• View in gallery
    Figure 1—

    Photograph of the left ear of a representative of a canine cadaver indicating the area for collection of a skin sample (black dashed circle). Inset—Photograph of a representative 2-cm-diameter skin sample that has been treated with synthetic canine cerumen before application of an otic gel.

  • View in gallery
    Figure 2—

    Mean ± SD values for absorption (A, C, and E) and cumulative absorption (B, D, and F) of 14C-terbinafine hydrochloride (A and B), 3H-betamethasone acetate (C and D), and florfenicol (E and F) through canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen. Notice that the scale on the y-axis differs among the panels.

  • View in gallery
    Figure 3—

    Mean ± SD cumulative absorption of 14C-terbinafine hydrochloride (triangles), 3H-betamethasone acetate (circles), and florfenicol (squares) through canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen.

  • View in gallery
    Figure 4—

    Mean ± SD concentration of 14C-terbinafine hydrochloride (A) and 3H-betamethasone acetate (B) in canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen. Values include the amount of each compound detected in tape strips obtained from the stratum corneum. Notice that the scale on the y-axis differs between panels.

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Impact of synthetic canine cerumen on in vitro penetration of auricular skin of dogs by florfenicol, terbinafine, and betamethasone acetate

Sarah EhlingDepartment of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Ronald E. BaynesDepartment of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Wolfgang BäumerDepartment of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Abstract

OBJECTIVE To determine the pharmacokinetics of florfenicol, terbinafine, and betamethasone acetate after topical application to canine auricular skin and the influence of synthetic canine cerumen on pharmacokinetics.

SAMPLE Auricular skin from 6 euthanized shelter dogs (3 females and 3 neutered males with no visible signs of otitis externa).

PROCEDURES Skin adjacent to the external opening of the ear canal was collected and prepared for use in a 2-compartment flow-through diffusion cell system to evaluate penetration of an otic gel containing florfenicol, terbinafine, and betamethasone acetate over a 24-hour period. Radiolabeled 14C-terbinafine hydrochloride and 3H-betamethasone acetate were added to the gel to determine dermal penetration and distribution. Florfenicol absorption was determined by use of high-performance liquid chromatography–UV detection. Additionally, the effect of synthetic canine cerumen on the pharmacokinetics of all compounds was evaluated.

RESULTS During the 24-hour experiment, mean ± SD percentage absorption without the presence of synthetic canine cerumen was 0.28 ± 0.09% for 3H-betamethasone acetate, 0.06 ± 0.06% for florfenicol, and 0.06 ± 0.02% for 14C-terbinafine hydrochloride. Absorption profiles revealed no impact of synthetic canine cerumen on skin absorption for all 3 active compounds in the gel or on skin distribution of 3H-betamethasone acetate and 14C-terbinafine hydrochloride.

CONCLUSIONS AND CLINICAL RELEVANCE 3H-betamethasone acetate, 14C-terbinafine hydrochloride, and florfenicol were all absorbed in vitro through healthy auricular skin specimens within the first 24 hours after topical application. Synthetic canine cerumen had no impact on dermal absorption in vitro, but it may serve as a temporary reservoir that prolongs the release of topical drugs.

Abstract

OBJECTIVE To determine the pharmacokinetics of florfenicol, terbinafine, and betamethasone acetate after topical application to canine auricular skin and the influence of synthetic canine cerumen on pharmacokinetics.

SAMPLE Auricular skin from 6 euthanized shelter dogs (3 females and 3 neutered males with no visible signs of otitis externa).

PROCEDURES Skin adjacent to the external opening of the ear canal was collected and prepared for use in a 2-compartment flow-through diffusion cell system to evaluate penetration of an otic gel containing florfenicol, terbinafine, and betamethasone acetate over a 24-hour period. Radiolabeled 14C-terbinafine hydrochloride and 3H-betamethasone acetate were added to the gel to determine dermal penetration and distribution. Florfenicol absorption was determined by use of high-performance liquid chromatography–UV detection. Additionally, the effect of synthetic canine cerumen on the pharmacokinetics of all compounds was evaluated.

RESULTS During the 24-hour experiment, mean ± SD percentage absorption without the presence of synthetic canine cerumen was 0.28 ± 0.09% for 3H-betamethasone acetate, 0.06 ± 0.06% for florfenicol, and 0.06 ± 0.02% for 14C-terbinafine hydrochloride. Absorption profiles revealed no impact of synthetic canine cerumen on skin absorption for all 3 active compounds in the gel or on skin distribution of 3H-betamethasone acetate and 14C-terbinafine hydrochloride.

