Chapter 4.3

The effect of ketoconazole on whole blood and skin ciclosporin concentrations in dogs

Laura L. Gray*, Andrew Hillier*, Lynette K. Cole* and Päivi J. Rajala-Schultz†

Departments of *Veterinary Clinical Sciences and †Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA

Correspondence: Laura L. Gray, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH 43210, USA. E-mail: gray.704@osu.edu

Background – Ciclosporin (CSA) is approved for the treatment of canine atopic dermatitis. Ciclosporin is metabolized by liver cytochrome P450 enzymes, a process inhibited by ketoconazole (KTZ).

Hypothesis/Objectives – The aims of this study were to determine skin and blood CSA concentrations when CSA was administered alone at 5.0 (Treatment 1) or 2.5 mg/kg (Treatment 2) and when CSA was administered at 2.5 mg/kg concurrently with KTZ at 5 (Treatment 3) or 2.5 mg/kg (Treatment 4). We hypothesized that skin and blood CSA concentrations in Treatment 1 would not differ from those obtained with T3 or T4.

Animals – In a randomized cross-over study, six healthy research dogs received each of the treatments (Treatment 1,2,3 and 4) once daily for 7 days.

Methods – After the first, fourth and seventh dose for each treatment, a peak and trough skin punch biopsy sample and whole blood sample were collected and analysed with high-performance liquid chromatography–tandem mass spectrometry. Data were analysed using a repeated measures approach with PROC MIXED in SAS. Pairwise comparisons were performed with least squares means and Tukey-Kramer adjustment for multiple comparisons.

Results – Mean blood CSA concentrations in Treatment 1 were not different from those in Treatment 2 or 4, but were less than in Treatment 3. Mean skin CSA concentrations in Treatment 1 were greater than in Treatment 2, not different from those in Treatment 4, and less than those in Treatment 3.

Conclusions and clinical importance – Administration of CSA and KTZ concurrently at 2.5 mg/kg each may be as effective as CSA alone at 5.0 mg/kg for treatment of canine atopic dermatitis.

Introduction

Ciclosporin (CSA) is an immune-modulating drug currently labelled in the USA as Atopica^® (Novartis Animal Health, Greensboro, NC, USA) for use in the treatment of canine atopic dermatitis (CAD).¹ As a calcineurin inhibitor, the primary mechanism of action of CSA is prevention of transcriptional activation of genes responsible for interleukin-2 production, a necessary step for full activation of the T-helper cell pathway.^2,3 The absence of interleukin-2 synthesis prevents the activation and proliferation of T cells, in addition to the secondary synthesis of cytokines involved in CAD, such as interleukin-4, interleukin-5, interleukin-8 and interferon-γ.² A systematic review and meta-analysis of 10 controlled clinical trials, enrolling approximately 800 dogs, provided strong evidence for the efficacy of CSA in the treatment of CAD and concluded that the efficacy of CSA was comparable to that of oral corticosteroids.⁴ The clinically effective dose of CSA for the treatment of CAD has been determined to be 5mg/kg orally once daily.^5,6 Treatment with CSA is typically maintained lifelong (as with all treatments for CAD), and 40-50% of dogs have continued control of their disease with every other day dosing at 5 mg/kg after 4-8 weeks of induction daily dosing.⁴

The cost of therapeutic agents as well as potential adverse effects are important factors for consideration, particularly when coupled with the fact that CAD commonly manifests at a young age and requires lifelong treatment. Long-term administration of Atopica^® on a daily or every other day basis, particularly in medium-sized to large dogs, can be cost prohibitive. The cost of Atopica^® was listed as a reason for discontinuing treatment in 10% (five of 51) of dogs with CAD being treated for at least 6 months in a study of long-term use of CSA.⁷ The authors are not aware of any published studies assessing the effect of drug cost on owners’ initial choice of therapy for their dog with CAD.

