Preliminary study on carprofen concentration measurements after transcutaneous treatment with Vetdrop® in a microfracture joint defect model in sheep

Nonsteroidal anti-inflammatory drugs (NSAID’s) are commonly used in human and veterinary medicine to treat inflammatory processes and to relieve mild to moderate pain. Most NSAIDs inhibit the activity of the cyclooxygenase enzymes (COX1 and COX2), which produce prostaglandins. Prostaglandins are important factors for the pathogenesis of inflammation, swelling, pain and fever [1]–[3]. These prostaglandins also fulfil various physiological functions, such as mucosal defence through prevention and promotion of healing of mucosal erosions. The administration of NSAID’s can be accompanied by miscellaneous side effects, ranging from gastrointestinal mucosal damage, aplastic anaemia, and inhibition of thrombocyte aggregation to increased cardiovascular risks and renal failure [4]–[7]. Transcutaneous application of NSAID’s could potentially be beneficial when treating circumscribed inflammatory processes, due to the fact that systemic concentrations of the drugs are lower, thereby reducing the incidence of side effects. This concept is supported by findings from previous studies where the topical application of NSAID’s led to lower plasma levels as compared to systemic administration, whilst still maintaining efficacy [8]–[10].

The skin constitutes a barrier for nocive influences and therapeutic agents. Therefore, much effort has been invested into overcoming this barrier for therapeutic purposes. As such, the development of carriers to aid in the transport of therapeutic compounds across such barriers represents a possible way of skin penetration [11].

MedDrop Technology AG (Thundorf, Switzerland) has developed a treatment system (Vetdrop®) employing carriers for transcutaneous application of pharmaceutical agents, including the NSAID carprofen.

Carprofen was used in this study based on its capacity to provide analgesia after orthopaedic procedures [12],[13] in sheep. In addition, carprofen may have a positive influence on the healing of cartilage in low concentrations [14]. Benton et al. [15] showed in an in vitro study that the administration of 1 and 10 μg/ml carprofen resulted in significantly higher glycosaminoglycan synthesis and cartilage matrix production, whereas a concentration of 20 μg/ml carprofen blocked this effect.

The current study was based on the hypothesis that the Vetdrop® Technology would allow the transcutaneous transport of carprofen into the stifle joint, which should be measureable in synovial fluid samples after application.

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.

In this study, we concentrate on reporting of the results of the carprofen receiving Groups 3 (VECA) and 4 (VECHCA) and show that the Vetdrop® System allows for transcutaneous delivery of carprofen. The results of the other 3 Groups are not addressed because they did not receive carprofen, but their results are reported elsewhere (Dissertation Nathalie Fouché, 2012).

In Groups 3 (VECA) and 4 (VECHCA) detectable carprofen concentrations were found in synovial fluid of the stifle as well as in the systemic blood circulation, although systemic concentrations were lower compared to control animals treated with carprofen intravenously.

The concentration of carprofen and some pharmacokinetic values could be determined, but direct comparisons with the control group (iv. application) were not considered totally appropriate. Pharmacokinetic calculations are based on the assumption that drugs are administered into a central compartment and subsequently distributed into peripheral compartments. This sequence is inverted with transcutaneous administrations and as such, pharmacokinetic algorithms may not fully apply in the case of transcutaneous delivery. Nevertheless, the standard pharmacokinetic formulas may help to interpret the data.

Factors, such as anatomical site, age, sex and skin disease influence the consistency of the epidermis and consequently the ability of substances to penetrate the skin barrier. Furthermore, drug delivery is also dependent on the pathways of skin permeation. Generally, it is considered that drug components can either pass the epidermis through a combination of transport routes including appendages or via a trans- or an intracellular route, and that the relative contribution of each of these is dependent on the physicochemical properties of the formulation and the properties of the skin.

Carprofen is a commonly used NSAID in veterinary medicine and was thus considered an ideal study material for the purposes of the current study. It has strong analgesic, antipyretic and antiphlogistic effects [17]–[19], although the exact mechanism of action of carprofen is not yet clear [20]. In vitro assays performed, using canine and equine chondrocytes demonstrated carprofen as having an inhibitory effect on prostaglandin-E2-synthesis [15],[21].

In our study, both Groups 3 (VECA) and 4 (VECHCA) reached the peak plasma level (57.1 – 342 ng/ml) between 18 – 20 hours after the first application.

In a transdermally administered fentanyl patch (2.05 μg/kg/h) study in 21 sheep, Ahern et al. [22] observed peak plasma concentrations between 4 and 24 h (0.62 – 2.73 ng/ml). Although the peak plasma concentration levels in the current study were wide ranging, they were always reached.

After the 2^nd application, peak plasma level rose up to 5.7-fold higher (up to 572 ng/ml) as compared to the initial application. This indicated that carprofen accumulated in the body. Nevertheless, the measured plasma concentration was consistently lower as compared to the intravenous administration of carprofen (up to 42 μg/ml, 10 min after 3^rd application, = 48 h after the 1^st application).

The carprofen concentration in the synovial fluid of group 3 (VECA) reached between 48 – 49% of the carprofen plasma level and in group 4 (VECHCA) 58 -59%. Group 4 (VECHCA) accumulated noticeably more carprofen in the synovial fluid as compared to group 3 (VECA). This effect may have been related to the additional chito-oligosaccharids in group 4 (VECHCA).

Analysing transdermally administered pharmaceutical agents raises the question as to whether the active ingredients reach their target location through direct penetration of the target tissue or through redistribution from systemic blood circulation Mills et al. [23] stated that topically applied NSAIDs can penetrate into deeper tissues and into the synovial fluid, and that local blood redistribution is contributing to this effect. They suggested that certain NSAIDs might increase the local blood flow and assist in transporting the applied drugs deeper into the tissue prior to their distribution into the systemic blood circulation. Indeed, Singh and Roberts [24] demonstrated that local tissue penetration of NSAIDs was possible up to a depth of 3 – 4 mm. However, Radermacher et al. [25] observed that the distribution of topically applied diclofenac into the synovial fluid was achieved through the blood supply and that direct penetration was minimal.

In this preliminary study we showed that the measured synovial concentration was always lower than the plasma concentration. Nevertheless, carprofen reached concentrations in the joint that are considered as being admissible for long-term therapy. Whether carprofen is transported directly into the joint or redistributed after systemic circulation is not yet clear and further investigations are needed. Therefore, in a main study a larger pharmacokinetic analysis needs to be set up.