CONCLUSIONS AND CLINICAL RELEVANCE 3H-betamethasone acetate, 14C-terbinafine hydrochloride, and florfenicol were all absorbed in vitro through healthy auricular skin specimens within the first 24 hours after topical application. Synthetic canine cerumen had no impact on dermal absorption in vitro, but it may serve as a temporary reservoir that prolongs the release of topical drugs.

Otitis externa is the most common disease of the external ear canal in dogs and is most likely secondary to underlying diseases. It accounts for up to 20% of dogs examined because of dermatologic problems.1–3 In healthy ears, cerumen contributes to maintaining the barrier function of the cutaneous epithelium by serving as a lubricant, water repellent, and trap for foreign substances (eg, dust and pollen). In otitic ears, the amount of cerumen increases, whereas the lipid content and hydrophobicity decrease, which in turn leads to impairment of the natural barrier function.4,5 An otic gel containing florfenicol, terbinafine, and betamethasone acetate is commercially available for the treatment of dogs with acute otitis externa and acute exacerbation of recurrent otitis externa associated with susceptible strains of bacteria (Staphylococcus pseudintermedius) and yeast (Malassezia pachydermatis).6 The formulation dissolves in cerumen and is slowly eliminated from the ear via mechanical action.6 Cleaning of the external ear canal is recommended prior to initial application, but cleaning should not be repeated until the end of treatment to allow contact of the gel with the ear canal.6

The penetration behavior of drugs differs among regions of the body as a result of differences in thickness of the stratum corneum, hair follicle density, friction, and other local differences.7–10 Additionally, interaction with skin lipids and cerumen also influences the penetration behavior of drugs through the skin.11 Appropriate ear cleaners and treatments must be chosen for otitis externa. Effective removal of cerumen is important for assessment of the integrity of the tympanic membrane and to allow penetration of drugs. However, cerumen may remain in the external ear canal and can reduce treatment effectiveness. Studies have not been conducted to evaluate the impact of cerumen on skin penetration.

The primary objective of the study reported here was to determine the amount of florfenicol, 14C-terbinafine hydrochloride, and 3H-betamethasone acetate that penetrated canine auricular skin over a 24-hour period, as determined by use of flow-through diffusion cells. A secondary objective was to assess the effect of synthetic canine cerumen on skin penetration and depth of drug penetration.

Materials and Methods

Sample

Intact auricular skin samples were collected from 6 euthanized shelter dogs (3 females [sexual status unknown]) and 3 neutered males). Dogs were euthanized for reasons unrelated to the study reported here and had no visible signs of otitis externa. Subjects were mixed-breed dogs that weighed between 15 and 30 kg and were 1 to 5 years old. Harvest of tissue from euthanized dogs did not require approval by an institutional animal care and use committee. The study was conducted in accordance with published guidelines12,13 unless otherwise stated.

In another study,14 investigators divided the external ear canal into 4 levels; the location for sample collection in the study reported here was in the third of those 4 levels. Investigators of the other study14 found that the density of hair follicles did not differ between the widest part of the pinna and the most proximal part of the annular cartilage. Therefore, an area of auricular skin located as close to the external opening of the ear canal as possible was selected for harvest (Figure 1). This area was chosen because a commercially available otic gela containing florfenicol, terbinafine, and betamethasone acetate can be used for the treatment of dogs with otitis externa.

Figure 1—
Figure 1—

Photograph of the left ear of a representative of a canine cadaver indicating the area for collection of a skin sample (black dashed circle). Inset—Photograph of a representative 2-cm-diameter skin sample that has been treated with synthetic canine cerumen before application of an otic gel.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.333

Pinnae were removed within 1 hour after dogs were euthanized. They were wrapped in gauze soaked in saline (0.9% NaCl) solution and stored overnight at 4°C. On the basis of the in vitro part of the guidelines for the testing of chemicals,13 no additional testing for skin integrity was deemed necessary. Skin adjacent to the external opening of the ear canal was carefully cleaned with a cotton-tipped swab and 1% soap solutionb to avoid increased percutaneous absorption (wash-in effect).15 Each pinna was affixed on polystyrene foam, and the skin adjacent to the external opening of the ear canal was split to a thickness of 200 to 500 μm with an electric dermatome.c Thickness of the split skin was included in the calculation for each sample. Then, a circular disk (2 cm in diameter) of each piece of skin was removed with a punch, placed into a polytetrafluoroethylene diffusion cell, and secured in place by use of a screw-cap dosing device to provide a dosing surface area of 0.636 cm2.

