samples.
• Pathogens such as E. coli, Staphylococcus spp., and Acinetobacter spp. were common in SSIs and exhibited elevated resistance rates.
• Referral practices reported higher proportions of resistant isolates compared to first-opinion practices, potentially due to prior antimicrobial exposure in referred cases.
• Staphylococcus spp., showed greater resistance in referral settings, highlighting the need for targeted stewardship in these environments.

Therapeutic Challenges:
• High resistance rates among isolates highlight the need for judicious antimicrobial use and reliance on culture and sensitivity testing to guide therapy.
• The lack of effective treatment options for Enterococcus spp. and other MDR pathogens underscores the critical role of surveillance in identifying emerging resistance threats.

Question 45

Prevalence of antimicrobial-resistant Enterobacteriaceae in equine hospitals

Answer 45

• AMR phenotypes, including AmpC β-lactamase, extended-spectrum β-lactamase (ESBL), and fluoroquinolone resistance, were significantly more prevalent after 48 hours of hospitalization.
• High prevalence of resistant Enterobacteriaceae bacteria in hospitalized horses, with variations between hospital settings (OSUGEC and KYEH).
• Hospital environments, particularly high-traffic areas like general-purpose wards and isolation units, were key reservoirs for AMR bacteria.
• Resistant bacteria were isolated from diverse surfaces, including stalls, floor drains, and feeding areas.

•Nosocomial and Cross-Contamination Risks: Evidence of clonal relationships between isolates from feces and hospital surfaces suggests cross-contamination.
• Horses hospitalized in high-risk areas or treated with broad-spectrum antimicrobials had higher odds of carrying AMR bacteria.

• Frequent use of critically important antimicrobials (e.g., carbapenems, fluoroquinolones) in hospital settings contributes to selection pressure.
• Differences in biosecurity and cleaning protocols between hospitals influenced AMR prevalence and dissemination.

Hospitalization Risks:
• Extended hospitalization increases AMR carriage risks, highlighting the need to minimize hospitalization duration where possible.
• Horses admitted with prior antimicrobial exposure were more likely to harbor resistant bacteria.
• Hospital surfaces can act as reservoirs and vectors for resistant bacteria, emphasizing the need for rigorous cleaning and disinfection practices.
• Biosecurity measures, including isolation and cohorting of high-risk patients, are critical to reduce nosocomial infection risks.

Question 46

Determination of the pharmacokinetic-pharmacodynamic cut-off values of marbofloxacin in horses to support the establishment of a clinical breakpoint for antimicrobial susceptibility testing

Answer 46

PK/PD Cut-Off Values:
• A PK/PD cut-off (PK/PDCO) value of 0.0625 mg/L for Gram-negative pathogens and 0.125 mg/L for Gram-positive pathogens was determined for marbofloxacin in horses.
• These values represent the maximum MICs for which 90% of the horse population can achieve the target fAUC/MIC ratio of 72 hours (critical for antimicrobial efficacy).
Variability in Drug Disposition:
• The study revealed significant variability in marbofloxacin disposition among horses, with a between-subject variability (BSV) of 42% for plasma clearance.
• Diseased horses exhibited a 23% reduction in plasma clearance compared to healthy horses, increasing drug exposure.
MIC Distributions:
• MIC90 for E. coli was 4 mg/L, while MIC90 for Streptococcus equi was 2 mg/L, indicating differences in susceptibility among pathogens.
• PK/PDCO values suggest that marbofloxacin may be effective against Enterobacteriaceae but less so against Streptococcus equi.

Influence of Dose and Pathogen Type:
• Higher doses (10 mg/kg) resulted in proportionally greater drug exposure compared to the standard 2 mg/kg dose.
• The efficacy against Gram-negative bacteria is better supported by the PK/PD data, particularly at the lower PK/PDCO value of 0.0312 mg/L for a more conservative target of 125 hours.

Clinical Implications:
• Marbofloxacin shows promise for treating infections caused by Gram-negative pathogens (E. coli), particularly when MIC values are low (<0.0625 mg/L).
•Its utility against Gram-positive pathogens (Streptococcus equi) is limited by higher MICs, necessitating alternative treatments for these cases.
• A dose of 2 mg/kg achieves effective concentrations for most Gram-negative pathogens but may require adjustment for severe infections or resistant strains.

