The Effects of Formulation on the
systemic Availability of Itraconazole in Pigeons

Susan E. Orosz, PhD, DVM,
Edward C. Schroeder, DVM, MS,
Sherry K. Cox, MS, Sonia Doss, LATg, and
Donita L. Frazier, DVM, PhD

Abstract: The purpose of this study was to examine the pharmacokinetic disposition of itraconazole in Columba livia after administration of two formulations for 1 or 14 days. In two treatment groups we used the protocol most often used in clinical practice: 5 mg/kg itraconazole given as granules from commercially obtained capsules gavaged with orange juice (groups 1 and 3). The other treatment groups received the drug dissolved in 0.1 N HCl, diluted with orange juice, and gavaged with food (groups 2 and 4). Group 4 had higher tissue concentrations than all other groups of the biologically active metabolite, hydroxyitraconazole, but not the parent drug. Concentrations of hydroxyitraconazole were higher than concentrations of the parent drug in all tissues examined. Concentrations of the parent drug and its active metabolite in tissues, but not plasma, were equal to or exceeded minimum inhibiting concentrations for Aspergillus spp. isolated from humans and, presumably, for birds. This suggests that therapeutic concentrations of itraconazole may be achieved in tissues, including the central nervous system, when administered with food at the dosage given group 4 (5 mg/kg q24h, dissolved in 0.1 N HCl). These factors may help determine the clinical efficacy of itraconazole in the treatment of aspergillosis.

Introduction

Itraconazole, a water insoluble, lipophilic triazole, is an antifungal drug that is used in human patients who are intolerant of amphotericin B. Its pharmacokinetics does not vary in humans with renal failure, suggesting that it is safe to use in these patients. Itraconazole works by interfering with cytochrome P450 and by inhibiting the conversion of langosterol to ergosterol, an important component of the fungal cell membrane.' Fewer and more transient side effects occur with itraconazole than with amphotericin B. Unlike ketoconazole, itraconazole does not appear to alter sex steroid hormone synthesis.

In mammals, itraconazole is metabolized in the liver and excreted primarily in the bile; studies show that it is best absorbed at a low pH and that oral absorption is enhanced by administration with a meal. Itraconazole is highly bound to plasma protein; however, with repeated dosing, concentrations in mammalian tissues other than in physiologically privileged sites are 2 to 10 times greater than in Plasma. In humans, clearance is prolonged, with a half-life (T,J of 15 to 20 hours after a single dose and a T, of 30 to 35 hours with multiple dosing. Steady-state concentration is achieved in humans within 10 to 14 days. Itraconazole is metabolized by oxidative degradation of the dioxolane and piperazine ring and by aliphatic oxidation and N-dealkylation of the 1-methylpropyl group. At least one metabolite that occurs in human beings, bydroxyitaconazole, has antifungal effects.

Because its adverse effects are minimal and often transient, itraconazole is well tolerated during long-term use. It is often used in immunosuppressed patients with a variety of systemic mycoses, including aspergillosis. In birds, it has been administered orally for the treatment of aspergillosis and candidiasis. It has been used successfully in the treatment of candidal tracheitis in a blue and gold macaw (Ara ararauna) and a candidal infection of the uropygial gland in a king penguin (Aptenodytes patagonicus).

Itraconazole has also been used in the treatment of aspergillosis in the respiratory tree of gentoo penguins (Pygocelis paptia) and in combination with clotrimazole in raptors.

The pharmacokinetics of itraconazole in birds is largely unexplored. Anatomic and physiologic differences in the gastrointestinal tract suggest that drug absorption may differ between birds and mammals. The ability of birds to metabolize itraconazole to the active metabolite hydroxyitraconazole has not been determined. In this study, tissue concentrations of itraconazole and hydroxyitraconazole were measured in domestic pigeons (Columba livia) given itraconazole (5 mg/kg PO q24h) for either I or 14 days. These times were chosen on the basis of the drug's half-life in mammals and the time required to reach steady-state blood concentrations in human patients. Treatment groups consisted of fasted birds given the drug in its commercially available form (granules) with orange juice and birds that received the drug dissolved in acid, diluted with orange juice, and gavaged with food.

