Single-dose pharmacokinetics of orally administered

terbinafine in bearded dragons (Pogona vitticeps)

and the antifungal susceptibility patterns
of Nannizziopsis guarroi
Michael S. McEntire, DVM1; Jennifer M. Reinhart, DVM, PhD1; Sherry K. Cox, PhD2; Krista A. Keller, DVM1*

1Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Champaign-Urbana, Urbana, IL
2Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN
*Corresponding author: Dr. Keller (kak@illinois.edu)
https://doi.org/10.2460/ajvr.21.02.0023

OBJECTIVE
To identify the antifungal susceptibility of Nanniziopsis guarroi isolates and to evaluate the single-dose pharmaco-
kinetics of orally administered terbinafine in bearded dragons.
ANIMALS
8 healthy adult bearded dragons.
PROCEDURES
4 isolates of N guarroi were tested for antifungal susceptibility. A compounded oral solution of terbinafine (25 mg/
mL [20 mg/kg]) was given before blood (0.2 mL) was drawn from the ventral tail vein at 0, 4, 8, 12, 24, 48, 72,
and 96 hours after administration. Plasma terbinafine concentrations were measured with high-performance liquid
chromatography.
RESULTS
The antifungal minimum inhibitory concentrations against N guarroi isolates ranged from 4,000 to > 64,000 ng/mL
for fluconazole, 125 to 2,000 ng/mL for itraconazole, 125 to 2,000 ng/mL for ketoconazole, 125 to 1,000 ng/mL for
posaconazole, 60 to 250 ng/mL for voriconazole, and 15 to 30 ng/mL for terbinafine. The mean ± SD peak plasma
terbinafine concentration in bearded dragons was 435 ± 338 ng/mL at 13 ± 4.66 hours after administration. Plasma
concentrations remained > 30 ng/mL for > 24 hours in all bearded dragons and for > 48 hours in 6 of 8 bearded
dragons. Mean ± SD terminal half-life following oral administration was 21.2 ± 12.40 hours.
CLINICAL RELEVANCE
Antifungal susceptibility data are available for use in clinical decision making. Results indicated that administration
of terbinafine (20 mg/kg, PO, q 24 to 48 h) in bearded dragons may be appropriate for the treatment of dermato-
mycoses caused by N guarroi. Clinical studies are needed to determine the efficacy of such treatment.

B earded dragons (Pogona vitticeps) are common

companion lizards in the United States and are
commonly infected with Nannizziopsis guarroi, the
causative agent of yellow-fungus disease.1–9 Nan-
nizziopsis spp are a genus of keratinophilic fungi
that cause cutaneous lesions in lizards ranging from
mild scale discoloration and dysecdysis to severe,
disfiguring, and ulcerative lesions leading to inva-
sive mycosis and death.1–9 Recent molecular charac-
terization has led to several nomenclature changes;
infection with this pathogen has been previously re-
ported under the names Chrysosporium guarroi and
Chrysosporium anamorph of Nannizziopsis vriesii
(CANV).3,7–9 At this time, genetic sequencing sug-
gests that most cases of CANV in companion rep-
tiles are caused by N guarroi.3 However, many fungal
pathogens within the genera Nannizziopsis, Ophid-
iomyces, Parananniziopsis, and Emydomyces were
previously assigned to the CANV group.3,7,9,10
Despite its recognition as an emerging disease in
reptilian patients, there is limited understanding of
the best treatment for N guarroi.3,5,8 Antifungal sus-
ceptibility information exists for 32 CANV isolates11;
however, given the recent nomenclature changes,
interpretation of these data is challenging because
isolates previously assigned to the CANV group may
have been members of various other genera.3,7–10
Few antifungal agents have been investigated for
treatment of N guarroi in bearded dragons. The
azole class of antifungals are the most commonly
used, although they have been associated with hep-
atotoxicity.1,2,11 Voriconazole has been shown to be