Perfusion system and topical application of drugs and synthetic canine cerumen

Skin flow-through experiments were conducted in a 2-compartment polytetrafluoroethylene flow-through diffusion cell system16 to evaluate penetration of the drugs in the otic gel over a 24-hour period. The decision was made to analyze all 3 drugs of the otic gel in the same experiment. Unfortunately, only 2 isotopes were commercially available and could be used for concurrent measurements.

Radiolabeled14C-terbinafine hydrochlorided and radiolabeled 3H-betamethasone acetatee were added to the topical gel to yield a target dose of 1 μCi/200 μL for each diffusion cell. Specific activity is a measure of radioactivity to mass. The molecular weight of terbinafine is 291.43 g/mol; specific activity of the 14C-terbinafine hydrochloride was 59 mCi/mmol (4.939 μg/μCi). The molecular weight of betamethasone acetate is 434.50 g/mol; specific activity of the 14C-terbinafine hydrochloride was 40,000 mCi/mmol (0.011 μg/μCi). Mixing the nonradiolabeled solution with the radiolabeled solution resulted in a new specific activity for each radioactive compound.

The amount of ethanol was the limiting factor for dilution of the radiolabeled compounds. A 1% solution of ethanol was used, and 50 μL of the radio-ligand dilution (mixture of both ligands) was added to 5 mL of the otic gel. One 6-mL syringe contained otic gel, and another 6-mL syringe contained the radio-ligands. The syringes were connected with a 2-cm-long piece of tubing, and the compounds were mixed in this closed system by pushing the plunger of each syringe to transfer the solution from one syringe into the other for 10 minutes. The first 500 μL of radiolabeled solution in the syringe (preadministration sample) and the amount remaining in the syringe after administration (postadministration sample) were used to measure radioactivity of the mixture. The applied doses were normalized on the basis of the amount of radioactive ligand.

The 200-μL dose of the otic gel spiked with 3H-betamethasone acetate and 14C-terbinafine hydrochloride was applied to the left auricular skin sample. The package insert for the otic gel indicated that each 1-mL dose contained 10 mg of terbinafine, 10 mg of florfenicol, and 1 mg of betamethasone acetate. Because the dose volume for the present study was 200 μL/diffusion cell, 2 mg of terbinafine, 2 mg of florfenicol, and 0.2 mg of betamethasone acetate were administered.

Skin samples obtained from the right ears were treated with 10 μL of synthetic canine cerumen. We prepared synthetic canine cerumen by mixing 9.16% squalene, 16.62% fatty acid ester, 19.98% sterol ester, and 54.24% triglycerides as described elsewhere.17 The synthetic canine cerumen was kept at 37°C so that it would be in a liquid state for application with a pipette; treated skin samples were then stored at 4°C to allow the wax to solidify, after which they were placed in the chambers. Each skin sample was treated with 200 μL of the otic gel spiked with the radio-ligands. Diffusion cells were then occluded with plastic paraffin filmf to prevent evaporation of the volatile compounds.

The dermal side of the skin disks was perfused with perfusion medium (modified Krebs-Ringer buffer) with Krebs-Ringer bicarbonate buffer solution (pH, 7.4) that contained NaCl (6.89 g/L), KCl (0.36 g/L), CaCl2 (0.28 g/L), KH2PO4 (0.16 g/L), MgSO4• 7H2O (0.30 g/L), NaHCO3 (2.75 g/L), dextrose (1.2 g/L), and bovine serum albumin (45.0 g/L). The medium also contained amikacin (250 mg/mL), penicillin G (250,000 U/mL), and sodium heparin (1,000 U/mL). The pH was maintained between 7.3 and 7.5, and the medium mimicked the in vivo oncotic pressure. This perfusion medium was chosen on the basis of the lipophilicity of the active ingredients in the otic gel (log10 of the partition coefficient was 5.9 for terbinafine hydrochloride, 2.8 for betamethasone acetate, and 0.8 for florfenicol). Perfusate and diffusion cell temperatures were maintained at 37°C by use of a constant temperature circulator.g Flow rate was maintained at approximately 4 mL/h by use of a peristaltic pump.h A sample of perfusate (1 mL) was obtained before administration (time 0) to ensure there was no radioactive contamination of the apparatus; additional samples (n = 12) were then collected in borosilicate glass scintillation vials at 15-minute intervals for the first 2 hours after administration, at 1-hour intervals for the next 6 hours, and at 4-hour intervals until the end of the 24-hour experimental period. Perfusate samples were stored frozen at −20°C until analysis.

