Absorption of Transdermal and Oral Cyclosporine in Six Healthy Cats

Introduction

Cyclosporine is a drug commonly used in veterinary dermatology that has proven efficacy in the treatment of canine atopic dermatitis.^1–3 Cyclosporine has also been used for the treatment of several feline dermatoses, including allergic dermatitis.^4–6 As a cyclic oligopeptide macrolide, cyclosporine inhibits activation of numerous cells that produce cytokines involved in allergic reactions and the cutaneous immune response.^1–9 More specifically, interleukin-2 production is inhibited, thereby decreasing activation and proliferation of T lymphocytes and decreasing their associated immune responses.⁹ Cyclosporine is available in capsule and liquid formulation but either the feline patient’s behavior or temperament can often make it difficult for an owner to administer either preparation.^10,11 Transdermal medications presumably avoid that obstacle and provide the additional advantages of avoidance of first-pass hepatic metabolism and decreased gastrointestinal side effects.^10–12 The ideal transdermal medication should be nonirritating to the skin, have a low molecular weight (approximately 500 Daltons), with a low melting point for increased solubility in skin lipids, and a relatively high (but balanced) partition coefficient.^11–15 Adequate solubility in oil and water is optimal for transdermal medications.^11–15 Cyclosporine is cyclic in structure, lipophilic, has a molecular weight of 1,202 Daltons, and has a relatively high melting point, as well as an unbalanced partition coefficient.^12,16,17 Those characteristics led to the hypothesis that cyclosporine may not be systemically absorbed after transdermal application. Transdermal and topical formulations of cyclosporine have shown little systemic and local absorption in humans, but in vitro and in vivo murine studies with penetration enhancers have shown increased absorption.^{12–14,16,18–21} Among the penetration enhancers that appear to facilitate transdermal delivery are lecithin vesicular carriers.¹⁶

Anecdotally, transdermally applied cyclosporine has been used to treat feline dermatoses. In 2009 and 2010, a large compounding pharmacy that provides a national internet service compounded 127 and 123 prescriptions for transdermally applied cyclosporine written by 32 and 33 veterinarians, respectively (written communication, Bruce Dell, BS, RPh, Roadrunner Pharmacy, Phoenix, AZ). To the authors’ knowledge, there is no scientific evidence to support the use of transdermal cyclosporine in cats.

The objective of this study was to compare cyclosporine blood concentrations measured after multiple doses of a transdermally applied cyclosporine compounded in a penetration enhancer, pluronic lecithin organogel (PLO), versus oral administration of the FDA-approved canine cyclosporine product in six healthy cats. The hypothesis was that the median cyclosporine blood concentrations achieved in cats receiving cyclosporine in a compounded transdermally applied PLO gel would achieve < 25% of concentrations achieved after oral administration.

Materials and Methods

This prospective, positively controlled, cross-over study was performed from August 2010 to September 2010 at a private practice specialty veterinary hospital located in Salt Lake City, Utah. Six client-owned, apparently healthy cats housed strictly indoors participated in the study. This study was approved by the medical directors of the private practice hospital where it was performed. Written informed consent was provided by each owner. Prior to drug administration, each cat was deemed healthy based on history, physical examination, complete blood count, serum biochemistry analysis, and urinalysis. All cats were negative for immunoglobulins G and M Toxoplasma gondii antibodies by enzyme-linked immunosorbent assay. No changes of diet or environment were allowed for any of the cats during the study. Cyclosporine^a capsules at a dose of approximately 5 mg/kg were given orally q 24 hr for 7 days. The FDA approved canine cyclosporine was used for this study because feline-approved cyclosporine was not available at the time this study was performed. The actual oral dose varied based on the limitation of capsule size (25 mg). After 7 days of oral cyclosporine administration, each cat underwent a 2 wk washout period where no cyclosporine was administered. This washout period was considered adequate based on the elimination half-life of cyclosporine in cats (8.2 hr).²²

Cyclosporine was applied transdermally for 21 days in the second phase of the study. A gel was prepared with cyclosporine as a pure powder substrate^b and was mixed into PLO^c. A single preparation of the transdermal cyclosporine was used throughout the study. The PLO components were mixed with cyclosporine with geometric dilution by a compounding pharmacist (Bruce Dell, BS, RPh, Roadrunner Pharmacy, Phoenix, AZ) 1 wk prior to distribution. The transdermal cyclosporine was formulated following guidelines set by the Professional Compounding Centers of America.²³ The finished preparation was labeled with a beyond use date of 180 days after preparation. The target concentration of the final product was 250 mg/mL, and the product was dispensed in 1 mL syringes. The applied dose was intended to deliver 25 mg of cyclosporine, a dose equivalent to the oral dose, in 0.1 mL of gel.

