Plasma concentrations of itraconazole, voriconazole, and terbinafine when delivered by an impregnated, subcutaneous implant in Japanese quail (Coturnix japonica).
Key words: aspergillosis, itraconazole, voriconazole, terbinafine, subcutaneous implant, avian, Japanese quail, Coturnix japonica
Aspergillosis is the most common avian mycosis and typically causes either acute disease or chronic, debilitating disease and mortality in psittacine birds, water birds, and raptors. (1-3) Chronic aspergillosis is the more common form, and affected birds exhibit voice changes, anorexia, biliverdinuria, lethargy or depression, dyspnea, emaciation, and occasionally, ataxia or paralysis if the central nervous system is involved. (4) Aspergillus fumigatus is the most common causative agent, accounting for approximately 95% of infections, followed by Aspergillus flavus and other species. (5)
During the past 3 decades, an array of pharmaceutical agents have been used to treat aspergillosis with varying degrees of success, including amphotericin B. enilconazole, clotrimazole, itraconazole, fluconazole, flucytosine, and voriconazole. (6-9) Itraconazole and voriconazole are currently the drugs of choice for treatment, (9) and a relatively high level of clinical success in treating the disease has been realized in recent years, especially with voriconazole. A study in falcons found that 95% and 100% of Aspergillus isolates were susceptible to voriconazole concentrations of [less than or equal to]0.38 [micro]g/mL ([less than or equal to]380 ng/ mL) and [less than or equal to]1 [micro]g/mL ([less than or equal to]1000 ng/mL), respectively. (10) The same study found that 79% of Aspergillus isolates were susceptible to itraconazole at concentrations <0.75 [micro]g/mL (<750 [micro]g/mL), whereas the remaining isolates had mean inhibitory concentrations >1 [micro]g/mL (>1000 ng/mL).
Terbinafine (Lamisil, Novartis, East Hanover, NJ, USA) was released in 1996 for treatment of human mycotic nail infections (11) but has received limited assessment and clinical use as a potential therapeutic agent in avian species. (12-14) It is a member of a new class of antifungal agents, the allylamines, which have a different mechanism of action (squalene epoxidase inhibitor) than the azoles have. (15-16) The mean inhibitory concentration for various Aspergillus species ranges from 0.005-5 [micro]g/mL (5-5000 ng/mL). (17)
Regardless of their efficacy, all presently available agents and formulations require once or twice daily PO administration schedules, and systemic fungal infections typically require long-term therapy, often 2-6 months. Although azoles and terbinafine may be given orally, usually by loading a powder form into a gelatin capsule and concealing it in a food item that is eaten and swallowed whole, there are problems with acceptance, absorption, and consistency in administration on a daily basis for the duration of the treatment period. A recent study in red-tailed hawks (Buteo jamaicensis) found that oral voriconazole may need to be given q8h to maintain targeted therapeutic concentrations (18); such frequent handling for an extended treatment period is stressful in animals and may contribute to negative treatment outcomes.
Slow-release implants have been used in a variety of situations to provide long-term delivery of therapeutic agents, and guidelines have been developed for testing release profiles. (19) Recently, a terbinafine-impregnated implant was evaluated in vitro and was found to release drug for approximately 18 weeks. (20) In this study, we evaluated the plasma concentrations of itraconazole, terbinafine, and voriconazole delivered via impregnated, subcutaneous implants in Japanese quail (Coturnix japonica). Japanese quail are commonly used in research as models for other avian species.
Materials and Methods
The impregnated implant investigated in this study is a novel delivery method of antifungal medication and is a modification of the Ferretonin implant manufactured by Melatek, LLC (Middleton, WI, USA). The Ferretonin implant delivers melatonin to a ferret (Mustela putorius juro) for approximately 6 months with no significant side effects. This implant is the size of a microchip transponder (4 mm X 2 mm X 1 mm) and can be placed subcutaneously with a syringe (Fig 1). Implants for this study were manufactured by Melatek, LLC, to contain a prescribed amount of itraconazole, voriconazole, or terbinafine (instead of melatonin) for the trial. Other than replacing the active ingredient in the implant, no other changes in formulation were made. Each implant contained only 1 antifungal drug; blank (control) implants with no drug were also used.
Six-week-old, male Japanese quail were obtained from a commercial source and maintained in group housing at The Raptor Center at the University of Minnesota. The mean (SD) weight for birds before study inclusion was 268 g ([+ or -] 33.5 g). Animals were housed in battery-type wire cages that measured 42 cm X 20 cm X 56 cm in an isolation room held at a constant 20[degrees]C (68[degrees]F). Birds were acclimated for 1 week before implant placement. Initially, birds were maintained on a 14-hour light : 10-hour dark photoperiod, but that schedule led to aggression problems as the birds matured sexually. Photoperiod was reduced to a nonstimulatory photoperiod (8-hour light : 16-hour dark) on day 21. The quail were fed a commercial layer hen ration (Sprout Game Bird Ration, Mills Fleet Farm, Appleton, WI, USA) and provided water ad libitum. Animals were euthanatized at the end of all study procedures, in accordance with the American Veterinary Medical Association's Guidelines for the Euthanasia of Animals. All procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee and complied with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.
