C. Belloli

Pharmacokinetics of imidocarb dipropionate in horses after intramuscular administration

The objective of this study was to determine the pharmacokinetic behaviour of imidocarb in horses following a single i.m. injection at the dose commonly administered to treat Babesia caballi infections or to prevent babesiosis. Eight horses were injected i.m. with a single dose of 2.4 mg imidocarb dipropionate/kg bwt and blood, faecal, urine and milk samples were collected. For imidocarb determination, a high-performance liquid chromatographic method (HPLC) was used after weak cation-exchange solid phase, or liquid-liquid, extraction procedures. Twelve hours after treatment, no detectable plasma concentrations were recorded in any of the treated animals. The distribution and elimination patterns of the drug suggested that it is quickly sequestrated in some storage tissues and remains in the body for a long time. Its prolonged presence in the body may confer a reservoir effect to imidocarb in some tissues, therefore making it undetectable in the plasma of animals but sufficient to produce its described therapeutic and prophylactic activities.

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1.

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1--1.0-8.0 microM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 microM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC50 at 48 h = 12.00 +/- 2.66 microM; MTT: CC50 at 72 h = 20.42 +/- 7.30 microM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.

Identification of Functional α-Adrenoceptor Subtypes in the Bovine Female Genital Tract During Different Phases of the Oestrous Cycle

The concentration and functionality of the α-adrenoceptor (α-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of α1-ARs was observed, whereas two distinct classes of α2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) α2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all α-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of α1-ARs was significantly (p < 0.05) higher than that of α2-ARs. By contrast, there were significantly (p < 0.05) more α2-ARs than α1-ARs in the myometrium. As far as α2-ARs are concerned, LA α2-ARs were significantly (p < 0.05) higher than HA α2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for α1-ARs was prazosin > phentolamine > yohimbine, whereas for α2-ARs the order of potency was yohimbine ≥ phentolamine > prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in α-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to α-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of α-ARs.

A rapid and simple method for the separation of pure lymphocytes from horse blood.

A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further centrifugation at 250 xg for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n = 15) contaminating platelets. A suspension of 85.96 +/- 2.20% pure granulocytes (93.23 +/- 1.74% neutrophils; n = 14) with minimal contamination by erythrocytes and high viability (93.11 +/- 1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible.

Identification of functional β-adrenoceptors in Caco-2 cell membranes

R. Odore1*, P. Badino1, A.L. Stammati2, I. De Angelis2, F. Zucco3, C. Belloli4 and G. Re1 1Department of Animal Pathology, Division of Veterinary Pharmacology and T oxicology, University of T orino, Grugliasco (T o); 2L aboratory of Compared T oxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome; 3Institute of Biomedical T echnologies, CNR, Rome; 4Department of Animal Health and Welfare, Division of Pharmacology and T oxicology, University of Bari, Valenzano (Ba), Italy *Correspondence: Department of Animal Pathology, Division of Veterinary Pharmacology and T oxicology, University of T orino, V ia L eonardo da V inci 44, 10095 Grugliasco (T o), Italy E-mail: odore@veter.unito.it

Effects of endotoxin and influence of cyclooxygenase-2 on β-adrenergic mediated relaxation in isolated equine digital artery.

The effects of endotoxin on β-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA β-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of β-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA β-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

Investigation of the metabolic activity of a bovine mammary epithelial cell line (BME-UV1)

The metabolic activity of a mammary epithelial cell line (BME-UV1) was evaluated on monolayers exposed, in serum free medium, to different concentrations (2-4-8 μM) of aflatoxin B1 (AFB1), a mycotoxin eliminated into milk especially as hydroxylated metabolite aflatoxin M1 (AFM1). After 4, 8, 12, 24 h of treatment, a dose and time dependent production of AFM1 has been detected. As the enzymes involved in the hydroxylation of AFB1 in bovine hepatocytes are mainly CYP1A and CYP3A, the results suggest that BME-UV1 express CYP450 isoenzymes which metabolize AFB1 thus representing a potential model for the investigation of the metabolic activity of bovine mammary epithelial tissue.

