steady-state plasma concentrations of itraconazole ...

Journal of Zoo and Wildlife Medicine 34(2): 171–178, 2003 Copyright 2003 by American Association of Zoo Veterinarians

STEADY-STATE PLASMA CONCENTRATIONS OF ITRACONAZOLE AFTER ORAL ADMINISTRATION IN KEMP’S RIDLEY SEA TURTLES, LEPIDOCHELYS KEMPI Charles A. Manire, D.V.M., Howard L. Rhinehart, B.A., C.V.T., Gennethel J. Pennick, M.T., Deanna A. Sutton, Ph.D., Robert P. Hunter, M.S., Ph.D., and Michael G. Rinaldi, Ph.D. Abstract: Pharmacokinetic studies of antifungal agents in reptiles are uncommon. Itraconazole, which has been used prophylactically in juvenile sea turtles suffering from hypothermia (cold stunning) on a regular basis, was evaluated for steady-state plasma concentrations. Five Kemp’s ridley sea turtles (Lepidochelys kempi) receiving itraconazole at several dosages in a rehabilitation program had blood collected within 24 hr to estimate dosing frequency. Subsequently, serial blood samples of Kemp’s ridley sea turtles that were given itraconazole at several dosages for 30 days to treat cold stunning were collected at various intervals to evaluate itraconazole plasma concentrations. Tissue samples were collected from one Kemp’s ridley that died during rehabilitation. Plasma concentrations of itraconazole (and of hydroxyitraconazole [OH-ITRA], one of its major bioactive metabolites) were determined using a modified, validated reverse-phase high-performance liquid chromatography technique. Itraconazole concentrations in tissues were determined by bioassay to be far greater than the plasma concentrations measured in any of the turtles. At a 15-mg/kg dosage, the half-life (t1/2) was 75 hr for itraconazole and 55 hr for OH-ITRA. All dosages produced adequate concentrations in some turtles, but consistent therapeutic concentrations were produced only at 15 mg/kg q72hr and 5 mg/kg s.i.d., with the latter producing the highest plasma concentrations. Key words: Itraconazole, hydroxyitraconazole, reptile, Kemp’s ridley sea turtle, Lepidochelys kempi, pharmacokinetics.

INTRODUCTION Cold stunning (extended hypothermia) commonly causes fall and winter sea turtle strandings in the eastern U.S.32,34 In affected turtles that survive, chronic immune compromise seriously threatens long-term survival and may lead to systemic infections, so prophylactic and therapeutic broad-spectrum antimycotic and antimicrobial agents are commonly administered during rehabilitation. There are few published pharmacokinetic studies on sea turtles or other reptiles, so rational dosage regimen design is difficult. The orally administered triazole antimycotic drug itraconazole inhibits 14-demethylase in fungal membranes. It has high lipid solubility, and in humans, where it shows high tissue–plasma ratios,5 it is used prophylactically in immune-compromised human patients as well as for a wide range of dermatomycoses and disseminated mycotic infections.9 From the Sea Turtle Rehabilitation Hospital, Mote Marine Laboratory and Aquarium, 1600 Ken Thompson Parkway, Sarasota, Florida 34236, USA (Manire, Rhinehart); the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA (Pennick, Sutton, Rinaldi); and the Zoological Pharmacology Laboratory, Department of Anatomy and Physiology, Kansas State University, 129 Coles Hall, Manhattan, Kansas 66506, USA (Hunter). Correspondence should be directed to Dr. Manire.