Analysis of perfusate samples for radiolabeled 14C-terbinafine hydrochloride and 3H-betamethasone acetate

At the end of the perfusion period, a cotton-tipped swab was used to remove any remaining gel from the surface of the auricular skin; the swab samples were placed into a liquid scintillation vial, and skin disks were transferred to waxed paper. One milliliter of each perfusate sample was pipetted into a new scintillation vial, and 15 mL of scintillation cocktaili was added to each vial. The solution remaining in the syringe and the surface swab samples were placed in vials, and 15 mL of scintillation cocktail was added to each vial. Skin adjacent to the area after removal of the auricular skin sample was placed in a 20-mL scintillation vial; 2 mL of 3% KOH solution was added, and the mixture was maintained overnight at 50°C to dissolve the tissue. Then, 15 mL of scintillation cocktail was added to the dissolved tissue. Each of these samples was analyzed 3 times on a liquid scintillation counterj for determination of total 14C and 3H. The mean of the 3 values was used to calculate the unabsorbed amount of each dose. The counting protocol was used to separate counts for 3H from counts for 14C, and values were reported as the number of disintegrations per minute, whereby 2,220,000 disintegrations/min equaled 1 μCi.

Analysis of perfusate samples for florfenicol

The manufacturer of the commercially available otic gel has reported that low plasma concentrations (1 to 42 ng/mL) of florfenicol have been detected after multiple-dose experiments.6 Because the otic gel was applied only once in the present study, we were concerned that UV detection would be insufficiently sensitive. An isotope for florfenicol was not commercially available for concurrent measurements, and there were small perfusate samples during the first 2 hours of the experiment; therefore, it was decided to use the technique of HPLC-UV for measurement of florfenicol concentrations, rather than to repeat the experiment with other dogs.

Stock solutions of florfenicol (1, 5, 10, 50, 100, 500, and 1,000 μg/mL) were prepared by dissolving florfenicolk (purity, 98%) in methanol. Stability in methanol was determined in preliminary experiments (data not shown), and accuracy of the standard curves ranged from 99.5% to 103.4% in 3 separate experiments. On each day of analysis, stock solutions were further diluted with perfusate fluid to yield a 7-point standard curve (0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μg/mL). Perfusate samples for florfenicol analysis were not mixed with scintillation cocktail; they were directly processed for HPLC-UV analysis. One milliliter of perfusate or standard solution was filtered through extraction columnsl by the application of vacuum. Columns were washed with 1 mL of a solution of water:methanol (95:5); samples were eluted with 1 mL of 100% methanol, evaporated at 40°C for 20 minutes, and reconstituted in 200 μL of the mobile phase (water:acetonitrile [75:25]). Mean ± SD accuracy of measurement for florfenicol was 107 ± 9.4%, and coefficient of variation was 8.7%. Samples were quantified with matrix-matched calibration plots. All samples were assayed by use of the mobile phase (water:acetonitrile [75:25]) on a C18 columnm with a guard column.n Detection and quantification were performed at 223 nm with commercial software.o Samples were held at 40°C. Injection volume was 40 μL. Run time was 7 minutes; peak elution was detected at 3.0 minutes. The limit of quantification for perfusate samples was 0.015 μg/mL, and the limit of detection was 0.01 μg/mL.

Evaluation of depth of penetration in skin specimens

A cotton-tipped swab was used to swab each skin disk with a 1% soap solution to remove remaining otic gel. Samples were obtained for the 2-cm-diameter disks by use of tape strips. An investigator used their thumb to press a tape strip onto the skin disk; the process was repeated with a new piece of tape until 6 tape samples had been obtained. All tape strips were pooled and dissolved in 10 mL of ethyl acetate; 1 mL of the solution was pipetted into a scintillation vial, and 15 mL of scintillation cocktailj was added.

The stratum corneum was not analyzed separately; it was added to the perfused skin to provide a profile of drug penetration. A 10-mm biopsy punch was used to remove the perfused center of each skin disk after the 24-hour experimental period. These punch specimens were used for evaluation of the depth of penetration. Punch specimens were fixed in a water-soluble compoundp in an aluminum foil container; care was used to ensure that the entire epidermal surface was in contact with the bottom of the foil while the contents were frozen on dry ice. Each frozen sample was placed in a cryomicrotome,q and 20-μm-thick slices were cut parallel to the surface of the skin. The entire punch specimen was cut into slices. Four slices were placed in each scintillation vial to ensure there was enough radioactivity to obtain a reading above the background value; 15 mL of scintillation cocktail was added, and samples were processed as previously described. Depth of penetration values were determined in 80-μm increments, starting at the surface of the skin.

