The study was conducted according to the Swiss legal requirements for animal protection and welfare (TschG 455) and received ethical approval by the federal veterinary authorities ‘Kantonale Tierversuchskommission Zürich’ (permission No 193/2009). It was part of a larger-scale project published and described elsewhere [16]. In brief, 28 sheep with a mean age of 2.5 ± 0.5 years and a body weight average of 59.4 ± 10.2 kg underwent microfracture surgery on the medial condyle either on the left or right stifle joint.
The animals were allocated to 6 different treatment groups. Groups 1 – 5 were treated using the Vetdrop® system and group 6 received carprofen intravenously. The transcutaneous treatment system (Vetdrop®) was developed for the transcutaneous application of pharmaceutical substances. The system consists of an oxygen generator and an application system, which is used in conjunction with specially developed vehicle solutions. The oxygen generator extracts oxygen from the atmosphere and this highly concentrated oxygen serves as a propellant. The oxygen is first stored in a pressure container and during treatment, the oxygen flows through a pressure reducing valve and a treatment tube to the application device.
The applicator serves as a nano-dispersion-device consisting of a drug reservoir, which lies within a gas tank. The pharmaceutical ingredients are filled into the drug reservoir through a port, surrounded by the oxygen gas tank. The oxygen combines with carrier substance under pressure and propels it through the diffuser. The diffuser assembly utilizes the Venturi-Effect (fluid pressure decreases in response to a constricted area of flow), in order to disperse the carrier. The size of the droplets lies in the range of nanometers. The size is regulated via a needle lace, which alters the width of the port.
The employed carriers are a proprietary product of Arvine Pharma AG (MedVital Serum, Arvine Pharma AG, Thundorf Switzerland). The vehicles are based on oil-in-water or water-in-oil carriers. The active ingredients are incorporated in water and oil phases.
Group 1 (VE) was treated with only vehicle, group 2 (VECH) with vehicle and chito-oliogosaccharids (2%), group 3 (VECA) with vehicle and carprofen (5.6%, total dose 0.5 – 0.6 mg/kg), group 4 (VECHCA) with vehicle, chito-oligosaccharids (2%) and carprofen (6.7 ± 15%, total dose 0.5 – 0.6 mg/kg), group 5 (S) was transcutaneously sham treated and group 6 (CA) was intravenously treated with carprofen (5%). This group received once per day 4 mg/kg BW carprofen intravenously (Rimadyl®, Pfizer AG, Zürich, Switzerland) during 4 days.
The transcutaneously treated groups were subjected to 18 applications, 3 applications per week at 15 minutes each for a total of six weeks (Figure 1). For drug delivery, the applicator was held in a distance of approximately 1 cm from the skin in an angle of 90° degrees. The areas of skin around the stifle joint were treated in an area of approximately 10 cm2 on the medial side of the limb, medial to the surgical wound and an equally sized area lateral to the surgical wound.
For the measurements of carprofen concentration presented here, 2 sheep from groups 3 (VECA), 4 (VECHCA) and 6 (CA) were included. The first transcutaneous treatment was performed one day prior to surgery (Day 0). The day of surgery was termed day 1. The transcutaneous treatments were performed on days 0, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33, 36, 38 and 40. After the first two transcutaneous treatments (day 0 and 3) the blood samples were taken from groups 3 and 4 at −5, 15, 20, 30, 60 minutes and 2, 3, 6 and 18 hours in relation to the application. In addition, blood samples were taken on days 5, 12, 19, 26, 33 and 40, always 6 hours after the transcutaneous application.
Synovial fluid was collected during surgery and afterwards once a week during 6 weeks using ultrasound guidance (Figure 2). For this purpose the sheep were sedated with Medetomidine (20ug/kg BW, Dorbene, Dr. E. Graeub AG, Switzerland). They were placed on their hindquarters and positioned in a more lateral position in order to expose the medial articular pocket of the limb that underwent surgery.
Additionally, blood samples were taken for comparison. The blood samples from the intravenously treated group 6 (CA) were taken on day 1, 2, 3, 4, and 5 at time points −5, 10, and 60 minutes and 3, 6 and 12 hours after the iv. carprofen administration. Two further blood samples were taken on day 8 and 12.
All blood samples were taken from the jugular vein. The first 4 ml of blood was discarded and the remaining 6 ml of blood was filled into a lithium heparin tube and centrifuged for 20 minutes at 1500 U/min. The resulting plasma was transferred into labelled sterile 1.5 ml eppendorf tubes and stored at −20°C for batch analysis.
Synovial fluid samples were obtained using a 5 ml syringe (Braun, Omnifix®) and a 18 G needle (Terumo®, Terumo Europe N.V., Belgium) and were stored in pre-labelled sterile 1.5 ml eppendorf tubes. Each sample was wrapped in aluminium foil and stored at −20°C for later batch analysis (Interlabor Belp AG, Belp, Switzerland). The carprofen concentrations were measured using high performance liquid chromatography (HPLC) in connection with Tandem - Mass Spectroscopy (HPLC-MS/MS). The liquid chromatography (Dionex UltiMate 300, Software Dionex Chromatography MS Link Vers. 2.7.0.2251) was used to separate the mixture of compounds into single components and the Mass-Spectroscopy (Triple Quadrupol Sciex API 4000, Software Analyst Vers. 1.5.1) to identify and quantify the components.