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Received: 4 March 2020    Revised: 28 May 2020    Accepted: 7 June 2020

DOI: 10.1111/jvp.12888

ORIGINAL ARTICLE

Clinical pharmacokinetics and outcomes of oral fluconazole

therapy in dogs and cats with naturally occurring fungal disease

Kate KuKanich1  | Butch KuKanich2  | Zhoumeng Lin2  | Amy J. Rankin1 |

Andrew S. Hanzlicek3 | Jean-Sebastien Palerme4 | Jonathan Bach5 | Audrey K. Cook6 |
Amy Juracek1 | Hyun Joo2

1
Department of Clinical Sciences, College
of Veterinary Medicine, Kansas State Abstract
University, Manhattan, KS, USA This multi-institutional study was designed to determine the clinical pharmacokinet-
2
Department of Anatomy and Physiology,
ics of fluconazole and outcomes in client-owned dogs (n = 37) and cats (n = 35) with
College of Veterinary Medicine, Kansas
State University, Manhattan, KS, USA fungal disease. Fluconazole serum concentrations were measured. Pharmacokinetic
3
Center for Veterinary Health Sciences, analysis was limited to animals at steady state (≥72 hr of treatment). The mean (range)
Oklahoma State University, Stillwater, OK,
USA
body weight in 31 dogs was 25.6 (2.8–58.2) kg and in 31 cats was 3.9 (2.4–6.1) kg
4
Department of Veterinary Clinical Sciences, included in pharmacokinetic analyses. The dose, average steady-state serum con-
College of Veterinary Medicine, Iowa State centrations (C SS), and oral clearance in dogs were 14.2 (4.5–21.3) mg/kg/d, 26.8
University, Ames, IA, USA
5 (3.8–61.5) µg/mL, and 0.63 ml min−1 kg−1, respectively, and in cats were 18.6 (8.2–
Department of Medical Sciences, School
of Veterinary Medicine, University of 40.0) mg/kg/d, 32.1 (1.9–103.5) µg/mL, and 0.61 ml min−1 kg−1, respectively. Random
Wisconsin-Madison, Madison, WI, USA
6
inter-animal pharmacokinetic variability was high in both species. Two dogs had near
Department of Small Animal Clinical
Sciences, Texas A&M University, College twofold increases in serum fluconazole when generic formulations were changed,
Station, TX, USA suggesting lack of bioequivalence. Median C SS for dogs and cats achieving clinical
Correspondence remission was 19.4 and 35.8 µg/ml, respectively. Starting oral doses of 10 mg/kg
Butch KuKanich, 1620 Denison Ave, Coles q12h in dogs and 50–100 mg total daily dose in cats are recommended to achieve
228, Manhattan, KS 66506.
Email: kukanich@ksu.edu median C SS associated with clinical remission. Due to the large pharmacokinetic vari-
ability, individualized dose adjustments based on C SS (therapeutic drug monitoring)
Funding information
Mark Derrick Canine Research Fund and treatment failure should be considered.

KEYWORDS

canine, feline, fluconazole, population pharmacokinetics, systemic fungal disease

1 |  I NTRO D U C TI O N
Fluconazole is commonly administered orally to treat systemic fun-
gal infections in dogs and cats. It works by inhibiting ergosterol
synthesis in the fungal cell membrane. In veterinary medicine, it
was first studied for cryptococcosis in cats, and many dosing rec-
ommendations have been extrapolated from that initial research
(Malik, Wigney, Muir, Gregory, & Love, 1992). It is now routinely
used to treat blastomycosis, coccidioidomycosis, and histoplasmo-
sis in dogs and cats (Arbona, Butkiewicz, Keyes, & Shubitz, 2020;
Ludwig, Hanzlicek, KuKanich, & Payton, 2018; Mazepa, Trepanier,
& Foy, 2011; Reinhart, KuKanich, Jackson, & Harkin, 2012; Wilson,
KuKanich, Hanzlicek, & Payton, 2018). Recommended doses of flu-
conazole range from 5 to 10 mg/kg PO every 12–24 hr for dogs and
10 mg/kg or 50 mg/cat every 12–24 hr (Arbona et al., 2020; Ludwig
et al., 2018; Malik et al., 1992; Mazepa et al., 2011; Papich, 2011;
Reinhart et al., 2012; Wilson et al., 2018). However, minimal pharma-
cokinetic (PK) data are available to support dosing recommendations,
and the majority of available PK data come from healthy animals
which may not predict the appropriate dosages for diseased animals
(Craig, Ramzan, & Malik, 1994; Humphrey, Jevons, & Tarbit, 1985;
Malik et al., 1992; Vaden, Heit, Hawkins, Manaugh, & Riviere, 1997).

