Topical Administration of Spironolactone-Loaded Nanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits.

The objective was to investigate whether mineralocorticoid receptor antagonism using a novel topical micellar formulation of spironolactone could prevent glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. Spironolactone micelles (0.1%, w/v) with a mean number weighted diameter of 20 nm were prepared using a pegylated copolymer (mPEG-dihexPLA) and showed a preliminary stability of at least 12 months at 5 °C. Preclinical studies in New Zealand white rabbits demonstrated that the 0.1% spironolactone micellar formulation was well-tolerated since no reaction was observed in the cornea following multiple daily instillation over 5 days. As expected, the preclinical studies also confirmed that dexamethasone significantly delayed epithelial wound healing as compared to untreated control (percentage re-epithelialization after day 4: 84.6 ± 13.9% versus 99.5 ± 1.0% for the control, p < 0.05). However, the addition of the 0.1% spironolactone micellar formulation significantly improved the extent of re-epithelialization, countering the dexamethasone induced delayed wound healing with a percentage re-epithelialization that was statistically equivalent to the control (96.9 ± 7.3% versus 99.5 ± 1.0%, p > 0.05). The biodistribution study provided insight into the ocular metabolism of spironolactone and hence the relative contributions of the parent molecule and its two principal metabolites, 7α-thiomethylspironolactone and canrenone, to the observed pharmacological effects. Comparison of the efficacies of spironolactone and potassium canrenoate (a water-soluble precursor of canrenone) in overcoming the dexamethasone-induced delayed wound healing confirmed that the former had greater efficacy. The results pointed to the greater potency of 7α-thiomethylspironolactone over canrenone as a mineralocorticoid receptor antagonist, which explained its superior ability in countering the glucocorticoid-induced overactivation that was responsible for the delayed wound healing. In conclusion, the preliminary results supported the above-mentioned hypothesis suggesting that coadministration of mineralocorticoid receptor antagonists to patients under glucocorticoid therapy might prevent the deleterious effects of glucocorticoids on complex corneal wound healing processes.

Association between sensitisation and pain-related behaviours in an experimental canine model of osteoarthritis.

Evaluation of nociceptive sensitisation in canine osteoarthritis studies has been poorly reported, or even related to other clinical symptoms. In 16 dogs, peak vertical force (PVF), subjective pain assessment using 3 scales, sympathetic stress response with electrodermal activity (EDA) measurement, and behavioural changes with video analysis and telemetered motor activity were quantified at baseline (D-7), and 28 and 56 days post transection of the cranial cruciate ligament. As markers of central sensitisation, selected spinal cord biomarkers (substance P and transthyretin) were quantified at D56. Electrical withdrawal thresholds on the stifle and the tail were measured as indicative of peripheral and central quantitative sensory testing (QST) sensitisation, respectively. The effects of vehicle administration (n=8) were compared with tiludronate (2mg/kg subcutaneously, q2 week, starting at D0) administration. Generalized estimated equations tested the association between the behavioural and physiological methods and QST sensitisation, and therefore the sensitivity of the methods for detecting treatment efficacy. Compared to tiludronate, at D56, vehicle-treated dogs had increased spinal substance P (P=0.01), concomitant decreased transthyretin (P=0.02), and (compared to baseline) demonstrated peripheral and central QST sensitisation, which was not present for tiludronate. Only PVF, the spontaneous behaviour "walking with full weight-bearing," and EDA were associated with occurrence of QST sensitisation and indicated significant tiludronate analgesic efficacy after inclusion of central QST sensitisation as a predictor variable in the statistical model. This study establishes the strong interest to implement QST as a predictor of canine osteoarthritis pain symptoms explained by pain sensitisation.

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model.

INTRODUCTION: The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA). METHODS: Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE(2) and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05. RESULTS: A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE(2) (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone. CONCLUSION: Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Treatment with tiludronic acid helps reduce the development of experimental osteoarthritis lesions in dogs with anterior cruciate ligament transection followed by reconstructive surgery: a 1-year study with quantitative magnetic resonance imaging.

OBJECTIVE: to investigate over a 1-year period in dogs that underwent extracapsular stabilization surgery (ECS) following anterior cruciate ligament (ACL) transection: whether reconstructive surgery could prevent osteoarthritis (OA) progression and whether treatment with the bisphosphonate tiludronic acid (TA) could improve the chronic evolution of OA structural changes. METHODS: ACL transection was performed on dogs on Day 0 and ECS on Day 28. Dogs were randomly divided into 2 groups: 15 received placebo and 16 were treated with TA (2 mg/kg subcutaneous injection) on Days 14, 28, 56, and 84. Magnetic resonance images were acquired on Days -10, 26, 91, 210, and 357, and cartilage volume was quantified. At sacrifice (Day 364), cartilage from femoral condyles and tibial plateaus was macroscopically and histologically evaluated. Expression levels of MMP-1, -3, -13, ADAMTS-4, -5, BMP-2, FGF-2, IGF-1, TGF-ß1, collagen type II, and aggrecan were determined using real-time RT-PCR. RESULTS: the loss of cartilage volume observed after ACL transection stabilized following ECS. Thereafter, a gradual gain occurred, with the cartilage volume loss on the tibial plateaus reduced at Day 91 (p < 0.02) and Day 210 (p < 0.001) in the TA-treated dogs. At sacrifice, TA-treated dogs presented a reduction in the severity of macroscopic (p = 0.03 for plateaus) and histologic (p = 0.07 for plateaus) cartilage lesions, had a better preserved collagen network, and showed decreased MMP-13 (p = 0.04), MMP-1 and MMP-3 levels. CONCLUSION: our findings indicate that in dogs with ACL transection, ECS greatly prevents development of cartilage volume loss. Treatment with TA provided an additional benefit of reducing the development of OA lesions.