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the most efficacious and the least toxic of this group
(14% mortality) in the treatment of what was at the
time termed CANV in bearded dragons, compared
with itraconazole (70% mortality).11 Despite clinical
and microbiological evidence of resolved infections,
the literature also supports a high rate of recurrence
of latent infections following treatment with drugs
in the azole class.2,6,12 Clients and clinicians are un-
derstandingly dissatisfied with current treatment
options for N guarroi given the expensive nature of
azoles, their variable toxicity rates, and the high like-
lihood of infection recrudescence. There is a need to
establish the susceptibility patterns of molecularly
confirmed isolates of N guarroi and to investigate
safer, more efficacious antifungals for treatment of
this disease.
Terbinafine hydrochloride is a synthetic allyl-
amine antifungal agent, originally developed for use
in the treatment of human mycotic nail infections.13–16
Terbinafine is fungicidal by inhibiting squalene epox-
idase, an enzyme required for ergosterol synthesis.
Accumulation of squalene within fungal cells leads to
cell death.13–16 Terbinafine is an attractive option for
antifungal therapy because there are rare reports of
toxicosis in other species owing to its keratinophilic
nature.16 The accumulation of terbinafine within ke-
ratinized tissues makes it a particularly attractive
treatment option for dermatomycoses.13–16 Pharma-
cokinetic studies for terbinafine have been reported
for several zoological mammal and avian species
and a single reptile species, the cottonmouth snake
(Agkistrodon piscivorous).17–23 Nebulization of ter-
binafine has become an effective treatment of an-
other important cutaneous reptile fungal pathogen,
Ophidiomyces ophidiocola, in venomous snakes, for
which oral administration is both dangerous and un-
reliable.17 Thus, terbinafine may represent an alter-
native antifungal treatment for N guarroi infection in
bearded dragons, but the susceptibility of the fun-
gus to terbinafine and the pharmacokinetics of the
drug must be established first.
The objectives of the study reported here were
to establish the antifungal susceptibility patterns of
molecularly confirmed N guarroi from clinical iso-
lates and to determine the pharmacokinetics of a
single dose of terbinafine following oral administra-
tion in bearded dragons. We hypothesized that oral
administration of terbinafine would provide plasma
drug concentrations that exceeded the minimum in-
hibitory concentration (MIC) against N guarroi and
be safe in bearded dragons.
Materials and Methods
Animals
This study was carried out with the approval of
the University of Illinois Institutional Animal Care and
Use Committee (protocol No. 17243). Eight bearded
dragons (5 males and 3 females) with ages ranging
from 2 to 3 years and body weights ranging from
256 to 384 g that were maintained as a teaching and
research colony were used for the study. Standard
AJVR | MARCH 2022 | VOL 83 | NO. 3
bearded dragon housing and husbandry was pro-
vided by the University of Illinois in accordance with
Institutional Animal Care and Use Committee proto-
col No. 18056.24 In brief, each bearded dragon was
individually housed in a front-opening 91.45 X 71.12
X 45.72-cm enclosure (V332 Vision Cage; LLL Rep-
tile and Supply Co) located within a temperature-
controlled room (24 to 25.5 °C). The daily diet in-
cluded a variety of dark leafy greens (collard greens,
kale, turnip greens, and mustard greens) with addi-
tional shredded vegetables (carrots, sweet potato,
and berries) supplemented with calcium carbonate
powder and twice weekly offering of gut loaded king
worm (Zophobas morio) larvae (High-calcium Crick-
et Diet; Fluker’s Farms and NOW Foods). Each enclo-
sure had a dedicated heat lamp and a dedicated UV
emitting bulb (both UVA and UVB) situated above
the habitat. Twelve hours of darkness were provided
daily with a basking temperature (Zoo Med Labora-
tories Inc) reaching 35 to 37 °C. The animals were
determined to be free of significant clinical disease
on the basis of normal activity, appetite, and urinary
and fecal outputs.
Culture and morphologic identification
of N guarroi isolates
Fungal isolates were derived from clinical sam-
ples of 2 bearded dragons and 2 green iguanas
(Iguana iguana). Three isolates were obtained from
patients evaluated at the William R. Pritchard Vet-
erinary Medical Teaching Hospital at University of
California-Davis, and 1 isolate was obtained as a sub-
mission from an outside private practice to the Vet-
erinary Microbiology Laboratory at the University of
California-Davis. All isolates were derived from clini-
cal patients with skin lesions considered consistent
with N guarroi infection (crusting and ulceration) as
either skin biopsies (2 isolates) or samples obtained
during necropsy (1 isolate). The isolate submitted by
a private practice was derived from skin and tissue,
but it was unknown whether the samples were ob-
tained before or after death.
Samples were inoculated onto inhibitory mold
agar (Hardy Diagnostics) and dermatophyte test
and rapid sporulation media (Derm Duet; Hardy Di-
agnostics) then incubated at 30 °C under ambient
atmosphere. Plates were examined daily for fungal
growth and fungal colonies subcultured to potato
flake agar (Biological Media Services, University of
California-Davis) and incubated as above. Prelimi-
nary identification of fungal isolates as N guarroi was
made by morphologic microscopic examination of
a tape preparation stained with lactophenol cotton
blue stain (Hardy Diagnostics) from the subculture.9
Molecular identification
Fungal DNA was obtained from each culture by
use of a NaOH extraction protocol.25 In brief, fresh
mycelium was added to 200 μL of 0.5M NaOH in a
1.5-mL microcentrifuge tube, ground by use of a
UV-sterilized pestle, incubated at 4 °C for 20 min-
utes, centrifuged at 16,873 X g for 2 minutes; 5 μL
of the resulting supernatant was added to 495 μL of
257
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100mM Tris HCl buffered with NaOH to a pH of 8.5 to
8.9 (Tris-HCl-DNA extraction solution). A PCR assay
(PTC 200 thermal cycler; Bio-Rad Laboratories Inc)
was performed in which the total reaction volume
was 25 μL (12.5 μL of a commercially available master
mix [GoTaq Green Master Mix; Promega Corp]; 1.2 μL
each of 10μM primer internal transcribed spacer
[ITS] 1F and ITS4, 3 μL of the Tris-HCl-DNA extrac-
tion solution, and 7.1 μL of DNA-free water) and the
thermal cycle parameters were initial denaturation at
94 °C for 2 minutes, 30 cycles of 94 °C for 30 sec-
onds, 55 °C for 45 seconds, and 72 °C for 1 minute
with a final extension step of 72 °C for 10 minutes.
Gel electrophoresis (1% Tris-borate-EDTA agarose
gel stained with ethidium bromide) was used to ver-
ify the presence of a PCR product before purifica-
tion with a clean-up system (Wizard SV Gel and PCR
Clean-Up System; Promega Corp). A cycle sequenc-
ing kit (BigDye Terminator version 3.1; Applied Bio-
systems Inc) was used to sequence the ITS in 1 di-