J vet Pharmacol Therap. 2020;00:1–10. wileyonlinelibrary.com/journal/jvp© 2020 John Wiley & Sons Ltd     1 |
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2       KUKANICH et al.
A pilot fluconazole PK study in dogs was performed by Humphrey
et al. (1985) using 2 male littermate Beagles weighing 13 kg, receiv-
ing 10 mg/kg fluconazole both PO (via esophageal tube) using a solu-
tion not commercially available (0.15 M hydrochloric acid-ethanol,
50% [vol/vol]; pH 2.4) and IV in a crossover design. Important data
gained from this study were that oral bioavailability of fluconazole in
dogs appeared excellent (essentially completely absorbed with that
experimental formulation), it has low protein binding and uniform
tissue distribution, and the major route of excretion is through renal
clearance. Two small studies have assessed PK parameters of fluco-
nazole in cats. Craig et al. (1994) administered 100 mg fluconazole
to 6 adult healthy cats IV and PO confirming complete oral bioavail-
ability in cats as found in dogs. A study by Vaden et al. (1997) dosed
7 healthy adult male research cats with PO and IV fluconazole at
50 mg per cat and found that this dose achieved penetration into
cerebrospinal fluid, aqueous humor, and epithelial lining fluid.
A population PK study enrolls the target population having natu-
rally occurring disease, including a widely variable population of cli-
ent-owned pet dogs and cats rather than a homogeneous population
of research animals (Muñana, Otamendi, Nettifee, & Papich, 2018;
Papich, 2017). It is ideal to consider patient variability and its re-
flection on PK parameters when making dosing recommendations
for clinical patients. Malik et al. (1992) evaluated fluconazole serum
concentrations in 19 cats with naturally occurring cryptococcosis
4–8 hr after receiving fluconazole orally and found a wide variation
of serum fluconazole concentrations (15–80 µg/ml) with the major-
ity of cats administered 50 mg PO every 12 hr. However, that study
did not measure other PK parameters, perform population PK mod-
eling, or assess the relationship between dose and serum concentra-
tion, or other patient variables and the timing of sample collection
was variable (4–8 hr after dosing).
This study aimed to (a) determine fluconazole pharmacokinetics
in dogs and cats using a population of animals with fungal disease,
(b) determine whether individual patient characteristics could pre-
dict variability in fluconazole serum concentrations, and (c) make
improved therapeutic dose recommendations of fluconazole in dogs
and cats using the pharmacokinetic data and patient variability.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Animals
Dogs and cats were eligible for enrollment if they had confirmed or
suspected naturally occurring fungal disease and were being treated
with FDA-approved oral fluconazole. Cases were recruited from the
veterinary teaching hospitals at Kansas State University, Oklahoma
State University, Iowa State University, University of Wisconsin
and Texas A&M University, from May 2015 through January 2018.
Informed client consent was collected from each enrolled pet owner,
and the Institutional Animal Care and Use Committee at Kansas
State University and collaborating universities approved this study.
Animals were cared for by veterinarians following best veterinary
practices at veterinary teaching hospitals during their enrollment.
Dogs and cats with suspected fungal illness were considered eli-
gible as long as fluconazole was prescribed by the treating veteri-
narian. This allowed enrollment of patients while diagnostic tests
were pending and cases of localized disease such as optic neuritis
that were challenging to confirm; data from patients with suspected
but unconfirmed fungal disease were only used to determine PK pa-
rameters, not for clinical efficacy determination. Animals included
in PK analysis had to be at steady-state serum drug concentrations,
defined a priori as ≥72 hr of treatment. Animals that were subse-
quently diagnosed with nonfungal disease were excluded from PK
and efficacy analyses.
2.2 | Drug administration and sample collection
Diagnosis of fungal disease, decision to prescribe fluconazole,
and fluconazole dosing protocol were made by the patient's treat-
ing veterinarian, not the research investigators or collaborators.
Fluconazole treatment was not altered in any way for the purpose of
this study. Data recorded for the study included patient signalment,
known or suspected disease, body weight and body condition, dose,
frequency, and duration of fluconazole, formulation (brand name
versus generic; tablet, capsule, suspension), time and date of most
recent fluconazole administration, whether fluconazole was admin-
istered at home or at hospital, fed or fasted, time and date of blood
draw/s, suspected adverse events, concurrent diseases (including
creatinine and bilirubin when available), and concurrent medications.
When fluconazole was administered at home, clients were called by
a research collaborator with a reminder to record the exact time of
administration and whether administered with food or fasted. For a
population PK study, infrequent sampling from a large population of
patients is used with up to 3 samples per dose of fluconazole (such as
4 hr, 8 hr, 12 hr postpill) permitted for this study, in contrast to large
numbers of samples from a small number of patients (Riviere, 1999).
Blood sampling times were flexible and either occurred at opportun-
istic times, such as when patients had blood sampled for other rea-
sons (routine chemistry monitoring) or were performed specifically
for the study. Approximately one milliliter whole blood was collected
at each sampling period and allowed to clot, spun and separated into
serum, and then stored frozen at −20°C or −70°C until processing.
Serum samples were batched and mailed to Kansas State
University for fluconazole and creatinine measurements every
6 months. Both fluconazole (24 months) and creatinine (8–12 months)
have been shown to be stable when serum is stored frozen (Askenazi
et al., 2014; Liew, Loh, Tan, & Peh, 2012; Thoresen, Tverdal, Havre, &
Morberg, 1995). Creatinine concentration was determined by a ref-
erence laboratory (Kansas State Veterinary Diagnostic Laboratory,
Manhattan KS). Fluconazole serum concentrations were measured
using a liquid chromatography (Acquity UPLC H-Class UPLC, Waters
Corporation) with triple quadrupole mass spectrometry detec-
tor (TQD, Waters Corporation) as previously described (KuKanich,
KuKanich, Rankin, & Locuson, 2019) (Table S1).
KUKANICH et al.
For patients with a well-documented, confirmed diagnosis of his-
toplasmosis, blastomycosis, cryptococcosis, or coccidioidomycosis,
medical records were reviewed to determine treatment outcome
