pharmacokinetics, efficacy, and safety of voriconazole ...

PHARMACOKINETICS, EFFICACY, AND SAFETY OF VORICONAZOLE AND ITRACONAZOLE IN HEALTHY COTTONMOUTHS (AGKISTRODON PISCIVORUS) AND MASSASAUGA RATTLESNAKES (SISTRURUS CATENATUS) WITH SNAKE FUNGAL DISEASE Author(s): Dana M. Lindemann, D.V.M., Matthew C. Allender, D.V.M., M.S., Ph.D., Dipl. A.C.Z.M., Marta Rzadkowska, B.S., Grace Archer, B.S., Lauren Kane, D.V.M., M.S., Eric Baitchman, D.V.M., Dipl. A.C.Z.M., Elizabeth A. Driskell, D.V.M., Ph.D., Dipl. A.C.V.P., Caroline T. Chu, D.V.M., M.S., Kuldeep Singh, D.V.M., M.S., Ph.D., Dipl. A.C.V.P., Shih-Hsuan Hsiao, D.V.M., MS., Ph.D., John M. Sykes IV, D.V.M., Dipl. A.C.Z.M., and Sherry Cox, M.S., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 48(3):757-766. Published By: American Association of Zoo Veterinarians https://doi.org/10.1638/2016-0179.1 URL: http://www.bioone.org/doi/full/10.1638/2016-0179.1

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

Journal of Zoo and Wildlife Medicine 48(3): 757–766, 2017 Copyright 2017 by American Association of Zoo Veterinarians

PHARMACOKINETICS, EFFICACY, AND SAFETY OF VORICONAZOLE AND ITRACONAZOLE IN HEALTHY COTTONMOUTHS (AGKISTRODON PISCIVORUS) AND MASSASAUGA RATTLESNAKES (SISTRURUS CATENATUS) WITH SNAKE FUNGAL DISEASE Dana M. Lindemann, D.V.M., Matthew C. Allender, D.V.M., M.S., Ph.D., Dipl. A.C.Z.M., Marta Rzadkowska, B.S., Grace Archer, B.S., Lauren Kane, D.V.M., M.S., Eric Baitchman, D.V.M., Dipl. A.C.Z.M., Elizabeth A. Driskell, D.V.M., Ph.D., Dipl. A.C.V.P., Caroline T. Chu, D.V.M., M.S., Kuldeep Singh, D.V.M., M.S., Ph.D., Dipl. A.C.V.P., Shih-Hsuan Hsiao, D.V.M., MS., Ph.D., John M. Sykes IV, D.V.M., Dipl. A.C.Z.M., and Sherry Cox, M.S., Ph.D.

Abstract: Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several freeranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations .250 ng/ml (voriconazole) and .1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations .250 ng/ml for 12–24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1–17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02–1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanoxt) was evaluated in seven apparently healthy freeranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species. Key words: Agkistrodon piscivorus, cottonmouth, Eastern massasauga rattlesnake, itraconazole, Sistrurus catenatus, voriconazole.

INTRODUCTION Snake fungal disease (SFD; Ophidiomyces ophiodiicola) has been diagnosed in free-ranging colu-

From the Wildlife Epidemiology Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Drive, Urbana, Illinois 61802, USA (Lindemann, Allender, Rzadkowska, Archer, Kane); Zoo New England, 1 Franklin Park Road, Boston, Massachusetts 02121, USA (Baitchman); Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln, Urbana, Illinois 61802, USA (Driskell, Chu, Singh, Hsiao); Wildlife Conservation Society, Zoological Health Program, 2300 Southern Boulevard, Bronx, New York 10460, USA (Sykes); and the Department of Biomedical and Diagnostic Sciences, University of Tennessee, 2407 River Drive, Knoxville, Tennessee 37996, USA (Cox). Correspondence should be directed to Dr. Lindemann ([email protected])

brid8,23 and viperid snakes.1,2,19,29 The SFD clinical syndrome associated with O. ophiodiicola results in facial swelling and disfiguration, scale discoloration, granulomas, and dysecdysis.1,2,19,29 Lesions are typically restricted to the epidermis, dermis, hypodermis, and skeletal muscle of the head and cervical region in affected snakes,2,20 but have been noted elsewhere on the body.19,20,29 SFD causes widespread morbidity and mortality across the eastern United States, specifically reported as a significant threat to endangered Eastern massasaugas (Sistrurus catenatus) in Illinois.2–4 Overall, data are lacking about antifungal efficacy and safety in snakes with SFD, making further investigation of paramount importance prior to future outbreaks. Triazole antifungals have been used to treat fungal infections in reptiles with varying success.11,20,24 Triazole antifungals selectively disrupt fungal ergosterol synthesis through inhibition of cytochrome P-450–dependent 14-alpha sterol de-

