Comparison of an injectable toltrazuril-gleptoferron and an oral toltrazuril + injectable gleptoferron in piglets: Hematinic activities and pharmacokinetics.

Iron deficiency anemia (IDA) and cystoisosporosis are the most common clinical conditions of fast-growing piglets. Until now, IDA and cystoisosporosis have been managed by intramuscular injection of iron complexes (such as dextran or gleptoferron) and oral administration of toltrazuril. Recently, a new combination product containing toltrazuril and gleptoferron for intramuscular application (Forceris®) has been registered. The objective of this study was to compare the pharmacokinetic profiles of toltrazuril and its main metabolite, toltrazuril sulfone, following a single oral (Baycox®) or intramuscular (Forceris®, a toltrazuril-iron combination product) administration at 20 mg/kg to young suckling piglets. The orally treated piglets were also supplemented with iron (Gleptosil®), and the hematinic activities were compared. Piglets in both groups received comparable doses. The peak concentration (Cmax) of toltrazuril after intramuscular administration was 11% lower than that after oral administration (p = .376). However, the exposure to toltrazuril (AUC) was significantly increased (40% higher) when toltrazuril was administered intramuscularly (p = .036). The Cmax and AUC values of the active metabolite, toltrazuril sulfone were 39% and 34% higher, respectively, after intramuscular administration (p = .007 and 0.008, respectively). Piglets in both groups were properly protected against IDA. In conclusion, a higher relative bioavailability of toltrazuril is observed when toltrazuril is administered intramuscularly.

Absorption and Distribution of Toltrazuril and Toltrazuril Sulfone in Plasma, Intestinal Tissues and Content of Piglets after Oral or Intramuscular Administration.

Piglet coccidiosis due to Cystoisospora suis is a major cause of diarrhea and poor growth worldwide. It can effectively be controlled by application of toltrazuril (TZ), and oral formulations have been licensed for many years. Recently, the first parenteral formulation containing TZ in combination with iron (gleptoferron) was registered in the EU for the prevention of coccidiosis and iron deficiency anemia, conditions in suckling piglets requiring routine preventive measures. This study evaluated the absorption and distribution of TZ and its main metabolite, toltrazuril sulfone (TZ-SO2), in blood and intestinal tissues after single oral (20 mg/kg) or single intramuscular (45 mg/piglet) application of TZ. Fifty-six piglets were randomly allocated to the two treatment groups. Animals were sacrificed 1-, 5-, 13-, and 24-days post-treatment and TZ and TZ-SO2 levels were determined in blood, jejunal tissue, ileal tissue, and mixed jejunal and ileal content (IC) by high performance liquid chromatography (HPLC). Intramuscular application resulted in significantly higher and more sustained concentrations of both compounds in plasma, intestinal tissue, and IC. Higher concentrations after oral dosing were only observed one day after application of TZ in jejunum and IC. Toltrazuril was quickly metabolized to TZ-SO2 with maximum concentrations on day 13 for both applications. Remarkably, TZ and TZ-SO2 accumulated in the jejunum, the primary predilection site of C. suis, independently of the administration route, which is key to their antiparasitic effect.

Population Pharmacokinetics and Pharmacodynamics Modeling of Torasemide and Furosemide After Oral Repeated Administration in Healthy Dogs.

Torasemide is a loop diuretic licensed in dogs for cardiogenic pulmonary oedema. The aim of this pharmacokinetic-pharmacodynamic (PK/PD) study was to define an optimally effective dosage regimen based on preclinical data. In a first study, 5 dogs received once-daily oral torasemide (0, 0.1, 0.2, 0.4, 0.8 mg/kg/day) for 14 days. A second study compared once-daily oral torasemide (0, 0.1, 0.2, 0.3, 0.4 mg/kg/day) to twice-daily furosemide (1, 2, 4, 8 mg/kg/day). For all doses of the second study, 11 dogs received a first day of treatment, followed by a 3 day washout and resumed daily treatment for 10 days (until Day 14). Blood and urine were collected to measure urinary torasemide excretion and plasma torasemide concentrations and daily diuresis and natriuresis. Torasemide PK was linear. After rapid absorption (Tmax 0.5-1 h), 61% of the bioavailable torasemide was eliminated unchanged in urine. Diuresis and natriuresis observed with torasemide were similar to the ones obtained after furosemide (daily dose-ratios: 1/20 to 1/10). The average diuresis increased from baseline (220 ± 53 mL/day for 10 kg dogs) to 730 ±120 mL after the first torasemide administration and up to 1150 ± 252 mL after 10 administrations at the highest dose. At higher doses (≥0.3 mg/kg/day), daily diureses after 10 diuretic treatment-days were higher than Day 1 and variable between dogs; in contrast, diureses remained constant over time and less variable for doses up to 0.2 mg/kg/day. Natriuresis peaked after the first day and decreased dramatically after the 2nd treatment-day then stabilized to a value close to baseline, except for 0.4 mg/kg/day. Urinary torasemide excretion predicted pharmacodynamics better than plasma concentrations. The decrease in natriuresis observed was successfully modeled using a resistance mechanism; this is likely due to a reabsorption of sodium which did not seem however to affect the volume of urine excreted. For a daily target diuresis of 460 mL/dog/day in severe pulmonary oedema (net fluid loss 240 mL/dog/day), a computed dose of 0.26 mg/kg/day (3.5 mg/kg/day furosemide-equivalent) was selected for clinical studies. Due to high inter-individual variability in diureses at doses ≥0.3 mg/kg, higher doses should be limited to 3-5 days to avoid supra-clinical effects in high responders.