Change in vascular smooth muscle response to 5-HT due to short- or long-term endothelial denudation of the bovine digital vein.

Several chronic progressive vascular diseases, such as laminitis, show vasocontractile dysfunction that might evolve into reperfusion injury and/or vessel structural remodelling, which may be traced back to aberrant endothelial function. In the present study, the vasomotor responses of bovine digital veins (BDVs) to 5-hydroxytryptamine (5-HT) were investigated in blood vessels, with and without endothelium present, and in samples deprived of endothelium before or after overnight incubation in tissue culture medium, to evaluate the effects of short- and long-term endothelial damage on vascular smooth muscle (VSM) reactivity. No significant effects were observed in the blood vessels tested immediately after the removal of endothelium. In contrast, a significant increase in VSM reactivity to 5-HT was seen in vessels incubated without endothelium. This long-term change in smooth muscle reactivity was prevented by exposure to the nitric oxide (NO) donor nitroprusside (P < 0.01), suggesting that the long-term lack of inhibitory control exerted by endothelium-derived NO is involved in increased VSM reactivity. The RhoA/ROCK pathway inhibitor fasudil reduced VSM hyper-contractility to ~65% (P < 0.001), the superoxide dismutase-mimetic tempol normalised the vascular response and the non-selective COX-inhibitor indomethacin exerted a moderate inhibitory effect (P < 0.05). Thus, over-activation of the RhoA/ROCK pathway and production of reactive oxygen species could account for VSM hyper-reactivity, triggered by long-term endothelium-deprivation in BDVs, suggesting that these biochemical mechanisms are potential targets for controlling the progressive vasocontractile dysfunction of digital veins in animals affected with laminitis.

Oral and intravenous administration of nimesulide in the horse: rational dosage regimen from pharmacokinetic and pharmacodynamic data

REASONS FOR PERFORMING STUDY The selective COX-2-inhibitor nimesulide is used extra-label in equine veterinary practice as an anti-inflammatory agent. However, there are no data on which to base the rational use of the drug in this species. OBJECTIVES To determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. METHODS The pharmacokinetics of nimesulide in the horse after oral administration (1 mg/kg bwt), and oral and i.v. administration (1.5 mg/kg bwt) were investigated, effects of feeding status on bioavailability determined, and plasma protein binding of the drug and its principal metabolites measured. Compartmental and noncompartmental pharmacokinetic analyses were performed. The plasma concentration-time profile was used, together with in vitro literature data on nimesulide inhibition of COX isoforms, to determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. RESULTS AND CONCLUSIONS The findings suggest that 1.5 mg/kg bwt may produce adequate clinical effects, and the dosing interval should be 12-24 h depending on condition severity. However, at that dose, the concentration in the animal exceeds the in vitro IC50 for both isoforms, so that COX-1/COX-2 selectivity is lost and side-effects due to COX-1 inhibition are a possibility. Nimesulide should therefore be used with caution in equine clinical practice.

A comparative study between responses of isolated bovine and equine digital arteries to vasoactive mediators

Hemodynamic perturbations, partly resulting from abnormal vasoconstriction of digital vessels, have been implicated in the pathogenesis of bovine and equine laminitis. This study compared the responsiveness of isolated bovine (BDA) and equine (EDA) digital arteries to pharmacological agents that stimulate receptor systems involved in the regulation of normal vessel tone. The role of the endothelium and the short- and longer-term effects of an experimentally induced endothelial damage were also evaluated. Species-related differences were found in the vessel reactivity to all of the receptor agonists tested. In intact BDA, as compared to intact EDA, norepinephrine was a more effective vasoconstrictor, 5-hydroxytryptamine a more effective but less potent vasoconstrictor, isoproterenol a less effective vasodilator and carbamylcholine a less potent vasodilator. In BDA, but not in EDA, the contractile responses to norepinephrine and 5-hydroxytryptamine were enhanced immediately after endothelium removal. However, the contractile reactivity of denuded BDA returned to basal values following overnight incubation. The differences suggest species specificity for the pathophysiology of digital vasomotor tone and function in horses and cattle.