Statistical analysis

Although each diffusion cell received 200 μL of the otic gel, each diffusion cell did not receive the target dose of 1 μCi of the 14C or 3H radiolabel. Because the radiolabeled compounds were not introduced homogeneously into the otic gel, the radioactive dose used for calculations was estimated from the total amount of radioactivity recovered for each diffusion cell (ie, the dose was all of the radioactivity recovered from the perfusate samples, postadministration dose, dosing device, surface swab specimens, tape strips, punch specimens, and adjacent skin samples). One skin sample, which received no treatment with canine synthetic cerumen, was excluded from the analysis because of leaking of the otitic gel through the skin (samples without canine cerumen treatment, n = 5; samples with synthetic canine cerumen treatment, 6).

Absorption was the sum of the total mass in all of the perfusate samples collected over the entire 24hour period (cumulative absorption). The mass as well as the percentage dose were calculated for all samples. The amount in the penetrated skin equaled the amount detected in the dosed skin without the amounts in the stratum corneum tape strips, and the total amount penetrated included the tape strips with the stratum corneum. Steady-state flux was determined by use of the linear portion of the cumulative absorption-versus-time curve. The pseudo–steady-state slope was calculated by use of early time points (1 to 4 hours) for florfenicol. The value for Kp was calculated from the ratio of the steady-state flux to the surface concentration and was expressed as the log Kp. Results were reported as mean ± SD. When applicable, values were rounded to 2 or 3 decimal places on the basis of the lowest values. Unpaired t tests were performed by use of commercial statistical software.r Values were considered significant at P < 0.5.

Results

Skin absorption

Absorption of 14C-terbinafine hydrochloride and 3H-betamethasone acetate increased constantly over the 24-hour period (Figure 2). Treatment with synthetic canine cerumen had no influence on cumulative absorption of 14C-terbinafine hydrochloride (1,103.06 ng without treatment vs 1,17374 ng with treatment) and 3H-betamethasone acetate (560.75 ng without treatment vs 660.62 ng with treatment; Table 1). Florfenicol absorption peaked approximately 2 hours after administration, with no influence of synthetic canine cerumen treatment on mean cumulative absorption (128.63 ng without treatment vs 147.26 ng with treatment). There was a greater percentage absorption of 3H-betamethasone acetate (0.28% of the dose without treatment vs 0.33% of the dose with treatment), compared with that for the other 2 compounds over the 24-hour period (14C-terbinafine hydrochloride, 0.06% of the dose without treatment vs 0.06% of the dose with treatment; florfenicol, 0.06% of the dose without treatment vs 0.07% of the dose with treatment; Figure 3). However, there were no differences in the percentages with and without synthetic canine cerumen treatment.

Figure 2—
Figure 2—

Mean ± SD values for absorption (A, C, and E) and cumulative absorption (B, D, and F) of 14C-terbinafine hydrochloride (A and B), 3H-betamethasone acetate (C and D), and florfenicol (E and F) through canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen. Notice that the scale on the y-axis differs among the panels.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.333

Figure 3—
Figure 3—

Mean ± SD cumulative absorption of 14C-terbinafine hydrochloride (triangles), 3H-betamethasone acetate (circles), and florfenicol (squares) through canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.333

Table 1—

Mean ± SD values for in vitro evaluation of absorption and penetration for the 24-hour period after topical application of an otic gel containing florfenicol, terbinafine hydrochloride, and betamethasone acetate to auricular skin samples obtained from 6 euthanized dogs.

 14C-terbinafine hydrochloride3H-betamethasone acetateFlorfenicol
VariableWithoutWithWithoutWithWithoutWith
Dose (μg)2001.70 ± 1.082002.57 ± 1.159200.00 ± 0200.01 ± 0200.00 ± 0200.01 ± 0
Absorption (% of dose)0.06 ± 0.020.06 ± 0.020.28 ± 0.090.33 ± 0.120.06 ± 0.060.07 ± 0.08
Absorption (ng)1,173.74 ± 446.191,103.06 ± 465.39560.75 ± 177.66660.62 ± 318.33128.63 ± 110.40147.26 ± 151.08
Steady-state flux (ng/cm2/h)87.23 ± 36.48100.77 ± 55.6953.48 ± 17.1262.06 ± 26.35617.99 ± 446.31569.79 ± 498.18
Log Kp (cm/h)−5.09 ± 0.18−5.05 ± 0.23−4.29 ± 0.15−4.23 ± 0.16−4.30 ± 0.31−4.42 ± 0.49
Cmax (ng/mL)NANANANA165.99 ± 140.21132.80 ± 92.97
Tmax (h)NANANANA2.000 ± 02.167 ± 0.41
Stratum corneum tape strips
  Absorption (% of dose)0.50 ± 0.201.03 ± 0.770.31 ± 0.130.62 ± 0.60NDND
  Absorption (μg)10.08 ± 4.0620.58 ± 15.360.62 ± 0.271.24 ± 1.18NDND
Penetrated skin (% of dose)*0.23 ± 0.140.22 ± 0.170.20 ± 0.150.20 ± 0.22NDND
Penetrated skin (μg)*4.56 ± 2.794.34 ± 3.430.39 ± 0.300.40 ± 0.45NDND
Total penetration (% of dose)1.34 ± 0.362.06 ± 0.551.30 ± 0.241.79 ± 0.26NDND
Total penetration (Hg)26.82 ± 7.2641.33 ± 10.982.59 ± 0.483.59 ± 0.52NDND