Sacubitril/valsartan (LCZ696) significantly reduces aldosterone and increases cGMP circulating levels in a canine model of RAAS activation.

Simultaneous blockade of angiotensin receptors and enhancement of natriuretic peptides (NP) by the first-in-class angiotensin receptor neprilysin (NEP) inhibitor sacubitril/valsartan constitutes an effective approach to treating heart failure. This study examined the effects of sacubitril/valsartan (225 and 675 mg/day) vs. placebo, sacubitril (360 mg/day), valsartan (900 mg/day), and benazepril (5 mg/day) on the dynamics of the renin-angiotensin-aldosterone system (RAAS) and the NP system in dogs. Beagle dogs (n = 18) were fed a low-salt diet (0.05% Na) for 15 days to model RAAS activation observed in clinical heart failure. Drugs were administered once daily during the last 10 days, while the effects on the RAAS and NPs were assessed on Day 1, 5, and 10. Steady-state pharmacokinetics of the test agents were evaluated on Day 5. Compared with placebo, sacubitril/valsartan (675 mg) substantially increased cGMP circulating levels, while benazepril and valsartan showed no effect. Additionally, sacubitril/valsartan (675 mg) and valsartan significantly increased plasma renin activity, angiotensin I and angiotensin II concentrations. Finally, sacubitril/valsartan (both doses), and valsartan significantly decreased plasma aldosterone vs. placebo. Systemic exposure to valsartan following sacubitril/valsartan 675 mg administration was similar to that observed with valsartan 900 mg administration alone. Sacubitril/valsartan favorably modulates the dynamics of the renin and NP cascades through complementary NEP and RAAS inhibition.

Pharmacokinetic/Pharmacodynamic Modeling of Renin-Angiotensin Aldosterone Biomarkers Following Angiotensin-Converting Enzyme (ACE) Inhibition Therapy with Benazepril in Dogs.

PURPOSE: The objective of this research was to provide a comprehensive description of the effect of benazepril on the dynamics of the renin-angiotensin aldosterone system (RAAS) in dogs. METHODS: Blood specimens for renin activity (RA), angiotensin II (AII), and aldosterone (ALD) quantitation in plasma were drawn from 12 healthy adult beagle dogs randomly allocated to 2 treatment groups: (i) benazepril 5 mg PO, q24 h (n: 6) and (ii) placebo (n: 6), in a cross-over design. A mechanism-based pharmacokinetic/pharmacodynamic model, which includes the periodic nature of RA, AII, and ALD during placebo treatment and the subsequent changes in dynamics following repeated dosing with benazepril, was developed. RESULTS: The disposition kinetics of benazepril active metabolite, benazeprilat, was characterized using a saturable binding model to the angiotensin converting enzyme. The modulatory effect of benazeprilat on the RAAS was described using a combination of immediate response models. Our data show that benazepril noticeably influences the dynamics of the renin cascade, resulting in a substantial decrease in AII and ALD, while increasing RA throughout the observation span. CONCLUSIONS: The model provides a quantitative framework for better understanding the effect of ACE inhibition on the dynamics of the systemic RAAS in dogs.

Influence of feeding schedules on the chronobiology of renin activity, urinary electrolytes and blood pressure in dogs.

