**4. Discussion**

The aim of the study was to determine fentanyl plasma concentrations in rabbits from patches applied at three different locations with the final aim to refine (postoperative) analgesia. A standard skin preparation and patch application protocol was applied to avoid inconsistent results as previously reported [16]. For the different locations, fentanyl plasma concentrations were measured, and practicability of patch application, maintenance and removal were assessed. To the authors' knowledge, the current study is the first to report fentanyl plasma concentrations in rabbits following patch application at different locations.

The results of this study indicate that the patch placed on the neck provided fentanyl plasma concentrations higher than 0.5 ng/mL from 6 to 72 h after application. This plasma concentration was reached earlier and peak concentrations were higher when the patch was applied on the neck compared to both ear sites. However, preparation of this location was the most complex due to the high density of fur. Additionally, animal handling was needed to check the patch adhesion on the skin. Furthermore, patch removal was accompanied by aversive reactions and lead to mild skin erythema. The cause of the erythema associated with patch application seemed to be due to the occlusive nature of the patch systems [24]. Cutaneous irritation was often encountered and limits the duration of time, a patch can be worn at a single site. However, the adhesive agen<sup>t</sup> on the tape may be causally related, too.

For patches applied on the outer ear surface analgesic plasma fentanyl concentrations were measured from 9 to 48 h, whereas skin preparation was easier than in group 3. We hypothesize, that a reason for the difference in fentanyl absorption might be owed to the influence of a difference in body temperature, as it is described in several former studies. In fact, a temperature of 40 ◦C can raise drug delivery up to one third [8,25]. Rabbits can regulate their ear blood flow, which might lead to a lower drug delivery due to a lower body temperature in contrast to the neck. Depending on how long potent analgesia is requested, the application on the outer surface of the ear pinna might still be relevant for the attachment of fentanyl patches. There is a study published by Christou et al. using sheep, in which the change of fentanyl patches after 24 and 72 h is performed to investigate the fentanyl

plasma concentrations thereafter [26]. It was shown that a patch change lead to an extended period of analgesia without a lack of insufficient pain managemen<sup>t</sup> in between and a higher peak concentration due to pre-loading. Additionally, the peak concentration increased in that study, although we assume that this does not represent an advantage, as long as a certain threshold plasma concentration is exceeded permanently. Most likely the incidence of side effects will increase with rising values.

An obvious finding of our study is that it is inappropriate to apply the fentanyl patch on the inner surface of the ear. While practicability was good and the upcoming burden for the rabbit low, fentanyl plasma concentrations were below the predefined threshold, which implies the unsuitability of this location. Low plasma concentrations in this group might be attributable to poor drug penetration due to endogenous and exogenous influences such as differing skin conditions as described in former studies [24,27]. Additionally, rate of manipulation was higher in this group, which probably favored detaching of the patches in 5 of 6 animals. Furthermore, fixation of the patch with tape was more difficult due to the concave anatomy of the ear pinna, which probably also facilitated detachment. However, frequent checks to ensure that the patch is still in place were necessary in all groups.

Individual peak values of animals of group 1 and 3 showed a high variability within the study period. Probably several individual factors, like different pH, variable depot formation in the stratum corneum and dermis, different body temperatures and differences in cutaneous blood flow, influenced this outcome as previously reported [24].

The neck has been already described for patch application in rabbits, but a detailed description of skin preparation and patch application process have not been reported [15]. In the present study, the method of application was accurately planned and described to avoid variations in drug absorption due to differences in site preparation. Riviere and Papich showed that abrasion removes the stratum corneum barrier and can alter the transdermal flux [24]. Foley and coworkers investigated plasma fentanyl concentrations in rabbits using different methods of hair removal. For rabbits with clipped hair, comparable to the study described here, they reported a peak of 1.11 ± 0.32 ng/mL at 24 h and a decrease to 0.77 ± 0.21 ng/mL 72 h after patch application [15]. This is lower than the peak values in this study, even though fentanyl patches with a half dose rate were used: 3.60 μg/kg/h in this study versus 6.67 μg/kg/h reported by Foley and coworkers. One possible explanation for the better uptake is the use of alcohol to degrease the skin and ameliorate the drug uptake in this study. Two other studies reported the use of alcohol to degrease the skin prior to patch application in sheep [8,26]. In other studies involving dogs and cats, no degreasing with alcohol was performed [18,28,29].

The main limitation of this study is that rabbit-specific fentanyl plasma concentration able to provide analgesia is unknown. In humans, the minimum effective plasma concentration for analgesia is assumed to range between 0.5 and 2.0 ng/mL [11]. Previous veterinary studies extrapolated this range to animals such as sheep, rabbits and minipigs [15,16,23]. In former human studies, wide ranges of 0.23 to 3 ng/mL were described [13,30]. In humans, contrasting evidence has been reported about the relationship between respiratory adverse effects and fentanyl plasma concentration. While in one study concentrations above 2.0 ng/mL were suggested to cause respiratory depression [31]. In another study no correlation between fentanyl plasma concentrations and side effects was found [32]. In our study, no obvious adverse effects were observed, although concentrations above 2.0 ng/mL were measured. However, there was no evaluation of heart rate and respiratory frequency, which are known among others to be influenced at an early stage [33]. In cats, a plasma concentration higher than 2.2 ng/mL was observed to cause dysphoric behavior [17]. No dysphoria was observed in the rabbits of the present study.

Further limitations of this study are the use of only female rabbits. In humans, no difference in fentanyl uptake between male and female patients has been shown. To our knowledge, this has not been investigated ye<sup>t</sup> in animals [34]. Another limitation is the use of a commercially available enzyme-linked immunosorbent assay (ELISA) developed to analyze human samples and no second test was performed to verify the results. In the literature the use of several different tests for analysis of fentanyl plasma concentration has been reported. A human ELISA was used in sheep, while no details

were provided about the ELISA method applied in the study by Gilbert et al. [16,26]. In contrast to this, tests as the liquid chromatography–MS and gas chromatography–MS methods served for analysis in humans as well as in sheep [16,30,35]. Besides these tests, radioimmunoassays (RIA) are commonly used for analysis of plasma or serum fentanyl concentrations, too [29,36].

Concerning the timing of fentanyl patch application in experimental settings, some additional aspects need to be considered. Based on the findings of the present study, at least 6 h are needed for the fentanyl patch to provide a potentially analgesic plasma concentration. This implies that additional analgesics are needed to cover this period if the patch is applied during preparation for surgery. Some authors recommend patch application 12–24 h prior to surgery in dogs and sheep to achieve su fficient analgesia for surgery [7,12,37]. However, earlier application also means more stress for the rabbit, as an additional handling session accompanied by sedation would be needed. Another important aspect is the desired duration of pain treatment with opioids, which will depend on the invasiveness of surgery as well as administration of other pain medication (e.g., NSAIDs). This has to be defined for each study or patient and adapted as needed. Patches can be removed earlier or a second patch could be applied to extend the analgesic e ffect. As already mentioned, the e ffects of a patch change were investigated in sheep [26], while no comparable studies have been performed in rabbits so far.