rection with the ITS5 primer on a high-throughput
capillary sequencer (3730XL; Applied Biosystems)
at the Roy J. Carver Biotechnology Center at the
University of Illinois. Sequences were examined and
manually corrected with sequence analysis software
(Sequencher version 5.1; Gene Codes Corp). Culture
identity was confirmed through nBLAST analysis of
the ITS1-5.8-ITS2 region using the National Center
for Biotechnology Information database.
Antifungal susceptibility
All plated isolates were sent on Sabouraud dex-
trose agar (Remel) to an external laboratory (Fungus
Testing Laboratory, Department of Pathology and
Laboratory Medicine, University of Texas San Anto-
nio, San Antonio, Texas) for antifungal susceptibil-
ity testing. Each isolate was broth dilution tested
against fluconazole, ketoconazole, itraconazole, ny-
statin, posaconazole, terbinafine, and voriconazole
as described.26
Compounded terbinafine
An oral solution (concentration, 25 mg/mL) of
terbinafine was compounded from commercially
available tablets (Sigma-Aldrich; 250 mg of terbin-
afine/tablet) crushed and dissolved in commercially
available suspending vehicles (Ora-Plus and Ora-
Sweet; Paddock Laboratories) using a previously
published27 compounding recipe that ensures a
stable suspension for 28 days. The time from com-
pounding to administration was < 48 hours.
Pharmacokinetic sampling and analysis
Pilot studies—To determine an appropriate
dose and sampling protocol, 2 pilot studies were
performed. Three lizards were randomly assigned
by pulling numbers from a hat to received 5 mg of
terbinafine/kg by gavage, and blood (0.2 mL) was
collected from the ventral tail vein at 0 (immediate-
ly after), 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after
terbinafine administration. A second group of 3 dif-
ferent lizards randomly assigned by pulling numbers
from a hat received 20 mg of terbinafine/kg by ga-
258 AJVR | MARCH 2022 | VOL 83 | NO. 3
vage, and blood (0.2 mL) was collected from the ven-
tral tail vein at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours
after drug administration. To determine the duration
of sampling needed to evaluate clearance, a fourth
lizard received 20 mg of terbinafine/kg by gavage,
and blood (0.2 mL) was collected from the ventral
tail vein at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72,
84, and 96 hours. The total amount of blood drawn
was less than 0.8% of the body weight for each indi-
vidual animal. Blood was collected into lithium hepa-
rin microtainers (Becton Dickinson), kept on ice until
centrifuged (Eppendorf AG22331 Centrifuge; Brink-
mann Instruments Inc) within 1 hour after collec-
tion, after which plasma was harvested and stored at
–20 °C until analysis.
Single-dose pharmacokinetics—Four months
following the pilot study, 8 lizards, including the 7
lizards used in the pilot study, received a single oral
dose of terbinafine (20 mg/kg), administered by ga-
vage directly into the stomach (total volume admin-
istered, 0.22 to 0.31 mL). Immediately after terbin-
afine gavage, a slurry of commercially available in-
tensive care diet (Intensive Care Herbivore; Emeraid
LLC; fed at 1.5% of body weight; total volume admin-
istered, 4.1 to 5.9 mL) was administered via gavage
needle. Blood (0.2 mL) was collected from the ven-
tral tail vein at 0, 4, 8, 12, 24, 48, 72, and 96 hours
after drug administration. The total amount of blood
drawn was less than 0.8% of the body weight for each
subject. Blood was collected into lithium heparin mi-
crotainers, kept on ice until centrifuged within 1 hour
after collection, after which plasma was harvested
and stored at –20 °C until analysis.
Terbinafine analysis
Plasma samples were analyzed with a reverse-
phase high-performance liquid chromatography
method.28 The system consisted of a 2695 sepa-
rations module, 2487 absorbance detector, and
computer equipped with software (Empower 3 Chro-
matography Software; Waters Corp). Terbinafine
was extracted from plasma samples using liquid ex-
traction. Briefly, previously frozen plasma samples
were thawed and vortexed, and 100 µL was trans-
ferred to a clean screw-top test tube followed by 75
µL internal standard (butenafine, 1.0 µg/mL). Hex-
ane (3 mL) was added, and the tubes were rocked
for 20 minutes and then centrifuged for 20 minutes
at 1,000 X g. The organic layer was transferred to a
clean tube and evaporated to dryness with nitrogen
gas. Samples were reconstituted in 250 µL of mobile
phase and 100 µL was analyzed.
The compounds were separated on a C18 column
(Symmetry Shield C18 Chromatography Kit; Waters
Corp; 4.6 X 100 mm; 5 µm). The mobile phase was a
mixture of 20mM phosphoric acid with 0.1% triethyl-
amine adjusted to pH 3.0 (A) and acetonitrile (65:35;
B). The flow rate was 1.1 mL/min with the column
maintained at ambient temperature (22 °C). Absor-
bance was measured at wavelength of 224 nm.
Standard curves for plasma analysis were pre-
pared by fortifying untreated, pooled plasma with
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terbinafine to produce a linear concentration range
of 5 to 1,500 ng/mL. Calibration samples were pre-
pared in the same manner as plasma samples. Aver-
age recovery for terbinafine was 95%, the intra-assay
variability ranged from 4.5% to 8.5%, and the interas-
say variability ranged from 0.93% to 8.5%. The lower
limit of quantification was 5 ng/mL.
Statistical analysis
Continuous parameters were summarized as the
mean ± SD. For the main study, noncompartmental
pharmacokinetic analysis was performed with com-
mercial software (Phoenix WinNonLin; Certara LP).
Reported parameters included the terminal rate
constant, terminal half-life (t1/2λ), maximum plasma
concentration (Cmax), time to maximum plasma con-
centration, area under the concentration-versus-
time curve (AUC) from time 0 to the last measured
concentration, AUC from time 0 to infinity, AUC from
the last measured time extrapolated to infinity and
expressed as a percentage of the total AUC, and
mean residence time (MRT). Accumulation ratios
(ARs) were calculated at dosing intervals of 24 and
48 hours using the following formula:
es in appetite or attitude despite the frequent han-
dling and venipuncture.
Antifungal susceptibility test results
Susceptibility test results for the 4 N guarroi
isolates against the antifungals fluconazole, itra-
conazole, ketoconazole, nystatin, posaconazole,
terbinafine, and voriconazole were summarized
(Table 1). On the basis of the susceptibility test
results of the 4 N guarroi isolates to terbinafine,
the target terbinafine concentration threshold for
the pharmacokinetic study was set at > 30 ng/mL.
Pharmacokinetics
Pilot studies—In the initial pilot study (terbin-
afine dose, 5 mg/kg), only 1 of the 3 bearded drag-
ons achieved plasma terbinafine concentrations > 30
ng/mL starting at approximately 4 hours and lasting
for < 8 hours after drug administration. In the sec-
ond pilot study (terbinafine dose, 20 mg/kg), all 4
bearded dragons achieved plasma terbinafine con-
centrations > 30 ng/mL by 0.5 to 8 hours after drug
administration and maintained concentrations > 30
ng/mL for 48 hours.

