In vitro and in vivo binding of phenylbutazone and related drugs to equine feeds and digesta

In vitro and in vivo studies of phenylbutazone binding to equine ingesta and digesta were undertaken. In vitro binding to chopped hay and powdered pony nuts in buffer solutions at 37 degrees C was found to be time-, concentration- and pH-dependent. Percentage binding generally increased with time, decreased with concentration and varied with buffer pH in an unpredictable manner. Other non-steroidal anti-inflammatory drugs (NSAIDs) also bound to hay, the degree of binding being less for meclofenamate and least for flunixin in comparison with phenylbutazone. Phenylbutazone became bound to digesta collected from eight regions of the gastrointestinal tract when they were spiked with a concentration of 1 mg.10 g-1 digesta, the amounts ranging from 80.0 per cent (duodenum) to 99.6 per cent (stomach). Binding also occurred to equine digesta following the oral administration of phenylbutazone (4.4 mg.kg-1) to three ponies. It was concluded that drug uptake by and release from equine ingesta and digesta were probably adsorptive and desorptive processes. The clinical significance of the findings for the use of NSAIDs in equine medicine was considered.     

Pharmacokinetics of trimethoprim/sulphachlorpyridazine in horses after oral, nasogastric and intravenous administration

In the present study, the pharmacokinetic parameters of a trimethoprim/sulphachlorpyridazine preparation following intravenous administration, administration by nasogastric tube and administration with concentrate were determined in the horse. Eight adult horses were dosed at 1 week intervals in a sequentially designed study at a dose of 5 mg/kg trimethoprim (IMP) and 25 mg/kg sulphachlorpyridazine (SCP) on all occasions. Plasma concentrations of both drugs were measured serially for 48 h. Pharmacokinetic parameters of clinical importance (distribution and elimination half-lives, clearance, bioavail-ability, volume of distribution) were determined both for TMP and SCP. Following intravenous administration, the volume of distribution at steady-state (V_d(33) was significantly larger for TMP (1.51 ± 0.25 L/kg than for SCP (0.26 ± 0.05 L/kg. The clearance was 7.73 ± 2.26 mL/min-kg for TMP and 2.64 ± 0.48 mL/min·kg for SCP. For both TMP and SCP, mean peak plasma concentrations (C_max) and the bioavailabilities (F) were reduced significantly when the drugs were mixed with concentrate (ct) as compared with those after nasogastric administration (ngt) (F_ct= 44.3 ± 10.7% vs. F_ngt= 68.3 ± 12.5% for TMP; F_ct= 46.3 ± 8.9% vs. F_ngt= 67.3 ±13.7% for SCP). Following the administration of TMP and SCP mixed with concentrate, the plasma concentration—time curves showed a biphasic absorption pattern in all horses. The first peak occurred 1–2 h and the second peak 8–10 h after administration of the combination preparation. Based on the pharmacokinetic data obtained and the published in vitro sensitivity data, it may be predicted that TMP and SCP given intravenously or by nasogastric tube at a dose of 5 mg/kg and 25 mg/kg respectively and a dosage interval of 8–12 h would result in sufficiently high plasma concentrations for effectiveness against susceptible bacteria. The single oral administration of TMP and SCP mixed with concentrate did not result in effective plasma concentrations. Further studies are needed to investigate whether higher plasma concentrations would be achieved by a multiple dosing scheme for several days.     

In vitro and in vivo evaluation of hypericin for photodynamic therapy of equine sarcoids

The therapeutic potential of the photodynamic compound, hypericin, in the treatment of equine sarcoids was evaluated. The in vitro cytotoxicity was assessed using three equine cell lines and the observed phototoxic effect was comparable to that on different highly sensitive human cell lines and significantly influenced by the energy density used although independent of the cell type. The in vivo antitumoural action of photodynamic therapy using hypericin was evaluated on three equine sarcoids in a donkey. Four intratumoural injections were given and the tumours were illuminated daily during 25 days. An 81% reduction in tumour volume was obtained at the end of therapy and 2 months later, a 90% reduction was observed. Further experimental work should be performed, but these results suggest that photodynamic therapy using hypericin has a potential for the non-invasive treatment of equine sarcoids.     

In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis

REASON FOR PERFORMING STUDY: While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. OBJECTIVES: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. METHODS: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). In addition, 12 yearling horses were treated with PPVO and whole blood RNA samples were prepared at regular intervals to assess effects on in vivo cytokine gene expression. Six of those yearlings were later treated with saline and served as treatment controls. Nine additional yearlings were injected intradermally with a single dose and their injection sites biopsied at 24 and 48 h for cytokine expression. RESULTS: In vitro culture of PBMC with PPVO led to a significant increase in IFNalpha and IFNbeta gene expression compared to mock-stimulated cultures. In addition, expression of IFNgamma and TNFalpha was significantly higher in PBMC stimulated with PPVO and Con A, than those stimulated with Con A alone. No changes were observed in IL-18 gene expression in vitro. Treatment of horses with a 3-dose regimen of PPVO resulted in elevation of IFNgamma gene expression, which was detected 24 h after the first dose and declined thereafter. Intradermal inoculation led to increased expression of IFNgamma along with IFNbeta, IL-15 and IL-18. CONCLUSIONS: Together these results indicate that PPVO stimulated IFNgamma production both in vitro and in vivo. Increased cytokine expression could account for its immunomodulatory activity. POTENTIAL RELEVANCE: The absence of adverse reactions and clear indications of increased expression of cytokine gene expression supports previous clinical uses for this immune modulator in those situations when increased expression of IFNgamma is warranted.     

