Pharmacokinetic Properties of Itraconazole in
Blue-Fronted Amazon Parrots
(Amazona aestiva aestiva)

Susan E. Orosz, PhD, DVM, ~ Donita L. Frazier, DVM, PhD,
Edward C. Schroeder, MS, DVM, ~ Sherry K. Cox, MS, ~ Dorcas 0. Schaeffer, DVM,
Sonia Doss, BS, LATg, and Patrick J. Morris, DVM

Abstract: We examined the pharmacokinetic disposition of itraconazole and its active metabolite, hydroxy itraconazole, in blue-fronted Amazon parrots (Amazona aestiva aestiva) on days I and 14 of treatment with itraconazole at two different dosages (5 or 10 mg/kg PO q24h). Itraconazole from commercially available capsules was dissolved in 0. 1 N HCl, diluted with orange juice, and gavaged with food. The plasma concentration of itraconazole did not change with repeated dosing at 5 mg/kg over 14 days, but was increased by the last day of administration at 10 mg/kg. Both dosages achieved plasma concentrations that equal or exceed the minimum inhibitory concentration for most Aspergillus species. The plasma concentration of the parent drug and the area under the plasma concentration versus time curve were higher at the 5 mg/kg dose in the Amazon parrots when compared with concentrations achieved in pigeons in a previous study. Although tissue concentrations of drug were not determined in our study, these results may reflect either greater gastrointestinal absorption or less distribution to tissues in the Amazon parrots compared with pigeons. Our study suggests that therapeutic concentrations of itraconazole should be achieved in the plasma of Amazon parrots given 5 mg/kg q24h. However, the clinician should note that failure of the avian patient to respond to this dosage may indicate that a dosage of 10 mg/kg q24h is required to achieve therapeutic concentrations in tissues that are not highly perfused.

Introduction
The treatment of fungal infections in mammals has been enhanced by the development of new antifungal drugs. A number of new azoles, synthetic compounds with one or more five-membered rings, are currently available for clinical use. The azoles can be divided into two groups: the imidazoles, which are represented by clotrimazole, miconazole, and ketoconazole; the triazoles, represented by itraconazole and fluconazole. These drugs act by inhibiting cytochrome P450-dependent ergosterol synthesis and cytochrome c oxidative and peroxidative enzymes. This disruption of enzymic processes ultimately leads to fungal cell death. Itraconazole has improved activity against fungi (Aspergillus spp, Sporothrix schenkii) and yeasts (Histoplasma, Blastomyces, and Coccidioides spp) when compared with ketoconazole. Itraconazole has been useful in the treatment of human patients who are intolerant of amphotericin B, which is the only fungicidal drug currently available. Compared with amphotericin B, itraconazole is much less toxic in humans, with fewer than 3% of patients exhibiting transient side effects during therapy.' It has been used in human patients with renal failure without changes in pharmacokinetic values, which suggests that itraconazole is safe in these patients.

Itraconazole is a lipophilic triazole, and its absorption is enhanced when administered with a fatty meal .7,10-12 Drug concentrations achieved in tissues are 2 to 10 times greater than in plasma, except for physiologically privileged sites such as the eye and the brain.' Absorption is facilitated by a low stomach pH. 11,14 Itraconazole is highly plasma protein bound. Its half-life (t'12) in humans after administration of a single dose is 15 to 20 hours; after multiple doses, the half-life is 30 to 35 hours. Steady-state plasma concentrations are achieved after 10 to 14 days of therapy in normal human volunteers .