Single-dose pharmacokinetics—The mean ± SD

plasma terbinafine concentration was plotted over
time (Figure 1) and the pharmacokinetic parameters
where τ is the proposed dosing interval. The
were summarized (Table 2) for all 8 bearded dragons.
maximum concentration at steady state and
The median (range) plasma terbinafine concentra-
concentration just prior to the next dose at
tion at each sample time were also summarized for
steady state were calculated from the AR and
male and female bearded dragons separately (Table
single-dose data.
3). For all 8 bearded dragons, the plasma terbinafine
concentration exceeded the target MIC (30 ng/mL) by
Results 4 hours after drug administration and remained about
the target MIC for > 24 hours. For 6 of the 8 bearded
Bearded dragons dragons (3 males and 3 females), the plasma terbin-
One bearded dragon died following completion afine concentration remained above the target MIC for
of the main study. Postmortem analysis of that ani-
mal revealed a focal gastric perforation with local-
ized peritonitis, consistent with iatrogenic trauma
from gavage; no other gross or histologic lesions
were observed in any of the tissues. No adverse
effects were observed in any of the other bearded
dragons during any of the experiments, and none
of the 3 females laid eggs during the month after
study completion. There were no perceived chang-

Table 1—Antifungal minimum inhibitory concentrations

(MICs; ng/mL) for each of 4 Nannizziopsis guarroi iso-
lates obtained from 2 bearded dragons (Pogona vitti-
ceps; BD) and 2 green iguanas (Iguana iguana; GI) with
clinical skin infections.
N guarroi isolate
Antifungal BD 1 BD 2 GI 1 GI 2 Figure 1—Mean ± SD plasma terbinafine concentration
over time following gavage administration of a single
Fluconazole 4,000 64,000 32,000 > 64,000 dose of terbinafine (20 mg/kg) to 8 bearded dragons
Itraconazole 125 1000 2,000 1,000 (Pogona vitticeps). The target plasma concentration for
Ketoconazole 125 200 2,000 2000 terbinafine (30 ng/mL) selected on the basis of culture
Posaconazole 125 250 500 1,000 and susceptibility results for 4 Nannizziopsis guarroi
Voriconazole 60 125 125 250 isolates obtained from 2 bearded dragons and 2 green
Nystatin 4,000 4,000 2,000 2,000 iguanas (Iguana iguana) with clinical skin infections is
Terbinafine 30 30 15 30 also indicated (dotted line).

