Table_1_Histamine-Induced Conjunctivitis and Breakdown of Blood–Tear Barrier in Dogs: A Model for Ocular Pharmacology and Therapeutics.pdf

Conjunctival inflammation disturbs the blood–tear barrier and thus affects the tear film stability and composition. We aimed to develop a non-invasive and reliable method to induce conjunctivitis in dogs, a large animal model for translational work on ocular surface disease in humans. Six beagle dogs underwent a randomized, vehicle-controlled, balanced crossover trial—on six separate days, one eye received topical artificial tears (vehicle), while the other eye received one of six concentrations of histamine solution (0.005–500 mg/ml). At sequential times after eyedrop administration, a conjunctivitis score was given to each eye based on the degree of palpebral and bulbar conjunctival hyperemia and chemosis, ocular pruritus, and discharge. Total protein content (TPC) and serum albumin were quantified in tear fluid at baseline and 20 min. Additionally, 13 dogs presenting for various ophthalmic diseases with associated conjunctivitis were examined. Experimentally induced conjunctivitis developed rapidly (<1 min) following topical histamine administration and lasted for 1–3 h (four lowest doses) to 6–8 h (two highest doses). The severity of conjunctivitis was dose-dependent. Histamine was overall well tolerated, although transient blepharitis, aqueous flare, and ocular hypertension occurred in a few dogs receiving histamine ≥375 mg/ml. TPC and serum albumin levels increased in tears of eyes receiving histamine ≥1.0 mg/ml, being significantly higher than vehicle and baseline in eyes receiving histamine ≥375 mg/ml. Lacrimal albumin levels were also increased in 13 dogs with naturally acquired conjunctivitis, up 2.7–14.9 fold compared to contralateral healthy eyes. Histamine-induced conjunctivitis represents a robust model for translational work on the ocular surface given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring ocular diseases. Histamine solutions of 1, 10, and 375 mg/ml induce mild, moderate, and severe conjunctivitis in dogs, respectively. Leakage of serum albumin in tear fluid of eyes with conjunctivitis suggests a breakdown of the blood–tear barrier.</p

Data_Sheet_1_Kinetics of Fluorescein in Tear Film After Eye Drop Instillation in Beagle Dogs: Does Size Really Matter?.PDF

The study aimed to determine the impact of drop size on tear film pharmacokinetics and assess important physiological parameters associated with ocular drug delivery in dogs. Two separate experiments were conducted in eight healthy Beagle dogs: (i) Instillation of one drop (35 μl) or two drops (70 μl) of 1% fluorescein solution in each eye followed by tear collections with capillary tubes from 0 to 180 min; (ii) Instillation of 10 to 100 μl of 0.1% fluorescein in each eye followed by external photography with blue excitation filter (to capture periocular spillage of fluorescein) and tear collections from 1 to 20 min (to capture tear turnover rate; TTR). Fluorescein concentrations were measured in tear samples with a fluorophotometer. The TTR was estimated based upon non-linear mixed-effects analysis of fluorescein decay curves. Tear film pharmacokinetics were not superior with instillation of two drops vs. one drop based on tear film concentrations, residual tear fluorescence, and area under the fluorescein-time curves (P ≥ 0.163). Reflex TTR varied from 20.2 to 30.5%/min and did not differ significantly (P = 0.935) among volumes instilled (10–100 μl). The volumetric capacity of the canine palpebral fissure (31.3 ± 8.9 μl) was positively correlated with the palpebral fissure length (P = 0.023). Excess solution was spilled over the periocular skin in a volume-dependent manner, predominantly in the lower eyelid, medial canthus and lateral canthus. In sum, a single drop is sufficient for topical administration in dogs. Any excess is lost predominantly by spillage over the periocular skin as well as accelerated nasolacrimal drainage.</p

RAS-Fingerprint^™ analytes at baseline (combined D0 and D21) and post-treatment (D7 and D28 combined) with spironolactone at 2 mg/kg/day and 4 mg/kg/day combined at 07:00 (T1) in 10 healthy purpose-bred Beagle dogs.