Otic gel was applied to skin samples without (n = 5) and with (6) prior treatment with synthetic canine cerumen.

Represents the value for the skin to which the otic gel was applied, except for the value for the stratum corneum tape strips.

Represents absorption plus the value for the stratum corneum tape strips plus the value for the adjacent skin plus the value for the penetrated skin.

NA = Not applicable. ND = Not determined.

Cmax and Tmax

Values for Cmax and Tmax were determined only for florfenicol on the basis of its finite absorption and flux profile (Figures 2 and 3). Treatment with synthetic canine cerumen did not affect Cmax (132.80 ng/mL without treatment vs 165.99 ng/mL with treatment) and Tmax (2 hours without treatment vs 2.2 hours with treatment) of florfenicol (Table 1).

Skin flux

Mean steady-state flux was comparable for 14C-terbinafine hydrochloride (87.23 ng/cm2/h without treatment vs 100.77 ng/cm2/h with treatment) and 3H-betamethasone acetate (53.48 ng/cm2/h without treatment vs 62.06 ng/cm2/h with treatment). For florfenicol, the pseudo–steady-state flux (569.79 ng/cm2/h without treatment vs 617.99 ng/cm2/h with treatment) was almost 10 times the values for the other 2 drugs. However, there were no differences in flux for florfenicol with and without synthetic canine cerumen treatment.

Kp

The log Kp values for 14C-terbinafine hydrochloride, 3H-betamethasone acetate, and florfenicol (−5.05, −4.23, and −4.27 cm/h, respectively) were comparable during the 24-hour period, although terbinafine appeared to be less permeable (almost a 10-fold difference) than the other 2 drugs. No significant difference was detected in the mean log Kp of all 3 drugs in skin with or without synthetic canine cerumen treatment during the 24-hour period (Table 1).

Skin penetration

Tape strips were deemed to represent the amount of the compounds in the upper third of the stratum corneum. This estimation was based on thickness of the stratum corneum of the ear pinnae of 6.58 to 15.09 μm reported in another study9 as well as results of a study18 in which the use of 10 tape strips completely removed the stratum corneum in the axilla and groin area. Skin penetration was assessed only for terbinafine and betamethasone because these were the only radiolabeled compounds that facilitated chemical analysis at 24 hours after administration. Evaluation of the patterns suggested that treatment with synthetic canine cerumen resulted in an almost 2-fold increase in retention of these drugs (14C-terbinafine hydrochloride, 1.03% of the dose without treatment vs 0.5% of the dose with treatment; 3H-betamethasone acetate, 0.62% of the dose without treatment vs 0.31% of the dose with treatment) in the stratum corneum (Table 1). Penetration of 14C-terbinafine hydrochloride into the remaining epidermis and dermis at 24 hours reached mean values of 4.56 μg (0.23% of the dose) in skin samples without synthetic canine cerumen treatment and 4.34 μg (0.22% of the dose) in skin samples with synthetic canine cerumen treatment. Penetration of 3H-betamethasone acetate reached mean values of 0.39 and 0.40 μg without and with synthetic canine cerumen treatment, respectively, which was approximately 0.20% of the dose for both drugs. Therefore, most of the radiolabeled compounds were found in the stratum corneum, compared with the epidermis (Figure 4). Overall, skin absorption for all 3 compounds and the skin penetration depth for 3H-betamethasone acetate and 14C-terbinafine hydrochloride were not significantly different between healthy canine auricular skin with and without synthetic canine cerumen treatment.