A pilot study, involving the 3 antifungals at 3 different concentrations each, was conducted. Forty birds were used as part of the pilot study. Birds were divided into 10 groups: 4 birds in each of 9 treatment groups and 4 birds maintained as controls with no implants. A single implant was placed in each bird subcutaneously over the dorsum between the scapulae. Each implant contained either itraconazole, voriconazole, or terbinafine; drug amounts in the implant were 0.5, 3, or 8 mg. Blood (0.3 mL) was collected from the jugular vein of each bird just before and at 2, 7, 21, 42, and 56 days after placement of the implant. After collection, blood was spun, and the plasma was separated and frozen at -80[degrees]C. Samples were stored for [less than or equal to]3 months and then shipped on dry ice to the Pharmacology Laboratory at the University of Tennessee's College of Veterinary Medicine (Knoxville, TN, USA).
Twenty-one birds were used as part of the full study. A single implant containing 1 of the 3 drugs was placed in each bird subcutaneously over the dorsum and between the scapulae. Three birds had blank (control) implants; the remaining 18 birds were divided into 3 groups of 6. Birds in each group received an implant with 24 mg of itraconazole, voriconazole, or terbinafine. A blood sample (0.3 mL) was collected from the jugular vein of each bird just before and at 2, 7, 21, 42, and 56 days after placement of the implant. After collection, samples were spun, and plasma was separated and frozen at -80[degrees]C. Samples were stored for [less than or equal to]3 months and then shipped on dry ice to the Pharmacology Laboratory at the University of Tennessee's College of Veterinary Medicine.
Plasma voriconazole samples were analyzed with reverse-phase high-performance liquid chromatography (HPLC). Voriconazole was purchased from Toronto Research Chemicals (Toronto, ON, Canada) and was 99% pure, whereas diazepam was purchased from Spectrum Chemical (Brunswick, NJ, USA) and was also 99% pure. The system consisted of a 2695 separations module, a 2487 absorbance detector, and a computer equipped with Empower software (Waters, Milford, MA, USA [Empower Software Solutions, Orlando, FL, USA]). Voriconazole was extracted from plasma samples by liquid extraction. Briefly, previously frozen plasma samples were thawed and vortexed, and 100 [micro]L was transferred to a clean, screw-top test tube, followed by the addition of 50 [micro]L of internal standard (1.0 [micro]g/mL diazepam). Hexane (3 mL) was added, and the tubes were rocked for 20 minutes and then centrifuged for 20 minutes at 1000g. The organic layer was transferred to a clean tube and evaporated to dryness with nitrogen gas. Samples were reconstituted in 250 [micro]L mobile phase, and 100 [micro]L was analyzed.
The compounds were separated on a Symmetry Shield [C.sub.18] (4.6 X 100 mm, 5 [micro]m) column with a Symmetry Shield [C.sub.18] guard column (Waters). The mobile phase was a mixture of 1) 20 mM phosphoric acid with 0.1% triethylamine adjusted to pH 3.0, and 2) acetonitrile (at a 66:34 ratio). The flow rate was 1.3 mL/min, and the column temperature was ambient. Absorbance was measured at 255 nm.
Standard curves for plasma analysis were prepared by fortifying untreated, pooled quail plasma with voriconazole to produce a linear concentration range of 10-2500 ng/mL. Calibration samples were prepared exactly the same as plasma samples. The quality-control samples used were 15, 350, 750, and 1700 ng/mL. The intra-assay accuracy for the method ranged from 93% to 100%, whereas the precision ranged from 3.7% to 7.7%. The inter-assay accuracy ranged from 83% to 100%, and the precision ranged from 6.8% to 9.9%. The lower limit of quantification was 10 ng/ mL.
Terbinafine plasma samples were analyzed by reverse-phase HPLC with liquid extraction.21 Terbinafine was purchased from US Pharmacopeia (Washington, DC, USA) and was 99% pure, whereas butenafine was purchased from Toronto Research Chemicals and also was 99% pure. Standard curves for plasma analysis were prepared by fortifying untreated, pooled quail plasma with terbinafine to produce a linear concentration range of 5-1500 ng/mL. The quality-control samples used were 15, 350, and 1200 ng/mL The intra-assay accuracy for the method ranged from 91% to 96%, whereas the precision ranged from 2.2% to 9.2%. The inter-assay accuracy ranged from 85% to 96%, and the precision ranged from 2.7% to 8.6%. The lower limit of quantification was 5 ng/mL.