As the cost of Atopica^® may be a limiting factor for its use, methods to reduce the dose of CSA without loss of clinical efficacy have been evaluated. Ciclosporin is primarily metabolized in the liver by cytochrome P450 enzymes 2B11 and 3A12/26.^8,9 Consequently, research thus far has focused on pharmacotherapeutic manipulation of this enzyme system by co-administration of drugs known to be P450-enzyme inhibitors. Recent studies indicate that neither metoclopramide nor cimetidine, both P450 inhibitors, have any effect on the pharmacokinetic profile of concurrently administered CSA.^10,11 Grapefruit juice and powdered whole grapefruit may variably affect the pharmacokinetic parameters of CSA in dogs.^11,12

Ketoconazole (KTZ) is a potent inhibitor of numerous cytochrome P450 enzymes, including CYP 2B11 and 3A12/26.^8,9 Numerous in vitro and in vivo studies have demonstrated that co-administration of KTZ with CSA results in increased whole blood CSA concentrations in dogs.^13–18 The inhibitory effect of KTZ on CSA clearance from blood was determined to be dose dependent, with the critical KTZ dosage range identified as 2.5-10 mg/kg daily.¹⁸ A study on normal research beagles investigating the dose of KTZ necessary to maintain whole blood CSA trough levels between 400 and 600 mg/mL showed that KTZ at 13.6 mg/kg daily enabled a 75% reduction of CSA dose, with estimated monetary savings of 57.8% at that time, and KTZ at 4.7mg/kg daily reduced CSA dose by 38% and thus reduced cost by 23.8%.¹⁴ These results have been applied clinically in studies on the efficacy of combination KTZ and CSA for the treatment of canine perianal fistulas, with excellent short-term results and a significant decrease in the dose of CSA required for clinical efficacy. One study involving 12 dogs with perianal fistulas showed that doses of KTZ between 5 and 11 mg/kg daily allowed a 50-75% dose reduction of CSA while maintaining target CSA trough levels, and all 12 dogs had at least short-term resolution of clinical signs.¹⁶ This reduced dose equalled a monetary saving of 35-71% when compared with the cost of treatment with CSA alone. Further studies in dogs with perianal fistulas had concurring results; a dose of KTZ of 10 mg/kg daily allowed for an 80% reduction of CSA administration, and doses of KTZ of 5.3-8.9 mg/kg twice daily enabled a reduction of CSA dose equalling a monetary saving of 70% compared with CSA alone.^15,17

As CSA is a lipophilic drug, it distributes widely in tissue and has been reported in the skin at concentrations up to 10 times higher than blood concentrations in humans.^19,20 An abstract of an unpublished study in dogs dosed with CSA at 3.8 mg/kg once daily for 14 days found that skin levels of CSA were 2.5-6.4 times higher than the whole blood CSA levels, and that depletion of CSA is slower from the skin than from the blood.²¹ Studies examining tissue levels of CSA are sparse and are primarily toxicological or postmortem studies.

Concurrent use of KTZ and CSA for treatment of CAD has been suggested by veterinary dermatologists at continuing education meetings in North America.^22–26 However, we are not aware of any published studies that have evaluated the effects of KTZ on CSA skin levels in dogs, or any published clinical trials of the efficacy of this combination of drugs for the treatment of CAD. The specific aims of this study were to determine skin and whole blood CSA concentrations when CSA was administered alone at a recommended (5.0 mg/kg/day) and a subtherapeutic dose (2.5 mg/kg/day) and when administered at a subtherapeutic dose (2.5 mg/kg/day) concurrently with KTZ at two different doses (2.5 or 5.0 mg/kg/day). We hypothesized that when CSA was administered alone at the recommended dose (5.0 mg/kg/day), the skin and whole blood CSA concentrations would not differ significantly from those obtained with subtherapeutic CSA dosing (2.5 mg/kg/day) concurrently with either dose (2.5 or 5.0 mg/kg/day) of KTZ.

Materials and methods

The experimental protocol was approved by the Institutional Animal Use and Care Committee (IACUC) of The Ohio State University.