and, if adequate outcome information was available, correlated
with fluconazole PK concentration as a measure of clinical efficacy.
For the purpose of this study, a successful treatment outcome was
defined as achieving clinical remission of fungal disease with flu-
conazole therapy, and treatment failure was defined as inability to
achieve remission. Clinical remission was defined as resolution of
all previous clinical signs of fungal disease on physical or ophthal-
mic examination or radiography; inactive ophthalmic lesions or
static radiographic changes (taken 1 month apart) were also consid-
ered remission (Mazepa et al., 2011; Reinhart et al., 2012; Wilson
et al., 2018).
2.3 | Population pharmacokinetic analysis with
a nonlinear mixed effects modeling approach
A population PK analysis with nonlinear mixed effects (NLME) mod-
eling approach using established methods was performed to fit
the serum fluconazole concentration data to a compartmental PK
model using commercial software Phoenix NLME TM (version 8.0,
Certara USA, Inc.) similar to previous studies (Mould & Upton, 2012;
Muñana et al., 2018; Papich, 2017; Sikina, Bach, Lin, Gehring, &
KuKanich, 2018).
Various model structures with different error models (i.e., ad-
ditive, multiplicative, and additive + multiplicative) were tested to
determine the best fit base model. The extended least-squares,
first-order conditional estimation (FOCE-ELS) algorithm with in-
teraction was used to fit the data to the model using steady-state
conditions with a dose interval at 12 hr to calculate the population
PK parameters. The selection of the final model was guided by
goodness-of-fit plots (e.g., observed versus predicted plasma con-
centrations, weighted residuals versus predicted concentrations,
and weighted residuals versus time), statistical significance between
models using −2LL (twice the negative log-likelihood), Akaike in-
formation criterion (AIC, a goodness-of-fit measure based on the
log-likelihood adjusted for the number of parameters [degrees of
freedom] in the fit), Bayesian information criterion (BIC, similar to
AIC but penalizes model complexity more heavily), and coefficient
of variation (CV%) of parameter estimates. The model was selected
based on the following considerations: superior goodness-of-fit
plots, less model complexity, less uncertainty of parameter esti-
mates, and/or smaller AIC and BIC values.
An exponential error model was employed to describe inter-indi-
vidual (inter-subject) variability (IIV, variance of a parameter among
different subjects) using the following equation:
Pi = Ppop × expnPi (1)
where Pi is the parameter of interest for an individual i, Ppop is the
theta (θ) value, the typical value for the estimate of the population
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mean of the parameter of interest, and nPi is the eta (η) value for
the individual for the parameter of interest. The eta values were as-
sumed to be independent and have a normal distribution with a mean
of zero and variance of ω2. An additive error model was selected to
describe the residual random error (ε) of the data, which represents
the residual intra-individual (intra-subject) variability with a mean of
zero, and a variance of σ 2, using the following equation:
Cobs_ij = Cpred_ij + 𝜀ij (2)
where Cobs_ij is the observed serum concentration of fluconazole for
subject number i at time j for the individual, Cpred_ij is the model-pre-
dicted concentration for subject i at time j, and εij is the residual error
value adjustment for subject i at time j. The present analysis used Dose
ID as subject number identifier because some subjects had serum con-
centration data on different days across a wide interval.
After the final base model was identified, covariate analyses
were performed to determine whether there were factors that may
explain the variability of clearance (Cl) for dogs and cats, respec-
tively. The following potential covariates were examined for both
dogs and cats, including body weight (kg), age (year), sex (male and
female), serum creatinine, serum total bilirubin, and co-treatment
with glucocorticoid or intravenous fluid.
A simple stepwise covariate search was performed to identify
significant covariates in dogs and cats. Results were considered
statistically significant, and a covariate was included with the final
model if the inclusion of the covariate resulted in a decrease of
−2LL value that was associated with a p value < .01. This process
was repeated until all significant covariates were accounted for. If
significant covariate(s) were identified, then a backward elimination
step was performed to eliminate covariate(s) on parameters whose
removal produced the smallest reduction in goodness-of-fit less than
the user-specific threshold value of p  < .001. The final model was
determined based on the stepwise covariate search results.
The final model for dogs included the covariate of body weight
on the parameter Cl was described the equation:
Cl = tvCl × BWdCldBW × expnCl (3)
where Cl is the estimate of clearance for the individual i, tvCl is the
population estimate for clearance, dCldBW is the derivative of the pa-
rameter Cl value with respect to body weight, and nCl is the eta of the
individual i for the clearance. The final model without any covariates
for cats was described using equation (1) presented above.
The performance and stability of the final PK models for dogs
and cats, respectively, were evaluated by examining the goodness-
of-fit plots and by a bootstrap analysis. The bootstrap samples were
collected through random resampling by replacing the original data-
set to generate another dataset with the same sample size as the
original but with a different combination of subjects. The bootstrap
resampling analysis was repeated 100 times using Phoenix NLME™.
If the parameter estimates fell into the 95% confidence intervals (CIs)
from the bootstrap analysis, the model was considered unbiased and
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4       KUKANICH et al.
stable. The predictive accuracies of the final models were confirmed
using the visual predictive check with 10 observations simulated for
each subject by showing that most of the observations fell within the
95th and 5th quartile range of the predicted concentrations.
3 |   R E S U LT S
3.1 | Enrolled patients
Thirty-seven dogs were enrolled in the study, accounting for 102
serum samples (median 2 samples per dog, range 1–11). Age of dogs
ranged from 6 months to 12 years old, (median 5 years old), with
10/37 dogs < 3 years old and 12/37 dogs ≥ 8 years old. There were
18 spayed female dogs, 4 intact female dogs, 11 castrated male dogs,
and 4 intact male dogs. Thirty-one dogs were purebred (5 German