Analysis for itraconazole and its active metabolite hydroxy itraconazole was performed with a modified HPLC method. (22) Itraconazole was purchased from US Pharmacopeia and was 99% pure, whereas both hydroxy itraconazole and the internal standard were purchased from Janssen Pharmaceutical (Beerse, Belgium), and both of those were 99% pure. Standard curves for plasma analysis were prepared by fortifying untreated quail plasma with itraconazole and hydroxy itraconazole to produce a linear concentration range of 10-5000 ng/mL. The quality-control samples used were 15, 350, 1200, 3500 ng/mL The itraconazole intra-assay accuracy ranged from 85% to 93%, whereas the precision ranged from 0.7% to 1.8%. The hydroxy itraconazole intraassay accuracy ranged from 80% to 90%, whereas the precision ranged from 1.0% to 4.2%. The itraconazole inter-assay accuracy ranged from 86% to 92%, and the precision ranged from 0.6% to 10%. The hydroxy itraconazole inter-assay accuracy ranged from 79% to 90%, whereas the precision ranged from 1.9% to 9.2%. The lower limit of quantification was 5 ng/mL.
No adverse effects associated with implant placement occurred in any of the birds. For the pilot study, measured drug concentrations (in ng/ mL) for each animal are shown in Table 1. One animal in the itraconazole 0.5-mg group was removed from the study before the day 21 collection because of injury from interbird aggression. For the full study, measured drug concentrations (in ng/mL) for each animal are shown in Table 2. None of the itraconazole or voriconazole implants were found to release sufficient drug to achieve targeted concentrations at any point during the study; targeted terbinafine concentrations were reached at some time points but were inconsistent. In the full study, 1 bird was removed from the voriconazole group on day 54 because of injury, 2 days before the end of the study; a blood sample was collected on that day as a final sample.
Aspergillosis is one of the most common fungal diseases of birds. Although medications exist that can cure these infections, treatment failures still occur. The stage of disease at diagnosis and the difficulty of long-term administration of antifungal medications can reduce the success rates of treatment in clinical cases. This study attempted to identify an alternative administration method of antifungal medication that would reduce handling and associated stress of diseased animals but still maintain therapeutic concentrations of drug over an extended period.
Various models of implants have previously been developed and used with success, often in ophthalmic conditions in people. (23,24) One study found that subcutaneous deslorelin acetate implants were useful in the treatment of adrenal disease in ferrets. (25) The implant used in this study was based on a product that has been successfully used to reduce the clinical signs associated with adrenal disease in ferrets and to increase fur growth in mink (Neovison vison). (26) The implant itself was the same as the commercially marketed product (Ferretonin), but instead of melatonin, the selected antifungal medication was added to the implant matrix.
Unfortunately, the release of the drug from the implants in this study was inconsistent across the examined period and generally below targeted concentrations, thereby limiting clinical application. Previous studies have shown itraconazole and voriconazole to be effective in treating Japanese quail infected with aspergillosis, (27,28) but similar studies examining terbinafine have not, to our knowledge, been reported. Pharmacokinetic studies have examined terbinafine in other avian species and have determined dose recommendations for oral and nebulized administration. (12-14) Targeted terbinafine concentrations were reached in some animals at various time points during the pilot study, but when a greater amount of drug was put into the implant in the full study, a higher, more consistent plasma concentration was not achieved. Although our in vitro study showed a relatively stable release of terbinafine during the examined period, that was not the case in this in vivo study. (20) Factors such as uneven distribution of the drug in the implant, which could lead to sporadic or no release of the drugs, or the dynamic physiologic nature of a living animal may have contributed to the inconsistent plasma concentrations of the 3 drugs examined. Additionally, the low aqueous solubility of itraconazole and terbinafine may have contributed to their inconsistent release from the implant or absorption by the bird. Future studies might examine implants impregnated with other drugs, such as antibiotics, but currently, the antifungal implants examined in this study cannot be recommended for clinical use in Japanese quail or other avian species.
Acknowledgments: This research was funded in part by the Morris Animal Foundation (grant D14ZO-824) and the Association of Avian Veterinarians. We thank Misty Bailey for editing the manuscript and Tim Cairns of Melatek LLC for manufacturing the implants.
Marcy J. Souza, DVM, MPH, Dipl ABVP (Avian), Dipl ACVPM, Patrick Redig, DVM, PhD, and Sherry K. Cox, MS, PhD
From the Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Dr. Knoxville. TN 37996, USA (Souza. Cox); and The Raptor Center, College of Veterinary Medicine, University of Minnesota, 1920 Fitch Ave, St. Paul, MN 55108. USA (Redig).