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> 48 hours. Accumulation ratios and predicted steady ly useful data for antifungal selection in managing
state concentrations are were summarized (Table 4). these cases.
A previous study evaluated the antifungal sus-
Discussion ceptibility of 32 CANV isolates derived from a variety
of lizards.11 In that study,11 the in vitro MICs ranged
The data presented in this study support that from 31 to 1,600 ng/mL for itraconazole, voricon-
terbinafine orally administered at a dose of 20 mg/ azole, and amphotericin B and from 10 to 500 ng/mL
kg was sufficient to reach the target MIC (30 ng/ for terbinafine and were more variable than the MICs
mL) for N guarroi and may be useful for treatment determined for the molecularly confirmed N guar-
of infections caused by N guarroi. Additionally, the roi isolates evaluated in the present study. It is likely
antifungal susceptibilities of the N guarroi isolates that the CANV isolates evaluated in that study11 rep-
assessed in this study differed from those obtained resented various fungal species within the genera
for other Onygenalean fungi and provided clinical- Nannizziopsis and Paranannizziopsis.3,7–9
Antifungal susceptibility has been investigated for
Table 2—Values for noncompartmental pharmacoki- other Onygenalean fungal pathogens. Ophidiomyces
netic parameters after gavage administration of terbi- ophidiocola, the causative agent of snake fungal dis-
nafine (20 mg/kg) to 8 bearded dragons. ease and also previously assigned to the CANV com-
Parameter Mean ± SD Range plex, has a similar antifungal susceptibility pattern
(ie, the MIC was 500 to 1,000 ng/mL for itraconazole,
Cmax (ng/mL) 434.85 ± 338.27 153–1,158 250 to 500 ng/mL for ketaconzole, 500 ng/mL for
tmax (h) 13 ± 4.66 8–24
t1/2λ (h) 21.24 ± 12.40 5.21–39.50 posaconzole, 250 ng/mL for voriconazole, and 15 ng/
λz (h) 0.049 ± 0.039 0.018–0.13 mL for terbinafine)29 as the N guarroi isolates of the
AUC0–last 10,085.97 ± 8,355.24 3,682–29,896 present study. Conversely, Emydomyces testavorans, a
(h•ng/mL) newly described fungus associated with shell mycosis
AUC0–∞ 11,364.19 ± 9,812.74 3,874.18–34,853.5 in aquatic freshwater turtles, has a different antifungal
(h•ng/mL)
AUC%extrap (%) 10.05 ± 5.90 3.59–17.99
susceptibility pattern (ie, the MIC was 1,000 to 2,000
MRT (h) 25.78 ± 9.53 11.84–40.40 ng/mL for fluconazole, 60 to 250 ng/mL for itracon-
azole, < 30 to 125 ng/mL for posaconazole, < 30 ng/mL
AUC0–last = Area under the concentration-versus-time curve for voriconazole, and 15 to 250 ng/mL),10 compared
from time 0 to the last measured concentration. AUC0–∞ = Area
under the concentration-versus-time curve from time 0 to in- with that for the N guarroi isolates of the present study.
finity. AUC%extrap = Area under the concentration-versus-time Clinicians confronted with a reptilian patient with a cu-
curve from the last measured time extrapolated to infinity and taneous mycosis should pursue molecular confirmation
expressed as a percentage of the total area under the concen- of the etiologic agent and rely on isolate-specific anti-
tration-time curve. Cmax = Maximum plasma concentration. MRT fungal susceptibility data either reported in the litera-
= Mean residence time. tmax = Time to maximum plasma con-
centration. t1/2λ = Terminal half-life. λz = Terminal rate constant.
ture (when available) or from culture and susceptibility
results for selection of appropriate antifungal therapy.
The terbinafine pharmacokinetics determined
Table 3—Median (range) of plasma terbinafine concen- for the bearded dragons of the present study dif-
tration (ng/mL) in male (n = 5) and female (3) bearded fered significantly from those in other species. The
dragons following gavage administration of a single time to maximum plasma concentration of terbin-
dose of terbinafine (20 mg/kg). afine for bearded dragons (13 ± 4.66 hours) was pro-
Time (h) Male Female longed, compared with that in cats30 (1.33 hours),
0 0 (0) 0 (0)
horses31 (1.7 hours), Greyhounds31 (2.0 hours), red-
4 51 (29–116) 309 (94–397) tailed hawks23 (Buteo jamaicensis; 3.4 hours), Afri-
8 96 (84–158) 511 (253–670) can penguins21 (Spheniscus demersus; 4.0 hours),
12 178 (36–378) 634 (265–1,158) and Hispaniolan Amazon parrots19 (Amazona ven-
24 77 (57–271) 234 (79–424) tralis; 6.4 hours). This suggests that absorption of
48 49 (0–95) 61 (40–202)
terbinafine by bearded dragons was delayed rela-
72 27 (0–51) 16 (0–133)
96 16 (0–33) 12 (0–87) tive to other species, likely owing to differences in
gastrointestinal physiology and the lower metabolic

Table 4—Accumulation ratios (ARs) and the predicted maximum concentration at

steady state (Cmax,ss) and concentration just prior to the next dose at steady state (Cτ,ss)
following gavage administration of terbinafine (20 mg/kg) to 8 healthy bearded drag-
ons assuming 24- and 48-dosing intervals.
Parameter 24-hour dosing interval 48-hour dosing interval
AR 1.87 ± 0.69 (1.04–2.91) 1.30 ± 0.29 (1.00–1.76)
Cmax,ss (ng/mL) 871.51 ± 1,030.67 (281.75–3,368.94) 593.26 ± 605.77 (205.92–2,034.05)
Cτ,ss (ng/mL) 348.86 ± 382.50 (80.30–1,233.53) 100.14 ± 111.70 (0–354.82)
Values represent the mean ± SD (range).