Materials and Methods

Male and female purpose-bred pigeons (C. livia) were housed in stainless steel pigeon cages. The pigeons were fed a measured amount of pigeon seed, and water was provided ad libitum. Complete blood cell counts, plasma chemistry profiles (i.e., total protein, albumen, glucose, calcium, phosphate, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, uric acid, lactate dehydrogenase, creatine kinase, and cholesterol), and plasma bile acid concentrations were determined in all birds before and after treatment. Food and water consumption was monitored throughout the treatment period. All birds were weighed at least two times during the treatment period.

The pigeons were divided into four groups of 8 to 13 birds each. Itraconazole was administered by gavage (5 mg/kg) into the crop. Groups I and 3 received granules obtained from commercial capsules mixed with 2 ml orange juice; for groups 2 and 4, the granules were first dissolved in 0.1 N HC1 (50 mg/ml) by sonication and then diluted with orange juice (1: 10) to a concentration of 5 mg/ml. Birds in groups I and 3 were fasted overnight (4 PM to 9 AM) before drug administration. The drug was administered once daily for 14 days in groups 3 and 4. Birds in groups 2 and 4 were also gavaged with 5 ml of gruel consisting of equal parts of Emeraid I ® (Lafeber Co., Cornell, IL, USA) and Gerber HiPro baby cereal (Gerber Products Co., Fremont, MI, USA) mixed with warm tap water (25:50, v/v/v) following administration of the drug. Birds in groups I and 2 received the drug only on day 1; birds in groups 3 and 4 received the drug once daily for 14 days.

Blood (0.7 ml) was collected from the medial metatarsal or basilic veins for plasma itraconazole and hydroxyitraconazole analyses at 1 and 8 hours after final gavage on day 1 (groups 1 and 2) or day 14 (groups 3 and 4) of treatment. Heparinized blood was centrifuged immediately after collection and plasma was frozen at -80'C until analysis. The pigeons were euthanatized with Beuthanasiaa (Schering-Plough Animal Health Corp., Kenilworth, NJ, USA) on either day 1 or day 14, 8 hours after the drug was administered, and tissue samples were collected for determination of parent drug and metabolite concentrations. Tissue samples included abdominal, caudal thoracic, and cranial thoracic air sacs, brain, small intestine, lung, liver, and kidney.

Itraconazole and hydroxyitraconazole were analyzed by high-performance liquid chromatography (HPLC). Pure itraconazole and the internal standard, R51012, were purchased from Janssen Research Diagnostics, Inc. (Flanders, NJ, USA). Hydroxyitraconazole was kindly provided by R. Woestenborghs (Janssen Research Foundation, Beerse, Belgium). Briefly, the HPLC analytical system consisted of a 600E solvent delivery system, a model 700WISP autosampler, an RCM 8 mm X 10 cm cartridge holder equipped with a Novapak C8 cartridge (4-µm particle size), and a Novapak GuardPak 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 and tissues 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 using- 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 less than 10%, respectively.

Statistically significant differences (P < 0.05) in plasma and tissue concentrations between treatment groups were determined by pairwise t-tests (Statistical Analysis System, Cary, NC, USA). The linear relationships between plasma and tissue concentrations of drug and metabolite were evaluated by determination of Pearson correlation coefficients (Statistical Analysis System).

Results

The plasma concentrations 1 and 8 hours after administration on day 1 were 152 97 ng/ml and 128 ± 42 ng/ml (group 1; mean standard error of the mean), and 104 ± 93 -ng/ml and 108 ± 91 -µg/ml (group 2), respectively. Concentrations at corresponding times on day 14 were 54 ± 50 ng/ ml and 157 ± 104 ng/ml (group 3), and 513  ± 288 ,ng/ml and 305 ± 122 ng/ml (group 4).

On day 1, the plasma itraconazole concentration in group 1 (birds given itraconazole as granules in orange juice) was not significantly different at both 1 and 8 hours after administration from that in group 2 (birds given drug dissolved in acid followed by food). After 14 days of drug administration, plasma concentration was significantly higher at both 1 and 8 hours in group 4 (birds given drug dissolved in acid followed by food) compared with group 3 (birds given itraconazole as granules in orange juice) (Fig. 1). The plasma itraconazole concentration 1 and 8 hours after drug administration was significantly higher on day 14 (group 4) compared differ on day 14 (group 3) compared to day 1 (group 1 ).