pointers, 4 Labrador retrievers, 4 miniature schnauzers, 2 huskies, 2
golden retrievers, 2 German shepherds, and 1 each of 12 additional
breeds), and 6 were of mixed breed. The range of body weight of
enrolled dogs was 2.8–58.2 kg. Dogs had confirmed diagnoses of
histoplasmosis (12), blastomycosis (8), coccidioidomycosis (3), cryp-
tococcosis (2), protothecosis (1), and aspergillosis (1). Three dogs had
either presumed or confirmed fungal ulcerative keratitis. Two dogs
were diagnosed with optic neuritis and chorioretinal lesions con-
sistent with systemic fungal disease; both dogs had negative fungal
testing (Histoplasma urine EIA and Cryptococcus latex agglutination).
One dog had cutaneous fungal disease diagnosed with cytology dur-
ing therapy for immune-mediated hemolytic anemia. Two dogs had
diffuse interstitial lung disease, and 2 dogs had enlarged tracheal
bronchial nodes with pyogranulomatous inflammation seen cyto-
logically; all four of these dogs had negative fungal testing, including
Histoplasma urine EIA (3 dogs), Coccidioides serum antibody (2 dogs),
and Blastomyces urine EIA (1 dog), and all 4 dogs were treated and
responded with long-term antifungal therapy.
Thirty-five cats were enrolled in the study, accounting for 107
serum samples (median 2 samples per cat, range 1–12). Age of cats
ranged from 7 months to 15 years old (median 7 years old), with
5/35 cats < 3 years old, and 17/35 cats ≥ 8 years old. There were
2 Siamese, 1 sphinx, and 1 ragdoll, 1 Maine Coon, and 30 domes-
tic short or long-haired cats. Fourteen cats were female spayed, 20
were male castrated, and 1 was male intact. Range of body weight
of enrolled cats was 2.4–6.7 kg. Cats had confirmed fungal diagno-
ses of histoplasmosis (32) and cryptococcosis (1). One cat received
fluconazole until tularemia was confirmed, and one cat received flu-
conazole while diagnostics were pending but was ultimately believed
to have feline infectious peritonitis.
3.2 | Concurrent diseases
In addition to fungal disease, 3 dogs had concurrent heart murmurs,
and one dog each had concurrent arthritis, atopy, dental disease, der-
matitis, hypoadrenocorticism, hypothyroidism, immune-mediated
hemolytic anemia, keratoconjunctivitis sicca, testicular mass, and
urinary incontinence. Additionally, 1 cat had diabetes mellitus, 2 had
hypercalcemia, and 7 had heart murmurs (1 confirmed hypertrophic
cardiomyopathy).
3.3 | Fluconazole administered
Dogs received a median fluconazole dose of 13.5 mg kg−1 day−1
(range 4.5–22.5 mg kg−1 day−1). One dog was administered the total
dose once daily (q24h) whereas the remainder (n  = 36) received it
divided into two doses per day (q12h). All dogs received human ge-
neric FDA-approved tablet formulation. The majority of canine doses
were administered with food (68%), with the remaining reported as
fasted (24%) or unknown (8%).
Cats received a median fluconazole dose of 17.5 mg kg−1 day−1
(range 8.2–40.0 mg kg−1 day−1). Five cats were administered the
total dose once daily (q24h) whereas the remainder (n = 30) received
it divided into two doses per day (q12h). Three cats were admin-
istered 40 mg/ml oral suspension of FDA-approved human generic
fluconazole. One cat received brand name human approved tablets
(Diflucan, Pfizer, New York, NY), while remaining cats received vari-
ous human FDA-approved generic fluconazole tablets. The majority
of feline doses were administered with food (58%), with the remain-
ing reported as fasted (41%) or unknown (<1%).
3.4 | Concurrent medications
Eleven dogs (18 serum samples) received concurrent glucocorticoid
therapy with fluconazole (median prednisone equivalent dose of
0.5 mg kg−1 day−1). Eight cats (10 serum samples) received concur-
rent glucocorticoid therapy (median prednisolone equivalent dose
of 0.4 mg kg−1 day−1). Nine dogs (23 serum samples) and 4 cats (5
serum samples) received concurrent intravenous fluids. An assort-
ment of other concurrent medications was administered (Table S2),
with only S-adenosylmethionine and amoxicillin–clavulanate being
administered to more than 3 dogs, and mirtazapine being adminis-
tered to more than 3 cats.
3.5 | Renal and hepatic laboratory results
Concurrent serum creatinine concentration was measured on
101/102 canine serum samples (1 sample had inadequate sample
size) and was ≥1.4 mg/dl in 3 dogs (9 samples, range 1.4–2.0 mg/
dl). Concurrent serum creatinine was measured on 103/107 feline
serum samples (4 had inadequate sample size) and was ≥1.6 mg/dl in
10 cats (21 samples, range 1.6–3.1 mg/dl). Complete staging of renal
disease was not a component of this study, and urine specific gravity
was not available for all patients.
When a chemistry profile was submitted on the same day as
sample collection, liver values were recorded. Total bilirubin was
KUKANICH et al.
elevated in four dogs (12/96 samples, range 0.8–8.6 mg/dl) and 7
cats (11/104 samples, range 0.4–7.4 mg/dl). Alanine transferase
(ALT) enzyme activity was increased in 12 dogs with a median of
1.9x the upper end of the reference interval (range 103–690 U/L).
Although comparisons to pretreatment ALT could be useful for dogs
with elevated ALT, at the time of initiating fluconazole therapy three
dogs were being administered systemic prednisone, one dog had
immune-mediated hemolytic anemia and was receiving cyclosporine
and mycophenolate in addition to prednisone, one dog was being ad-
ministered topical prednisone acetate, two dogs had been switched
from itraconazole to fluconazole due to hepatotoxicity, and one dog
had histoplasmosis confirmed on hepatic cytology. The dog with the
highest ALT was receiving concurrent prednisone (1.5 mg kg−1 day−1)
and had elevated ALP (878 U/L) at the time of study sampling. Three
cats had increased ALT activity (range 326–1738 U/L); none had he-
patic cytology or histopathology performed.
3.6 | Suspected fluconazole adverse effects
Veterinarians reported suspected adverse effects attributable to
fluconazole in 4 dogs and 7 cats. They included decreased appetite
(4 cats, 1 dog), increased ALT (3 cats, 1 dog), focal alopecia (2 cats),
and hypoadrenocorticism, ocular discharge, dry skin, and malaise (1
dog each). One cat who received 33.7 mg kg−1 day−1 (50 mg q 12 hr)
fluconazole had an increase in ALT activity from pretreatment nor-
mal value of 125 U/L–1738 U/L in 3 weeks. After 1 week at reduced
dose of 17.1 mg kg day−1 (50 mg q 24 hr), ALT was 251 U/L, and it was
normal (79 U/L) 3 months later. A second cat had ALT increase from
pretreatment normal value of 134 U/L to ALT 326 U/L after 2 days of
fluconazole at 21.0 mg kg day−1 but was confirmed to have tularemia.
The third cat had been receiving fluconazole for 7 months prior to
his elevation in ALT (536 U/L); at this time, he was deemed in remis-
sion, discontinued fluconazole, and ALT returned to normal gradually
