Objective-To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples. Samples-Blood samples obtained from a healthy 6-month-old calf. Procedures-Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography–tandem mass spectrometry. Results-Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition. Conclusions and Clinical Relevance-Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Objective-To investigate age-related and regional differences in estimated metabolite concentrations in the brain of healthy dogs by means of magnetic resonance spectroscopy (MRS). Animals-15 healthy Beagles. Procedures-Dogs were grouped according to age as young (n = 5; all dogs were 2 months old), adult (5; mean age, 4.5 years), or geriatric (5; all dogs were 12 years old). Imaging was performed by use of a 1.5-T MRI system with T1- and T2-weighted spin-echo and fluid-attenuated inversion recovery sequences. Signal intensity measurements for N-acetyl aspartate, creatine, choline, and lactate-alanine (the spectroscopic peaks associated with alanine and lactate could not be reliably differentiated) were determined with MRS, and areas under the spectroscopic peaks (representing concentration estimates) were calculated. Ratios of these metabolite values were compared among age groups and among brain regions with regression analysis. Results-The choline-to-creatine ratio was significantly higher in young dogs, compared with other age groups. The N-acetyl aspartate-to-choline ratio was significantly lower in young dogs and geriatric dogs than in adult dogs. When all age groups were considered, the choline-to-creatine ratio was significantly higher and N-acetyl aspartate-to-choline ratio was significantly lower in the frontal lobe than in all other regions. The N-acetyl aspartate-to-creatine ratio was significantly lower in the cerebellum than in other regions. Conclusions and Clinical Relevance-Metabolite ratios varied significantly among age groups and brain regions in healthy dogs. Future studies should evaluate absolute concentration differences in a larger number of dogs and assess clinical applications in dogs with neurologic diseases.

A high rate of mortality, expense, and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. The purpose of this study was to determine, using a flow-cytometric assay, the 50% T-cell inhibitory concentration (IC50) of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) in canine lymphocytes stimulated with concanavalin A (Con A). Whole blood was collected from 5 privately owned, healthy dogs of various ages, genders, and breeds. Peripheral blood mononuclear cells, obtained by density-gradient separation, were cultured for 72 h with Con A, a fluorochrome-tagged cell proliferation dye, and various concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM), and A77 1726 (1, 5, 10, 25, 50, and 200 μM). After incubation, the lymphocytes were labeled with propidium iodide and an antibody against canine CD5, a pan T-cell surface marker. Flow cytometry determined the percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants. The mean (± standard error) IC50 was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Inhibition of T-cell proliferation by the 4 immunosuppressants was demonstrated in a concentration-dependent manner, with variability between the dogs. These results represent the initial steps to tailor this assay for individual immunosuppressant protocols for dogs with immune-mediated disease.

Objective: To evaluate the pharmacokinetics and clinical efficacy of budesonide in dogs with inflammatory bowel disease (IBD). Animals: 11 dogs (mean ± SD age, 5.7 ± 3.9 years; various breeds and body weights) with moderate or severe IBD. Procedures: Each dog received a controlled-release formulation of budesonide (3 mg/m2, PO, q 24 h) for 30 days (first day of administration was day 1). The concentration of budesonide and its metabolite (16-α-hydroxyprednisolone) was measured via liquid chromatography–tandem mass spectrometry in plasma and urine samples obtained on days 1 and 8 of treatment. On those days, plasma samples were obtained before the daily budesonide administration and 0.5, 1, 2, 4, and 7 hours after drug administration, whereas urine samples were obtained after collection of the last blood sample. A clinical evaluation was performed on the dogs before onset of drug administration and on days 20 and 30 after start of drug administration. Results: The highest plasma concentration of budesonide and 16-α-hydroxyprednisolone on day 1 was detected at 1 hour and at 2 hours after drug administration, respectively. After standardization on the basis of specific gravity, the ratio between urinary concentrations of budesonide and 16-α-hydroxyprednisolone was 0.006 and 0.012 on days 1 and 8, respectively. The clinical response was adequate in 8 of 11 dogs. Conclusions and Clinical Relevance: Budesonide was rapidly absorbed and metabolized in dogs with IBD. The drug gradually accumulated, and there was an adequate therapeutic response and no adverse effects.