There was a significant increase in aldosterone (P Table 3). Spheres show relative concentrations of angiotensin peptides. Blue spheres generally signify that the angiotensin peptide’s action is inert, red indicates predominantly vasoconstrictive and pro-fibrotic effects, and green indicates vasodilatory and anti-fibrotic actions. Enzymes are shown as blue connecting lines between peptides.</p

Data_Sheet_1_Albumin in Tears Modulates Bacterial Susceptibility to Topical Antibiotics in Ophthalmology.PDF

Bacterial keratitis is a serious and vision-threatening condition in veterinary and human patients, one that often requires culture and susceptibility testing to adjust therapy and improve clinical outcomes. The present study challenges the antimicrobial susceptibility testing (AST) paradigm in ophthalmology, enabling more accurate in vitro-to-in vivo translation by incorporating factors normally present during host-pathogen interactions in clinical patients. Thirty bacteria (10 Staphylococcus pseudintermedius, 10 Streptococcus canis, 10 Pseudomonas aeruginosa) were isolated from canine patients with infectious keratitis. For each isolate, commercial plates (Sensititre™ JOEYE2) were used to assess the minimal inhibitory concentration (MIC) of 17 different antibiotics in the absence (0% albumin, control) or presence of canine albumin (0.01–2%). For Staphylococcus pseudintermedius, the experiment was repeated with actual tear fluid collected from canine eyes with ocular surface inflammation. Kruskal-Wallis, Wilcoxon signed rank test and Spearman's correlation tests were used for statistical analysis. Clinical outcomes were unfavorable in selected canine patients with bacterial keratitis (e.g., globe perforation, graft dehiscence) despite standard AST (i.e., 0% albumin in test medium) confirming that most corneal infections (93%) were susceptible to ≥1 topical antibiotics used at the initial visit. Albumin levels ≥0.05% increased MICs in a dose-dependent, bacteria-specific, and antibiotic-specific manner. No significant differences (P = 1.000) were noted in MICs of any antibiotic whether albumin or tear fluid was added to the Mueller-Hinton broth. Percent protein binding inherent to each antibiotic was associated with clinical interpretations (Spearman's rho = −0.53, P = 0.034) but not changes in MICs. Albumin in tears impacted the efficacy of selected ophthalmic antibiotics as only the unbound portion of an antibiotic is microbiologically active. The present findings could improve decision making of clinicians managing bacterial keratitis, reduce development of antimicrobial resistance, influence current guidelines set by the Clinical and Laboratory Standards Institute, and serve as a reference for bacteriological evaluations across medical fields and across species.</p

Effect of spironolactone dosage (combined 2 mg/kg/day and 4 mg/kg/day) on RAS-Fingerprint^™ analytes in 10 healthy purpose-bred Beagle dogs at baseline (combined D0 and D21) and post-treatment (combined D7 and D28) using a cross-over study design at 07:00 (T1) prior to morning dosing.

Data are presented as median (IQR) in pM/L for AngI, AngII, aldosterone, Ang1-7, Ang1-5, AngIII, AngIV, and PRA-S and as a ratio for ACE-S, AA2, and ALT-S. P value compares overall treatment to baseline.</p

Effect of spironolactone dosage (2 mg/kg/day vs. 4 mg/kg/day) on RAS-Fingerprint^™ analytes in 10 healthy purpose-bred Beagle dogs at combined baseline (D0 T1, D0 T2, D21 T1, and D21 T2) and post-treatment (D7 T1, D7 T2, D28 T1, and D28 T2) using a cross-over study design.

Data are presented as median (IQR) in pM/L for AngI, AngII, aldosterone, Ang1-7, Ang1-5, AngIII, AngIV, and PRA-S and as a ratio for ACE-S, AA2, and ALT-S. P value represents comparison of percent change from baseline between the two dosing groups.</p

RAS-Fingerprint^™ analytes on Day 21 of the study (baseline) vs. Day 28 of the study (post-treatment with spironolactone at 2 mg/kg/day and 4 mg/kg/day combined) at 07:00 (T1) in 10 healthy purpose-bred Beagle dogs.