757

758

JOURNAL OF ZOO AND WILDLIFE MEDICINE

methylase.13,16,17,20,22 This disruption results in an abnormal cell membrane, altered membrane fluidity and function, and cessation of fungal growth.13,17,22 Itraconazole is highly protein bound to blood cells and plasma proteins, widely distributed to lipophilic tissues, and found in high concentrations in the nails and skin.17 It is available commercially as an oral suspension, capsule, and injectable.17 Voriconazole is a second-generation triazole antifungal derived from structural modification of fluconazole, which results in an extended spectrum of activity.13,16,22 Voriconazole is commercially available as a tablet, oral suspension, or injectable.13,16,22 Voriconazole12,30 and itraconazole6,11,17,20 have been used effectively to treat Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex in lizards. Furthermore, timber rattlesnakes (Crotalus horridus horridus) with SFD were treated with voriconazole tablets (10 mg/kg per cloaca three times a week for 4 wk) with no adverse effects noted,19 but drug concentrations were not measured and animals were lost to long-term follow-up. The pharmacokinetics of itraconazole10,18 and voriconazole15 have rarely been evaluated in chelonians and lizards, but no studies are available in snakes. Alzett osmotic pumps are miniature infusion pumps designed for continuous infusion of therapeutic agents to unrestrained animals.5,11 Because of the minimal handling required, this technique can facilitate treatment of dangerous and venomous animals, including vipers. Osmotic pumps are available in a variety of physical sizes with variable release rates and durations.5 The pump rate for each model is fixed; however, the dose of the drug delivered can be adjusted by changing the drug concentration loaded into the pump.5 Factors to consider when selecting a pump model include animal size, route of compound administration (subcutaneous vs. intraperitoneal), delivery rate, duration of administration, and dose.5 Ambient temperature and osmolality are also important variables in heterotherms because the pump infusion rate is dependent on the osmotic gradient and the body temperature of the animal.5 Osmotic pumps have been used in reptiles to administer a variety of medications;21,26,27 however, delivery of triazole antifungals has not been previously attempted in any snake species. To the authors’ knowledge, there have been no studies evaluating the pharmacokinetics or safety of itraconazole or voriconazole in any snake species. Oral and intravenous antifungal therapy is less practical for the treatment of venomous snakes and, therefore, was not pursued in this study. The

goals of this study were to obtain preliminary pharmacokinetic data for subcutaneous injection of voriconazole and cloacal administration of itraconazole in healthy cottonmouths, which will establish future research directions for the effective treatment of SFD. In addition, subcutaneous osmotic-pump delivery of voriconazole was attempted for treatment of SFD in two pitviper species.

MATERIALS AND METHODS Animals Eleven clinically healthy free-ranging adult cottonmouths (Agkistrodon piscivorus) (C1–C11) were acquired from an abundant population in southern Illinois. These animals were transported to the University of Illinois and were maintained in a climate-controlled room at 68–718F in Neodesha cages (Neodesha Plastics, Inc., Neodesha, Kansas 66757, USA) with a basking site. The snakes were offered prekilled rodents every 1–2 wk and maintained on newspaper that was changed as needed. Prior to the start of the study, each cottonmouth underwent a complete physical examination to measure mass (g) and confirm that there were no abnormalities consistent with SFD (see above). The mean 6 standard deviation body weight of the snakes was 0.266 6 0.071 kg, with a range of 0.178–0.421 kg. A 6-mo washout period occurred between voriconazole and itraconazole drug trials. Following completion of the study, the surviving cottonmouths were euthanized humanely and tissues collected for itraconazole concentration determination. Concurrently, two Eastern massasaugas and a timber rattlesnake diagnosed with SFD based on clinical signs and quantitative polymerase chain reaction (qPCR) results were treated with voriconazole via subcutaneous osmotic pumps. The Eastern massasaugas were housed in Neodesha cages with a 40-W heat bulb on one side of the enclosure, providing an approximate temperature gradient of 70–858F. Similar to the massasaugas, the timber rattlesnake was held in a clinical setting with a temperature gradient from the mid-70s to upper 80s 8F in the basking area. All activities were approved by the Institutional Animal Care and Use Committee at the University of Illinois (IACUC protocol number 14031 and 14093). Experimental design Study 1—in vitro susceptibility: To establish a list of appropriate antifungals with activity against

LINDEMANN ET AL.—PHARMACOKINETICS OF TRIAZOLES IN PITVIPERS

Ophidiomyces, pure cultures from two infected Eastern massasaugas were submitted for antifungal susceptibility testing by the Clinical Laboratory and Standards Institute (CLSI) microtiter dilution method9 (The Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center, San Antonio, Texas 78229, USA). Each isolate was subjected to testing for: itraconazole, posaconazole, voriconazole, ketoconazole, clotrimazole, miconazole, terbinafine, terconazole, and griseofulvin. Study 2—subcutaneous voriconazole: Three cottonmouths (C1–C3) were used in a pilot study to determine an appropriate dosing and sampling protocol for the full subcutaneous study. A single subcutaneous injection of voriconazole (Vfend I.V.t, Pfizer Inc., New York, New York 10017, USA; reconstituted to 10 mg/ml) was administered at doses of 5 mg/kg (two animals) and 10 mg/kg (one animal). Based on the results obtained during the pilot study, four additional animals (C4–C7) were administered a single subcutaneous dose (5 mg/kg) of voriconazole in the full study. Venipuncture was performed via the caudal tail vein for both the pilot and full study, which were performed 4 wk apart. Blood (up to 0.3 ml) was collected in a 3-ml syringe with a 23-ga needle at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 hr following the single subcutaneous injection of voriconazole. Samples were placed in a lithium heparin microtainer (Becton Dickinson, Franklin Lakes, New Jersey 07417, USA) and centrifuged immediately; the plasma was placed in a cryovial, and stored at 208C. Tissue samples from individuals that died were harvested and stored at 208C. Samples were transported on dry ice for analysis (Pharmacology Lab, Department of Biomedical & Diagnostic Sciences, University of Tennessee Veterinary Teaching Hospital, Knoxville, Tennessee 37996, USA). Study 3—osmotic-pump voriconazole: Two Eastern massasaugas29 (M1, M2) diagnosed with SFD by qPCR1 and culture were treated with voriconazole subcutaneous osmotic pumps (Alzet miniosmotic pump model 2002, DURECT Corp., Cupertino, California 95014, USA). Voriconazole (22.2 mg/ml; 0.2 ml total volume) was loaded into the osmotic pump. The Eastern massasaugas were anesthetized with isoflurane (5%) in 100% oxygen with the use of a facemask and maintained on 1– 3% isoflurane. The osmotic pump (2.4-cm length; 0.08-cm outer diameter; 21 ga) was placed subcutaneously through an 8-mm skin incision with the use of local anesthesia (lidocaine) and the