Figure 4—
Figure 4—

Mean ± SD concentration of 14C-terbinafine hydrochloride (A) and 3H-betamethasone acetate (B) in canine auricular skin over a 24-hour period. Skin samples were obtained from 6 euthanized dogs. Otic gel was applied to skin samples with (solid line [n = 6]) or without (dotted line [5]) prior treatment with synthetic canine cerumen. Values include the amount of each compound detected in tape strips obtained from the stratum corneum. Notice that the scale on the y-axis differs between panels.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.333

Discussion

Pharmacokinetics of florfenicol, terbinafine, and betamethasone acetate in a topical gel applied to canine auricular skin and the influence of treatment with synthetic canine cerumen were evaluated in vitro. Evaluation of the absorption profiles revealed no impact of synthetic canine cerumen for any of the 3 compounds and no impact on dermal penetration of 3H-betamethasone acetate and 14C-terbinafine hydrochloride within the 24-hour experimental period. During that period, the highest absorption (as a percentage of the dose) was for 3H-betamethasone acetate (0.33% of the dose), which was followed by 14C-terbinafine hydrochloride and florfenicol. Each of the 3 compounds was applied as an infinite dose (an amount of test preparation applied to the skin whereby a maximum absorption rate of the test substance [per unit area of skin] is achieved and maintained), which was confirmed for 14C-terbinafine hydrochloride and 3H-betamethasone acetate. In comparison, there was an unexpected decrease in florfenicol absorption and flux after 2 hours with and without synthetic canine cerumen treatment. This contradicts the assumption and flux profile of an infinite dose.19 More likely, a finite dose (an amount of test preparation applied to the skin whereby a maximum absorption rate of the test substance may be achieved for a time interval but is not maintained) of florfenicol was applied, although the 200-μL dose was an infinite dose for the other compounds. Concurrent delivery of hydrophilic and lipophilic drugs in a gel is a complex challenge, and water-soluble molecules such as florfenicol may remain in the delivery material because of the high water content.20,21

It is possible the compounds reacted with each other; however, to evaluate this would require that the compounds be tested separately. In vitro tests have been conducted by the manufacturer6 to determine the fractional inhibitory concentration index; there was no interference among the 3 active compounds. However, data from in vitro studies do not determine events that happen on the skin surface or whether a film will form. We detected florfenicol in the perfusate within the first hour after application, a peak at 2 hours, and a decrease thereafter. The aforementioned hypothesis may have been unique for florfenicol. In contrast, constant concentrations of radiolabeled ligands were measured throughout the 24-hour period. Therefore, we concluded that the radiolabeled ligands were constantly released from the applied otic gel.

Saturation of the medium could be ruled out owing to the constant flux of the perfusate through the system. Other experimental issues (eg, leakage) also did not apply because of the slow increase and common peak at 2 hours. Dermal absorption is primarily a diffusion-driven process. In healthy skin, the stratum corneum and possibly the cerumen can form a reservoir that may prolong release of a drug, which would make the drug systemically available again.22,23 This could have explained the high amounts of 14C-terbinafine hydrochloride and 3H-betamethasone acetate found in the tape strips. Skin treated with synthetic canine cerumen had approximately 2 times as much of the substances in the tape strips, compared with the amount for untreated skin. A possible explanation was that synthetic canine cerumen enhanced the depot function of the stratum corneum, although the difference was not significant during the 24-hour experiment. Unfortunately, time periods > 24 hours cannot be tested in vitro because of loss of integrity of the skin.12

The manufacturer has performed a multiple-dose pharmacokinetics study6 to evaluate the extent of systemic absorption of the otic gel. Healthy mixed-breed dogs were used, and they received 0, 1X, and 5X the labeled dose for a total of 6 administrations in 5 weeks. Analysis of the data revealed systemic absorption of all active compounds during the first 2 to 4 days after administration, but with low plasma concentrations (range, 1 to 42 ng/mL).6 The in vitro study reported here confirmed systemic availability of all 3 compounds; however, the percentage of the absorbed doses was extremely low (range, 0.05% to 0.33%).

To estimate the steady-state plasma concentration for these drugs, one can substitute the value of 0.0000531 cm/h for Kp in the following equation: Css = A × C × Kp/(Vd × Ke), where Css is the plasma concentration at steady state, A is the area of topical application, C is the concentration applied, Vd is the volume of distribution, and Ke is the elimination constant after IV administration to dogs in another study.24 Use of the Kp data for florfenicol in the present suggested that the plasma concentration at steady state would be approximately ≤ 1.0 ng/mL if both ears of a dog (20 cm2) were exposed to two 10-mg/mL doses, assuming a volume of distribution of 17 L and elimination constant of 0.63/h. This is in light of the fact that only 0.4% to 2.3% of the 3H-betamethasone acetate and 14C-terbinafine hydrochloride penetrated the skin and that most of the dose was still present in the otic gel after 24 hours. Nevertheless, evaluation of tolerance before and after ACTH stimulation revealed a decrease in cortisol concentrations, which indicated that betamethasone acetate is absorbed and enters the systemic circulation.6 The manufacturer also detected systemic drug concentrations,6 which is similar to the absorption of terbinafine hydrochloride and florfenicol in the present in vitro study. There was a slight elevation in alanine transaminase activity (3 dogs) as well as minimal or mild microscopic hepatocellular vacuolation (2 dogs) when 5 times the recommended dose of the otic gel was administered over a 5-week period.6 Because of the sample preparation used to determine the depth of penetration for the radiolabeled terbinafine hydrochloride and betamethasone acetate, samples could not be analyzed with HPLC to determine florfenicol concentrations. The absorbed amounts of florfenicol in the present in vitro study were close to the limit of quantification and were only approximately 0.06% of the applied dose.