over 3 months as the cat remained in remission. Only 1 cat had flu-
conazole discontinued (switched to itraconazole) due to a potential
adverse effect (alopecia) which began resolving within a month of
the medication change. One dog, a 10 yr miniature Schnauzer, who
had been receiving fluconazole (18.2 mg kg−1 day−1) for 9 months for
disseminated histoplasmosis developed hypoadrenocorticism that
was suspected to be either an adverse effect of the fluconazole or
the secondary to the histoplasmosis infection.
3.7 | Pharmacokinetic analysis
Only dogs and cats reaching a steady state of fluconazole and di-
agnosed with confirmed or clinically suspected fungal disease were
included in the population PK analysis. Thirty-one dogs with a
mean (range) body weight 25.6 (2.8–58.2) kg and 31 cats with body
weight 3.9 (2.4–6.1) kg were included in the final PK analyses. The
dose-normalized serum concentrations in dogs (Figure 1) and cats
(Figure 2) were variable. The mean (range) dose was 14.2 (4.5–21.3)
|
      5
100
Fluconaz ole Plasma Concentration ( µg /ml)
80
60
40
20
0 6 12 18 24
Time (hr)
F I G U R E 1   Dose-normalized fluconazole plasma concentrations
from dogs (n = 31) with suspected or confirmed fungal infections
after repeated oral administrations at steady-state conditions
1000
Fluconaz ole Plasma Concentration (µg/ml)
100
10
1
0 6 12 18 24
Time (hr)
F I G U R E 2   Dose-normalized plasma fluconazole concentrations
from cats (n = 31) with suspected or confirmed fungal infections
after repeated oral administrations at steady-state conditions
mg/kg/d in dogs and 18.6 (8.2–40) mg/kg/d in cats. The mean
(range) steady-state serum concentrations (C SS) in dogs were 26.8
(3.8–61.5) µg/mL with an oral clearance (Cl/F) of 0.65 (0.16–2.24)
mL/min/kg based on Cl/F = Dose/C SS, and mean (range) C SS in cats
was 32.1 (1.9–103.5) µg/mL with an Cl/F of 0.62 (0.14–2.24) mL/min/
kg based on Cl/F = Dose/C SS.
The final population PK model used for the analysis was a
one-compartment model with first-order absorption and first-order
elimination. Stepwise covariate search results showed that body
weight was a significant covariate for the dog model, but other tested
potential covariates (i.e., age, sex, serum creatinine, serum bilirubin,
and co-treatment with glucocorticoid or intravenous fluid) were not
clinically relevant. Therefore, body weight was included as a contin-
uous covariate in the final dog model (Figure 3). For the cat model,
none of the tested variables (including body weight) was found to
be significant; therefore, the base model without any covariates was
used as the final model. Goodness-of-fit plots from the final dog
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6       KUKANICH et al.
and cat models are presented in Figure 4a,b, respectively. Excellent
correlations between the observed and the model-predicted serum
concentration values were observed for both models. The PK param-
eter estimates collected from the bootstrap analysis are provided in
Table 1. The bootstrap analysis results showed that the 95% CIs of all
parameters were generally centered around the parameter mean es-
timates. The mean values collected from the bootstrap analysis were
comparable to parameter mean estimates from the final models, and
overall, these results suggest that the final models are stable. The
Cl/F determined by the population PK model for dogs was 969 ml/
hr, equivalent to 0.63 ml min−1 kg−1 compared to 0.65 ml min−1 kg−1
determined by the simple equation Cl/F  = Dose/C SS. Likewise the
Cl/F in cats determined by the population PK model was 143 ml/
−1 −1 −1 −1
hr, equivalent to 0.61 ml min  kg compared to 0.62 ml min  kg
determined by the simple equation Cl/F  = Dose/C SS. Steady state
F I G U R E 3   Scatter plot of covariate of body weight versus eta
(random variable) for the primary parameter of clearance in the dog
model indicating a linear relationship between body weight and
clearance in dogs
was defined as 3 days based on previous PK studies demonstrating
an approximate 14-hr half-life in dogs and cats (Craig et al., 1994;
Humphrey et al., 1985) and confirmed with our results (Table 1).
Two dogs had noticeable dose-normalized increases in serum
fluconazole concentration when they switched oral generic for-
mulations, both from Citron Pharma (East Brunswick) to Glenmark
Pharmaceuticals (Mahwah, NJ, USA). In one dog, serum fluconazole
concentrations ranged 24.6–30.0 µg/ml (4 samples over a month),
prior to switching formulations, then increased to 51.6–56.9 µg/ml
(7 samples over 6 months). A second dog had serum fluconazole
concentrations ranging 15.3–24.7 µg/ml (3 samples over 5 months)
prior to switching, and then, fluconazole concentration increased to
42.0–61.7 µg/ml (6 samples over 4 months). One of these dogs had
GI histoplasmosis; no other dog with GI histoplasmosis had similar
changes in plasma concentrations.
3.8 | Clinical outcome
Of the twelve dogs with confirmed histoplasmosis, 7 achieved
clinical remission with fluconazole. Of the remaining 5 dogs, one
responded well to subsequent itraconazole administration; one was
euthanized within a week of diagnosis due to fulminant disseminated
histoplasmosis; one died within 20 days of diagnosis after develop-
ing acute-onset seizures after beginning a course of metronidazole,
no necropsy was performed; one died at home 3 months after di-
agnosis while still taking fluconazole (no necropsy was performed);
and one was euthanized after 7 months due to progressive disease.
Inconsistent outcome data are available for dogs with other fungal
infections. Of dogs with confirmed histoplasmosis (N  = 11), blas-
tomycosis (N  = 3), cryptococcosis (N  = 1), and coccidioidomycosis
(N  = 1) and adequate follow-up information, the relationship be-
tween remission status and the serum fluconazole is presented in
Figure 5. The median (range) fluconazole C SS for remission was 19.4
(8.1–79.0) µg/mL compared to 12.0 (3.5–31.6) µg/mL for dogs not
achieving remission.
Of the 32 cats with confirmed histoplasmosis, 24 achieved docu-
mented clinical remission. An additional cat was euthanized 9 months
after diagnosis, shortly after discontinuing fluconazole; remission was
F I G U R E 4   Goodness-of-fit plots of
serum fluconazole concentrations in
dogs and cats. Comparisons between
observed versus individual-predicted
fluconazole concentrations in the serum
of dogs (a) and cats (b) after repeated
oral administrations to fluconazole
(25–500 mg/dog and 25–50 mg/cat) at
12-hr intervals
KUKANICH et al.       7|
TA B L E 1   Final parameter values for the population pharmacokinetic models for fluconazole in dogs (n = 31) and cats (n = 31) with
suspected or confirmed fungal infections after repeated oral administrations at steady-state conditions

