K. Lunsford

Oral cyclosporine treatment in dogs: a review of the literature.

Cyclosporine is an immunomodulatory drug used to treat an increasing spectrum of diseases in dogs. Cyclosporine is a calcineurin inhibitor, ultimately exerting its inhibitory effects on T‐lymphocytes by decreasing production of cytokines, such as interleukin‐2. Although, in the United States, oral cyclosporine is approved in dogs only for treatment of atopic dermatitis, there are many other indications for its use. Cyclosporine is available in 2 oral formulations: the original oil‐based formulation and the more commonly used ultramicronized emulsion that facilitates oral absorption. Ultramicronized cyclosporine is available as an approved animal product, and human proprietary and generic preparations are also available. Bioavailability of the different formulations in dogs is likely to vary among the preparations. Cyclosporine is associated with a large number of drug interactions that can also influence blood cyclosporine concentrations. Therapeutic drug monitoring (TDM) can be used to assist in attaining consistent plasma cyclosporine concentrations despite the effects of varying bioavailability and drug interactions. TDM can facilitate therapeutic success by guiding dose adjustments on an individualized basis, and is recommended in cases that do not respond to initial oral dosing, or during treatment of severe, life‐threatening diseases for which a trial‐and‐error approach to dose adjustment is too risky. Pharmacodynamic assays that evaluate individual patient immune responses to cyclosporine can be used to augment information provided by TDM.

Cyclosporine A affects the in vitro expression of T cell activation-related molecules and cytokines in dogs.

Cyclosporine is a powerful immunosuppressive drug that is being used with increasing frequency to treat a wide range of immune-mediated diseases in the dog. To date, ideal dosing protocols that will achieve immunosuppression with cyclosporine in dogs remain unclear, and standard methods that can measure effectiveness of immunosuppression have not been established. The aim of our study was to evaluate the effects of in vitro cyclosporine exposure on a panel of molecules expressed by activated T cells to ascertain their potential as biomarkers of immunosuppression in dogs. Blood was drawn from six healthy dogs, and peripheral blood mononuclear cells (PBMC) were isolated and activated. Half of the cells were incubated with 200 ng/mL cyclosporine prior to activation, and the other half were not exposed to cyclosporine. Samples were analyzed using flow cytometry, and the expression of intracellular cytokines IL-2, IL-4, and IFN-γ was evaluated after 6, 12, and 24h of drug exposure. Each cytokine exhibited a time-dependent suppression profile, and all but two samples activated in the presence of cyclosporine showed lower cytokine expression than untreated controls. We also evaluated the expression of the surface T cell activation molecules CD25 and CD95 by flow cytometry after 36 h of drug exposure. Expression of these surface molecules decreased significantly when activated in the presence of cyclosporine. Our results suggest that suppressed expression of the markers related to T cell activation could potentially be utilized as an indicator of the efficacy of cyclosporine therapy in dogs.

Pharmacodynamic monitoring of canine T-cell cytokine responses to oral cyclosporine.

BACKGROUND Pharmacodynamic assays measure the immunosuppressive effects of cyclosporine on T-cells and offer an alternative assessment of efficacy in individual patients. OBJECTIVE To assess the immunosuppressive effects of high and low dosage cyclosporine on canine T-cells and to develop a novel testing system for individualized dose adjustment. ANIMALS Seven healthy female Walker hounds. METHODS Experimental study using a paired comparison design. Flow cytometry was used to measure T-cell expression of IL-2, IL-4, and IFN-γ. Cytokine expression 8 days after oral administration of high and low dosages of cyclosporine was compared to baseline and washout values, respectively. The high dosage was initially 10 mg/kg q12h and was then adjusted to attain established immunosuppressive trough blood drug concentrations (>600 ng/mL). The low dosage was 5 mg/kg q24h. RESULTS High dosage cyclosporine resulted in significant decreases in IL-2 and IFN-γ expression (P = .0156, P = .0156), but not IL-4 expression (P = .2188). Low dosage cyclosporine was associated with a significant decrease in IFN-γ expression (P = .0156), while IL-2 expression was not affected (P = .1094). CONCLUSIONS AND CLINICAL IMPORTANCE T-cell function is suppressed at trough blood drug concentrations exceeding 600 ng/mL, and is at least partially suppressed in some dogs at low dosages. Direct evaluation of T-cell function could be an effective, more sensitive alternative to measuring blood drug concentrations for monitoring immunosuppressive therapy.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs

OBJECTIVE To determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. STUDY DESIGN Randomized, blocked, crossover design with a 14-day washout period. ANIMALS Healthy intact female Walker Hounds aged 1-6 years and weighing 20.5-24.2 kg. METHODS Dogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg(-1), PO, every 12 hours), carprofen (4.4 mg kg(-1), PO, every 24 hours), meloxicam (0.2 mg kg(-1), PO, on the 1st day then 0.1 mg kg(-1), PO, every 24 hours), and deracoxib (2 mg kg(-1), PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B(2) was also measured before and during administration of each drug. RESULTS All NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg(-1) every 12 hours), carprofen (4.4 mg kg(-1) every 24 hours), meloxicam and deracoxib, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Oral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding.

Pharmacokinetics of subcutaneous low molecular weight heparin (enoxaparin) in dogs.

Unfractionated heparin has been the standard heparin used in human and veterinary medicine for its anticoagulation effect; however, it has a complex pharmacodynamic profile that requires close monitoring. Low molecular weight heparins have a more predictable bioavailability, allowing standardized dosing without individual patient monitoring. This project was designed to a) evaluate the pharmacokinetics of the subcutaneous (SC) administration of the low molecular weight heparin, enoxaparin, in dogs using anti-Xa activity as a marker of plasma enoxaparin concentrations and b) to establish the dose necessary to maintain activity within an established target range. Enoxaparin at 0.8 mg/kg SC q 6 hours consistently maintained target levels of anti-Xa activity in normal dogs without evidence of hemorrhagic complications.

Platelet Cyclooxygenase Expression in Normal Dogs

BACKGROUND Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. HYPOTHESIS/OBJECTIVES The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. ANIMALS Eight female, intact hounds. METHODS A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. RESULTS Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. CONCLUSIONS AND CLINICAL IMPORTANCE Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness.

In vitro and in vivo assessment of platelet function in healthy dogs during administration of a low-dose aspirin regimen

OBJECTIVE To assess the in vitro and in vivo platelet function of healthy dogs during administration of a low-dose aspirin regimen. ANIMALS 16 dogs. PROCEDURES Dogs received aspirin (1 mg/kg, PO, q 24 h) for 7 days. Blood and urine samples were collected before (day 1; baseline) and on days 3 and 7 of the low-dose aspirin regimen. Platelet function was evaluated by use of turbidimetric and conventional impedance aggregometry, multiple-electrode impedance aggregometry, a platelet function analyzer (PFA), and determination of urine 11-dehydro-thromboxane B2 concentration. Turbidimetric aggregometry results were compared with the results obtained by the other 4 methods. Fourteen days after cessation of aspirin, platelet-rich plasma was incubated with acetylsalicylic acid and platelet function was assessed by turbidimetric aggregometry to determine whether this technique could accurately identify dogs that responded to the low-dose aspirin regimen. RESULTS Of the 16 dogs, 13 had turbidimetric and conventional impedance aggregometry results that were decreased by > 25% from baseline on days 3 and 7, and 4 and 7 dogs had PFA closure times > 300 seconds on days 3 and 7, respectively. The median urine 11-dehydro-thromboxane B2 concentration-to-creatinine concentration ratio decreased by 49% between days 1 and 7. Turbidimetric aggregometry results were correlated with conventional impedance aggregometry results. There was poor agreement between the turbidimetric aggregometry and PFA results. The multiple-electrode impedance aggregometry protocol failed to reliably detect aspirin-induced platelet dysfunction. In vitro incubation of platelet-rich plasma with acetylsalicylic acid followed by turbidimetric aggregometry did not predict whether dogs responded to the low-dose aspirin regimen. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the response to a low-dose aspirin regimen varied among healthy dogs.