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Caption: Figure 1. Subcutaneous implant (to the right under the microchip) used for antifungal drug delivery in Japanese quail (Coturnix japonica). A penny is shown for size comparison.
Table 1. Plasma concentrations of antifungal drug in Japanese quail after administration via impregnated subcutaneous implant. A total of 36 quail were used during the pilot study; each drug and dose combination was administered to 4 birds. Each value represents the concentration of drug measured in a single animal. No hydroxyitraconazole, the metabolite of itraconazole, was detected in any samples from birds with itraconazole implants. Plasma concentration, ng/mL Day Drug Dose, 0 2 mg Itraconazole 0.5 D, D, D, D D, D, D, D 3 D, D, D, D D, D, D, D 8 D, D, D, D D, D, D, 17 Terbinafine 0.5 D, D, D, D D, D, D, 63 3 D, D, D, D D, D, 57, 104 8 D, D, D, D D, D, 39, 58 Voriconazole 0.5 D, D. D, D D, D, D, D 3 D, D, D, D D, D, D, D 8 D, D, D, D D, D, D, D Plasma concentration, ng/mL Day Drug Dose, 7 21 mg Itraconazole 0.5 D, D, D, B D, D, D (a) 3 D, D, B, B D, D, D, 14 8 D, D, D, 92 D, D, D, D Terbinafine 0.5 D, D, D, D D, D, D, 37 3 D, 32, 55, 57 D, D, 41, 49 8 D, D, 33, 47 D, D, D, 57 Voriconazole 0.5 D, D, D, 22 D, D, 37, 38 3 D, D, 20, 30 D, 30, 36, 44 8 D, D, 14, 37 17, 49, 59, 65 Plasma concentration, ng/mL Day Drug Dose, 42 56 mg Itraconazole 0.5 D, D, D (a) D, D, D (a) 3 D, D, D, D D, D, D, D 8 D, D, D, D D, 23, 110, 212 Terbinafine 0.5 D, B, B, 13 B, B, 12, 30 3 D, D, B, 32 B, 16, 17, 20 8 D, D, 10, N 12, 24, 26, N Voriconazole 0.5 D, D, D, D D, D, 28, 47 3 B. B, B, 33 D, D, D, 40 8 B, 6, 27, 52 D, D, 31, 36 Abbreviations: D indicates nothing detected in the sample; B, concentration was below the level of quantification ([less than or equal to] 5 ng/mL for voriconazole; [less than or equal to] 10 ng/mL for terbinafine and itraconazole); N, no sample was analyzed for 1 bird on days 42 and 56 in the terbinafine 8-mg group. (a) One bird from the itraconazole 0.5 mg group was removed from the study because of illness before the day-21 blood collection. Table 2. Plasma concentrations of antifungal drugs in Japanese quail after administration via impregnated, subcutaneous implant. A total of 21 birds were used during the full study; control implants were administered to 3 birds, and each drug (24 mg) was administered to 6 birds. Each value represents the concentration of drug measured in a single animal. No hydroxyitraconazole, the metabolite of itraconazole, was detected in any samples from birds with itraconazole implants. Plasma concentration, ng/mL Day Implant 0 2 Control D, D, D D, D, D Itraconazole D, D, D, D, D, D, D, B, D, B 22, 222 Voriconazole D, D, D, D, 38, 44, 53, 65, D, M 78, 134 Terbinafine D, D, D, D, D, B, B, B, 5, D, D 42 Plasma concentration, ng/mL Day Implant 7 21 Control D, D, D D, D, D Itraconazole D, D, B, B, D, D, D, B, 81, 99 B, 42 Voriconazole 16, 30, 31, 34, D, 28, 39, 59, 41, 88 65, 105 Terbinafine D, D, B, B, D, D, D, B, B, B B, B Plasma concentration, ng/mL Day Implant 42 56 Control D, D, D D, D, D Itraconazole D, D, D, B, D, D, D, D, B, 107 D, B Voriconazole D, 14, 32, 32, D, D, 11, 24, 44, 113 91, 104 Terbinafine D. D. D. B, D, D, D, D, B, 100 D, B Abbreviations; D indicates nothing detected in the sample; B, concentration was below the level of quantification ([less than or equal to] 5 ng/mL for voriconazole; [less than or equal to] 10 ng/mL for terbinafine and itraconazole); M, missing sample.
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|Title Annotation:||Original Study|
|Author:||Souza, Marcy J.; Redig, Patrick; Cox, Sherry K.|
|Publication:||Journal of Avian Medicine and Surgery|
|Date:||Jun 1, 2017|
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