Typical Bootstrap Bootstrap 97.5%

Species Parameter Unit population value Omega mean CV% 2.5% Cl Cl

Dog Ka 1/hr 0.999 1.059 1.005 0.91 0.990 1.020

V/F mL 19,874 1.058 20,003 1.45 19,712 20,799
Cl/F mL/hr 969 1.057 973 1.97 941 1,017
stdev0 321 NA 298 33.375 242 556
dCldBW 0.00223 NA 0.0035 247.16 −0.0119 0.0214
T ½ (calculated) hr 14.5

Cat Ka 1/hr 0.988 0.995 0.99 11.02 0.866 1.085

V/F mL 3,070 0.995 3,107 11.53 2,694 3,709
Cl/F mL/hr 143 0.995 143 8.13 115 160
stdev0 288 NA 336 85.44 243 590
T ½ (calculated) hr 14.6

Note:: These values are not weight-normalized. Omega, between-subject variability or inter-individual variability; Bootstrap mean, diagnostic analysis
to better understand the precision of estimates and stability of the model through random sampling and replacement; Cl, confidence interval; Cl/F,
clearance per fraction of the dose absorbed; dCldBW, the derivative of the clearance value with respect to body weight; Ka, oral absorption rate
constant; NA, not available or not applicable; stdev0, residual error; T1/2, half-life; V/F, volume of distribution per fraction of the dose absorbed.
90 100
Fluconazole serum concentration (µg/ml)