Itraconazole is used in veterinary mycoses that are unresponsive to more common antimycotics.13 Its pharmacokinetic properties have been studied in birds,15,17,21,22,30 cats,4 chameleons (Anolis sp.),23 and spiny lizards (Sceloporus sp.),7 and it has been evaluated for efficacy in dogs,1 horses,2 and bottlenose dolphins (Tursiops truncatus).27,28 Because all sea turtle species are either endangered or threatened, controlled pharmacokinetic studies are difficult to perform, and dosages of itraconazole in reptiles have been extrapolated from dog and spiny lizard dosages.1,7,24 We studied steady-state itraconazole plasma concentrations in juvenile Kemp’s ridley sea turtles (Lepidochelys kempi) to assist with appropriate dosing for this species. MATERIALS AND METHODS Animals All turtles were individually housed in recirculating, filtered seawater tanks with a minimum volume of 1,500 L per turtle maintained at 25–278C. Turtles weighed 1.5–4.7 kg on admission. During rehabilitation, they were given itraconazole in capsule form with a meal (squid or capelin) until all antibiotics had been discontinued for a minimum of 14 days. The preliminary group of five juvenile Kemp’s ridley sea turtles (sex unknown) was rehabilitated for cold stunning after a mass stranding in the Cape Cod area during the winter of 1999–2000. All of

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Table 1. Antibiotic regimens with itraconazole dose information for each Kemp’s ridley sea turtle used during the preliminary portion of this study.

Turtle ID

Turtle Turtle Turtle Turtle Turtle

Itraconazole dosage (daily) (mg/kg)

B D E J K

24.4 22.3 5.4 5.2 4.9

Antibiotics in previous 14 days

Concurrent antibiotics

Trimethoprim sulfaa Trimethoprim sulfa Trimethoprim sulfa Rifampin/oral gentamicind Rifampin

Rifampinb Rifampin Metronidazolec Same Metronidazole

Sulfamethoxazole and trimethoprim, USP, Teva Pharma. USA, Sellersville, Pennsylvania 18960, USA. Rifampin USP, Eon Labs, Laurelton, New York 11413, USA. Metronidazole USP, Sidmark Laboratories, Inc., East Hanover, New Jersey 07936, USA. d Genta-fuse 100, Burns Veterinary Supply, Inc., Rockville Center, New York 11570, USA. a

b c

them were on antibiotics (Table 1) and oral itraconazole capsules (Sporanox, Janssen Pharmaceutica, Beerse, Belgium; 5 mg/kg s.i.d. [n 5 3] or 22–25 mg/kg s.i.d. [n 5 2]). After at least 30 days of itraconazole therapy and during ongoing treatment with itraconazole, blood was collected into anticoagulant blood tubes (Lithium Heparin Vacutainer, Becton-Dickinson, Franklin Lakes, New Jersey 07417, USA), using external jugular venipuncture at 4, 8, and 12 hr, and 18 or 24 hr after a dose of itraconazole. Because of the small size of these turtles, these samples were not taken serially on the same day but were taken over several days. The blood was centrifuged, and the separated plasma was stored at 2208C until analyzed. One of these turtles died subsequently while still receiving oral itraconazole (11 hr after the last dose). Lung, liver, and kidney samples were collected approximately 25 hr after the last dose for the determination of tissue itraconazole concentra-

tions. This turtle had received itraconazole (5.4 mg/ kg s.i.d.) for 145 days. Tissue samples were stored at 2708C until analyzed. Subsequently, nine other juvenile Kemp’s ridley sea turtles (sex unknown) in the same size–age class as the first group and also from Cape Cod the following winter underwent rehabilitation for cold stunning and were given oral itraconazole capsules at three different dosages for at least 30 days before plasma concentrations of itraconazole and its major metabolite, hydroxyitraconazole (OH-ITRA), were determined. Some of these turtles received antibiotics and itraconazole concomitantly (Table 2), but none received rifampin, which increases the rate of itraconazole metabolism in humans.16 These turtles were given itraconazole at three dosages (three turtles per dosage): 5, 10, and 15 mg/kg p.o. q72hr. At least 30 days after initiation of treatment, blood samples were collected from all turtles just before dosing (and 72 hr after the pre-

Table 2. Antibiotic regimens with itraconazole dose information for each Kemp’s ridley sea turtle used during the steady-state portion of the study.