759

incision was closed with 2-0 PDS in a horizontal mattress pattern. These animals were held in a clinical setting, which allowed for thermoregulation, rather than at a constant temperature typical of laboratory settings. To calculate a projected adjusted pump rate, an average body temperature was assumed to be between 70 and 858F. The pump rate (QT) was adjusted by the following formula,5 taking into account the specified pumping rate at 378C (Q0), temperature in 8C (T), and osmolality of the solution outside the pump (p ¼ 7.5 atm): QT ¼ Q0 0:135 eð0:054TÞ ½0:004p þ 0:03 With the use of these parameters, the adjusted pump rate was between 0.21–0.33 ll/hr, resulting in a dosage range of 1.02–1.6 mg/kg/hr for these two snakes. Simultaneously, a single timber rattlesnake (T1) diagnosed with SFD was treated with voriconazole via osmotic pump (Alzet mini osmotic pump model 2ML2, DURECT). Voriconazole (10 mg/ ml; 2 ml total volume) was loaded into the osmotic pump (4.6-cm length; 0.08-cm outer diameter; 21 ga). The adjusted pump rate was calculated with the use of the following formula:5 QT ¼ Q0 0:141 eð0:051TÞ ½0:007p þ 0:12 This resulted in a projected pump rate of 2.42– 3.5 ll/hr and a dosage range of 12.1–17.5 mg/kg/ hr. Blood was collected from the ventral coccygeal vein every 7–21 days, centrifuged immediately, plasma harvested, and stored at 808C until analysis. The osmotic pump was replaced at approximately 1 and 2 mo postimplantation. Study 4—per-cloaca itraconazole: The pharmacokinetics of a single per-cloaca dose of itraconazole (Sporanox 40 mg/ml oral suspension, Janssen Pharmaceuticals, Titusville, New Jersey 08560, USA; 10 mg/kg) was evaluated in seven apparently healthy free-ranging cottonmouths (C2–C4, C8–C11). Blood (up to 0.3 ml) was collected in a 3-ml syringe with a 23-ga needle at 0, 0.5, 2, 4, 8, 24, 48, 72, and 96 hr following a single per cloaca dose of itraconazole. Samples were placed in a lithium heparin microtainer (Becton Dickinson), centrifuged immediately, the plasma placed in a cryovial, and stored at 208C until sample analysis. Following euthanasia, tissue itraconazole concentrations in the kidney, liver, and skin were tested in all seven animals. Sample analysis: All analysis was performed by the Pharmacology Lab, Department of Biomedi-

760

Table 1.


Antifungal in vitro susceptibility data for isolates of Ophidiomyces from two Eastern massasaugas.

Antifungal drug

Isolate 1 (lg/ml)

Isolate 2 (lg/ml)

Target concentration (lg/ml)

Itraconazole Posaconazole Voriconazole Ketoconazole Clotrimazole Miconazole Terbinafine Terconazole Griseofulvin

1 0.5 0.25 0.5 ,0.03 0.06 0.015 8 1

0.5 0.5 0.25 0.25 ,0.03 0.06 0.015 4 1

1 0.5 0.25 0.5 0.03 0.06 0.015 8 1

cal & Diagnostic Sciences, University of Tennessee Veterinary Teaching Hospital. Voriconazole and itraconazole plasma samples were analyzed with the use of a reverse-phase high-performance liquid chromatography method (HPLC). The system consisted of a 2,695 separations module, a 2,475 fluorescence detector, and a computer equipped with Empower software (Waters Corporation, Milford, Massachusetts 01757, USA). Itraconazole was evaluated as previously described.7 Voriconazole was extracted from plasma samples with the use of liquid extraction. Briefly, previously frozen plasma samples were thawed and vortexed, and 100 ll was transferred to a clean screw-top test tube followed by 50-ll internal standard (1.0 lg/ml diazepam). Hexane (3 ml) was added and the tubes were rocked for 20 min and then centrifuged for 20 min at 1,000 g. The organic layer was transferred to a clean tube and evaporated to dryness with nitrogen gas. Samples were reconstituted in 250 ll of mobile phase and 100 ll was analyzed. The compounds were separated on a Symmetry Shield C18 (4.6 3 100 mm, 5 lm) column with a Symmetry Shield C18 guard column (Waters Corporation). The mobile phase was a mixture of 1) 20 mM phosphoric acid with 0.1% triethylamine adjusted to pH 3.0 and 2) acetonitrile (66 : 34). The flow rate was 1.3 ml/min and the column temperature ambient. Absorbance was measured at 255 nm. Standard curves for plasma analysis were prepared by fortifying untreated, pooled plasma with voriconazole to produce a linear concentration range of 10–5,000 ng/ml. Calibration samples were prepared exactly as plasma samples. Average recovery for voriconazole was 90%, and intra- and interassay variability ranged from 3.7 to 7.8% and 6.9 to 9.9%, respectively. The lower limit of quantification was 10 ng/ml. Pharmacokinetic analysis: Plasma concentrations were analyzed for each individual animal by noncompartmental analysis with the use of