The contribution of the renin-angiotensin-aldosterone system (RAAS) to the development of congestive heart failure (CHF) and hypertension (HT) has long been recognized. Medications that are commonly used in the course of CHF and HT are most often given with morning food for the sake of convenience and therapeutic compliance. However, biological rhythms and their responsiveness to environmental clues such as food intake may noticeably impact the effectiveness of drugs used in the management of cardiovascular disorders. Only sparse information about the effect of feeding schedules on the biology of the RAAS and blood pressure (BP) is presently available. Two studies were designed to explore the chronobiology of renin activity (RA), BP, renal sodium (UNa,fe) and potassium (UK,fe) handling in relation to meal timing in dogs. In a first experiment (Study a), blood and urinary samples for measurement of RA, UNa,fe and UK,fe were drawn from 18 healthy beagle dogs fed a normal-sodium diet at either 07:00, 13:00 or 19:00 h. In a second experiment (Study b), BP was recorded continuously from six healthy, telemetered beagle dogs fed a similar diet at 07:00, or 19:00 h. Data were collected throughout 24-h time periods, and analyzed by means of nonlinear mixed-effects models. Differences between the geometric means of early versus late time after feeding observations were further compared using parametric statistics. In agreement with our previous investigations, the results indicate that RA, UNa,fe, UK,fe, systolic, and diastolic BP oscillate with a circadian periodicity in dogs fed a regular diet at 07:00 h. A cosine model with a fixed 24-h period was found to fit the variations of RA, UK,fe and BP well, whereas cyclic changes in UNa,fe were best characterized by means of a combined cosine and surge model, reflecting a postprandial sodium excretion followed by a monotonous decay. Our data show that feeding time has a marked influence on the chronobiology of the renin cascade, urinary electrolytes, and BP. Introducing a 6- or 12-h delay in the dogs' feeding schedule caused a shift of similar magnitude (05:06 and 12:32 h for Studies a and b, respectively) in the rhythm of these biomarkers. In all study groups, RA and BP exhibited a marked fall just after food intake. The drop in RA is consistent with sodium and water-induced body fluid expansion, while the reduction of BP could be related to the decreased activity of renin and the secretion of vasodilatory gut peptides. An approximately 1.5-fold (1.2-1.6-fold) change between the average early and late time after feeding observations was found for RA (p < 0.0001), UNa,fe (p < 0.01) and UK,fe (p < 0.05). Postprandial variations in BP, albeit small (ca. 10 mmHg), were statistically significant (p < 0.01) and supported by the model-based analysis. In conclusion, the timing of food intake appears to be pivotal to the circadian organization of the renin cascade and BP. This synchronizing effect could be mediated by feeding-related signals, such as dietary sodium, capable of entraining circadian oscillators downstream of the master, light-dark-adjusted pacemaker in the suprachiasmatic nucleus.

Chronobiology of the renin-angiotensin-aldosterone system in dogs: relation to blood pressure and renal physiology.

The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in the regulation of blood pressure and volume homeostasis. Its contribution to the development of cardiovascular diseases has long been recognized. Extensive literature has shown that peptides of the RAAS oscillate with a circadian periodicity in humans, under strong influence of posture, sleep, and age. Although observations of time-variant changes in the renin cascade are available in dogs, no detailed chronobiological investigation has been conducted so far. The present studies were designed to explore the circadian variations of plasma renin activity (RA) and urinary aldosterone-to-creatinine ratio (UA:C) in relation to blood pressure (BP), sodium (UNa, UNa,fe), and potassium (UK, UK,fe) renal handling. Data derived from intensive blood and urine sampling, as well as continuous BP monitoring, were collected throughout a 24-h time period, and analyzed by means of nonlinear mixed-effects models. Differences between the geometric means of day and night observations were compared by parametric statistics. Our results show that variables of the renin cascade, BP, and urinary electrolytes oscillate with significant day-night differences in dogs. An approximately 2-fold (1.6-3.2-fold) change between the average day and night measurements was found for RA (p < 0.001), UA:C (p = 0.01), UK,fe (p = 0.01), and UNa (p = 0.007). Circadian variations in BP, albeit small (less than 10 mm Hg), were statistically significant (p < 0.01) and supported by the model-based analysis. For all variables but UNa and UNa,fe, the levels were higher at night than during the day. The data also indicate that blood pressure oscillates in parallel to the RAAS, such that, as opposed to healthy humans, BP does not drop at night in dogs. The postprandial decrease in RA is assumed to be related to body fluid volume expansion secondary to water and sodium intake, whereas the reduction of UA:C reflects aldosterone-stimulated secretion by the renin-angiotensin II pathway. UNa and UNa,fe peaked in the afternoon, about 7-8 h after food intake, which is consistent with the "impulse-response pattern" of sodium excretion described in previous publications. Finally, UK and UK,fe mirrored aldosterone-mediated potassium secretion in the kidney tubules. To describe the circadian variations of the various variables, two different mathematical representations were applied. A cosine model with a fixed 24-h period was found to fit the periodic variations of RA, UA:C, UK, UK,fe, and BP well, whereas changes in UNa and UNa,fe were best characterized by a surge model. The use of nonlinear mixed effects allowed estimation of population characteristics that can influence the periodicity of the RAAS. Specifically, sodium intake was found to interact with the tonic and the phasic secretion of renin, suggesting that varying feeding time could also impact the chronobiology of the renin cascade.