Itraconazole has been administered orally to a variety of avian patients; however, its use is rarely reported in the literature. Itraconazole was administered at a dosage of 8 mg/kg q24h for 29 days in a king penguin (Aptenodytes patagonicus) for treatment of ocular aspergillosis; and to two other penguins with uropygial gland candidiasis at a dosage of 10 mg/kg q24h for 20 days. Itraconazole was used unsuccessfully in a gentoo penguin (Pygocelis papua) with pulmonary aspergillosis at a dosage of 8.3 mg/kg q24h for 30 days, followed by 17 mg/kg q24h for 19 days. Death of this bird was attributed to Aspergillus organisms in the brain. Itraconazole has also been used in several different raptors, psittacines, and waterfowl at a dosage of 10 mg/kg q24h for 10 days. In a report by Joseph et al, itraconazole was administered to five birds that were also nebulized with clotrimazole for treatment of aspergillosis. No signs of toxicosis were noted in the birds in these studies; however, use of this drug may cause death in African grey parrots (Psittacus erithacus).

To date, avian pharmacokinetic studies have been limited to pigeons (Columba livia). Lumeij and coworkers suggested that itraconazole granules from commercially available capsules (Sporanox [Janssen Pharmaceutica, Titusville, NJ, USA] or Trisporal [Janssen Pharmaceutics B.V. Beerse, Belgium]), should be given at a dosage of 6 mg/kg q12h. However, if a therapeutic concentration is needed in the respiratory tract, 26 mg/kg ql2h was recommended. Drug toxicity at the proposed dosages was not assessed. In the second study, itraconazole was administered as one of two formulations: granules from the commercially available capsules were directly administered or the granules were dissolved in acid, diluted with orange juice, and given with food. Both formulations were administered at 5 mg/kg q24h in the pigeons. Results of this study suggested that the concentrations of itraconazole and its active metabolite, hydroxyitraconazole, in selected tissues were equal to or exceeded published minimum inhibitory concentration for Aspergillus spp isolated from humans, and presumably birds, with the acid formulation.

Anatomic and physiologic differences in the avian gastrointestinal tract suggest that drug absorption may differ between types of birds. The purpose of this study was to determine the pharmacokinetic properties of itraconazole in a common psittacine species, the blue-fronted Amazon parrot (Amazona aestiva aestiva).

Materials and Methods
Eight blue-fronted Amazon parrots (five females, three males), with body weights ranging from 306 to 424 g, were used in the study. The birds were individually housed in stainless steel cages and maintained at temperatures between 26'C (79'F) and 30'C (86'F). The photoperiod was controlled to maintain 12 hours of light. Water was provided ad libitum and the parrots were fed a regular diet of fresh vegetables and pelleted feed (Lafeber Co., Cornell, IL, USA) for the duration of the study. Before the study began, blood samples from all parrots were submitted for hematologic testing, plasma biochemical analysis, and measurement of serum bile acid concentrations. All results were within reference ranges. Blood tests were repeated on day 14 of drug administration.

Itraconazole was administered to each bird at two dosages, 5 mg/kg PO q24h and 10 mg/kg PO q24h, for 14 days in two separate studies. The two studies were separated by a period of 3 months. The itraconazole was dissolved in 0.1 N HC1 (50 mg/ml) by sonication, diluted with orange juice (1: 10) to a concentration of 5 mg/ml, and given by gavage followed by tubing with 5 ml of gruel consisting of equal parts of Emeraid I (Lafeber Co.) and Gerber HiPro baby cereal (Gerber Products Co., Fremont, M1, USA) mixed with warm tap water (25:25:50,V/V/V).

