Ashley M. Stokes

Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses?

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.

Pharmacokinetics of the injectable formulation of methadone hydrochloride administered orally in horses

Methadone hydrochloride is a synthetic mu-opioid receptor agonist with potent analgesic properties. Oral methadone has been successfully used in human medicine and may overcome some limitations of other analgesics in equine species for producing analgesia with minimal adverse effects. However, there are no studies describing the pharmacokinetics (PK) of oral opioids in horses. The aim of this study was to describe the PK of orally administered methadone (0.1, 0.2 and 0.4 mg/kg) and physical effects in 12 healthy adult horses. Serum methadone concentrations were measured by gas chromatography/mass spectrometry at predetermined time points for 24 h, and PK parameters were estimated using a noncompartmental model. Physical effects were observed and recorded by experienced clinicians. No drug toxicity, behavioural or adverse effects were observed in the horses. The disposition of methadone followed first order elimination and a biphasic serum profile with rapid absorption and elimination phases. The PK profile of methadone was characterized by high clearance (Cl/F), small volume of distribution (V(d)/F) and short elimination half-life (t(1/2)). The mean of the estimated t(1/2) (SD) for each dose (0.1, 0.2 and 0.4 mg/kg) was 2.2 (35.6), 1.3 (46.1) and 1.5 (40.8), and the mean for the estimated C(max) (SD) was 33.9 (6.7), 127.9 (36.0) and 193.5 (65.8) respectively.

Bioavailability and pharmacokinetics of oral and injectable formulations of methadone after intravenous, oral, and intragastric administration in horses

OBJECTIVE To characterize the bioavailability and pharmacokinetics of oral and injectable formulations of methadone after IV, oral, and intragastric administration in horses. ANIMALS 6 healthy adult horses. PROCEDURES Horses received single doses (each 0.15 mg/kg) of an oral formulation of methadone hydrochloride orally or intragastrically or an injectable formulation of the drug orally, intragastrically, or IV (5 experimental treatments/horse; 2-week washout period between each experimental treatment). A blood sample was collected from each horse before and at predetermined time points over a 360-minute period after each administration of the drug to determine serum drug concentration by use of gas chromatography-mass spectrometry analysis and to estimate pharmacokinetic parameters by use of a noncompartmental model. Horses were monitored for adverse effects. RESULTS In treated horses, serum methadone concentrations were equivalent to or higher than the effective concentration range reported for humans, without induction of adverse effects. Oral pharmacokinetics in horses included a short half-life (approx 1 hour), high total body clearance corrected for bioavailability (5 to 8 mL/min/kg), and small apparent volume of distribution corrected for bioavailability (0.6 to 0.9 L/kg). The bioavailability of methadone administered orally was approximately 3 times that associated with intragastric administration. CONCLUSIONS AND CLINICAL RELEVANCE Absorption of methadone in the small intestine in horses appeared to be limited owing to the low bioavailability after intragastric administration. Better understanding of drug disposition, including absorption, could lead to a more appropriate choice of administration route that would enhance analgesia and minimize adverse effects in horses.

Use of serial laminar tissue collection via biopsy in conscious healthy horses.

OBJECTIVE To determine the feasibility of performing serial laminar and skin biopsies on sedated horses and whether sampling affected adjacent tissues. ANIMALS 6 horses. PROCEDURES Laminar tissues were harvested via biopsy through the hoof wall from healthy conscious horses via sedation and regional anesthesia. Eight specimens were collected at 4 time points during 24 hours from a single foot. Laminar biopsy specimens were harvested with a 6-mm-diameter biopsy punch after burring through the horny corium to the stratum medium. Skin biopsy specimens were collected from an area proximal to the coronary band. All tissues were examined via light microscopy. Total RNA was extracted and quantified, and gene expression analysis was completed for 2 housekeeping genes and the inflammatory mediator cyclooxygenase-2. RESULTS Laminar and skin biopsies yielded adequate specimens for histologic and gene expression evaluation. There was no extension of inflammation or detectable damage to adjacent tissues during the 24-hour period in either laminar or skin specimens as judged via histologic findings and cyclooxygenase-2 expression. Lameness and discomfort induced by the procedure were minimal. CONCLUSIONS AND CLINICAL RELEVANCE Laminar biopsy provided a satisfactory method of collecting laminar specimens and allowed serial sampling of individual horses.