Validation of a dried blood spot LC-MS/MS approach for cyclosporin A in cat blood: comparison with a classical sample preparation.

A rapid, selective and sensitive bioanalytical method was developed and validated for cyclosporin A (CsA) in cat blood samples using dried blood spot (DBS) coupled with high pressure liquid chromatography hyphenated to positive electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). CsA was quantified using a structural analog cyclosporin D (CsD) as internal standard in multiple reaction monitoring mode (MRM) using the transitions 1220→1203 for CsA and 1234→1217 for CsD. A 5-µL blood aliquot was spotted onto a DBS card, then after a drying step, blood spots were punched out from the cards and extracted with MeOH before injection into a LC-MS/MS system. The linear range of the method was 5-2000 ng/mL, with accuracy and precision within the FDA acceptance criteria (i.e., ±15% and ±20% for the lowest level). In the study presented herein a comparison was made between this new methodology, based on the use of DBS, and a previously developed solid phase extraction (SPE) procedure, applied to blood samples from a cat pharmacokinetic study. Good correlation (r(2)=0.97) was demonstrated between the data obtained with the two methods. The DBS methodology revealed to be cheaper, faster, less solvent-consuming and requiring less blood volume from animals than the previous SPE method. Thus, the proposed HPLC-ESI-MS/MS method, based on DBS, has demonstrated to be a suitable and advantageous approach for the analysis of CsA in cat blood.

Population pharmacokinetic analysis of blood and joint synovial fluid concentrations of robenacoxib from healthy dogs and dogs with osteoarthritis.

PURPOSE: The purpose of this population analysis was to characterize the pharmacokinetic properties of robenacoxib in blood and stifle joint synovial fluid of dogs. METHODS: Data were obtained from two studies: 1) 8 healthy Beagle dogs in which an acute inflammation was induced by injection of urate crystals into one joint; 2) 95 dogs from various breeds diagnosed with osteoarthritis (OA). Robenacoxib concentrations in blood and synovial fluid were measured using a validated HPLC-UV and LC-MS method. Non-linear mixed effects modeling was performed using NONMEM6. RESULTS: A two-compartment pharmacokinetic model with linear elimination was developed to describe blood concentrations of robenacoxib. Blood clearance in healthy animals was found to be 75% higher than in OA dogs. Synovial fluid concentrations were modeled using an effect-compartment-type model predicting longer residence times in OA dogs compared to healthy Beagles (e.g. concentrations above the IC(50) for COX-2, respectively, 16 h vs. 10 h at 1.5 mg/kg). CONCLUSIONS: Robenacoxib was found to reside longer at the effect site (inflamed joint) compared to blood in both healthy and OA dogs. These results may explain the good efficacy observed with once-daily dosing in clinical trials and the high safety index of robenacoxib in dogs.

Cisplatin, gentamicin, and p-aminophenol induce markers of endoplasmic reticulum stress in the rat kidneys.

In vitro evidence of the involvement of the endoplasmic reticulum (ER) during drug-induced renal toxicity is accumulating. ER stress and ER-mediated cell death markers have been reported after exposure of renal cells to model toxicants and nephrotoxic drugs in various in vitro models, but in vivo experiments with clinically relevant nephrotoxic compounds are lacking. In order to determine the relevance of the in vitro findings, markers of ER stress (XBP1 messenger RNA processing and protein expression; GRP78 and GRP94 upregulation) and ER-mediated cell death (caspase-12 and calpain activation) were examined in kidney tissue of rats exposed to nephrotoxic doses of cisplatin (CIS), gentamicin (GEN), and p-aminophenol (PAP), a nephrotoxic metabolite of acetaminophen. XBP1 signaling was observed with all three drugs and was associated with increased expression of GRP94 and GRP78 in GEN- and PAP-treated animals, but surprisingly not after CIS exposure. m-Calpain expression was increased after 7 days of CIS treatment, whereas it was decreased in PAP-treated rats. Caspase-12 cleavage products were increased after CIS, GEN, and PAP administration. The results of this study demonstrate that three clinically relevant nephrotoxic drugs are all associated with changes in markers of ER stress and ER-mediated cell death in vivo. Further investigation is warranted to determine the role of the ER, the calpain system, and caspase-12 in drug-induced renal cell death.