Fluconazole serum concentration (µg/ml)

No remission 90 No remission
80
Remission Remission
70 80

70
60
60
50
50
40
40
g/ml
30
30
20 g/ml 20
16.2 µg/ml
g/ml
10 10

0 0
0 1 0 1
Remission status Remission status

F I G U R E 5   Relationship of serum fluconazole concentration to F I G U R E 6   Relationship of serum fluconazole concentration to

remission in dogs with confirmed fungal disease (histoplasmosis
N = 11, blastomycosis N = 3, cryptococcosis N = 1,
coccidioidomycosis N = 1), with solid circles representing dogs who
did not achieve remission and open circles representing dogs who
achieved remission
remission in cats with confirmed histoplasmosis, with solid circles
representing cats who did not achieve remission and open circles
representing cats who achieved remission

not properly documented, and with no necropsy, it remains unclear if 4 | D I S CU S S I O N

this cat had progressive disease, relapsed, or unrelated illness at the
time of death. Of the remaining cats, 1 cat switched to itraconazole
due to potential adverse effect (alopecia); 1 cat switched to itracon-
azole due to progressive disease; 2 were lost to follow-up; and 3 cats
either died or were euthanized due to progressive disease (5 days,
7 days, 19 months into therapy). In comparing cats with confirmed
histoplasmosis, the relationship between remission status and serum
fluconazole concentration is presented in Figure 6. The median
(range) fluconazole C SS for remission was 35.8 (6.0–95.2) µg/mL com-
pared to 16.2 (7.5–38.4) µg/mL for cats not achieving remission.
Fluconazole was expected to be an optimal oral drug for a popula-
tion PK study that would have low serum concentration variability
due to its excellent oral bioavailability in dogs and cats, long half-life,
and renal excretion. For this reason, it was anticipated that patient
factors (age, weight, renal function, etc.) could help predict serum
concentrations via a PK model, and such a model used to optimize
dosing recommendations. Therefore, it was unexpected to find such
high random variability in the serum concentrations of fluconazole in
this population of animals and little correlation with patient factors.
 |
8       KUKANICH et al.
The long half-life (~14 hr), frequent dose administration (q12 h)
and TMAX of 2–4 hr of fluconazole suggest relatively little fluctuation
in serum drug concentrations during a dose interval at steady state.
The opportunistic timing of blood sampling allowed better char-
acterization of steady-state serum concentrations and subsequently
Cl/F rather than the absorption rate. As such, the estimates of the
absorption rate constant, Ka, are not as robust as ideal, but the esti-
mates of Cl/F are robust. As treatment of fungal infections is chronic
and the half-life of fluconazole is long, Ka is expected to minimally
affect drug exposure due to the slow clearance of fluconazole.
The relationship Cl/F, dosage and average serum concentrations at
steady state (C SS) is described with the equation: Dosage = C SS * Cl/F
and emphasizes the importance of a robust estimate of Cl/F when
designing optimal drug dosing recommendations.
We expected that body weight would be a significant co-
variate in dogs due to the relatively large range of body weights,
2.8–58.2 kg included in the pharmacokinetic analyses. The results
(Figure 3) suggest a linear relationship and dosing by body weight
(mg/kg) in dogs is most appropriate. If a logarithmic relationship
was present, dosing based on body surface area may have been
more appropriate. Based on these data, an oral dose starting at
−1 −1
20 mg kg  day (or divided to 10 mg/kg q 12 h) is recommended to
exceed a mean C SS of 20 µg/ml. This would surpass the concentra-
tion associated with remission in dogs (C SS 19 µg/ml) and may tar-
get organisms with a minimum inhibitory concentration of 16 µg/
ml or less. This assumes that fluconazole distributes in tissues as
active drug in concentrations similar to serum concentrations as
previously demonstrated in mice (Humphrey et al., 1985). However,
with the large unaccounted variability in dogs, the dose may need
adjustment based on clinical effects including adverse effects or
lack of efficacy.
It is important to note there are no established antifungal break-
points for dogs and cats with any systemic fungal disease. In humans,
plasma concentrations exceeding the antifungal minimum inhibitory
concentration (MIC) are associated with better clinical outcomes
(Lepak & Andes, 2011). Most antifungals exhibit a time-dependent
effect on fungal organisms.
Body weight was not a significant covariate in cats, most likely
due to the small range of body weights, 2.4–6.1 kg. Therefore, me-
ticulous dosing is not needed in cats. A dose of 50 mg/cat/day for
cats weighing less than 4 kg, and 100 mg/cat/day in cats weighing
greater than 4 kg is recommended due to available dosage forms
(50 mg tablets). Many clinicians divide the total daily dose and ad-
minister q 12 h to decrease gastrointestinal adverse effects (i.e.,
25 mg PO q 12 h, or 50 mg PO q 12 h). This dose in cats produced
a mean Css of 32 µg/ml, very close to the median concentration
associated with clinical remission in cats (35.8 µg/ml). As such, this
dose may target organisms with an MIC of 32 µg/ml or less if serum
concentrations are directly related to the MIC of the organism. Like
dogs, large unaccounted pharmacokinetic variability was observed
in cats, and thus, the dose may need to be adjusted based on clinical
effects including adverse effects or lack of efficacy. Liquid suspen-
sions of FDA-approved fluconazole are available that can be used
in cats, but that degree of precision is not needed due to the large
variability of fluconazole exposure in cats. However, if it is easier to
administer a liquid formulation to a cat or increases the client com-