Animals

Six clinically normal adult laboratory dogs (foxhounds) 1-4 years of age were used in this study. All dogs had complete blood counts and serum biochemical profiles prior to study enrolment. Dogs were housed in the laboratory research facility at the veterinary college and were under the care of the University Laboratory Animal Resources (ULAR) staff. The animals were housed indoors, in individual concrete runs, in a temperature- and humidity-controlled environment. They were maintained on a diet of lams Mini Chunks (The lams Company, Cincinnati, OH, USA) or Teklad 25% Lab Dog Diet (Harlan Laboratories, Indianapolis, IN, USA), fed once a day, with occasional dog treats, and water ad libitum. During the study periods, food was available from 14.00 to 07.30 h.

All treatments (CSA and CSA + KTZ) were administered at 09.30 h to comply with the Atopica^® label recommendation that the drug should be administered at least 2 h before or after feeding because the bioavailability is better in fasted animals.²⁷ Vomiting for more than two consecutive dosing periods (48 h) was deemed a criterion for withdrawal from the study, as were signs of systemic illness (lethargy, fever, changes in complete blood counts or chemistry values above or below the reference range). Anti-emetics were not permitted because most of these agents are also metabolized by (or affect) the cytochrome P450 enzyme system.¹¹ The dogs were visually monitored daily, and any adverse events (including vomiting or diarrhoea/soft stool, as well as erythema, swelling or discharge from biopsy sites) were recorded. Biopsy sites that became infected were to be treated topically with chlorhexidine gluconate solution 2% (Phoenix™ Pharmaceutical Inc., St Joseph, MO, USA) and Triple antibiotic ointment^® (E. Fougera and Co., Melville, NY, USA) twice daily until completion of the study period, and then with 5.0-10.0 mg/kg generic cefpodoxime (Proxetil Putney Inc., Portland, ME, USA) once daily until resolution of the infection. Antibiotic administration was not permitted during the study period to avoid increasing the likelihood of vomiting or diarrhoea.

Sample collection

Whole blood samples for CSA analysis (1.0 mL) were collected via cephalic, lateral saphenous or jugular venipuncture with a 22-gauge needle. The blood was placed into a lavender-topped tube (EDTA tube) and gently mixed, then transferred to Eppendorf tubes (VWR International, Radnor, PA, USA) and stored frozen at -80°C until analysis.

Skin samples were collected from the dorsal or dorsolateral neck. These sites were chosen for ease of access and maximal skin thickness for sample analysis. Local anaesthesia was performed by injecting 0.5 mL of 2% lidocaine (Butler Schein™ Animal Health, Dublin, OH, USA) subcutaneously with a 25-gauge needle. After 5 min, the skin sample was collected with an 8 mm biopsy punch (Medichoice^® Tru Punch Disposable Biopsy Punch; Owens & Minor, Mechanicsville, VA, USA), and the site was closed with a single cruciate suture using 3-0 absorbable monofilament (3-0 PDS, Ethicon©; Novartis Animal Health, Greensboro, NC, USA). All subcutaneous tissue was trimmed from the skin sample, and the skin was placed in an Eppendorf tube and stored frozen at -80°C until analysis.

Study design

Prior to commencement of the study, whole blood and skin samples were taken for CSA analysis to ensure that each dog was beginning with CSA levels below the limit of detection. Completion of each of the four treatment periods detailed below was followed by a 14-day washout, at which time whole blood and skin samples were collected for CSA analysis to ensure that all CSA concentrations were below the level of detection prior to entering the next treatment period. Complete blood counts and serum biochemical profiles were performed on all dogs within 90 days prior to study enrollment, as well as prior to (day 0) and immediately following completion (day 8) of Treatments 3 and 4.

The six dogs were randomly assigned via a computer-generated list (using the dog’s research number) into two groups of three dogs each. One group was administered CSA at 5 mg/kg orally once daily for 7 days (Treatment 1), and the other group was administered CSA at 2.5 mg/kg orally once daily for 7 days (Treatment 2). Ciclosporin was administered as Atopica^®, which is available as 10, 25, 50 and 100 mg capsules. Combinations of these capsule sizes were used to dose the dogs as closely as possible to 2.5 and 5.0 mg/kg/day. After the first, fourth and seventh dose of CSA, a skin sample was collected at 4 h (estimated peak skin concentration) and at 24 h (estimated trough skin concentration) to determine skin CSA levels.²¹ After the first, fourth and seventh dose of CSA, a whole blood sample was collected at 1.4 h (peak whole blood concentration) and at 24 h (trough whole blood concentration) to determine whole blood CSA levels.²⁸ Peak concentration samples were thus collected on days 1, 4 and 7, while trough concentration samples were collected on days 2, 5, and 8.