Blood (0.6-0.8 ml) was collected into heparinized tubes from the jugular vein for plasma itraconazole and hydroxyitraconazole analyses at 1, 8, and 16 hours after itraconazole administration on day 1, and at 1, 8, and 24 hours after itraconazole administration on day 14. The blood samples were centrifuged immediately after collection and the plasma was frozen at -80'C until drug analysis. Itraconazole and hydroxyitraconazole were analyzed by high-performance liquid chromatography (HPLC). Pure itraconazole and the internal standard were purchased from Janssen Research Diagnostics (Flanders, NJ, USA), and hydroxyitraconazole was provided by Dr. R. Woestenborghs (Janssen Research Foundation, Belgium). Briefly, the HPLC analytical system consisted of a 600E solvent delivery system, a model 700WISP autosampler, a RCM 8-mm X 10-cm cartridge holder equipped with a Novapak C8 cartridge (4--mm particle size) and a Novapak Guard-Pak precolumn insert, a model 470 fluorescence detector (Waters, Milford, MA, USA), and a NEC Powermate computer (NEC, Foxborough, MA, USA). Itraconazole and hydroxyitraconazole were extracted from plasma with methanol. The HPLC mobile phase was an isocratic mixture of water: acetonitrile: diethylamine (42:58:0.005, v/v/v). Fluorescence of the parent drug and metabolite were measured by an excitation of 245 nm and emission of 380 nm. The sensitivity of the assay was 10 ng/ml for itraconazole and hydroxyitraconazole. The assay was validated by measuring blank plasma and tissues and plasma and tissues spiked with known amounts of itraconazole and hydroxyitraconazole. The intraassay and interassay coefficients of variation were less than 5% and 10%, respectively.

The data from all birds were combined to obtain pharmacokinetic analysis for the entire group because of the difficulty of collecting multiple blood samples from a single bird without inducing physiologic changes and the limitations of pharmacokinetic curve-fitting with only three blood samples. The plasma itraconazole and hydroxyitraconazole concentrations were fitted to equations by a commercially available software program (Rstrip, MicroMath Scientific Software, Salt Lake City, UT, USA). The areas under the plasma concentration versus time curves (AUCs) were calculated by the trapezoidal method. The Cmax is the calculated maximum serum concentration from the fitted concentration versus time curve, and half-life (t 1/2) = 0.693/Kel where Kel, is the elimination rate constant.

Results
The plasma concentrations of bile acids after drug administration were within the reference range in all birds. Results of the plasma biochemical analyses and hematologic testing also remained within reference ranges throughout the treatment period.

The concentration versus time data were fitted to a two-exponential model with the first term reflecting the absorption phase and the second term describing the elimination phase. The computer-predicted itraconazole concentration versus time curves on days I and 14 generated from data for the Amazon parrots given itraconazole at 5 mg/kg PO q24h and 10 mg/kg PO q24h are shown in Figure 1.

The pharmacokinetic values calculated from the concentration versus time curves are shown (Table 1). The AUC and the maximum plasma concentration (C max) did not change significantly with repeated dosing at 5 mg/kg; however, these values increased after daily dosing for 14 days at 10 mg/kg. As expected, the AUC was proportional to the dose given. The maximum plasma concentration was achieved approximately 4 to 5 hours after dosing on day 1, whereas this time increased to 6 to 7 hours by day 14.

The mean (±SD) plasma itraconazole concentrations 1, 8, and 16 hours after dosing with 5 mg/kg on day I were 56 ± 75, 1,220 ± 526, and 497 ± 504 ng/ml, respectively. Hydroxyitraconazole could not be detected in the plasma of parrots I hour after dosing with 5 mg/kg on day 1; however, the mean concentrations 8 and 16 hours after dosing were 196 ± 29 and 247 ± 93 ng/ml, respectively. On day 14, the 1-, 8-, and 24-hour concentrations were 225 ± 193, 1,364 ± 605, 173 ± 162 ng/ml itraconazole and 197 ± 81, 309 ± 82, and 258 ± 53 ng/ml hydroxyitraconazole, respectively. The mean plasma itraconazole concentrations 1, 8, and 16 hours after 10 mg/kg on day I were 92 -_ 106, 1,976 ± 754, and 867 ± 370 ng/ml, respectively. Corresponding itraconazole concentrations on day 14 were 1,547 t 1,321, 3,398 t 1,849, and 1,208 ± 1,305 ng/ ml. The much higher fluorescent peaks seen on the chromatograms of plasma samples from the parrots given the higher dose precluded clear resolution of the metabolite peak; therefore, hydroxyitraconazole concentrations could not be determined in these samples on day 14.