Turtle ID

Dosage (q72hr) (mg/kg)

ST0113 ST0165 ST0166 ST0167 ST0164 ST0164 ST0166 ST0167

5 5 10 10 10 15 15 15

Concurrent antibiotics

Trimethoprim sulfaa None Amikacin/Unasynd Amikacin/Unasyn None None Amikacin None

Antibiotics in previous 14 days

Amikacinb Oral gentamicinc None None Oral gentamicin None Amikacin/oral gentamicin Piperacilline/oral gentamicin

Sulfamethoxazole and trimethoprim, USP, Teva Pharma. USA, Sellersville, Pennsylvania 18960, USA. Amiject D, Burns Veterinary Supply, Inc., Rockville Center, New York 11570, USA. Genta-fuse 100, Burns Veterinary Supply, Inc. d Unasyn, Pfizer Corp., New York City, New York 10017, USA. e Pipracil, Lederle Piperacillin, Inc., Carolina, Puerto Rico 00987. a

b c

MANIRE ET AL.—ITRACONAZOLE IN SEA TURTLES

vious dosage). Blood was also collected from turtles given the two lower dosages at 3–4, 24, and 48 hr after receiving the dosage and from turtles given the highest dosage at 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hr after receiving the dosage. After blood was collected, plasma was separated and stored at 2208C until analyzed. Plasma analysis Calibration standards, controls, and turtle plasma samples were assayed for itraconazole and OHITRA using a modified and validated method for human and laboratory animal pharmacokinetic studies.35 The extraction recovery rate was approximately 90% for itraconazole and approximately 89% for OH-ITRA. The itraconazole interday coefficients of variation (CVs) for controls (0.05, 0.25, and 2.5 mg/ml) were 5.91%, 6.09%, and 4.98%, respectively. The OH-ITRA controls CVs (0.05, 0.25, and 2.5 mg/ml) were 7.60%, 8.94%, and 8.21%, respectively. Tissue analysis Tissue samples were analyzed with a modified bioassay of itraconazole levels using a test medium of yeast-nitrogen base agar inoculated with Candida kefyr.3 For controls, a 15% variation from the target values of 1.0 mg/ml (0.85–1.15 mg/ml) and 10.0 mg/ml (8.8–11.5 mg/ml) was allowed. Steady-state plasma concentrations analysis The steady-state plasma concentrations were evaluated with pharmacokinetic parameters using noncompartmental analysis. Area under the curve (AUCtlast), area under the first moment of the curve (AUMCtlast), peak concentration (Cmax), time at which peak concentration is attained (Tmax), mean residence time (MRT), apparent terminal rate constant (l), and half-life (t1/2) were calculated.8,31 The t1/2 and l were calculated after the 15-mg/kg dose only. The AUCtlast and AUMCtlast were determined using trapezoidal rule. RESULTS In the preliminary portion of the study, all sea turtles, regardless of dosage, maintained approximately the same plasma steady-state concentration of both itraconazole and OH-ITRA (Fig. 1). There were no obvious peaks or troughs in the plasma concentration of either itraconazole or OH-ITRA. The lowest itraconazole concentration determined for any of the five turtles, regardless of dosage, was 1.4 mg/ml.