Each dose was applied by the owner to the nonhaired portion of the pinna at the same time of the day (q 24 hr). Pinnae were alternated with each application. Owners were instructed to wear gloves provided by the author (R.M.) and to wash their hands after each application. The medication was to be stored in a cool, dry environment at room temperature (owners were instructed not to refrigerate the medication). The time and site of medication application was recorded each day in a medication log provided to all owners. Owners were also asked to log any possible adverse events associated with drug administration, including vomiting, diarrhea, lethargy, or irritation to the pinna.

Whole blood samples were collected 2 hr and 12 hr after the cyclosporine administration in ethylenediaminetetraacetic acid tubes by venipuncture using manual restraint in all cats after 7 days of orally administered cyclosporine and again after 7 days, 14 days, and 21 days of transdermally applied cyclosporine. Samples also were collected after the 2 wk oral drug washout period, which was prior to initiating the transdermal phase of the study. Samples were shipped on ice overnight within 24 hr of collection to a laboratory^d and were analyzed within 48 hr of arrival. Ethylenediaminetetraacetic acid whole blood samples were brought to room temperature then mixed by multiple inversions to assure homogeneity. Cyclosporine was detected in feline whole blood using an immunoassay^e on a general chemistry analyzer^f. The lower limit of quantitation was 25 ng/mL, and the upper limit was 2,100 ng/mL, with the ability to dilute up to 6,000 ng/mL. The coefficient of variation values were < 15% for the low range and < 5% in the high range for each assay performed.

To confirm the labeled concentration of the transdermally applied cyclosporine, the concentration of cyclosporine was evaluated in three randomly chosen samples of the transdermal cyclosporine. Gel samples were diluted with ethanol and water to the appropriate concentration (based on the calibration curve) then mixed with whole blood prior to quantitation using the same immunoassay indicated above.

Results

Study participants ranged in age from 1 yr to 7 yr, with a median of 18 mo. Four of the cats were spayed females and two were castrated males. Three cats were domestic shorthairs, two were domestic mediumhairs, and one was a domestic longhair. The cats’ weights ranged from 3.4 kg to 4.9 kg, with a median of 4.4 kg. The transdermally applied and orally administered cyclosporine doses ranged from 5.1 mg/kg to 7.4 mg/kg (median, 5.7 mg/kg), with an equivalent oral and transdermal dose for each individual cat.

Three randomly chosen 1 mL syringes of the cyclosporine compounded for transdermal application were analyzed to determine the actual concentration. The target concentration was 250 mg/mL. The mean cyclosporine concentration (± standard deviation) was 231 mg/mL (± 10 mg/mL), and the percent of the target concentration was 92.5% (± 4%). The concentration for each syringe was 228 mg/mL, 223 mg/mL, and 243 mg/mL, with the percent of the target concentration equaling 91%, 89%, and 97%, respectively. The actual average concentration of those randomly sampled transdermal medications was within 10% of predicted, although concentrations were consistently lower than target.

No cat exhibited any adverse events during the study, including irritation to the pinna, which was evaluated daily by the owner and weekly by one of the authors (R.M.). Verbal assurance was provided by each owner that the medication was applied properly, including amount and application site, and that the cyclosporine used for transdermal application was properly stored.

Cyclosporine blood concentrations measured 2 hr and 12 hr after oral administration and transdermal application of cyclosporine for each cat at all time points have been shown in Table 1. Cyclosporine blood concentrations were greater at 2 hr and 12 hr after oral administration than cyclosporine blood concentrations at 2 hr (P = 0.0051) and 12 hr (P = 0.0051) after transdermal application of cyclosporine on both day 7 and day 21. The median cyclosporine blood concentrations at 2 hr (55 ng/mL) and 12 hr (38 ng/mL) after transdermally applied cyclosporine was 2.5% and 3.1% of the median cyclosporine blood concentrations at 2 hr (2,208 ng/mL) and 12 hr (1,232 ng/mL) after orally administered cyclosporine, respectively. Regarding cyclosporine blood concentrations after transdermal administration, a significant difference could not be detected between either cyclosporine blood concentrations at 2 hr (P = 0.30) or 12 hr (P = 0.38) after transdermal application of cyclosporine when comparing day 7 to day 21, and no difference could be detected between cyclosporine blood concentrations at 2 hr and 12 hr (P = 0.270) on day 21.

TABLE 1 Cyclosporine Blood Concentrations After Oral and Transdermal Cyclosporine Administration in Six Cats*

*Cyclosporine blood concentrations (ng/mL) were measured at 2 hr and 12 hr following 7 days of orally administered cyclosporine after a 2 wk washout period, and 7 days, 14 days, and 21 days following transdermal administration.