There were significant increases in AngII, AngI, Ang1-5, and PRA-S (P Table 4). Spheres show relative concentrations of angiotensin peptides. Blue spheres generally signify that the angiotensin peptide’s action is inert, red indicates predominantly vasoconstrictive and pro-fibrotic effects, and green indicates vasodilatory and anti-fibrotic actions. Enzymes are shown as blue connecting lines between peptides.</p

Effect of spironolactone treatment (combined 2 mg/kg/day and 4 mg/kg/day) in 10 healthy purpose-bred Beagle dogs using a cross-over study design on select serum biochemistry variables and SAP between baseline (combined D0 and D21) and treatment (combined D7 and D28) at 07:00 (T1; prior to feeding or morning dosing).

Effect of spironolactone treatment (combined 2 mg/kg/day and 4 mg/kg/day) in 10 healthy purpose-bred Beagle dogs using a cross-over study design on select serum biochemistry variables and SAP between baseline (combined D0 and D21) and treatment (combined D7 and D28) at 07:00 (T1; prior to feeding or morning dosing).</p

Table_1_Exploring the benefits of in-diet versus repeated oral dosing of saracatinib (AZD0530) in chronic studies: insights into pharmacokinetics and animal welfare.DOCX

Saracatinib/AZD0530 (SAR), a Src tyrosine kinase inhibitor, mitigates seizure-induced brain pathology in epilepsy models upon repeated oral dosing. However, repeated dosing is stressful and can be challenging in some seizing animals. To overcome this issue, we have incorporated SAR-in-Diet and compared serum pharmacokinetics (PK) and brain concentrations with conventional repeated oral dosing. Saracatinib in solution or in-diet was stable at room temperature for >4 weeks (97 ± 1.56%). Adult Sprague Dawley rats on SAR-in-Diet consumed ~1.7 g/day less compared to regular diet (16.82 ± 0.6 vs. 18.50 ± 0.5 g/day), but the weight gain/day was unaffected (2.63 ± 0.5 g/day vs. 2.83 ± 0.2 g/day). Importantly, we achieved the anticipated SAR dose range from 2.5–18.7 mg/kg of rat in response to varying concentrations of SAR-in-Diet from 54 to 260 ppm of feed, respectively. There was a strong and significant correlation between SAR-in-Diet dose (mg/kg) and serum saracatinib concentrations (ng/ml). Serum concentrations also did not vary significantly between SAR-in-Diet and repeated oral dosing. The hippocampal saracatinib concentrations derived from SAR-in-Diet treatment were higher than those derived after repeated oral dosing (day 3, 546.8 ± 219.7 ng/g vs. 238.6 ± 143 ng/g; day 7, 300.7 ± 43.4 ng/g vs. 271.1 ± 62.33 ng/g). Saracatinib stability at room temperature and high serum and hippocampal concentrations in animals fed on SAR-in-Diet are useful to titer the saracatinib dose for future animal disease models. Overall, test drugs in the diet is an experimental approach that addresses issues related to handling stress-induced variables in animal experiments.</p

Effect of spironolactone dosage (combined 2 mg/kg/day and 4 mg/kg/day) in 10 healthy purpose-bred Beagle dogs using a cross-over study design on RAS-Fingerprint^™ analytes at each sampling period at 12:00 (T2) 5-hours after oral dosing of spironolactone.

The sampling periods include two baseline sampling periods at Day 0 (D0) and Day 21 (D21) as well as following two seven-day treatment periods at Day 7 (D7) and Day 28 (D28). Data are presented as median (IQR) in pM/L for AngI, AngII, aldosterone, Ang1-7, Ang1-5, AngIII, AngIV, and PRA-S and as a ratio for ACE-S, AA2, and ALT-S. P value compares overall treatment to baseline at each specific timepoint (D0 vs. D7 and D21 vs. D28).</p