Following a 14-day washout period, the same two groups of three dogs were then randomly assigned to receive either CSA at 2.5 mg/kg and KTZ at 5.0 mg/kg orally once daily for 7 days (Treatment 3) or CSA at 2.5 mg/kg and KTZ at 2.5 mg/kg orally once daily for 7 days (Treatment 4). Ketoconazole (Teva, Sellersville, PA, USA) was administered in the generic form of 200 mg tablets (or part thereof) or as a compounded solution of the tablets per Trissel’s formulary (prepared by the facility’s pharmacy) in order to ensure dosing as close to 2.5 or 5.0 mg/kg as possible.²⁹ The compounded solution was not used for the 5.0 mg/kg KTZ dose owing to the large volume required. The CSA and the KTZ were administered concurrently. After the first, fourth and seventh dose of CSA and KTZ, a skin sample was collected at 4 h (estimated peak skin concentration) and at 24 h (estimated trough skin concentration) to determine skin tissue CSA levels. After the first, fourth and seventh dose of CSA and KTZ a whole blood sample was collected at 1.4 h (peak whole blood concentration) and at 24 h (trough whole blood concentration) to determine whole blood CSA levels. Peak concentration samples were thus collected on days 1, 4 and 7, while trough concentration samples were collected on days 2, 5 and 8.

Cross-over

After a 14-day washout period, the two groups of dogs then followed a full cross-over study design. Thus, at completion of the study each of the six dogs had received each of the four treatments in random order.

Ciclosporin analysis

Skin and whole blood samples were shipped overnight on dry ice to iC42 Bioanalytics (UC Denver, Denver, CO, USA) for analysis. Samples were shipped and analysed in two batches; the first sent half-way through the study and the second at study completion.

Ciclosporin in EDTA whole blood and tissue was quantified using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) following the procedures as previously described. ³⁰ All tissues were weighed. Tissues were pulverized under liquid nitrogen, and 100 mg of the frozen tissue powder was measured into 1 mL of the 0.5 mol/L potassium phosphate buffer (pH 7.4) and homogenized. Homogenates were prepared as 200 μL aliquots for extraction.

For protein precipitation, 800 μL of ZnSO₄·7H₂O (17.28 g/L) in 30:70 (v/v) HPLC grade water/methanol containing the deuterated internal standard ciclosporin-D4 (50 ng/mL) was added to 200 μL aliquots of EDTA whole blood and tissue homogenates. Samples were vortexed for 2.5 min and then centrifuged at 13,000g for 10 min at 4°C. The supernatant was transferred into glass HPLC vials for analysis.

The extracts were analysed using an LC/LC-MS/MS system (HPLC Agilent 1100 Series, Applied Biosystems and Sciex API 4000 triple quadruple mass spectrometer, Carlsbad, CA, USA). For on-line sample clean-up, 20 μL supernatant was loaded and cleaned on a 4.6 mm × 12.5 mm, 5 μm, Eclipse XDB-C8 column (Agilent, Santa Clara, CA, USA) using a mobile phase of 20% methanol and 80% 0.1% formic acid at a flow rate of 5 mL/min for 1 min. Then the column switching valve (Rheodyne, Cotati, CA, USA) was activated and the analytes were back-flushed onto the analytical column (4.6 mm × 150 mm, 5 μm, Eclipse Zorbax XDB -C8, Agilent), which was kept at 65°C. A gradient was used from 87% methanol and 13% 0.1% formic acid to 100% methanol in 2.0 min at a flow rate of 1mL/min and held at 100% for 1.5 min. The mass spectrometer was run in the positive MRM (multiple reaction monitoring) mode. The declustering potential(DP) was set to 131 V. Detection of the ions was performed by monitoring the transitions of m/z 1224.6 → 1112.4 for ciclosporin [M+Na⁺] and m/z 1228.6 → 1112.4 for the deuterated internal standard ciclosporin-D4 [M+Na⁺]. The collision energy (CE) was 85 eV.