In vitro and ex vivo pharmacodynamics of selected non-steroidal anti-inflammatory drugs in equine whole blood

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential COX-2 inhibitor. Eltenac was a preferential COX-2 inhibitor in vitro but un-selective in the ex vivo study. Naproxen maintained its non-selectivity both in vitro and ex vivo. These findings have demonstrated that in vitro studies may not accurately predict in vivo NSAID selectivity for COX and should be confirmed using an ex vivo whole blood assay.     

In vitro quality assessment of two tropical shrub legumes in relation to their extractable tannins contents

The in vitro dry matter digestibility (IVDMD) of the two leguminous tree fodders Gliricidia sepium and Calliandra calothyrsus which differ in their tannin content was examined by the rumen simulation technique. Extractable condensed tannin (CT) concentrations ranged from 0.57% in G. sepium to 5.05% in C. calothyrsus using the butanol-HCl extraction. On the basis of their respective CT contents, G. sepium was classified as containing traces of CT, whereas C. calothyrsus had medium amounts of CT. Polyethylene glycol (PEG) was added at different concentrations (0, 1.5, 5 and 10 g/100 g plant material substrate) to assess the effect of tannins on IVDMD. The IVDMD was higher for G. sepium (range: 60-65%) than for C. calothyrsus (39.5-53.5%). In vitro gas production and IVDMD increased with increased PEG concentrations especially for C. calothyrsus. A non-significant response to increasing PEG concentrations for IVDMD of G. sepium confirms PEG binding with the tannins. On the basis of these results, it is assumed that G. sepium has a higher nutritive value than C. calothyrsus. Good relationship between PEG binding and the improvement of IVDMD confirms the usefulness of this technique for improving the nutritive value of tanniniferous tropical browses.     

Phagocytic function of equine neutrophils exposed to Mycoplasma felis in vitro and in vivo

Neutrophils were isolated from the peripheral blood of adult equids (group 1) and were purified on a density gradient of polyvinylpyrrolidone-coated silica gel. A bactericidal assay was developed, using an equine skin isolate of Staphylococcus epidermidis as target bacterium in medium containing pooled fresh equine serum for opsonization. Significant (P less than 0.05) killing was observed after 60 or 120 minutes' incubation. Reduction in bactericidal function of blood neutrophils was not found after incubation with a virulent strain of Mycoplasma felis for 30 or 60 minutes. Similarly, the function of blood and pleural neutrophils of ponies with M felis-induced pleuritis (group 2) was not different from the function of similar neutrophils from sham-inoculated control ponies (group 3). The number of viable M felis organisms was markedly reduced in the presence of fresh equine serum and equine-specific antibodies, but not when the serum was heat inactivated. Equine neutrophils did not phagocytize M felis. Seemingly, M felis did not impair bactericidal activity of equine neutrophils. Therefore, this mechanism does not predispose equids to bacterial pleuritis.     

In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis

REASONS FOR PERFORMING STUDY: Elevated plasma homocysteine (HCy) concentration is a risk factor for cardiovascular diseases associated with endothelial dysfunction, including the human digital ischaemic disease, Raynaud's phenomenon. HYPOTHESIS: HCy causes dysfunction of equine vascular endothelium and elevated plasma concentrations predispose to laminitis. OBJECTIVES: To determine 1) the concentration of HCy in vitro, which inhibits equine vascular endothelial cell function and 2) any association between risk of laminitis and plasma HCy concentration. METHODS: Endothelial function was studied by measuring endothelium-dependent vasodilatory responses of the equine isolated perfused digit and basal nitric oxide (NO) production by cultured equine digital vein endothelial cells (EDVECs). Total plasma HCy (tHCy) concentrations were measured in samples collected in the winter and spring from normal ponies and ponies predisposed to laminitis. RESULTS: HCy (10 and 100 micromol/l) inhibited endothelial function and, at concentrations above 100 micromol/l, inhibited NO production by EDVECs. Plasma tHCy concentration ranged from 13 to 14.7 micromol/l. There was no effect of season or disease status on the concentration measured. CONCLUSIONS: In vitro, HCy was shown to interfere with endothelial cell function at physiologically relevant concentrations. No evidence was found for an association between risk of laminitis and high plasma concentrations of HCy. POTENTIAL RELEVANCE: Elevated plasma HCy concentrations could adversely affect endothelial cell function and mangement regimens that lead to increases in plasma HCy concentration should be avoided in ponies predisposed to laminitis.     

In vitro responses of equine digital vessels to dopamine and fenoldopam.