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Discussion

This study demonstrates that consistently low cyclosporine blood concentrations were achieved in the majority of cats after transdermal application of cyclosporine in PLO. In contrast, the cyclosporine blood concentrations 2 hr and 12 hr after orally administered cyclosporine were consistently detected and were well above recommended concentrations in all six cats. That data support the authors’ hypothesis. Specifically, median cyclosporine blood concentrations either 2 hr or 12 hr after 7 days and 21 days of transdermal application of cyclosporine were not within 25% of the cyclosporine blood concentrations achieved either 2 hr or 12 hr after 7 days of orally administered cyclosporine. Only 3 of the 36 cyclosporine blood concentrations evaluated during the 3 wk period of transdermal application reached the target range of 25% of the cyclosporine blood concentrations achieved after oral administration. That target was reached by cat 2 on day 14 of the transdermal application of cyclosporine 12 hr after transdermal application and cat 6 on day 21 of the transdermal application of cyclosporine 2 hr and 12 hr after transdermal application (Table 1). The characteristics of cyclosporine that make it a poor choice for transdermal delivery are likely the reason for inadequate cyclosporine blood concentrations in the remaining cats after transdermal application of cyclosporine in this study.

At the end of the study, the owner of cats 2–4, which were housed together, revealed that allogrooming was observed between the cats. Cats 2 and 4 were observed grooming each other on day 20 of the transdermal application of cyclosporine. The owner reported that allogrooming did not occur directly after application of the transdermal medication. Allogrooming was allowed as this would replicate a “real-life situation” in the home environment. Excluding the spuriously high result on day 14 in cat 2, the cyclosporine concentrations were consistently low in five of the six cats that received the transdermal formulation, indicating minimal absorption of transdermally applied cyclosporine.

Cat 6 was a 7 yr old spayed female domestic shorthair that was housed individually. Cat 6’s cyclosporine blood concentrations after transdermal application and oral administration of cyclosporine were consistently higher than the other five cats. Determining the basis for the marked variability in cyclosporine blood concentrations, particularly with transdermal application, is difficult. Both absorption and metabolism of cyclosporine are variable in humans, dogs, and cats after oral administration.^22,24 The added factor of transdermal application further contributes to the variability. One possible explanation for cat 6’s higher cyclosporine blood concentrations could be individual variation in metabolism.²² The significantly higher cyclosporine blood concentrations after transdermal application of cyclosporine in cat 6 compared with the other cats could also be explained by enhanced absorption of the transdermally applied cyclosporine in that particular cat. It is possible that a clinical benefit would be present with cat 6 due to its high cyclosporine blood concentration. Future studies with more study participants is needed to determine if other cats could achieve that concentration of cyclosporine after transdermal application and also to determine if clinical benefit is achieved.

The cyclosporine applied transdermally was formulated in PLO, composed of lecithin mixed with isopropyl palmitate and poloxamer, which acts as a solubilizing agent, surfactant, and emulsifier.^25,26 PLO is commonly used as a penetration enhancer and vehicle for transdermal medications in human and veterinary medicine; however, there are limited studies demonstrating effective drug delivery.²⁵ Studies evaluating the transdermal absorption of other drugs compounded with PLO include amitriptyline, buspirone, dexamethasone, fluoxetine, and glipizide. Those studies and have shown varying levels of absorption, with none consistently resulting in therapeutic concentrations.^11,25–29 An exception appears to occur with methimazole for the treatment of hyperthyroidism in cats where clinical efficacy has been shown utilizing a transdermal formulation compounded with PLO, although even with that drug, absorption appears to be variable.^10,29 The stratum corneum acts as a barrier to the absorption of chemicals, and PLO has been implicated in causing skin irritation at the site of application that could increase absorption because of a disrupted stratum corneum.^14,28 In the current study, the owners were asked to alternate the pinna on which the medication was applied to decrease the possibility that repeat application of the transdermally applied cyclosporine formulated in PLO would lead to increased absorption due to irritation and disruption of the stratum corneum. A number of in vitro and in vivo studies in murine species have reported various methods that enhance cyclosporine movement across the skin.^18–22 The variable methods of those studies exemplify the difficultly encountered with transdermal delivery of cyclosporine. No study thus far has demonstrated effective delivery of cyclosporine using a simple PLO gel.

Although cyclosporine blood concentrations were monitored as the endpoint in this study, the very low concentrations did not necessarily indicate lack of efficacy. Because the relationship between cyclosporine blood concentrations and efficacy has not been established in veterinary dermatology, a well-designed clinical trial is warranted to determine if it is clinically efficacious to treat feline allergic disease with cyclosporine applied transdermally as either a PLO gel or with other penetration enhancers.²

One limitation of this study was the small number of cats involved in the study. Additional cats would have increased the power of the study. The use of client-owned cats was another limitation of the study. If the cats were either in a single cat household or a research colony, housed individually, the variable of allogrooming between cats would have been eliminated.

Conclusion

This study showed that when compared with oral administration, the absorption of cyclosporine compounded for transdermal application was limited. The penetration enhancer, PLO, was unable to overcome the characteristics of cyclosporine, making it a poor choice for transdermal delivery. Additional studies are needed to evaluate the clinical efficacy of transdermal cyclosporine, potentially with other penetration enhancers. Finally, based on the results of this study, the use of transdermal cyclosporine compounded in PLO in cats cannot be recommended at this time due to limited and variable absorption.