Question 47

Audit of antimicrobial use in eleven equine practices over a five-year period (2014-2018)

Answer 47

• Over the five-year period (2014–2018), the total antimicrobial use in the 11 equine practices decreased by approximately 50%.
• Notable reductions were observed for high-priority critically important antimicrobials (HPCIAs):
-Enrofloxacin use reduced by 38%.
-Ceftiofur use reduced by 95%.
-Cefquinome use was completely eliminated (100% reduction).
• There was an increase in the proportionate use of trimethoprim-sulphadiazine and doxycycline, while the use of cefquinome and other HPCIAs significantly declined.
• Procaine penicillin remained stable, comprising 5–6% of annual total antimicrobial use.

Contributing Factors to Reduction:
• Increased awareness of antimicrobial resistance (AMR) through educational campaigns such as the British Equine Veterinary Association’s (BEVA) Protect Me guidelines.
• Regulatory pressures and recommendations limiting HPCIA usage in veterinary practice.
• Intermittent availability of certain antimicrobials (e.g., ceftiofur and cefquinome) during the study period.

Question 48

Cannabinoid receptors are expressed in equine synovium and upregulated with synovitis

Answer 48

• CB1 and CB2 receptors were expressed in the synovial intimal layer of over 95% of healthy and osteoarthritic equine joints.
• The receptors were also identified in the subintimal blood vessels, indicating their involvement in joint physiology.
• Both CB1 and CB2 receptor expressions were significantly upregulated in synovial tissues with higher histologic synovitis grades.
• CB1 receptor expression in the synovial intima decreased with increasing severity of macroscopic cartilage degradation.

Potential Role in Pain and Inflammation:
• The ECS, particularly CB1 and CB2 receptors, may regulate inflammatory and immune responses in equine joints.
• Their role in reducing proinflammatory cytokine production and matrix degradation highlights their therapeutic potential.

Question 49

Residual effects of intra-articular betamethasone and triamcinolone acetonide in an equine acute synovitis model

Answer 49

• Triamcinolone acetonide exhibited sustained anti-inflammatory effects 14 days post-administration, reducing IL-6 and PTGS1 gene expression, lameness scores, serum amyloid A (SAA), and cortisol levels.
• Betamethasone showed more transient effects, with significant reductions in IL-6 and PTGS1 but over a shorter duration.
• Both corticosteroids suppressed cortisol levels, with triamcinolone causing a longer and more pronounced effect compared to betamethasone. This raises concerns about the suppression of the hypothalamic–pituitary–adrenal (HPA) axis, which could limit the horse’s ability to respond to stress or infections.

• Triamcinolone reduced lameness scores significantly more than betamethasone, suggesting superior residual pain-relieving properties.
• Horses treated with triamcinolone were sound by 12 hours post-LPS challenge, while betamethasone-treated horses showed less consistent improvement.
• Serum Amyloid A (SAA): SAA levels were significantly lower in horses treated with triamcinolone, further supporting its prolonged anti-inflammatory action.
• No delay in the SAA response post-LPS injection was noted for either corticosteroid, but residual effects were evident in triamcinolone-treated horses.

• Both corticosteroids impacted IL-6 (pro- and anti-inflammatory) and PTGS1 (cyclooxygenase-1) expression.
• Triamcinolone had a more sustained effect, while betamethasone’s impact was shorter-lived.

Question 50

Antimicrobial prophylaxis is not indicated for horses undergoing general anaesthesia for elective orthopaedic MRI

Answer 50

• The study identified a 5.6% incidence of post-anaesthetic fever in horses undergoing elective orthopaedic MRI under general anaesthesia. Most fevers (80%) were of unknown origin and self-resolved without evidence of infection.
• prophylactic antimicrobial use was associated with a 3.8 times higher odds of developing post-anaesthetic fever (p ≤ 0.001).
• Younger horses (1–4 years) were nearly three times more likely to develop fever compared to older horses (≥5 years).

• While pneumonia was diagnosed in 18% of febrile horses, 43% of fevers resolved with NSAIDs, and 26% resolved spontaneously without treatment.
• The findings suggest that many fevers were non-infectious and may have been due to transient inflammatory or thermoregulatory responses.

• The findings discourage routine prophylactic antimicrobial use for elective orthopaedic MRI cases, given the low infection risk and potential for adverse outcomes, including antimicrobial resistance.
• Early fever management should focus on monitoring and NSAIDs for symptomatic relief rather than immediate antimicrobial intervention.

Question 51

Rational dosage regimens for cephalothin and cefazolin using pharmacokinetics and pharmacodynamics analysis in healthy horses

Answer 51

• CET exhibited a higher clearance rate and shorter half-life compared to CEZ, indicating faster elimination.
• Both drugs demonstrated low serum protein binding in horses (CET: 19.9%, CEZ: 15.2%), resulting in a greater proportion of free drug available for antibacterial activity.
• MIC90 for CET and CEZ against Streptococcus zooepidemicus was 0.12 mg/L, and against Staphylococcus aureus was 0.5 mg/L.
• MICs for gram-negative bacteria (e.g., E. coli and Klebsiella spp.) were significantly higher (>4.0 mg/L for CET, ≤2.0 mg/L for CEZ).