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rate of bearded dragons. For the bearded dragons
of the present study, terbinafine was administered
with a meal because administration of terbinafine
with food enhances the absorption and increases
the Cmax for the drug in human patients.32 The inclu-
sion of a gavage-fed meal with the terbinafine was
added to the methods of the full study to facilitate
peak absorption. The Cmax (434 ± 338 ng/mL) for
the bearded dragons of the present study follow-
ing oral administration of terbinafine at a dose of 20
mg/kg was similar to the Cmax for African penguins
receiving terbinafine a dose of 15 mg/kg, PO (200
ng/mL)21; horses receiving terbinafine at a dose
of 20 mg/kg, PO (310 ng/mL)31; and Hispaniolan
Amazon parrots receiving terbinafine at a dose of
60 mg/kg, PO (353 ng/mL),19 but much lower than
the Cmax measured in red-tailed hawks (1,200 ng/
mL),23 cats (3,220 ng/mL),30 and Greyhound dogs31
(4,010 ng/mL) that received terbinafine at a dose
of 30 mg/kg, PO. The low Cmax may be the result of
slower absorption or differences in terbinafine dos-
ages or formulations administered among studies.
It is also possible that the oral bioavailability of ter-
binafine in bearded dragons is lower than that for
species with a higher Cmax.
The plasma t1/2λ of terbinafine (21.2 ± 12.4
hours) for the bearded dragons of the present
study was longer than that observed in cats30 (8.01
hours), Greyhound dogs31 (8.6 hours), horses31
(8.1 hours), and Hispaniolan Amazon parrots19 (8.7
hours) and similar to that observed in African pen-
guins21 (17.0 hours) and red-tailed hawks23 (17.5
hours). This may represent slow elimination of ter-
binafine because of slower metabolism of the drug
in bearded dragons, although it is unknown wheth-
er terbinafine undergoes hepatic biotransformation
in this species. The t1/2λ of terbinafine is also likely
influenced by its lipophilic and keratophilic nature,33
which results in a large volume of distribution as the
drug accumulates in peripheral tissues and slowly
redistributes back into the blood. The accumulation
of terbinafine in keratinized tissues (dermis, epider-
mis, nail, and hair) is observed in multiple species
and makes terbinafine an excellent choice for the
treatment of fungal dermatomycoses.20,34–37
In mammals, terbinafine is highly protein bound,
with > 99% plasma protein binding occurring in dogs,
rabbits, and humans.14,16,34 There are no published
data on the protein affinity of terbinafine in any
reptile species. However, it must be assumed that
a degree of protein binding does occur in bearded
dragons and a state of hypoproteinemia or hyper-
proteinemia will affect terbinafine pharmacokinetics.
Vitellogenesis results in elevated protein concentra-
tions in reproductively active females preparing for
egg laying.38 Given the time of year that the present
study was performed (March), the female bearded
dragons enrolled in the study may have been in an
active state of vitellogenesis, despite a lack of gross
lipemia in all blood samples and egg laying in the
month before and month after study completion.
Although the small number of bearded dragons en-
rolled in this study did not provide enough statistical
AJVR | MARCH 2022 | VOL 83 | NO. 3
power to compare the findings between males and
females, the higher plasma terbinafine concentra-
tions noted in the females may have represented sex
differences associated with seasonal plasma protein
levels. The quantification of plasma proteins was not
pursued in this study but may be worthwhile in fu-
ture studies. However, assessment of tissue or cel-
lular drug concentration is likely the most appropri-
ate method for determination of the required kinetic
parameters for drugs that are highly protein bound,
with a free fraction of < 20% in plasma.38 Clinicians
prescribing terbinafine should strongly consider per-
forming plasma protein quantification prior to and
during terbinafine treatment.
The presence of hepatic disease may alter ter-
binafine pharmacokinetics owing to alteration of
hepatic biotransformation or as a consequence of
hypoproteinemia. Although not evaluated in reptil-
ian species, results of mammalian studies14,16,34 sup-
port that terbinafine undergoes extensive hepatic
biotransformation through multiple enzymatic path-
ways. The bearded dragons of the present study did
not undergo diagnostic testing to ascertain hepatic
health but were deemed healthy on the basis of nor-
mal behavior, appetite, and outputs. Additionally,
the bearded dragon that died and underwent nec-
ropsy following completion of the study did not have
any gross or histologic evidence of hepatopathy. Two
other bearded dragons not enrolled in the study but
maintained in the same research colony as the study
animals were euthanized. One of those bearded
dragons was euthanized 1 year before study initia-
tion and was determined to have biliary lithiasis. The
other bearded dragon was euthanized 1 month after
study completion and was found to have an intes-
tinal intussusception. At the time this manuscript
was written, the 7 remaining bearded dragons of the
present study were alive and appeared healthy with-
out any signs of illness suggestive of hepatic disease
and were not being treated for any illness over 1 year
later. The findings of this study should be presumed
to represent terbinafine pharmacokinetics in healthy
bearded dragons, and clinicians utilizing terbinafine
should consider pursuing diagnostic testing to as-
sess hepatic health in clinically ill patients.
In the present study, pharmacokinetic analysis
confirmed that gavage administration of a single
dose (20 mg/kg) of terbinafine to bearded dragons
exceeded the MIC of terbinafine (30 ng/mL) neces-
sary to inhibit growth of N guarroi for at least 24
hours. The pharmacodynamic parameter most pre-
dictive of antibiotic efficacy (T > MIC, AUC/MIC, or
Cmax/MIC) has not been established for terbinafine