Discussion
In this study, itraconazole was dissolved in 0.1 N HCl, diluted with orange juice, and gavaged with food. A previous study showed that concentrations of itraconazole and its active metabolite, hydroxyitraconazole, were increased in pigeons given itraconazole by this formulation compared with administration of the commercially available itraconazole granules alone. A significant increase in bioavailability of itraconazole also occurs in human patients when the drug is taken with food. For this reason, birds were gavaged with food that could mimic the type of gavage feeding mixture used in clinical practice for an anorectic bird. Previous studies in humans have shown that absorption is reduced under conditions of low intragastric acidity. Grain-eating birds, such as chickens, are considered to have a gastric pH that is higher than mammals. The plasma concentrations of parent drug in the parrots in the present study were greater than previously reported in pigeons, consistent with a difference in drug absorption between the two species. This may result from a difference in gastric acidity between pigeons and Amazon parrots; however, to our knowledge this has not been documented.

Peak plasma concentration is achieved in humans 2.5 to 4 hours after dosing. Similarly, peak plasma concentration was observed 4 hours after dosing in pigeons. The maximum plasma concentration was achieved in the parrots approximately 5 hours after dosing on day 1; whereas this time increased to 6 to 7 hours by day 14.

The half-life of itraconazole in Amazon parrots after 14 days of dosing (6 to 7 hours) was somewhat shorter than the half-life previously reported for pigeons (13.3 and 8.7 hours). The half-life appeared to be shorter on day 14 of the 5-mg/kg treatment. This is probably a result of variability in drug concentrations at 8 hours followed by uniformly low concentrations at 16 hours. In contrast, there was considerable variability in drug concentrations at 16 and 24 hours in the other groups. Similar variability occurs in the drug concentrations in the plasma and tissues of pigeons and humans.

As expected, the AUC increased in proportion to the dose given. If the capacity of a drug elimination pathway is not exceeded, it takes four to five halflives to eliminate 95-97% of the drug. If dosing occurs at intervals more frequent than four to five half-lives, drug accumulation will occur. Itraconazole did not accumulate in the plasma with multiple dosing at 5 mg/kg q24h for 14 days as evidenced by equivalent AUCs and Cmaxs. In contrast, both AUC and Cmax were significantly increased after 14 days of dosing at 10 mg/kg q24h. With a half-life of 6 hours, drug accumulation is expected to occur if doses are given more frequently than every 24 to 30 hours. Drug accumulation occurred with multiple 10-mg/kg doses because a large amount of drug remained in the plasma at the time of each subsequent dose. Drug accumulation also occurs after multiple dosing in humans .

The in vitro sensitivities of all organisms to itraconazole vary considerably depending on varied media (solid agar vs broth dilutions), incubation time, temperature, and pH of the media used by different laboratories. The minimum inhibitory concentrations of itraconazole for different Aspergillus isolates vary between 0.01 and 10 ug/ml; however, the range is most often between 0.1 and 1.0 ug/ml. The minimum inhibitory concentrations for different isolates of Candida range between 0.001 and 128 ug/ml, with most isolates having sensitivities between 0.2 and 2.0 ug/ml. Although the relationship between in vitro minimum inhibitory concentrations and therapeutic plasma and tissue concentrations has not been clearly defined, failure to reach an adequate plasma concentration of itraconazole has been implicated as the major risk factor for developing fatal fungal infections in severely neutropenic patients.The acceptable plasma concentration was considered to be > 250 ng/ml. Other studies revealed that a serum concentration < 5 ug/ml in humans with Aspergillus infection and < I ug/ml in humans with cryptococcal meningitis is associated with an unfavorable outcome.