The lower limit of quantification for ciclosporin in EDTA whole blood was 5.0 ng/mL, and for tissues 25 ng/g (0.025 ng/mg). The assay was linear over three orders of magnitude. The interassay accuracy was between 85 and 115%, and total imprecision was ≤17%. There were no matrix interferences, carry-over or ion suppression. Both batches of samples were reported with assay specific quality control data and calculated interassay accuracy.

Sample size estimation

A pilot study with two dogs was performed prior to the study. Results from the pilot study were used for a power calculation, using a power of 80% and significance level of 0.05, which indicated that six dogs would be needed in order to detect a significant difference in CSA concentrations in skin using the trough value from day 8 between treatment groups 1 and 2. The preliminary data suggested that differences when comparing the additional treatment groups were greater, and would require less than six dogs to achieve statistical significance (Stata 10; Stata Corp., College Station, TX, USA).

Statistical analysis

Daily CSA blood and skin concentrations were considered the outcomes in the data analyses using a repeated measures approach with PROC MIXED in SAS (version 9.2; SAS Institute Inc., Cary, NC, USA). Compound symmetry covariance structure was used to account for the nonindependence of the repeated observations from individual dogs. The effects of day, treatment and order of treatments on skin and blood CSA concentrations were initially assessed and then treatment-day interaction was also tested. If the treatment-day interaction was significant, analyses were further stratified by treatment; and blood and skin CSA concentrations between the days were compared within each treatment, separately for days with presumed peak (days 1, 4 and 7) and trough values (days 2, 5 and 8). Pairwise comparisons between days and between treatments were performed by obtaining least squares means and using Tukey-Kramer adjustment to account for multiple comparisons. The correlation between skin CSA and blood CSA values was assessed using Spearman correlation coefficients. For all analyses, values of P ≤ 0.05 were considered statistically significant.

Results

All treatments were administered as intended (Table 1) except for one dog (no. 38), that received medications in ~15 g (1 tablespoon) of canned food because the dog was resistant to manual pilling. The interassay accuracy of the CSA HPLC-MS/MS according to comparison with internal quality controls was reported for the two batches of samples, with each batch containing samples of skin and whole blood from all four treatment groups. The first batch analysis showed that blood CSA detection accuracy ranged from 98.3 to 104%, and skin accuracy ranged from 95.7 to 115%. The second batch showed that blood CSA detection accuracy ranged from 86.7 to 102%, and skin accuracy ranged from 93.6 to 117%. None of the skin and whole blood samples collected prior to entry into the study or prior to each treatment (following the 14-day washout period) had detectable levels of CSA.

Table 1. Target and measured dosage range of ciclosporin administered alone or in combination with ketoconazole to six healthy dogs

Descriptive statistics for the measured (unadjusted) whole blood and skin CSA concentrations for all treatment groups by day are presented in Table 2. Using the repeated measures model and evaluating for fixed effects, the treatment (Treatment 1, 2, 3 or 4), the day (day 1, 2, 4, 5, 7, 8) and the treatment-day interaction each had a significant effect on skin and whole blood CSA concentrations (F-test, P < 0.0001). However, the order in which treatments were received did not have a significant effect on whole blood or skin CSA concentrations (F-test, P = 0.854 and F-test, P = 0.756, respectively).