Mean itraconazole concentration in the pulmonary air sacs and in the kidneys was significantly higher after 14 days of drug administration in group 4 than after a single dose in group 2, but not in group 3 compared with group 1 (birds given the drug in orange juice only). Concentration in the other tissues was not significantly different on day 1 compared with day 14. On both days 1 and 14, mean tissue itraconazole concentrations were not significantly different between the groups (Table 1). On day 1, plasma itraconazole concentrations at and 8 hours were significantly correlated to drug concentrations in the liver and small intestine in group 2, and in the liver and air sacs in group 1.
On day 14, plasma itraconazole concentrations were significantly correlated with drug concentration in the air sac at 1 and 8 hours and with concentration the small intestine at 1 hour in group 4. Plasma concentration was also significantly correlated with drug concentration in the kidney, lung, and brain 8 hours after drug administration in group 4. Plasma concentration was also significantly  correlated with drug concentration in the kidney, lung, and brain 8 hours after drug administration in group 4. No correlations were seen on day 14 between tissue and plasma concentrations of drug in group 3.

Hydroxyitraconazole concentration was consistently higher than concentration of parent drug in all tissues but not in plasma. Tissue concentration of hydroxyitraconazole was not significantly different between treatment groups on day 1; however, the metabolite concentration in the liver, kidney, brain, and small intestine was significantly higher in group 4 after 14 days of drug administration (Table 2). This trend was also seen in the lung and air sacs, but the differences were not statistically significant.

Mean concentration of the metabolite in the kidneys, liver, brain. and small intestine was higher on day 14 compared with day 1 of treatment in group 4. In contrast, the mean metabolite concentration increased only in the kidneys of group 3.

Plasma concentration of bile acids was within normal reference range for all birds in all treatment groups. Results of CBC and plasma biochemical C1 analyses were also within normal reference ranges.

Discussion

The plasma and tissue itraconazole concentrations necessary to eradicate fungal organisms in birds are not known. To date, in vitro antifungal susceptibility testing remains a research tool and cannot be relied upon to consistently provide clinically useful information because inconsistent culture methods have been used. Van Custem and colleagues reported the minimum inhibitory concentrations of itraconazole for Candida albicans and Aspergillus fumigatus in brain-heart infusion broth as 0.1 µg/ml (1,076 strains tested) and 0.01-1.0 µg/ml, respectively. In a separate study, Van Cutsem and Janssen concluded that 98.8% of the strains of A. fumigatus were sensitive at 10   µg/ml.

In vitro inhibitory concentrations of hydroxyitraconazole have not been described; however, the metabolite is likely to be equipotent or of greater potency than the parent drug. Antifungal plasma concentrations determined by bioassay (i.e., estimation of concentrations based on inhibition of fungal growth in vitro that represent both the parent drug and active metabolite(s) are typically two to three times the parent drug concentrations measured by HPLC. A subcommittee of the National Committee for Clinical Laboratory Standards has recently agreed upon a standardized method for in vitro susceptibility testing of yeast. A great need exists for evaluation of correlations between clinical response in birds and in vitro susceptibility testing.

Clinicians must rely on accumulated experience for guidance in administering antifungal drugs. A few studies have attempted to correlate efficacy with drug concentrations in human patients. Failure to achieve plasma concentrations greater than 250 ng/ml were observed to be the major risk factor for developing fatal fungal infections in severely neutropenic human patients. Other studies show that serum concentrations less than 5,000 ng/ml in patients with aspergillosis and less than 1,000 ng/ml in patients with cryptococcal meningitis infections are associated with an unfavorable outcome . In the present study, plasma concentrations of itraconazole on day I exceeded 250 ng/ml in only 1 of 10 birds in both treatment groups. Only one of the birds in group 3 had a plasma drug concentration greater than 250 ng/ml at 1 and 8 hours after drug administration on day 14. Of birds in group 4, 2 of 13 birds failed to achieve plasma concentrations greater than 250 ng/ml at 1 hour, and 3 of 13 birds failed to achieve concentrations greater than 250 ng/ml at 8 hours on day 14. However, only one bird had low plasma concentrations at both times. Only two blood samples were collected from each bird in this study; therefore, peak plasma concentrations may have been higher than the determined values. Lumeij et al found that the peak plasma concentration (1,130 ng/ml) in pigeons given 10.3 mg/kg of itraconazole granules occurred 4 hours after drug administration.