Objective: To determine plasma pharmacokinetics of penciclovir following oral and rectal administration of famciclovir to young Asian elephants (Elephas maximus). Animals: 6 healthy Asian elephants (5 females and 1 male), 4.5 to 9 years old and weighing 1,646 to 2,438 kg. Procedures: Famciclovir was administered orally or rectally in accordance with an incomplete crossover design. Three treatment groups, each comprising 4 elephants, received single doses of famciclovir (5 mg/kg, PO, or 5 or 15 mg/kg, rectally); there was a minimum 12-week washout period between subsequent famciclovir administrations. Serial blood samples were collected after each administration. Samples were analyzed for famciclovir and penciclovir with a validated liquid chromatography–mass spectroscopy assay. Results: Famciclovir was tolerated well for both routes of administration and underwent complete biotransformation to the active metabolite, penciclovir. Mean maximum plasma concentration of penciclovir was 1.3 μg/mL at 1.1 hours after oral administration of 5 mg/kg. Similar results were detected after rectal administration of 5 mg/kg. Mean maximum plasma concentration was 3.6 μg/mL at 0.66 hours after rectal administration of 15 mg/kg; this concentration was similar to results reported for humans receiving 7 mg/kg orally. Conclusions and Clinical Relevance: Juvenile Asian elephants are susceptible to elephant endotheliotropic herpesvirus. Although most infections are fatal, case reports indicate administration of famciclovir has been associated with survival of 3 elephants. In Asian elephants, a dose of 8 to 15 mg of famciclovir/kg given orally or rectally at least every 8 hours may result in penciclovir concentrations that are considered therapeutic in humans.

Objective-To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. Sample Population-Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. Procedure-The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). Results-All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures. Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF(1 alpha)) were measured in 10 Miniature Horses given 0.25 micrograms of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF(1 alpha) (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF(1 alpha)). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.

The cause of species difference in the susceptibility of erythrocytes to L-sorbose, and the difference in the hemolytic effect of sorbose on high potassium-containing (HK) and low potassium-containing (LK) canine erythrocytes were examined. L-sorbose was phosphorylated in canine erythrocytes, but not in human erythrocytes. Furthermore, sorbose-1-phosphate, a metabolite of L-sorbose, strongly inhibited the hexokinase of LK canine erythrocytes, but not that of HK canine erythrocytes. These results strongly indicated that inhibition of hexokinase by sorbose-1-phosphate in LK erythrocytes induced severe glycolytic limitation in these cells, resulting in hemolysis, and that HK erythrocytes are resistant to sorbose-induced hemolysis because these cells have a high hexokinase activity.

Plasma disposition and urinary recovery of sulfamethazine (SMZ), its N4-acetylated metabolite (N4AcSMZ), and 2 of its hydroxylated metabolites--5-hydroxysulfamethazine 5OHSMZ) and 6-hydroxymethylsulfamethazine (6CH2OHSMZ)--were determined in either sex of 4 animal species: rats, dwarf goats, rabbits, and cattle. Rats, rabbits, and dwarf goats had significant (P < 0.01) sex difference in SMZ plasma clearance. Male rats had higher plasma clearance than did female rats, and excreted higher amounts of the hydroxy metabolites and lower amounts of N4AcSMZ. The N4AcSMZ metabolite was predominant in plasma and urine of rabbits. Male rabbits had higher plasma clearance than did female rabbits, but differences in metabolite profile were not apparent. With regard to plasma SMZ elimination, the situation in goats was opposite to that in rats. Male goats had considerably lower clearance than did female goats. This was associated with a lower hydroxylation rate in males. Plasma half-life of SMZ in cows was lower than that in bulls, probably because of a smaller distribution volume in cows. Compared with elimination via urine, elimination via milk was negligible in cows. Significant differences in metabolite profiles were not found between bulls and cows. Similar to those in rats and mice, hormone-dependent xenobiotic metabolic pathways may exist in other species. Depending on species and xenobiotic compound residue concentrations of xenobiotics, their metabolites, or both may differ with sex of the animal, or may be altered after treatment with anabolic hormones.

Norfloxacin was given to 2 groups of chickens (8 chickens/group) at a dosage of 8 mg/kg of body weight, IV and orally. For 24 hours, plasma concentration was monitored serially after each administration. Another group of chickens (n = 30) was given 8 mg of norfloxacin/kg orally every 24 hours for 4 days, and plasma and tissue concentrations of norfloxacin and its major metabolites desethylenenorfloxacin and oxonorfloxacin were determined serially after the last administration of the drug. Plasma and tissue concentrations of norfloxacin, desethylenenorfloxacin, and oxonorfloxacin were measured by use of high-performance liquid chromatography. Pharmacokinetic variables were calculated, using a 2-compartment open model. For norfloxacin, the elimination half-life and the mean +/- SEM residence time for plasma were 12.8 +/- 0.59 and 15.05 +/- 0.81 hours, respectively, after oral administration and 8.0 +/- 0.3 and 8.71 +/- 0.23 hours, respectively, after IV administration. After single oral administration, norfloxacin was absorbed rapidly, with Tmax of 0.22 +/- 0.02 hour. Maximal plasma concentration was 2.89 +/- 0.20 micrograms/ml. Oral bioavailability of norfloxacin was found to be 57.0 +/- 2.4%. In chickens, norfloxacin was mainly converted to desethylenenorfloxacin and oxonorfloxacin. Norfloxacin parent drug and its 2 major metabolites were widely distributed in tissues. Considerable tissue concentrations of norfloxacin, desethylenenorfloxacin, and oxonorfloxacin were found when norfloxacin was administered orally (8 mg/kg on 4 successive days), The concentration of the parent fluoroquinolone in fat, kidneys, and liver was 0.05 micrograms/g on day 12 after the end of dosing.