Table 2. Descriptive statistics for ciclosporin concentrations in whole blood and skin

The adjusted mean whole blood CSA concentration across all days for Treatment 1 (307.5 ng/mL) was not significantly different from the adjusted mean whole blood CSA concentration for Treatment 2 (169.41 ng/mL, Tukey-Kramer, P = 0.14) or Treatment 4 (417.74 ng/mL, Tukey-Kramer, P = 0.136), when evaluated with a mixed-model approach considering treatment, day and treatment-day interaction. However, the adjusted mean whole blood CSA concentration for Treatment 1 was significantly lower than the adjusted mean whole blood CSA concentration for Treatment 3 (644.83 ng/mL, Tukey-Kramer, P = 0.0002). The adjusted mean whole blood CSA concentration for Treatment 3 was significantly higher than the adjusted mean whole blood CSA concentration for Treatment 4 (Tukey-Kramer, P = 0.0081), and the adjusted mean whole blood CSA concentration for Treatment 2 was significantly lower than that for Treatments 3 and 4 (Tukey-Kramer, P < 0.0001 and P = 0.0040, respectively).

Utilizing least squares means obtained from the mixed models, the daily mean peak whole blood CSA concentrations did not differ significantly from each other within any treatment group (Table 3). Likewise, the daily mean trough whole blood CSA concentrations were not significantly different within Treatment 3; however, they were lower on day 2 compared with day 5 and on day 2 when compared with day 8 in Treatments 1, 2 and 4 (Table 3).

Table 3. Least squares means obtained from the mixed models for daily peak and trough whole blood ciclosporin (CSA) concentrations for six healthy dogs

Using the mixed-model approach considering treatment, day and treatment-day interaction, the adjusted mean skin CSA concentration for Treatment 1 (0.61 ng/mg) was significantly higher than the adjusted mean skin CSA concentrations for Treatment 2 (0.262 ng/mg, Tukey-Kramer, P = 0.05), not significantly different from the adjusted mean skin CSA concentration for Treatment 4 (0.697 ng/mg, Tukey-Kramer, P = 0.895), but was significantly lower than the adjusted mean skin CSA concentration for Treatment 3 (1.236 ng/mg, Tukey-Kramer, P = 0.0006). The adjusted mean skin CSA concentration for Treatment 3 was also significantly higher than the adjusted mean skin CSA concentration for Treatment 4 (Tukey-Kramer, P = 0.0024), and the adjusted mean skin CSA concentration for Treatment 2 was significantly lower than that of Treatments 3 and 4 (Tukey-Kramer, P < 0.0001 and P = 0.0129, respectively).

Utilizing least squares means obtained from the mixed models, the mean daily peak skin CSA concentrations on day 4 were significantly higher than day 1 mean daily peak CSA concentrations in Treatments 1, 2 and 3, while day 7 mean daily peak CSA concentrations were significantly higher than those of day 1 within all four treatments (Table 4). The mean trough CSA concentrations on day 5 were significantly higher than those on day 2 for Treatments 1 and 4; day 8 mean trough CSA concentrations were significantly higher than day 2 mean trough CSA concentrations for Treatments 1, 2 and 4; and day 8 mean trough CSA concentrations were significantly higher than those of day 5 only for Treatment 4 (Table 4).

Table 4. Least squares means obtained from the mixed models for daily peak and trough skin ciclosporin (CSA) concentrations for six healthy dogs Peak skin CSA Trough skin CSA

The correlation between whole blood and skin CSA concentrations combining values from all treatments was moderate (Spearman correlation coefficient, r² = 0.6689). The correlation of whole blood and skin CSA values within each treatment group were moderate for Treatments 1 and 2 (Spearman correlation coefficient of 0.786 and 0.6881, respectively) and for Treatments 3 and 4 (Spearman correlation coefficient of 0.5623 and 0.5766, respectively).

One dog (no. 38) exhibited higher skin CSA concentrations than other animals, especially for the day-7 peak and day-8 trough of Treatment 3. Figures 1 and 2 show the whole blood CSA (Figure 1) and skin CSA concentrations (Figure 2) for dog no. 38 (red), as well as the concentrations for the other five dogs obtained during Treatment 3. Statistical significance was not affected by these values.

Figure 1. Ciclosporin (CSA) concentrations in whole blood for six healthy dogs (nos 32, 38, 62, 82, 94 and 97) treated with 2.5 mg/kg of CSA and 5.0 mg/kg of ketoconazole once daily (Treatment 3). Days 1, 4 and 7 represent peaks, while days 2, 5 and 8 represent troughs.

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