Phoenix 6.4 (Pharsight Corporation, Mountain View, California, USA). Values for elimination rate constant (kz), elimination half-life (t½), maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), area under the plasma concentration time curve from time 0 to infinity (AUC0–‘), and mean transient time (MTT) were calculated.

RESULTS Study 1—in vitro susceptibility: Evaluation of in vitro susceptibility identified concentrations .250 ng/ml (voriconazole) and .1,000 ng/ml (itraconazole) as being effective against O. ophiodiicola (Table 1). Note that isolate 2 displayed greater in vitro susceptibility to itraconazole (MIC .500 ng/ml) when compared to isolate 1 (MIC .1,000 ng/ml), where MIC means the minimum inhibitory concentration. These data were used to determine the target plasma and tissue concentrations for voriconazole and itraconazole in the pharmacokinetic studies. Study 2—subcutaneous voriconazole: Voriconaconcentrations after a single 5- or 10-mg/kg injection are shown in Table 2. One pilot animal (C1; 5 mg/kg dose) became lethargic and died within 2 hr of voriconazole injection. The two remaining pilot animals (C2 and C3) survived through the end of the study with no adverse effects noted. Both doses maintained plasma concentrations above the MIC (.250 ng/ml) for the first 12 hr after administration (Table 2); however, because of the adverse effects noted in C1 and similarity in plasma concentration profile between the two doses, the lower 5-mg/kg dose was used in the full study. In the full study, all animals showed relatively rapid absorption of voriconazole after subcutaneous injection with peak concentrations occurring within 1 hr in all except one animal (C7). Three of the four animals (C5–C7) died within 12 hr of

761


Table 2. Plasma concentration (ng/ml) of voriconazole after subcutaneous administration in cottonmouths at 5 mg/kg (n ¼ 6) and 10 mg/kg (n ¼ 1). Voriconazole plasma concentration (ng/ml) in cottonmouths Time

C1, 5 mg/kg

C2, 10 mg/kg

C3, 5 mg/kg

C4, 5 mg/kg

C5, 5 mg/kg

C6, 5 mg/kg

C7, 5 mg/kg

0 hr 0.25 hr 0.5 hr 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 48 hr 72 hr 96 hr 120 hr Survival

0 9,432 3,517 –a – – – – – – – – – No

0 2,826 6,405 6,534 5,812 6,353 4,248 3,955 311 52 NDb ND – Yes

0 5,063 2,805 2,728 2,516 3,876 1,891 985 100 58 30 ND – Yes

0 5,293 7,093 3,081 2,730 2,440 1,281 1,584 427 82 ND – – Yes

0 15,657 4,406 3,254 1,856 – – – – – – – – No

0 3,458 3,930 2,472 2,362 – – – – – – – – No

0 1,269 2,496 2,579 1,874 2,933 2,454 – – – – – – No

a b

No sample. ND, none detected.

voriconazole injection (Table 2). The clinical signs exhibited prior to death occurred approximately 60–100 min postinjection and included depression, lethargy, and loss of righting reflex. Torticollis was noted in C5. In the single surviving cottonmouth from the full study (C4), plasma concentrations remained above the in vitro susceptibility concentration for the first 24 hr after voriconazole administration. Complete gross postmortem examination and histopathology was performed for all four snakes that died during the pilot and full study. No cause of death and minimal abnormalities were observed in all tissues of the pilot animal (C1). Two of the full-study animals showed no gross abnormalities, whereas the third animal had coelomic effusion with petechial hemorrhages of visceral fat. Histopathology in all three full-study animals (C5–C7) demonstrated a mild to moderate multifocal acute degenerative myopathy of skeletal muscle in a region of the body wall and in the head. Liver, lung, kidney, and spleen were tested for voriconazole concentrations from one snake that died (C7), and skin was tested from two individuals (C6 and C7). The liver (11,472 ng/g) had the highest tissue concentration of voriconazole, followed by the kidney (10,264 ng/g), skin (4,836 ng/g in C6; 7,378 ng/g in C7), lung (5,536 ng/g), and spleen (5,088 ng/g). Study 3—osmotic-pump voriconazole: The voriconazole plasma concentration results for both Eastern massasaugas are shown in Table 3. Pathology revealed lesions consistent with pro-

gression of SFD in both animals M1 and M2. One animal (M1) was euthanized approximately 2 mo after initial osmotic-pump placement because of bilateral swelling of the mandible and deteriorating condition. Postmortem examination confirmed marked subcutaneous swelling of the mandible with multiple ulcerations of the oral mucosa. Notable histologic lesions consisted of severe heterophilic and granulomatous dermatitis, cellulitis, and osteomyelitis. Lesions were associated with numerous 3–5-lm diameter, septate, acute angle branching fungal hyphae. The second animal (M2) was found dead 12 days after Table 3. Voriconazole plasma concentrations in two Eastern massasaugas (M1 and M2) and one timber rattlesnake (T1) after osmotic-pump placement. Voriconazole plasma concentration (ng/ml) Time