Based on the minimum inhibitory concentration and these clinical studies, the maximum serum concentration achieved with either 5 or 10 mg/kg should be effective against most, but not all, Aspergillus species. Itraconazole administration at the higher dose should be effective against most Candida spp; however, some Candida spp are extremely resistant to itraconazole. Surprisingly, the mean itraconazole plasma concentration 8 hours after dosing with 5 mg/kg on day I was more than 10 times higher in the parrots in the present study than in pigeons. On day 14, the 8-hour concentration was four times higher in parrots compared with pigeons. In contrast to the present study, the plasma concentrations 1 and 8 hours after administration of itraconazole to pigeons on day 1 and 14 were below the minimum inhibitory concentrations for many Aspergillus and Candida isolates. Similarly, the plasma concentrations of itraconazole in parrots given 10 mg/kg on day 1 were approximately three times higher compared with Pigeons given the same dose. The plasma itraconazole concentrations in the parrots were greater than concentrations generally achieved in human patients. The peak plasma concentration following 100 and 200 mg given to human patients ranges between 130 and 600 ng/ml and 270 and 600 ng/ml, respectively.

The AUC was much greater in the parrots than previously reported for pigeons given 5 mg/kg (20,837 vs 3,487 ng/ml/hour). This difference could result from greater bioavailability in the parrots because of more complete absorption, less first-pass metabolism in the liver, or greater distribution of drug in the tissues of pigeons compared with parrots. The longer half-life reported in pigeons would support this latter explanation, because half-life is directly correlated to volume of distribution (t 1/2 = 0.693 Vd/Cl). It is unclear why a greater distribution of itraconazole to the tissues would occur in pigeons compared with parrots. Protein binding of itraconazole in plasma is 99.8% following a single dose given to human patients.' Plasma protein binding limits distribution of drugs to tissues; therefore, a difference in distribution between parrots and pigeons could result from differences in the affinity of plasma proteins for the drug or metabolite. Tissue drug and metabolite concentrations were not determined in the parrots in this study: therefore, we can neither confirm nor refute this explanation. To date, plasma protein binding of itraconazole has not been determined in birds.

In conclusion, several findings of this study are of particular clinical interest. The plasma concentration of itraconazole increased in proportion to the dose given. At 5 or 10 mg/kg, the predicted peak concentrations in the plasma equaled or exceeded the minimum inhibitory concentration for most species of Aspergillus. In contrast, the 5-mg/kg dose is unlikely to achieve a therapeutic plasma concentration of itraconazole for candidal infections. A factor that may be clinically important is the variability of plasma concentrations observed among birds in the present study. All avian patients should be closely monitored to determine if drug administration results in clinical improvement. Surprisingly, the plasma itraconazole AUC was much greater in the Amazon parrots than previously reported in pigeons. Although this may reflect greater gastrointestinal absorption, it may also reflect comparatively lower tissue concentrations in the Amazon parrots. Because tissue concentration is the most important determinant for cure of fungal infections, patients that do not respond to the 5-mg/kg q24h dosage should be given 10 mg/kg q24h. Similar to variability in plasma concentrations, tissue concentrations may also vary among birds.

All birds should be carefully monitored for signs of toxicosis during prolonged drug administration. Amazon parrots were given itraconazole for 14 days in our study; however, most antifungal therapeutic regimens suggest humans should take the drug for 6 months to 1 year. Close monitoring of patients is particularly important when administering itraconazole at the dosage of 10 mg/kg PO q24h, which in our study resulted in drug accumulation after 14 days. We have given itraconazole to Amazon parrots at this dosage for 3 months without signs of toxicosis (S. Orosz, unpubl. data). This dosage may apply for other Amazon parrots; however, anecdotal information on the death of African grey parrots" suggests that pharmacokinetic s should be studied in additional avian species.

Acknowledgments: We would like to thank Lillian E. Gerhardt, LVT for her assistance with this project. This study was funded by the Association of Avian Veterinarians, The American Federation of Aviculture, The Lafeber Company, and the Department of Comparative Medicine and the College of Veterinary Medicine. The University of Tennessee. It was conducted in facilities that are fully accredited by the American Association for the Accreditation of Laboratory Animal Care.

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