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Steady-state plasma concentrations study The plasma itraconazole and OH-ITRA concentrations were highly variable. The Cmax ranges for each dosage were 0.19–0.78 mg/ml (5 mg/kg), 0.13–0.98 mg/ml (10 mg/kg), and 0.81–1.07 mg/ml (15 mg/kg). The AUC, AUMC, and MRT results were nonlinear (Table 3). The plasma t1/2 at a dosage of 15 mg/kg p.o. q72hr was 75 hr for itraconazole and 55 hr for OH-ITRA. Plasma intraconazole concentrations peaked at 2 hr and again (at a higher level) at 12–24 hr (Fig. 2). Likewise, OH-ITRA concentration peaked at 2– 4 hr, with a second higher peak at 36–48 hr. The mean ratio of plasma itraconazole to OH-ITRA overall in the steady-state concentration study was 15.7 (61.34 SEM, n 5 54). Tissue concentrations For the turtle that died during treatment, the plasma concentrations at 24 hr after treatment (taken several weeks before death) were 2.13 mg/ml for itraconazole and 0.27 mg/ml for OH-ITRA. Tissue concentrations of itraconazole after death were 10.1 mg/gm in the lungs, 16.3 mg/gm in the liver, and 18.3 mg/gm in the kidney, which exceeded the plasma concentrations measured in any of the turtles. DISCUSSION The pharmacokinetic parameters of itraconazole have been studied in humans, where the major active metabolite OH-ITRA is found at twice the concentration of itraconazole in a steady-state condition in healthy volunteers.14 The itraconazole–OHITRA ratio can be extremely variable (0–10 with an average of 1.47), however.25 The half-lives of itraconazole and OH-ITRA in humans are 15–24 hr12 and about 14 hr,14 respectively, both after a single dose. In blue-fronted Amazon parrots (Amazona aestiva aestiva), itraconazole half-life is 6.1–7.2 hr, depending on dosage,21 and 13.3 hr in racing pigeons (Columbia livia domestica).17 In the spiny lizard, itraconazole half-life is 48 hr.7 For related azoles in reptiles, the half-life of ketoconazole is 13–14 hr in the gopher tortoise (Gopherus polyphemus),23 and for fluconazole it is 132–140 hr in the loggerhead sea turtle (Caretta caretta).19 In the Kemp’s ridley sea turtles of this study, plasma OH-ITRA concentrations were about 6% of plasma itraconazole concentrations, much lower than those in humans and other animals. It is possible that turtles and mammals metabolize itraconazole by different pathways, although the difference could also be the result of slower metabolism

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Figure 1. Log plasma itraconazole (ITRA) and hydroxyitraconazole (OH-ITRA) concentration–time curves during preliminary study. A. Itraconazole concentrations. B. Hydroxyitraconazole concentrations.

Table 3. Steady-state plasma concentration mean pharmacokinetic values (6SD) for itraconazole (ITRA) and hydroxyitraconazole (OH-ITRA) administered as oral capsules at three different dosages given once every 72 hr with a meal. n 5 3 per dosage. PK values

ITRA, 5 mg/kg

ITRA, 10 mg/kg

AUC AUMC MRT l (hr21) t1/2 (hr)

20.1 6 12.3 524 6 245 28 6 7.9

20.6 6 18.8 508 6 395 26 6 6.4

ITRA, 15 mg/kg

54.0 1,882 35 0.0092

6 11.9 6 433 6 0.65 6 0.002 75

OH-ITRA, 5 mg/kg

OH-ITRA, 10 mg/kg

2.18 6 1.06 56.9 6 17.3 29 6 9.0

1.84 6 2.08 48.0 6 46.8 29.1 6 9.0

OH-ITRA, 15 mg/kg

3.19 115 36.1 0.0127

6 1.22 6 42.9 6 0.72 6 0.0084 55


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Figure 2. Log plasma itraconazole (ITRA) and hydroxyitraconazole (OH-ITRA) concentration–time curves at three different dosages. Closed circles are ITRA and open circles are OH-ITRA. A. Dosage 5 mg/kg q72hr. B. Dosage 10 mg/kg q72hr. C. Dosage 15 mg/kg q72hr.