M1

M2

T1

Preimplant Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 12 Week 13

0 102 125 – 59a 90 85 70 76 69 – –

0 108 –b – – – – – – – – –

0 0a 66 279 – 826a 1,032 1,045 506 254a 1,020 444

a b

a

Osmotic pump placed or replaced. No sample.

a

762


Figure 1. Itraconazole and hydroxyitraconazole plasma concentration concentrations (mean 6 SD) after percloaca (n ¼ 7) administration in cottonmouths.

osmotic-pump placement. Gross examination revealed dysecdysis and multiple cutaneous crusts predominantly along the head and cervical region, as well as acute fibrinous pleuritis. Histologically, the skin was affected by a similar inflammatory process with intralesional fungal hyphae, similar to the first animal (M1). In addition to cutaneous lesions, this animal had severe granulomatous and heterophilic sinusitis and pleuropneumonia centered on fungal hyphae. Therapeutic plasma concentrations were not reached in either of the Eastern massasaugas (Table 3). In the single timber rattlesnake, therapeutic plasma concentration for SFD was reached (.250 ng/ml) approximately 2 wk after initial pump placement and remained above therapeutic levels for the entire 3-mo duration of treatment (Table 3). One week after implant placement, there was an initial 17% loss of body weight and hematocrit (HCT) decreased from 25% to 16% (reference range 14–49%).28 Thirty-four days after surgery, the snake shed, resulting in marked improvement of gross lesions. A 3-mm-diameter, slightly raised area of mild erythema remained beneath the left eye, compared to significant facial disfigurement that had been present previously. At day 58, gross improvement of the remaining facial lesion continued, all lost weight was regained with an additional 4% increase over day 0, and 16% HCT persisted. Biopsy of the lesion site at this time, however, remained qPCR positive for Ophidiomyces. At day 87, the third and final osmotic

pump was removed and HCT was found to have decreased further to 9%. Two months after implant removal, the small remaining facial lesion remained unchanged. Further treatments were not elected. The animal was released back to the wild and was subsequently found deceased the following spring, from unknown causes, and the body was not available for necropsy. Study 4—per-cloaca itraconazole: The mean plasma concentrations of itraconazole and the hydroxyitraconazole metabolite over time are shown in Figure 1. Derived pharmacokinetic variables are summarized in Table 4. Note that sufficient data points for modeling were only obtained from three animals and the program could not calculate half-life, elimination rate constant, or AUC for hydroxyitraconazole. The itraconazole mean maximum plasma concentration (Cmax) of 474 6 149 mg/ml was reached at 10.86 6 16.57 hr. The terminal half-life was 14.92 6 5.33 hr. Tissue concentrations for itraconazole and hydroxyitraconazole are shown in Table 5. Therapeutic plasma and tissue concentrations were not achieved based on in vitro data.

DISCUSSION Providing a therapeutic protocol for treatment of SFD remains challenging based on the results of this study. Subcutaneous voriconazole plasma and tissue concentrations were well above the in vitro susceptibility concentrations for the isolates used in this study, including the skin (target organ

763


Table 4. Pharmacokinetic parameters (mean 6 SD) after a single per-cloaca administration of itraconazole (10 mg/kg) in cottonmouths (n ¼ 3).a Pharmacokinetic parameter

Itraconazole, mean 6 SD

Terminal half-lifeb (hr) Elimination rate constant, kz (1/hr) Tmax (hr) Cmax (ng/ml) AUC0–‘ (hr*ng/ml) AUC0–8 (hr*ng/ml) MTT0–‘ (hr)

14.92 0.046 10.86 474 19,014 2,416 28.79

6 6 6 6 6 6 6

Hydroxyitraconazole, mean 6 SD

5.33 0.016 16.57 149 17,111 891 16.03

56 6 13.86 123 6 49

49.18 6 10.6

a Abbreviations: kz, elimination rate constant; Cmax, maximum plasma concentration, Tmax, time to maximum plasma concentration; AUC0–‘, area under the plasma concentration time curve from time 0 to infinity; AUC0–8, area under the plasma concentration time curve from time 0 to 8 hr; MTT, mean transient time. b Harmonic mean.