Dosing Recommendations:
CET:
• 22 mg/kg q8h for S. zooepidemicus infections.
• 22 mg/kg q4h or continuous infusion for S. aureus to achieve adequate exposure, as q4h administration is impractical for clinical settings.
CEZ:
• 10 mg/kg q12h for S. zooepidemicus.
• 10 mg/kg q8h for S. aureus.

Monte Carlo Simulations:
• Simulations predicted a high probability of target attainment (PTA ≥90%) for the above regimens, ensuring effective bacterial control.
• Simulations highlighted the need for more frequent dosing to combat gram-negative infections, which may lead to impractical regimens.

Question 52

Evaluation of the distribution of a mixture of mepivacaine and iopromide via local infiltration in equine cadaver skulls

Answer 52

Infraorbital nerve block using 10 mL was sufficient and minimized risks of excessive leakage (seen at higher volumes, into the maxillary sinus).

Question 53

Comparison of post-operative inflammatory response in horses undergoing elective castration treated preoperatively with ceftiofur crystalline free acid or procaine penicillin G

Answer 53

• There were no significant differences in quantitative or qualitative post-operative inflammatory markers (e.g., serum amyloid A (SAA), total nucleated cell count (TNCC), fibrinogen (FIB), and total protein (TP)) between the ceftiofur crystalline free acid (CCFA) and procaine penicillin G (PPG) groups.
• The slight difference in TNCC levels at Day 14 (lower in the CCFA group) was clinically insignificant.
• A higher rate of short-term complications was observed in the CCFA group (48%) compared to the PPG group (31%). However, all complications (e.g., incisional drainage, ventral edema, and subcutaneous tissue prolapse) were mild, self-limiting, and resolved by Day 8.
• SAA was identified as a more sensitive marker for detecting inflammation compared to TNCC, FIB, or TP.
• Elevated SAA levels correlated with increased preputial edema scores, highlighting its diagnostic value in assessing post-operative inflammation.

Question 54

Oral prednisolone achieves measurable concentrations in equine synovial fluid within 3 hours of administration: Preliminary observations

Answer 54

• Orally administered prednisolone was detected in the synovial fluid of both the radiocarpal and tibiotarsal joints within 3 hours of administration.
• Mean concentrations were 28.44 ng/mL (radiocarpal joint) and 20.33 ng/mL (tibiotarsal joint), with variability among horses.
• The detected concentrations were within ranges previously considered therapeutically effective for other corticosteroids in equine joints.
• This finding suggests oral prednisolone could be a viable alternative to intra-articular corticosteroid injections for managing joint inflammation in horses.
• Variability in prednisolone levels across individuals was consistent with its moderate bioavailability (50%-60%) and influenced by oral administration.
• no comment on clinical efficacy

Question 55

Clodronate detection and effects on markers of bone resorption are prolonged following a single administration to horses

Answer 55

• Clodronate persisted in blood for 14–175 days and in urine for up to 175 days post-administration, indicating a prolonged detection window.
• Detection times varied significantly between individual horses, influenced by factors such as bone turnover rates and exercise intensity.
• Clodronate binds strongly to hydroxyapatite in bone, serving as a reservoir that releases the drug over extended periods.
• The release of clodronate was influenced by osteoclastic activity, with exercise enhancing its liberation from bone.

Pharmacokinetic Variability:
• Terminal half-life ranged from 43.1 to 6814.8 hours (approximately 1.8–283.9 days), showcasing interindividual variability.
• High variability underscores the need for careful interpretation of pharmacokinetic data in therapeutic and regulatory contexts.
Biomarkers of Bone Resorption:
• CTX-1 (Carboxy-terminal Cross-linking Telopeptide of Type 1 Collagen): s increased in both treated and control groups, suggesting potential influence from exercise or biological variability.
• TRAcP5B (Type 5 Acid Phosphatase): TRAcP5B levels decreased significantly in the clodronate group starting on Day 35 and remained suppressed throughout the study. The reduction indicates decreased osteoclastic activity and aligns with clodronate’s mechanism of inhibiting osteoclast function.
• Prolonged suppression of TRAcP5B suggests sustained osteoclast inhibition, which could impair bone remodeling and healing in horses undergoing intensive training.
• The potential for prolonged analgesic effects, inferred from reductions in TRAcP5B, highlights its utility in managing chronic pain but warrants caution regarding misuse.