The in vitro responses of isolated vascular preparations of digital arteries and veins obtained from healthy anaesthetised horses were determined for dopamine and fenoldopam. The digital vessels were harvested, cut into 4 mm vascular segments, suspended in tissue baths and attached to force-displacement transducers. Dose-response studies between 10(-8) and 10(-4)M concentrations were performed for all drugs. The change in tension of each vascular ring was measured in grams of force. The reactivity between palmar and plantar digital vessels and baseline vascular responses were determined for dopamine. The vascular responses of dopamine were compared to in vitro data for other known vasoconstrictor agents. The mechanism of vasoconstriction induced by dopamine was further defined using prazosin, a specific competitive alpha-1 adrenoceptor antagonist. The vasodilating ability of fenoldopam, a dopamine-1 (DA-1) receptor agonist, was also determined using noradrenaline- preconstricted vascular segments from palmar digital vessels. The effective concentration to produce 50 per cent of the maximal response (EC50) and the maximal contraction in grams of force per milligram of the vascular ring (g/mg) were calculated. There were no differences in the reactivity between the palmar and plantar digital vessels. Dopamine produced intense constriction in arteries and veins but only at very high molar concentrations. Prazosin decreased significantly the sensitivity of the veins to dopamine (increased the mean EC50 values) but not the arteries. Prazosin had no effect on the maximal contractions of the vessels. Fenoldopam produced very little relaxation of either the arteries or veins. These results suggest that dopamine produces constriction in equine digital arteries and veins and that the constriction is only partially mediated by alpha-1 adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens

1. Three bacterial phytases derived from Bacillus, Escherichia coli or Klebsiella were compared with a phytase derived from Aspergillus niger in vitro and in vivo. 2. The in vitro results indicated that Aspergillus, E. coli and Klebsiella phytase displayed their activity optima in an acid pH range while Bacillus phytase did so in neutral pH. 3. The trials also revealed that only Bacillus phytase is more resistant to heat treatments, while E. coli and Klebsiella phytases are more stable against proteolytic inactivation. 4. In vivo phytases derived from Aspergillus, Bacillus, E. coli, Klebsiella or a combination of Bacillus and E. coli improved the utilisation of phosphorus (P balance) significantly to 0.54, 0.54, 0.55, 0.55 or 0.58, respectively, compared to 0.42 in the negative control. 5. The phytases used in this study seemed to be equally effective in improving P utilisation regardless of proposed intestinal site of activity. Combination of phytases acting in the gizzard with phytases acting in the intestine seems to be a promising way to further improving in vivo efficacy of phytases in poultry.     

In vitro culture of equine respiratory mucosa explants

An in vitro model of the upper respiratory tract of the horse was developed to investigate mechanisms of respiratory diseases. Four tissues of the upper respiratory tract of three horses were collected. Explants were maintained in culture at an air–liquid interface for 96 h. At 0, 24, 48, 72 and 96 h of cultivation, a morphometric analysis was performed using light microscopy, scanning electron microscopy and transmission electron microscopy. The explants were judged on morphometric changes of epithelium, basement membrane and connective tissue. Viability was evaluated using a fluorescent Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labelling (TUNEL) staining. No significant changes in morphometry and viability of any of the explants were observed during cultivation. Hence, the in vitro model may be useful to study infectious and non-infectious diseases at the level of the equine respiratory tract, with potential application to the development of vaccines and treatments for diseases of the respiratory tract.     

The osmolality of caecal contents in piglets following weaning

Pigs weaned for five days had caecal contents with significantly lower osmolalities than those of unweaned animals of the same age. Supplementation of a standard weaning diet with therapeutic levels of oxytetracycline to suppress the normal large intestinal microbial flora did not significantly further reduce osmolarity of caecal contents after weaning. This observation suggests that microbial activity in the large intestine may not be sufficiently developed by five days after weaning to have a major influence on absorptive processes at that site. Incomplete development of the large intestinal microflora may be one more factor which acts to predispose the young pig to diarrhoea after weaning.     

In vitro mechanical properties of equine tendons in relation to cross-sectional area and collagen content

The mechanical properties of the deep digital flexor tendon (DDFT), the superficial digital flexor tendon (SDFT) and the suspensory ligament (SL) of the hindlimb of the horse were studied in vitro. The tendons were observed at several morphologically distinct sites. The loaded tendon is homogeneously strained, in spite of large variations in cross-sectional area. Consequently the modulus of elasticity was inversely proportional to the corresponding cross-sectional area and ranged from 738 MPa (megaPascal, N mm-2) to 1398 MPa within the DDFT, from 1000 MPa to 1282 MPa within the SDFT and from 576 MPa to 669 MPa within the SL. The collagen content was inversely proportional to the cross-sectional area and proportional to the modulus of elasticity. This stresses the influence of tendon composition on the mechanical properties, and also demonstrates the difficulty in judging the strength of a particular tendon or site within a tendon from its cross-sectional area. The respective tendons ruptured at strains of 10.0 per cent (DDFT), 12.3 per cent (SDFT) and 11.0 per cent (SL). The influence of strain rate on the modulus of elasticity is small, and these tendons may therefore be considered as non-linear elastic structures. The average hysteresis is about 5 per cent.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.