for Ophidiomyces treatment in pitvipers). These data suggests that voriconazole may be an effective drug for treatment of SFD dermatitis; however, the small sample size used in this study and the adverse reactions noted warrant further investigation prior to its use in pitvipers. Based on the data collected in Study 2, there does not appear to be a clear relationship between the subcutaneous voriconazole dose administered (5 mg/kg vs 10 mg/kg sc) and adverse effects. The single snake receiving a 10-mg/kg sc dose (C2) in the pilot study survived, whereas four snakes receiving a 5-mg/kg sc dose died within 12 hr (Table 2). Similarly, there is no clear correlation between peak plasma concentration and death. For example, two of the snakes that died (C1 and C5) showed peak plasma concentrations (9,432 ng/ml and 15,657 ng/ml, respectively) at 0.25 hr, whereas the other two snakes that died (C6 and C7) had much lower peak plasma concentrations at 0.5 hr (3,930 ng/ml) and 4 hr (2,933 ng/ml), respectively. The surviving snakes (C2–C4) had intermediate peak plasma concentrations that occurred between 0.25 and 1 hr (Table 2). Finally, there was no association between the dose administered and peak plasma concentration (Table 2). Several snakes (C1, C4, and C5)

receiving the 5-mg/kg sc dose had higher peak plasma concentrations than the snake receiving the 10-mg/kg sc dose. It is important to note that only a single animal received voriconazole at 10 mg/kg sc; therefore, conclusions regarding dose safety should be made with caution. Three of the cottonmouths that died following subcutaneous voriconazole administration exhibited clinical signs including depression, lethargy, loss of righting reflex, and torticollis. Voriconazole toxicity has been recently reported in multiple penguin species with clinical signs including anorexia, lethargy, weakness, ataxia, paresis, apparent vision changes, seizure-like activity, and generalized seizures.14 In humans, the most frequently reported adverse effects associated with voriconazole administration include visual disturbance (ie, temporary altered color discrimination, blurred vision, appearance of bright spots, and photophobia), skin reactions (ie, rash and photosensitivity reactions), and hepatic enzyme elevation.13,16 Less-common side effects include headache, nausea, vomiting, diarrhea, abdominal pain, and visual hallucinations.13,16 Although no microscopic lesions were observed in the examined brains of the three full-study snakes that died after subcutaneous voriconazole

Table 5. Tissue itraconazole (I) and hydroxyitraconazole (H) concentrations (ng/g) in seven apparently healthy cottonmouths (C2–C4, C8–C11) after single per-cloaca administration of itraconazole (10 mg/kg). C2

Tissue

Kidney Liver Skin

C3

I

H

250 356 ND

ND 2,117 ND a

C4

b

C9

C10

C11

H

I

H

I

H

I

H

I

H

I

H

330 497 764

ND ND ND

171 42 –c

ND 205 ND

115 759 50

ND ND ND

130 71 318

ND ND –

100 BLQb 782

ND ND ND

ND ND ND

ND ND ND

ND, none detected. BLQ, below the limit of quantification (5 ng/g). c No sample. a

C8

I

764


administration, seizure-like activity resulting from neurotoxicity is suspected. Neurologic signs, including head tilt, have also been previously observed in a 3-yr-old Dumeril’s ground boa (Acrantophis dumerili) after it received voriconazole (8.6 mg/kg sc in 5 ml Lactated Ringer’s Solution [LRS] for 5 days). This snake was later found dead with its jaws locked on its mid body (Chinnadurai, pers. comm.). In addition, six 7mo-old Dumeril’s ground boas were treated prophylactically with voriconazole (10 mg/kg sc in 3–5 ml sterile saline) prior to shipment because of a history of fungal dermatitis. Within 30 min of voriconazole administration, these snakes demonstrated hyperesthesia, excessive muscle tone, twitching, and postured with mouths open. Four of these animals died within 24–48 hr of treatment, one survived, and another was later euthanized due to prolonged anorexia (Chinnadurai, pers. comm.). Similar to the cottonmouths in this study, none of these animals showed abnormalities in the brain upon postmortem examination, despite the neurologic signs observed prior to death. One possible explanation for the lack of lesions in the central nervous system on histopathology is the acute nature of toxicity, resulting in clinical signs and death before the development of brain lesions. Postmortem examination in three cottonmouths showed regions of acute degenerative myopathy. A case of voriconazole-associated myositis has been previously reported in a 34-yr-old woman being treated for pulmonary aspergillosis post–renal transplant.25 To the authors’ knowledge, this finding has not been previously reported in veterinary species and the significance of this lesion in the role of these animals’ deaths is unknown. In Study 3, osmotic-pump delivery of voriconazole resulted in therapeutic plasma concentrations in the single timber rattlesnake, but not in either of the Eastern massasauga rattlesnakes. According to manufacturer’s instructions, the osmotic-pump infusion rate is temperature dependent.5 The variable body temperature in heterothermic animals that are housed in a clinical setting with an ambient temperature gradient complicates the pharmacokinetics and, therefore, treatment of snakes with SFD. This is illustrated by the pharmacokinetic plasma concentrations observed in this osmotic-pump study. According to calculated pump rates based on a range of body temperatures, the timber rattlesnake received a drastically higher dosage (12.1– 17.5 mg/kg/hr) when compared to the Eastern massasauga rattlesnakes (1.02–1.6 mg/kg/hr).