in humans or animals.39,40 As this strategy is rarely
applied to antifungal agents, it is not known whether
terbinafine acts by a concentration- or time-depen-
dent mechanism. Evaluation of AR projections sug-
gested that, for bearded dragons, a dosing interval
of 24 to 48 hours may be appropriate to maintain
concentrations greater than the target MIC through-
out the course of treatment, but additional multi-
dose studies are needed to fully evaluate the phar-
macokinetics of terbinafine at steady state.
261
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 All 8 bearded dragons of the present study tol-
erated terbinafine well and no overt clinical signs
suggestive of toxicosis were observed. The admin-
istration of terbinafine via gavage was performed
to improve dosing accuracy but may have resulted
in the acute death of 1 study subject. Oral, rather
than gavage, administration of terbinafine is rec-
ommended for clinical patients. In human patients
receiving terbinafine, the most commonly reported
adverse effects are gastrointestinal disturbances,
headache, skin disorders, and taste abnormalities.
Reported adverse effects of terbinafine in veteri-
nary patients include transient periocular swelling
and erythema in dogs,31,39 regurgitation in avian
species,19 transient lassitude in cats,30 and tran-
sient head shaking and lip curling in horses.31 Care-
ful monitoring of clinical patients is warranted until
multidose pharmacokinetic and controlled efficacy
and safety trials can be pursued.
The present study had several important limi-
tations, including a small study population, lack of
multidose data, and lack of preliminary testing to
ascertain the hepatic health and plasma protein
concentration of the study subjects, all of which
could be addressed in future larger-scale studies.
Although the stability of terbinafine in frozen plas-
ma has been demonstrated in other species,23 it was
not investigated in plasma samples obtained from
the bearded dragons of the present study. Given the
accumulation of terbinafine within keratinized tis-
sues, tissue or cellular concentrations may be more
appropriate for determining the kinetic and dynam-
ic parameters needed to establish successful dos-
ing regimens. Terbinafine is primarily metabolized
in the liver14,16,34; however, the present study did
not evaluate terbinafine metabolites (carboxyter-
binafine, desmethyhydroxyterbinafine, hydroxyter-
binafine or n-desmethylterbinafine) in either the
pharmacokinetic or susceptibility experiments. It is
possible reptiles may metabolize terbinafine differ-
ently than avian and mammalian species or that N
guarroi may have differing susceptibility patterns to
terbinafine metabolites. Lastly, this study presented
preliminary data supporting sex differences in ter-
binafine pharmacokinetics that should be explored
in future studies.
In conclusion, administration of terbinafine (20
mg/kg, PO) every 24 to 48 hours may be useful in
treating infections caused by N guarroi in bearded
dragons by producing plasma concentrations greater
than the target MIC (30 ng/mL) established for the
drug in the present study. However, multiple-dose
pharmacokinetic and controlled treatment trials are
needed to confirm this recommendation. Given the
predicted concentrations at steady state, terbinafine
doses of 20 mg/kg, PO, administered at 24- or 48-
hour intervals are recommended for bearded drag-
ons; however, a lower dose may be sufficient for
24-hour dosing. Further research is needed to de-
termine the details of reptilian terbinafine metabo-
lism, protein binding, and to evaluate its accumula-
tion within keratinized tissues in both infected and
healthy reptiles.
262 AJVR | MARCH 2022 | VOL 83 | NO. 3
Acknowledgments
Supported by the University of Illinois College of Veteri-
nary Medicine Companion Animal Research Grant.
The authors thank Drs. David Sanchez-Migallon
Guzman, Joanne Paul-Murphy, and Barbara Byrne for their
assistance in obtaining the fungal isolates used for culture,
identification, and sensitivity in this study.
References
1. Abarca ML, Martorell J, Castellá G, Ramis A, Cabañes FJ.
Dermatomycosis in a pet inland bearded dragon (Pogona
vitticeps) caused by a Chrysosporium species related to
Nannizziopsis vriesii. Vet Dermatol. 2009;20(4):295–299.
2. Bowman MR, Paré JA, Sigler L, et al. Deep fungal derma-
titis in three inland bearded dragons (Pogona vitticeps)
caused by the Chrysosporium anamorph of Nannizziopsis
vriesii. Med Mycol. 2007;45(4):371–376.
3. Cabañes FJ, Sutton DA, Guarro J. Chrysosporium-related
fungi and reptiles: a fatal attraction. PLoS Pathog. 2014;10
(10):e1004367. doi:10.1371/journal.ppat.1004367
4. Le Donne V, Crossland N, Brandão J, et al. Nannizziopsis
guarroi infection in 2 inland bearded dragons (Pogona
vitticeps): clinical, cytologic, histologic, and ultrastruc-
tural aspects. Vet Clin Pathol. 2016;45(2):368–375.
5. Mitchell MA, Walden MR. Chrysosporium anamorph Nan-
nizziopsis vriesii: an emerging fungal pathogen of cap-
tive and wild reptiles. Vet Clin North Am Exot Anim Pract.
2013;16(3):659–668.
6. Schmidt-Ukaj S, Loncaric I, Spergser J, Richter B,
Hochleithner M. Dermatomycosis in three central bearded
dragons (Pogona vitticeps) associated with Nannizziopsis
chlamydospora. J Vet Diagn Invest. 2016;28(3):319–322.
7. Sigler L, Hambleton S, Paré JA. Molecular characteriza-
tion of reptile pathogens currently known as members
of the Chrysosporium anamorph of Nannizziopsis vriesii
complex and relationship with some human-associated
isolates. J Clin Microbiol. 2013;51(10):3338–3357.
8. Paré JA, Sigler L. An overview of reptile fungal patho-
gens in the genera Nannizziopsis, Paranannizziopsis
and Ophidiomyces. J Herpetol Med Surg. 2016;26(1-
2):46–53.
9. Stchigel AM, Sutton DA, Cano-Lira JF, et al. Phylogeny of
Chrysosporia infecting reptiles: proposal of the new fam-
ily Nannizziopsiaceae and five new species. Persoonia.
2013;31:86–100.