Calpain inhibition but not reticulum endoplasmic stress preconditioning protects rat kidneys from p-aminophenol toxicity.

p-Aminophenol (pAP, 225 mg/kg) administration to rats induced renal failure and has been associated with markers of endoplasmic reticulum (ER) stress, as well as calpain and caspase-12 activation in kidneys. To determine the importance of ER stress and calpain during pAP-induced nephrotoxicity, rats were pretreated with low, nontoxic, doses of ER stress inducers or with the selective calpain inhibitor PD150606 (3 mg/kg). Prior ER stress induced by tunicamycin and oxidized dithiothreitol did not result in protection against renal failure, but PD150606 administration was protective and decreased significantly the rise in creatinine and blood urea nitrogen observed after 24-h post-pAP administration. pAP-induced XBP1 upregulation was not modified by calpain inhibition, but a trend to lower GRP94 induction was determined, suggesting that pAP-induced ER stress was mostly calpain independent. In contrast, pAP-induced caspase-12 cleavage products were significantly decreased with PD150606 pretreatment, demonstrating that caspase-12 activation was calpain dependent. This study reveals the importance of calpain in pAP-induced renal failure. Further research with other nephrotoxicants needs to be performed to determine if calpain activation is a common feature of drug-induced renal failure.

Effect of endoplasmic reticulum stress preconditioning on cytotoxicity of clinically relevant nephrotoxins in renal cell lines.

The cytoprotection of LLC-PK1 cells afforded by endoplasmic reticulum (ER) stress preconditioning suggests that the ER plays an important role during drug-induced renal toxicity. However, in vitro studies have been largely limited to LLC-PK1 cells and model toxins. Therefore, we tested the hypothesis that cytoprotection following ER stress preconditioning is a common property of renal cell lines (LLC-PK1 (pig), NRK-52E (rat), HEK293 (human), MDCK (dog)) and extends to clinically relevant nephrotoxins. ER stress inducers (tunicamycin, thapsigargin and oxidized dithiothreitol (DTTox)) resulted in a dose-dependent increase in GRP78 and GRP94 stress protein expression, but the magnitude of induction was cell line- and inducer-dependent. Toxicity of the model toxins iodoacetamide and tert-butylhydroperoxide was modified by preconditioning. DTTox was effective in decreasing the toxicity in all cell lines, but protection was variable with tunicamycin and thapsigargin. Toxicity of clinically relevant drugs (cisplatin, gentamicin, glyoxylate, cyclosporine A, p-aminophenol) was significantly decreased in cells preconditioned by tunicamycin or DTTox. These results demonstrate that ER stress preconditioning offers cytoprotection against clinically relevant nephrotoxins in renal cell lines from multiple species, although there were qualitative and quantitative differences between the cell lines. These results support the hypothesis that ER is involved in drug-induced renal toxicity.

The endoplasmic reticulum in xenobiotic toxicity.

The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P450 enzymes involved in xenobiotic metabolism, as well as a number of conjugating enzymes. In addition to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-associated apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in molecular events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity will be discussed.

[Evolution of bacterial resistance to certain antibacterial agents in horses in a veterinary hospital]. / Evolution de la résistance bactérienne enverscertains agents antibactériens chez les chevauxdans un centre hospitalier vétérinaire.

Evolution of bacterial resistance to certain antibacterial agents in horses in a veterinary hospital. A total of 255 antibiograms, conducted according to the Kirby-Bauer method from bacterial isolates collected from horses at the Hôpital Vétérinaire d'Enseignement at the Université de Montréal between 1996 and 1998 were compared with the results obtained about 10 years ago with corresponding bacterial species. A significant increase in the percentage of strains resistant to trimethoprim-sulfamethoxazole (TMP-SXT), penicilline, tetracycline, and to gentamicin was observed. The percentages of isolates of Actinobacillus spp. and coagulase-positive staphylococci resistant to TMP-SXT increased from 0% to 17% and from 0% to 33%, respectively, (P < 0.05). The percentage of isolates of Actinobacillus spp resistant to penicilline also increased from 33% to 73% (P < 0.05). Regular monitoring of the evolution of bacterial resistance to antibacterial agents is indispensable in a hospital structure. The adoption of "good antibiotherapy practices" is essential to guarantee a decrease in the risks of selection and dissemination of a resistant bacterial strain.