The dosage difference likely explains why therapeutic plasma concentrations were reached in the timber rattlesnake, but not in the massasaugas. This finding underscores the importance of appropriate osmotic-pump selection, ambient temperature monitoring, and drug delivery rate in clinical cases of SFD treated with azoles via osmotic-pump delivery. Osmotic pumps have been used to deliver amikacin in corn snakes (Elaphe guttata),27 florfenicol and amikacin in Mojave rattlesnakes (Crotalus scutulatus),26 and gonadotropin-releasing hormone in green iguanas.21 Substantial local necrosis and death occurred in five Mojave rattlesnakes that were administered florfenicol via subcutaneously implanted osmotic pumps.26 Because of the advanced stage of disease of both of the Eastern massasaugas in the current study, mortality was not likely associated with voriconazole administration. In the timber rattlesnake, despite marked gross improvement, lesion biopsy remained qPCR positive for Ophidiomyces after 44 consecutive days of therapeutic plasma concentrations. Unfortunately, follow-up biopsy was not obtained prior to the eventual release of this animal, making interpretation of therapy effectiveness difficult. Persistent infection or the presence of killed organisms, requiring additional shed to clear remaining DNA from the skin, may explain the positive qPCR result. Because timber rattlesnakes are known to show marked gross improvement through ecdysis without antifungal treatment,19 it is impossible to draw conclusions regarding the effectiveness of voriconazole osmotic-pump delivery with data from only a single animal available. Further study is needed to investigate the safety and efficacy of this technique, including more animals and variable voriconazole concentration. Per-cloaca administration of itraconazole (10 mg/kg) in the cottonmouths in Study 4 did not reach therapeutic levels in the plasma or tissues. A study evaluating the pharmacokinetics of oral itraconazole in Kemp’s Ridley sea turtles concluded that consistent therapeutic concentrations were produced only at 15 mg/kg po q 72 hr and 5 mg/kg po every 24 hr.18 Itraconazole plasma and tissue concentrations were also investigated in spiny lizards following once-daily dosing (mean dose 23.5 mg/kg po every 24 hr for 3 days).10 The Cmax in that study was 2,480 ng/ml, much higher than the Cmax obtained in the current study; however, no adverse effects were noted.10 Further research is needed to determine effective itraconazole doses by the cloacal route while maintaining safety.


CONCLUSION The data presented in this report serve as an initial step towards understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. The variation in drug selection, dose, route of administration, fungal organism, and reptile species poses significant challenges when comparing the results obtained in this study to those in the current literature. Therapeutic plasma and tissue concentrations were reached with subcutaneous voriconazole administration at 5 mg/kg; however, significant adverse effects and death were noted in this portion of the study. In contrast, therapeutic drug concentrations were not reached with osmotic-pump voriconazole in Eastern massasaugas or per-cloaca itraconazole in cottonmouths. Multiple dose pharmacokinetics for these routes and additional routes of administration (po, iv) need to be investigated in pitviper species for treatment of SFD. Acknowledgments: Funding for this project was provided by a Competitive State Wildlife Grant from the Fish and Wildlife Service in the US Department of the Interior. The authors thank Dr. Sarah Baker, Dr. Michael Dreslik, and Dr. Chris Phillips for assistance in collecting the cottonmouths, Sasha Tetzlaff and Michael Ravesi for massasauga collection, Joan Bailey for sample analysis, Dr. Joe Martinez and Anne Stengle for assistance with handling and restraint of the timber rattlesnake, and Dr. Sathya K. Chinnadurai for contributing clinical case data to the discussion of this manuscript.

LITERATURE CITED 1. Allender MC, Bunick D, Dzhaman E, Burrus L, Maddox C. Development and use of a real-time polymerase chain reaction assay for the detection of Ophidiomyces ophiodiicola in snakes. J Vet Diagn Invest. 2015;27(2):217–220. 2. Allender MC, Dreslik M, Wylie S, Phillips C, Wylie DB, Maddox C, Delaney MA, Kinsel MJ. Chrysosporium sp. infection in Eastern massasauga rattlesnakes. Emerg Infect Dis. 2011;17(12):2383–2384. 3. Allender MC, Dreslik MJ, Wylie DB, Wylie SJ, Scott JW, Phillips CA. Ongoing health assessment and prevalence of Chrysosporium in the Eastern massasauga (Sistrurus catenatus catenatus). Copeia. 2013;2013(1):97– 102. 4. Allender MC, Raudabaugh DB, Gleason FH, Miller AN. The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential

765

impact on free-ranging snake populations. Fungal Ecol. 2015;17:187–196. 5. ALZETt—pump selection [Internet]. [cited 2016 March 20]. Available from http://www.alzet.com/ products/guide_to_use/pump_selection.html 6. Bowman MR, Pare´ JA, Sigler L, Naeser JP, Sladky KK, Hanley CS, Helmer P, Phillips LA, Brower A, Porter R. Deep fungal dermatitis in three inland bearded dragons (Pogona vitticeps) caused by the Chrysosporium anamorph of Nannizziopsis vriesii. Med Mycol. 2007;45(4):371–376. 7. Cox SK, Orosz S, Burnette J, Frazier D. Microassay for determination of itraconazole and hydroxyitraconazole in plasma and tissue biopsies. J Chromatogr B Biomed Sci Appl. 1997;702(1):175–180. 8. Dolinski AC, Allender MC, Hsiao V, Maddox CW. Systemic Ophidiomyces ophiodiicola infection in a free-ranging plains garter snake (Thamnophis radix). J Herpetol Med Surg. 2014;24(1):7–10. 9. Fothergill AW. Antifungal susceptibility testing: clinical laboratory and standards institute (CLSI) methods. In: Hall GS (ed.). Interactions of yeasts, moulds, and antifungal agents. Totowa (NJ): Humana Press; 2012. p. 65–74. 10. Gamble KC, Alvarado TP, Bennett CL. Itraconazole plasma and tissue concentrations in the spiny lizard (Sceloporus sp.) following once-daily dosing. J Zoo Wildl Med. 1997;28(1):89–93. 11. Gibbons PM. Advances in reptile clinical therapeutics. J Exot Pet Med. 2014;23(1):21–38. 12. Hellebuyck T, Baert K, Pasmans F, Van Waeyenberghe L, Beernaert L, Chiers K, De Backer P, Haesebrouck F, Martel A. Cutaneous hyalohyphomycosis in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of Nannizziopsis vriesii and successful treatment with voriconazole. Vet Dermatol. 2010;21:429–433. 13. Herbrecht R. Voriconazole: therapeutic review of a new azole antifungal. Expert Rev Anti Infect Ther. 2004;2(3):485–497. 14. Hyatt MW, Georoff TA, Nollens HH, Wells RL, Clauss TM, Ialeggio DM, Harms CA, Wack AN. Voriconazole toxicity in multiple penguin species. J Zoo Wildl Med. 2015;46(4):880–888. 15. Innis CJ, Young D, Wetzlich S, Whitcomb AJ, Tell L. Plasma concentrations and safety assessment of voriconazole in red-eared slider turtles (Trachemys scripta elegans) after single and multiple subcutaneous injections. J Herpetol Med Surg. 2014;24:28–35. 16. Johnson LB, Kauffman CA. Voriconazole: a new triazole antifungal agent. Clin Infect Dis. 2003;36(5): 630–637. 17. Keller KA. Itraconazole. J Exot Pet Med. 2011; 20(2):156–160. 18. Manire CA, Rhinehart HL, Pennick GJ, Sutton DA, Hunter RP, Rinaldi MG. Steady-state plasma concentrations of itraconazole after oral administration in Kemp’s ridley sea turtles, Lepidochelys kempi. J Zoo Wildl Med. 2003;34(2):171–178.

766


19. McBride MP, Wojick KB, Georoff TA, Kimbro J, Garner MM, Wang X, Childress AL, Wellehan Jr JF. Ophidiomyces ophidiodiicola dermatitis in eight freeranging timber rattlesnakes (Crotalus horridus) from Massachusetts. J Zoo Wildl Med. 2015;46(1):86–94. 20. Mitchell MA, Walden MR. Chrysosporium anamorph Nannizziopsis vriesii: an emerging fungal pathogen of captive and wild reptiles. Vet Clin North Am Exot Anim Pract. 2013;16(3):659–668. 21. Phillips JA, Alexander N, Karesh WB, Millar R, Lasley BL. Stimulating male sexual behavior with repetitive pulses of GnRH in female green iguanas, Iguana iguana. J Exp Zool. 1985;234(3):481–484. 22. Plumb DC. Plumb’s veterinary drug handbook. 8th ed. Stockholm (WI): PharmaVet Inc.; 2015. 1296 p. 23. Rajeev S, Sutton DA, Wickes BL, Miller DL, Giri D, Meter MV, Thompson EH, Rinaldi MG, Romanelli AM, Cano JF, Guarro J. Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta). J Clin Microbiol. 2009; 47(4):1264–1268. 24. Schmidt V. Fungal infections in reptiles—an emerging problem. J Exot Pet Med. 2015;24(3):267– 275. 25. Shanmugam VK, Matsumoto C, Pien E, Rosen J, Kumar P, Whelton S, Steen V. Voriconazole-associated myositis. J Clin Rheumatol. 2009;15(7):350–353.

26. Sykes J, Folland D, Bemis D. Osmotic pump delivery of florfenicol or amikacin in Mojave rattlesnakes (Crotalus scutulatus) with Salmonella arizonae osteomyelitis. In: Proc Conf Assoc Rept Amphib Vet; 2008. p. 29–30. 27. Sykes JM, Ramsay EC, Schumacher J, Daniel GB, Cox S, Papich M. Evaluation of an implanted osmotic pump for delivery of amikacin to corn snakes (Elaphe guttata guttata). J Zoo Wildl Med. 2006;37(3): 373–380. 28. Teare J (ed.). ISIS physiological reference intervals for captive wildlife: a CD-ROM resource. Bloomington (MN): International Species Information System; 2013. 29. Tetzlaff S, Allender M, Ravesi M, Smith J, Kingsbury B. First report of snake fungal disease from Michigan, USA involving massasaugas, Sistrurus catenatus (Rafinesque 1818). Herpetol Notes. 2015;8:31– 33. 30. Van Waeyenberghe L, Baert K, Pasmans F, van Rooij P, Hellebuyck T, Beernaert L, de Backer P, Haesebrouck F, Martel A. Voriconazole, a safe alternative for treating infections caused by the Chrysosporium anamorph of Nannizziopsis vriesii in bearded dragons (Pogona vitticeps). Med Mycol. 2010;48(6): 880–885. Accepted for publication 3 January 2017