The PhD thesis of Claire Underwood from the University of Queensland School of Veterinary Science, overseen by Chris Pollitt, Anton Middelberg and Simon Collins, was recently released online. The research is titled “Drug delivery to equine digital lamellar tissue.”
Abstract
Laminitis, a serious and debilitating disease of the equine foot, is a significant cause of wastage in the equine industry. There is no scientifically validated treatment and laminitis prophylaxis is currently limited to digital cryotherapy. Several key steps in laminitis pathophysiology have been elucidated, resulting in identification of drugs that may prevent laminitis (anti-laminitis drugs (ALDs)). Many of these pharmaceuticals are not suitable for systemic delivery but may be amenable to either local or nanoparticle delivery mechanisms. The aim of this thesis was to evaluate local, systemic and nanoparticle delivery mechanisms,using the matrix metalloproteinase (MMP) inhibitor marimastat, to establish a method of drug delivery that yields sustained therapeutic lamellar concentrations for experimental and potential clinical use.
Firstly, a method was developed for collecting lamellar interstitial fluid using ultrafiltration to enable determination of tissue drug concentrations. The ultrafiltration probe placement technique was developed using 16 cadaver limbs. Subsequently probes were placed in the lamellar tissue of 6 living horses. Lamellar ultrafiltrate (LUF) was collected continuously for 5 days at 55 [30-63] μL/h (median [interquartile range]).
In order to establish marimastat concentrations necessary for inhibition of lamellar MMPs,zymography was performed using lamellar homogenates incubated in increasing concentrations of marimastat. Based on this assay, target marimastat concentrations of 177 ng/mL were set (the concentration necessary to inhibit 90% of lamellar MMP-2 and MMP-9).
Four delivery mechanisms were evaluated for lamellar drug delivery; intraosseous infusion of the distal phalanx (IOIDP), constant rate systemic intravenous infusion (CRI), regional limb perfusion (RLP) and systemic intravenous bolus (SIVB). IOIDP and CRI with 0.53 mg/minute marimastat were performed on 5 horses. Steady-state lamellar ultrafiltrate marimastat concentrations (LUF[M]) were 139 [88-497] ng/mL and 136 [93-157] ng/mL respectively. LUF[M] were not consistently above those considered sufficient to inhibit lamellar MMPs in vivo. SIVB and RLP of 0.23 mg/kg marimastat were administered to 6 horses. The maximum LUF[M] were 264 [195-335] ng/mL and 61,200[16,713-67,746] ng/mL respectively. LUF[M] were greater than those considered necessary to inhibit lamellar MMPs for at least 6 h after RLP in all horses. Therefore, RLP was identified as the most suitable technique for administration of marimastat during experimentally induced laminitis.
In a following study, the suitability of RLP and lamellar ultrafiltration as a model for assessing the efficacy of candidate ALDs was investigated. Oligofructose was administered to 10 horses to induce laminitis. Treatment horses received 140 mg marimastat by RLP every six hours (n=6), control horses received saline by RLP every 6h. LUF[M] were consistently above the concentration considered necessary to inhibit lamellar MMPs in vivo. Therefore, RLP with concurrent measurement of LUF[M] was a suitable investigative model for delivery and quantification of marimastat concentrations in lamellar tissue during experimental laminitis development and may be suitable to investigate candidate ALDs. Marimastat did not prevent laminitis pathology in the oligofructose model, which suggests that proteases within the inhibitory spectrum of marimastat are not the key factor initiating laminitis pathology.
Nanoparticles can be harnessed for targeted drug delivery resulting in sustained high drug concentrations at the target site. They may offer a more clinically applicable solution for lamellar drug delivery. Production of a tailor-made nanoparticle- formulation for sustained local delivery of marimastat was investigated. Marimastat was loaded into 2 nanoparticle formulations; nanoemulsions and mesoporous silica nanoparticles. Loading efficiency was either low or the marimastat was rapidly released, and was thus deemed unproductive for further studies. The biodistribution of 99mTc-labelled liposomes was investigated to evaluate whether passively targeted nanoparticles have potential for lamellar drug delivery. 8.5 GBq 99mTc-liposomes were administered intravenously to 6 horses after dosing with oligofructose to induce laminitis and to 4 control horses. The percentage of the injected dose of liposomes that accumulated in the lamellar tissue of laminitis horses was significantly higher compared to controls (0.21 [0.14-0.3]% vs. 0.07 [0.06-0.11]%, p=0.02). The results of this study indicated liposomes have potential for sustained drug delivery to lamellar tissue. If necessary, once a successful ALD is identified, subsequent work should focus on utilising nanoformulations to optimise delivery, for clinical application.
The novel research described in this thesis demonstrated RLP was a suitable method for locally delivering candidate ALDs and developed ultrafiltration for sampling lamellar IF to ensure attainment of therapeutic lamellar concentrations. Although marimastat was not successful in preventing laminitis, the same investigative model can now be applied to evaluate the efficacy of alternative candidate ALDs. When laminitis pathogenesis can be effectively arrested by the efficacious delivery of a safe, potent ALD genuine progress will have been made.