10. Woodburn DB, Miller AN, Allender MC, Maddox CW, Terio
KA. Emydomyces testavorans, a new genus and species of
Onygenalean fungus isolated from shell lesions of fresh-
water aquatic turtles. J Clin Microbiol, 2019;57(2):1–11.
Doi:10.1128/JCM.00628-18
11. Van Waeyenberghe L, Baert K, Pasmans F, et al. Voricon-
azole, 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.
12. Johnson RS, Sangster CR, Sigler L, Hambleton S, Paré JA.
Deep fungal dermatitis caused by the Chrysosporium ana-
morph of Nannizziopsis vriesii in captive coastal bearded
dragons (Pogona barbata). Aust Vet J. 2011;89(12):515–
519.
13. Barot BS, Parejiya PB, Patel HK, Mehta DM, Shelat PK.
Drug delivery to the nail: therapeutic options and chal-
lenges for onychomycosis. Crit Rev Ther Drug Carrier
Syst. 2014;31(6):459–494.
14. Jain S, Sehgal VN. Terbinafine, a unique oral antifungal:
current perceptions. Int J Dermatol. 2000;39(6):412–423.
15. Keller KA. Therapeutic review: terbinafine. J Exot Pet
Med. 2012;21(2):181–185.
16. Krishnan-Natesan S. Terbinafine: a pharmacological and
clinical review. Expert Opin Pharmacother. 2009;10(16):
2723–2733.
Unauthenticated | Downloaded 06/04/23 02:33 PM UTC
17. Kane LP, Allender MC, Archer G, et al. Pharmacokinetics
of nebulized and subcutaneously implanted terbinafine in
cottonmouths (Agkistrodon piscivorus). J Vet Pharmacol
Ther. 2017;40(5):575–579.
18. Emery LC, Cox SK, Souza MJ. Pharmacokinetics of neb-
ulized terbinafine in Hispaniolan Amazon parrots (Am-
azona ventralis). J Avian Med Surg. 2012;26(3):161–
166.
19. Evans EE, Emery LC, Cox SK, Souza MJ. Pharmacokinetics
of terbinafine after oral administration of a single dose to
Hispaniolan Amazon parrots (Amazona ventralis). Am J
Vet Res. 2013;74(6):835–838.
20. Court MH, Robbins AH, Whitford AM, Beck EV, Tseng FS,
Reeder DM. Pharmacokinetics of terbinafine in little brown
myotis (Myotis lucifugus) infected with Pseudogymno-
ascus destructans. Am J Vet Res. 2017;78(1):90–99.
21. Bechert U, Christensen JM, Poppenga R, Le H, Wyatt J,
Schmitt T. Pharmacokinetics of orally administered ter-
binafine in African penguins (Spheniscus demersus) for
potential treatment of aspergillosis. J Zoo Wildl Med.
2010;41(2):263–274.
22. Souza MJ, Redig P, Cox SK. Plasma concentrations of itra-
conazole, voriconazole, and terbinafine when delivered by
an impregnated, subcutaneous implant in Japanese quail
(Coturnix japonica). J Avian Med Surg. 2017;31(2):117–122.
23. Bechert U, Christensen JM, Poppenga R, Fahmy SA, Redig
P. Pharmacokinetics of terbinafine after single oral dose
administration in red-tailed hawks (Buteo jamaicensis). J
Avian Med Surg. 2010;24(2):122–130.
24. Raiti P. Husbandry, diseases and veterinary care of the
bearded dragon (Pogona vitticeps). J Herpetological Med
Surg. 2012;22(3-4):117–131.
25. Osmundson TW, Eyre CE, Harden KM, Dhillon J, Gar-
belotto MM. Back to basics: an evaluation of NaOH and
alternative rapid DNA extraction protocols for DNA bar-
coding, genotyping, and disease diagnostics from fungal
and oomycete samples. Mol Ecol Resour. 2013;13(1):66–
74.
26. Alexander BD, Procop GW, Dufresne P, et al. Reference
Method for Broth Dilution Antifungal Susceptibility Test-
ing for Filamentous Fungi. 3rd ed. Clinical and Laboratory
Standards Institute; 2015. CLSI Standard M38.
27. Abdel-Rahman SM, Nahata MC. Stability of terbinafine
hydrochloride in an extemporaneously prepared oral sus-
pension at 25- and 4-degrees C. Am J Health Syst Pharm.
1999;56(3):243–245.
AJVR | MARCH 2022 | VOL 83 | NO. 3
28. Cox S, Hayes J, Hamill M, Martin A, Pistole N. Determining
terbinafine in plasma and saline using HPLC. J Liq Chro-
matogr Relat Technol. 2015;38(5):607–612.
29. Lindemann DM, Allender MC, Rzadkowska M, et al.
Pharmacokinetics, efficacy and safety of voriconazole
and itraconazole in healthy cottonmouths (Agkistro-
don piscivorus) and massasauga rattlesnakes (Sistrurus
catenatus) with snake fungal disease. J Zoo Wildl Med.
2017;48(3):757–766.
30. Wang A, Ding H, Liu Y, Gao Y, Zeng Z. Single dose phar-
macokinetics of terbinafine in cats. J Feline Med Surg.
2012;14(8):540–544. doi:10.1177/1098612X12442280
31. Williams MM, Davis EG, Kukanich B. Pharmacokinetics
of oral terbinafine in horses and Greyhound dogs. J Vet
Pharmacol Ther. 2011;34(3):232–237.
32. Nedelman J, Cramer JA, Robbins B, et al. The effect of
food on the pharmacokinetics of multiple-dose terbi-
nafine in young and elderly healthy subjects. Biopharm
Drug Dispos. 1997;18(2):127–138.
33. Hosseini-Yeganeh M, McLachlan AJ. Physiologically based
pharmacokinetic model for terbinafine in rats and humans.
Antimicrob Agents Chemother. 2002;46(7):2219–2228.
34. Jensen J. Pharmacokinetics of Lamisil in humans. J Der-
matolog Treat. 1990;1:(suppl 2) 15–18.
35. Faergemann J, Zehender H, Denouel J, Millerioux L. Levels
of terbinafine in plasma, stratum corneum, dermis, epi-
dermis (without stratum corneum), sebum, hair and nails
during and after 250 mg terbinafine orally once per day
for four weeks. Acta Derm Venereol. 1993;73(4):305–309.
36. Foust AL, Marsella R, Akucewich LH, et al. Evaluation of
persistence of terbinafine in the hair of normal cats after 14
days of daily therapy. Vet Dermatol. 2007;18(4):246–251.
37. Gimmler JR, White AG, Kennis RA, Cruz-Espindola C,
Booth DM. Determining canine skin concentrations of ter-
binafine to guide the treatment of Malassezia dermatitis.
Vet Dermatol. 2015;26(6):411–416.
38. Howard JG, Jaensch S. Haematology and plasma biochemis-
try reference intervals in wild bearded dragons (Pogona vitti-
ceps). Aust Vet J. 2021;99(6):236–241. doi:10.1111/avj.13060
39. Sakai MR, May ER, Imerman PM, et al. Terbinafine phar-
macokinetics after single dose oral administration in the
dog. Vet Dermatol. 2011;22(6):528–534.
40. Toutain PL, del Castillo JR, Bousquet-Melou A. The
pharmacokinetic-pharmacodynamic approach to a ra-
tional dosage regimen for antibiotics. Res Vet Sci.
2002;73(2):105–114.
263
Unauthenticated | Downloaded 06/04/23 02:33 PM UTC