This potential variability in antifungal drug concentration among individual avian patients may result in variability in clinical outcome. Low plasma concentration may lead the clinician to believe that a higher dosage may be required in some pigeons; however, consideration should be made regarding accumulation of the active metabolite. To date, no studies in avian species have attempted to describe correlation between plasma concentration of the hydroxy metabolite and the therapeutic outcome. The metabolite was not detectable in the plasma of all birds 1 and 8 hours after drug administration; therefore, a better indication of therapeutic outcome may be tissue concentrations of metabolite and parent drug In this study, tissue concentrations of the metabolite were much greater than either plasma or tissue concentration of parent drug. Dissolution of the commercially available granules in acid led to a high concentration of itraconazole in plasma, which, in turn, resulted in a higher hydroxyitraconazole tissue concentration.

Bioavailability and elimination of itraconazole are dose-dependent in humans. Higher dosages are thought to, result in saturation of first-pass metabolism in the liver. The peak plasma concentration in humans is more than twice as high after a 200-mg dose compared with a 100-mg dose. The half-life after a single dose of drugs in humans ranges from 17 to 25 hours, whereas the half-life is 30 to 40 hours after 14 days of dosing. The half-life of itraconazole after a single 10-mg/kg dose was 13.3 hours in pigeons. Altered half-life with repeated drug administration may be a reflection of enterohepatic recirculation of unchanged itraconazole as well as saturated metabolism pathways. Enterohepatic circulation has been reported in rats but has not been determined in humans or birds. In the present study, the itraconazole concentration was significantly higher in groups 2 and 4 compared with groups 1 and 3. Plasma concentrations were greater after 14 days of drug administration in group 4 compared with one dose in group 2 but not after 14 days in group 3 compared with one dose in group 1. This suggests that saturation of elimination pathways may have occurred in group 4, which absorbed the greater amount of drug. However, it is expected that elimination half-lives would be prolonged with saturation of the elimination pathways. Elimination half-lives were not determined, because only two blood samples were collected from each bird.

The currently marketed formulation of itraconazole is capsules containing itraconazole-coated lactose granules. Itraconazole is essentially insoluble in aqueous solutions at neutral pH; however, it is soluble in acidic solutions. Absorption of itraconazole is reduced under conditions of low intragastric acidity, such as in patients receiving the H2 antagonists cimetidine and ranitidine. The pH of the crop and stomach of birds is considerably less acidic than that of the mammalian stomach . Hydrochloric acid is secreted by the glandular stomach; however, ingesta typically remains within the glandular stomach for only a limited period of time and is often mixed with the contents of the ventriculus in a rotatory manner, resulting in a higher pH compared with mammals. In addition, dissolved drug may be adsorbed by the koilin lining of the gizzard, which consists of desquamated cells, merocrine secretions, and bile acids. In contrast to gastric absorption in mammals, it is unlikely that marked drug absorption occurs across the keratinlike epithelium of the gizzard of birds. Itraconazole is known to bind to keratin in skin for prolonged periods of time, and binding to the epithelium of the gizzard may preclude systemic availability. In human patients there is a significant increase in the bioavailability of itraconazole when it is taken with food. Birds with fungal infections are often anorexic. In this study, groups 1 and 3 were fasted to mimic this condition. In contrast, groups 2 and 4 mimicked birds that would be eating normally or tube fed in a veterinary hospital.

As expected, dissolution of itraconazole granules in acid resulted in significantly higher itraconazole concentrations in the blood. Although the drug concentration was higher in the lung, brain, and small intestine of birds on day 1, and in the lung, small intestine. and air sac on day 14 in birds in group 4 compared with group 3, the differences were not statistically significant. Tissue concentrations of hydroxyitraconazole were remarkably higher than concentrations of parent drug. Mean metabolite concentrations in all tissues were greater in group 4 compared with group 3; however, the differences in the lung and air sac were not statistically significant. The lack of significance is probably a reflection of the wide variability between birds. Surprisingly, the concentration of itraconazole in the lung of pigeons given the drug in a capsule at a higher dose (10 mg/kg) was lower than that reported in this study .