Gastroesophageal reflux and laryngeal dysfunction in a dog

CASE DESCRIPTION A 7-year-old neutered male Saint Bernard was evaluated because of a 6-month history of coughing, gagging, change in phonation, excessive panting, and chronic intermittent vomiting and diarrhea. CLINICAL FINDINGS Physical examination revealed no remarkable findings other than panting. Total thyroxine concentration and results of a CBC, serum biochemistry analysis, urinalysis, and thoracic radiography were within reference limits. A laryngeal examination revealed edema, erythema, and ulceration of the larynx and pharynx, with normal laryngeal movement. Results of bronchoscopy and cytologic examination of bronchoalveolar lavage fluid were diagnostic only for distal tracheitis. Esophagoscopy and an esophagography revealed esophagitis consistent with gastroesophageal reflux. Gastroduodenoscopy and histologic examination of biopsy specimens revealed Helicobacter colonization and lymphocytic or plasmacytic enteritis. TREATMENT AND OUTCOME Following treatment for gastroesophageal reflux and suspected Helicobacter infection with combination antacid and antimicrobial treatment, the dogs respiratory signs resolved but vomiting continued. Gastroduodenoscopy revealed complete resolution of the previous laryngitis, pharyngitis, and esophagitis. Treatment for the lymphocytic or plasmacytic enteritis was initiated with prednisone (1 mg/kg [0.45 mg/lb], p.o., q 12 h) and a novel protein diet. The previous treatment was also continued. Complete resolution of clinical signs was maintained 4 months after initiation of appropriate treatment. CLINICAL RELEVANCE Laryngeal dysfunction induced by gastroesophageal reflux as occurred in the patient described in this report is a previously undocumented association in the veterinary literature. This association could be a potential consideration in dogs with concurrent respiratory and gastrointestinal signs. The present report may provide a basis for further studies investigating this association.

The effects of cyclosporine on platelet function and cyclooxygenase expression in normal dogs.

BACKGROUND Cyclosporine has been shown to alter platelet plasma membranes and have a hypercoagulable effect in humans, leading to thromboembolic complications. HYPOTHESIS/OBJECTIVES Our hypothesis was that by modulating platelet reactivity, cyclosporine increases the risk of thromboembolic complications. The objective was to determine the effects of cyclosporine on primary hemostasis in normal dogs. ANIMALS Eight healthy, intact female dogs. METHODS A repeated-measures design utilized flow cytometry to evaluate platelet expression of platelet reactivity markers (P-selectin and phosphatidylserine) and COX-1 and COX-2 during the administration of 2 cyclosporine dosages (19 mg/kg q12h [immunosuppressive dosage] and 5 mg/kg q24h [atopy dosage]). Urine 11-dehydro-thromboxane-B(2) (11-dTXB(2) ) concentration was normalized to urine creatinine concentration, and platelet function was analyzed by PFA-100. RESULTS After a week of the immunosuppressive dosage, all platelet reactivity markers showed a significant decrease in mean fluorescent intensity (MFI). After the atopy dosage, only P-selectin and COX-2 MFI demonstrated a change from baseline, decreasing by 29% (P = .013) and 31% (P = .003), respectively. Urinary 11-dTXB(2) -to-creatinine ratio significantly increased at all time points during the immunosuppressive dosage, but no significant change occurred during administration of the atopy dosage. PFA-100 closure times using collagen/ADP cartridges increased by 62% (P = .008) with the immunosuppressive dosage and decreased by 45% with the atopy dosage (P = .035). No significant changes in closure times occurred with collagen/epinephrine cartridges. CONCLUSIONS AND CLINICAL IMPORTANCE Our study suggests that, similar to what is observed in humans, cyclosporine alters the platelet plasma membrane and increases thromboxane production in dogs, especially at immunosuppressive dosages.

Ultra-pure platelet isolation from canine whole blood

BackgroundSeveral research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs.ResultsUsing an optimized OptiPrep density gradient technique, platelet recovery was 51.56% with 99.99% platelet purity and leukocyte contamination of 100 leukocytes per 108 platelets, on average. Platelet samples were subjected to additional purification with CD45-labeled Dynabeads after density barrier centrifugation resulting in a 95-fold depletion of residual leukocytes. Platelets purified using these methods remained inactivated as assessed by Annexin V and P-selectin labeling with flow cytometry.ConclusionsThe use of OptiPrep density gradient is a quick method for obtaining highly purified platelet samples from low volumes of canine whole blood with minimal contamination. Additional depletion of residual leukocytes can be achieved using CD45-labeled beads. These platelet samples can then be used for many downstream applications that require ultra-pure platelet samples such as RNA and protein analysis.