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of the drug due to the low metabolic rate of reptiles or of different expression of cytochrome P-450 isoforms in sea turtle liver. Likewise, itraconazole can inhibit mixed-function oxidases, and this may have an impact on its metabolism in sea turtles.6 It is notable that a green sea turtle (Chelonia mydas) being treated at the same time and receiving 5 mg/ kg q24hr showed results very similar to those from the Kemp’s ridleys. The low metabolic rate of sea turtles may also account for the long elimination half-life of itraconazole and OH-ITRA compared with the values for mammals, birds, and even other reptiles. In addition, it is possible that environmental temperatures (which affects the metabolic rate in these reptiles) and compartmentalization of the drug in the tissues would have an effect on the elimination from the plasma in sea turtles.18 Dosage regimens Several studies reported that 0.5 mg/ml trough concentrations were necessary in neutropenic human patients to effectively prevent fungal infections.10,11 A similar concentration in immune-compromised sea turtles would probably be an effective dose. Likewise in animals for in vitro testing, Candida spp. and Cryptococcus neoformans with MIC # 0.12 mg/ml are considered susceptible and with MIC 5 0.25–0.50 mg/ml are considered susceptible depending on dose.26 Therefore, 0.5 mg/ml was estimated to be an effective trough concentration, although tissue levels would be expected to be much higher when plasma concentrations were 0.5 mg/ml. This would suggest that all doses used in this study can be effective in individuals, but a dosage of 15 mg/kg q72hr or 5 mg/kg s.i.d. produced consistent concentrations and 5 mg/kg s.i.d. produced the highest plasma concentrations. Side effects and drug interactions In humans, the most common side effects of itraconazole are abdominal pain, nausea, vomiting, and dyspepsia. Treatment should be monitored closely in patients with increased liver enzymes.20 Cholestasis with greatly increased bilirubin levels has been found in some humans on itraconazole therapy.33 Total lactate dehydrogenase (LDH) may increase in cetaceans with the use of itraconazole and ketaconazole.29 In addition, a long list of drugs involving the cytochrome P-450 CYP3A4 isoform enzyme must be avoided, closely monitored, or have reduced dosages when given concurrently with itraconazole.16 The dosages of itraconazole reported in this study were tolerated by all the turtles without ad-

verse clinical signs. Plasma LDH was markedly elevated during most of the rehabilitation process, but this occurred before the animals were given itraconazole for the first time and had decreased before discontinuation of itraconazole therapy. No clear connection with the itraconazole and the elevation in LDH was determined. No other hematologic or plasma chemistry changes attributable to itraconazole toxicity were detected. All the turtles in the steady-state plasma concentration portion of this study had been undergoing treatment for various medical conditions and infections but were near the end of the treatment at the time of the study. It is not likely that these treatments would have affected absorption or metabolism of itraconazole. CONCLUSIONS Itraconazole was clinically tolerated by the sea turtles in this study. Administration of itraconazole capsules, in dosages of 5–25 mg/kg s.i.d. to 5–15 mg/kg q72hr, produced plasma itraconazole concentrations equivalent to the recommended trough levels for humans and effective MICs for two groups of fungi (0.5 mg/ml). Itraconazole–OHITRA ratio at the above dosages was approximately 16:1. The low metabolic rate of sea turtles, and possibly other factors, increased not only the itraconazole–OH-ITRA ratio but also the half-life. The recommended dosage of itraconazole capsules is 5 mg/kg s.i.d., although 15 mg/kg q72hr produced equally consistent plasma concentrations that were somewhat lower. Acknowledgments: We thank Neil and Ron Chawkins of Symphony Electronics Corporation for a donation that made this study possible and Asha Modak of the Fungus Testing Laboratory for performing the high-performance liquid chromatography analyses on the plasma samples. We also thank David Smith, Petra Cunningham-Smith, Lisa Duffy, Eric Anderson, Jeannie Senn, Lynne Byrd, and many volunteers and interns who assisted with turtle rehabilitation. Dr. Andy Stamper and the staff of New England Aquarium provided initial treatment of the turtles, and the Massachusetts Audubon Society Wellfleet Bay Wildlife Sanctuary rescued the turtles under adverse conditions. LITERATURE CITED 1. Arceneaux, K. A., J. Taboada, and G. Hosgood. 1998. Blastomycosis in dogs—115 cases (1980–1995). J. Am. Vet. Med. Assoc. 213: 658–664. 2. Ball, M. A., W. C. Rebhun, J. E. Gaarder, and V. Patten. 1997. Evaluation of itraconazole dimethyl-sulfox-


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Indian River Lagoon system, Florida. Copeia 1989: 696– 703. 35. Woestenborghs, R., W. Lorreyne, and J. Heykants. 1987. Determination of itraconazole in plasma and animal tissues by high performance liquid chromatography. J. Chromatogr. 413: 332–337. Received for publication 18 September 2002