Although more than 99% of itraconazole is bound to plasma proteins after a single dose in humans, concentration in tissues typically exceeds plasma concentration with repeated dosing. In rats, very low concentrations are achieved in the brain, eye, and saliva after a single dose; however, the brain: plasma ratio of concentrations in dogs receiving itraconazole for 12 months was close to one. In this study, parent drug could not be detected in the brains of 3 of 8 or in 5 of 11 birds in group 3 and group 4, respectively. The hydroxy metabolite was present in all brain tissue samples analyzed. These concentrations of parent drug suggest that it should not be used for aspergillosis of the brain in birds. However, if the hydroxy metabolite is equipotent, it may be clinically useful. This is a difficult problem to address clinically because amphotericin B does not cross the blood-brain barrier well, and fluconazole is less effective against aspergillosis than against candidiasis.

The epithelial cells lining the respiratory tree of birds act as fixed macrophages, which considerably enhances uptake of foreign material compared with mammals. High concentrations of biologically active itraconazole have been shown to accumulate intracellularly in human alveolar macrophages, with little drug released from these cells after uptake. Itraconazole concentrations in bronchoalveolar lavage fluid and a bronchial biopsy specimen in a human patient given 100 mg on alternate days were 13-25 nmol/L and 1.7 pmol/mg tissue, respectively. The lung: plasma concentration ratios seen in this study were similar to those reported in humans, and the air sac: plasma ratios were similar to those reported for omentum in humans. Although the number of adipocytes is higher in omentum compared with air sacs, their epithelia are similar. Remarkably, the concentrations of the hydroxy metabolite ranged from 13 to 541 µg/g of lung tissue and from 6 to 184 µg/g of air sac tissue in group 4. In contrast, the respective ranges for group 3 were only 1-5 µg/g and 1.5-10 pg/g, respectively. The clinical response seen in avian aspergillosis patients treated with itraconazole dissolved in acid, diluted with orange juice, and gavaged with food, at The University of Tennessee College of Veterinary Medicine (UTCVM) is presumably a result of the high metabolite concentrations achieved.

Experimental and clinical studies indicate that prolonged treatment with antifungal agents is necessary for clinical success. Forty-two days after inoculation of mice with A. fumigatus and treatment with 5 mg/kg/day itraconazole, only 50% were culture negative. In this study, parent drug and metabolite concentrations and clinical toxicity were evaluated after only 14 days of drug administration; however, the only side effects observed in an orange-winged Amazon parrot (Amazona amazonica) treated with 5 mg/kg itraconazole for 1 year were transient anorexia and lethargy (Orosz, unpubl.). Birds are typically given the itraconazole in acid formulation for 4 to 6 months at UTCVM, with no adverse effects seen to date. Although no itraconazole-associated toxicities in birds have been reported in the literature, there is concern that the drug may have contributed to the deaths of African grey parrots (Psittacus erithacus) (Orosz, unpubl.). No pharmacokinetic data have been published for this species; however, these birds may either absorb more of the drug, metabolize the drug more efficiently thereby accumulating more of the metabolite, or the parent drug or metabolite(s) may be more toxic in this species. Drug disposition may also vary with disease states. Studies are needed to address these potentially serious complications.

This study demonstrates a number of clinically relevant aspects of antifungal therapy. There appears to be a biologically significant difference in tissue antifungal concentrations in birds given itraconazole dissolved in acid and gavaged with orange juice and food (group 4) compared with those receiving the granules in orange juice (group 3). The clinical effect seen may be due in part to this enhanced bioavailability as well as to the higher levels of the active metabolite accumulating in the tissues. The concentration of the parent drug plus its active metabolite in the lungs and pulmonary air sacs are above the minimum inhibitory concentrations reported for most Aspergillus organisms; however, the concentration of the parent drug alone was not therapeutic. Although the concentration of the parent drug was low or undetectable in the brain, the active metabolite accumulated there with repeated administration of the drug dissolved in acid. This suggests that therapeutic central nervous system concentrations may be achieved when the drug is administered in this manner. Another factor to consider is the variability of drug accumulation between individuals that was seen in this study. The pharmacokinetics may also vary with the species examined, particularly those with anatomically and physiologically distinct gastrointestinal tracts. Further studies are needed to clarify these issues in order to more effectively manage aspergillosis in avian species.

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

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