Evaluation of a wireless ambulatory capsule (SmartPill®) to measure gastrointestinal tract pH, luminal pressure and temperature, and transit time in ponies

REASONS FOR PERFORMING STUDY This study investigated the use of a wireless ambulatory capsule (WAC; SmartPill(®) pH.p GI Monitoring System) to determine WAC-gastric emptying time (GET) in ponies. OBJECTIVES To measure WAC-GET and compare it to those findings with GET assessed by nuclear scintigraphy (S-GET). HYPOTHESIS WAC-GET will be slower than S-GET, but will be significantly correlated. METHODS Seven healthy adult mixed-breed pony mares were used in this study. Feed was withheld for 12 h prior to the WAC administration. After administration, a complete-feed diet was fed to allow the WAC to pass into the stomach. Luminal pH, temperature and pressure were collected by a modified receiver secured to the pony. Once the pH reached a value of ≥ 8.0, it was determined that gastric emptying had occurred, and ponies were fed grass hay. After 5 days, data were downloaded and analysed using proprietary software. During the second period of the study, after at least 2 weeks, 4 of the ponies underwent a standard S-GET test. RESULTS The WAC was successfully administered, and data were collected from all ponies. The mean percentage of data packets collected by the receiver was 84.9 ± 3.51% (range 66.8-95.1%). Mean WAC-GET was 7.38 h (range 0.15-46.65 h). Mean gastric pH was 4.75 (range 2.07-6.99). Mean small intestinal transit time was 4.6 h. The mean pH for the small intestine was 8.0. The mean S-GET time (in hours) when 10% of the radioactive feed is present in the stomach (T-90%) was 2.3 h. The S-GET did not correlate significantly with the WAC-GET. CONCLUSIONS AND POTENTIAL RELEVANCE The WAC was safely administered to ponies, and data were collected using a modified receiver. The WAC-GET varied considerably between ponies, but was ≤ 3 h in 5 of the 6 ponies. The WAC used in this study provided a noninvasive technique that produced novel information about the pony gastrointestinal tract, but owing to the substantial variability in GET values and long transit time it may not be a reliable clinical tool at this time.

Transcriptional changes associated with recurrent airway obstruction in affected and unaffected horses.

OBJECTIVE To identify differentially expressed genes in pulmonary tissues of horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD), which is a form of recurrent airway obstruction (RAO), compared with those of unaffected horses. ANIMALS 6 horses with SPAOPD-RAO and 6 unaffected (healthy) horses. PROCEDURES Horses were assigned to 2 groups on the basis of medical history, clinical score, and transpleural pressure. Total RNA from each of the 5 lung lobes of each of the 6 SPAOPD-RAO-affected horses was extracted and pooled. Similarly, total RNA from unaffected horses was pooled. Differential display (DD) PCR assay was performed, and differentially expressed bands were purified and cloned into a plasmid vector. Plasmids were extracted from recombinant colonies, and purified DNA was sequenced. Genes of interest for RAO pathogenesis were identified. Real-time PCR assay was performed to confirm findings for the DD PCR assay. RESULTS 18 differentially expressed genes (17 upregulated and 1 downregulated) were identified. Three genes of particular interest were found to be altered (2 upregulated and 1 downregulated) in horses with SPAOPD-RAO by use of real-time PCR assay, and these findings matched the differential expression found by use of the DD PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE SPAOPD-RAO in horses is a multifactorial, complex disease involving several genes. Upregulated genes, particularly beta2-microglobulin, and the downregulated secretoglobin gene can serve as marker genes that may help to identify SPAOPD-RAO at an early age.