The aforementioned in vivo study6 and the in vitro study reported here were conducted with healthy dogs or the skin from healthy dogs. Compared with results for healthy skin, the skin barrier function is impaired in inflammatory conditions (eg, otitis externa) and absorption of drugs will be altered.5,6,25 In a previous study,16 eczematous dermatitis in a monkey led to a 2-fold increase of absorption of topically applied hydrocortisone. In humans, theophylline flux through the stratum corneum of lesional atopic dermatitis skin and nonlesional atopic dermatitis skin was approximately twice that for non–atopic dermatitis skin.26 The increase may have been attributable to a defective barrier, inflammation, blood flow, or temperature alterations, although it is known that removal of the stratum corneum will speed (up to 1,000 times as fast) the diffusion of small water-soluble molecules into the systemic circulation.27 Therefore, routine topical antimicrobial treatment of otitis externa in dogs may lead to low systemic plasma concentrations, which could increase the risk for bacteria and yeast to develop antimicrobial resistances.28,29

For the present in vitro study, 3H-betamethasone acetate, 14C-terbinafine hydrochloride, and florfenicol were all absorbed through healthy auricular skin within the first 24 hours after application. Synthetic canine cerumen had a minor impact on dermal absorption of the otic gel in vitro, but it may serve as a temporary reservoir that prolongs the release of drugs. Recently, in vitro studies30,31 of porcine skin revealed that dermal penetration of lipophilic and hydrophilic substances was increased in tape-stripped or abraded skin. The methods used in the present study could be beneficial for evaluating dermal absorption during inflammatory conditions.

Acknowledgments

Supported by Elanco Animal Health.

The authors declare that there were no conflicts of interest.

The authors thank Jim Brooks and Jim Yeatts for technical assistance.

ABBREVIATIONS

Cmax

Maximum concentration

HPLC

High-performance liquid chromatography

Kp

Permeability coefficient

Tmax

Time to maximum concentration

Footnotes

a.

Osurnia, Elanco Animal Health, Greenfield, Ind.

b.

Ivory dishwashing liquid, Procter and Gamble, Cincinnati, Ohio.

c.

Padgett Instruments Inc, Kansas City, Mo.

d.

Terbinafine-[14C] hydrochloride, American Radiolabeled Chemicals, St Louis, Mo.

e.

Betamethasone acetate [1,2,4-3H], American Radiolabeled Chemicals, St Louis, Mo.

f.

Parafilm, Bemis Com, Oshkosh, Wis.

g.

Brinkmann Inc, Westbury, NY.

h.

Watson-Marlow PumpPro, Watson Marlow, Wilmington, Mass.

i.

Bioscint liquid scintillation cocktail, National Diagnostics Inc, Atlanta, Ga.

j.

Tri-Carb 2910 TR, Perkin Elmer, Waltham, Mass.

k.

Sigma-Aldrich Corp, St Louis, Mo.

l.

Oasis HLB, 1 mL, 30 mg, Waters Corp, Milford, Mass.

m.

Cortecs, 2.7-μm column, 4.6 × 100 mm, Waters Corp, Milford, Mass.

n.

Waters Atlantis T3, 5-mm column, 4.6 × 20 mm, Waters Corp, Milford, Mass.

o.

Empower, version 2002, Waters Corp, Milford, Mass.

p.

OCT compound, Tissue-Tek, Sakura Finetek USA, Torrance, Calif.

q.

Cryocut microtome, American Optical Corp, Southbridge, Mass.

r.

GraphPad Prism 7, version 7.0b, GraphPad Software Inc, La Jolla, Calif.

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Contributor Notes

Drs. Ehling and Bäumer's present address is Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Free University of Berlin, 14195 Berlin, Germany.

Address correspondence to Dr. Ehling (sarah.ehling@fu-berlin.de).