pliance, then an FDA-approved fluconazole suspension can be used.
Compounded formulations of fluconazole, especially suspensions,
should be avoided due to lack of consistency with quality control
and potentially lack of stability (Laporte et al., 2017).
In both dogs and cats, a large unaccounted variability was doc-
umented (Figures 1 and 2), despite good model fit (Figure 4) be-
tween individual-predicted and observed concentrations. Other
covariates (age, sex, co-treatment with glucocorticoid, co-treat-
ment with intravenous fluid, and creatinine) produced clinically ir-
relevant changes in the pharmacokinetic modeling fit. Ideally, the
large degree of variability could be accounted for with inclusion
of additional covariates and dose adjustment made on clinical as-
sessments. The lack of additional relevant covariates could be due
to the relatively small sample size, missing data points, or multi-
ple variables in a single animal confounding the analysis. Based on
these data, therapeutic drug monitoring of individual patients could
result in dose adjustments to either minimize adverse effects if the
serum concentrations are high, or increase efficacy if the serum
concentrations are low. Steady-state serum concentrations in dogs
and cats are expected approximately 3 days (based on a 14-hr half-
life) after starting or adjusting oral fluconazole administration and
therapeutic drug monitoring could be implemented to refine dosing
in a specific patient.
Enrolled animals were treated with FDA-approved formulations.
The initial datasheets only differentiated generic and brand name
formulations. We did not anticipate differences within the formula-
tions, as they are bioequivalent in humans. However, bioequivalence
in humans does not always equate to bioequivalence in animals due
to differences in gastrointestinal tract anatomy and physiology. We
noted two dogs whose serum concentrations nearly doubled acutely
during therapy with no dosage adjustments. Review of the data re-
vealed both of the dogs were switched from a generic tablet formu-
lation made by Citron Pharma to a generic tablet formulation made
by Glenmark Pharmaceuticals. The most likely reason for the essen-
tially doubling serum concentration was due to higher bioavailability
of the Glenmark tablet. Unfortunately, we do not have manufacturer
data for all formulations used in the study, as some were dispensed at
the university hospitals, but others were dispensed from local phar-
macies. No other dogs followed a similar pattern of either chang-
ing formulation with these manufacturers that we are aware and no
other dogs in the study had a doubling or halving of serum concen-
trations without changes in dose. In reviewing the available data,
the following formulations were used in this study: Citron Pharma,
Glenmark Pharmaceuticals, Teva Pharmaceuticals (Sellersville, PA,
USA), and Harris Pharmaceuticals (Fort Myers, FL, USA). Therefore,
the tablet formulation by different manufacturers could be a con-
tributing factor to the large variability that was observed in dogs.
Further studies are indicated comparing formulations from different
manufacturers for bioequivalence in dogs. Based on these limited
data, the Glenmark tablet formulation might have twice the oral
KUKANICH et al.
bioavailability compared to the Citron formulation but would need
to be confirmed with bioequivalence studies.
The variability in the relationship between the steady-state
serum concentrations and clinical efficacy of fluconazole is probably
multifactorial. First, although this was a multi-year and multi-insti-
tutional study, the total number of cases enrolled for cats and dogs
with a defined diagnosis was low, leading to low robustness. Another
factor likely affecting a correlation is the presence of fulminant
disease in which even the most effective antifungal or high serum
concentrations could still result in treatment failure; two cats and
one dog were euthanized within 7 days due to severe disseminated
histoplasmosis. Re-exposure and reinfection could not be differen-
tiated from relapse in enrolled patients and would adversely affect
the correlation. Fungal susceptibilities were not collected for these
clinical isolates; therefore, it is unknown if the lack of response in
some individuals could have been due to resistant isolates. As with
other clinical trials, some patients were lost to follow-up and so the
clinical outcome in those patients could not be included. It also is
possible that some animals may never be “cured” due to other fac-
tors including poor immune function or sequestration of fungal or-
ganisms in inactive forms or protected environments. A much larger
study with inclusion of disease severity and fungal susceptibility
testing could result in better correlations of serum concentrations
to clinical outcome.
Elevations in ALT in patients with systemic fungal disease can
be from underlying hepatic infection, adverse effects of azoles, or
concurrent medications. One dog with increased ALT had confirmed
hepatic histoplasmosis, but it was beyond the scope of this study
to confirm hepatic involvement either at diagnosis or at the time of
serum sample collection if increased ALT was documented. Six other
dogs with increased ALT had complicating factors, such as concur-
rent prednisone therapy or history of increased ALT from itracon-
azole. The results show that even if confounded by some cases with
concurrent hepatic disease or concurrent medications, the increases
in ALT activity with fluconazole were infrequent and mild to moder-
ate in nature in both dogs and cats and resolved with dosage adjust-
ments or switching to itraconazole.
In conclusion, fluconazole was well tolerated in dogs and cats.
Initial fluconazole dosages in dogs should start a 20 mg/kg/d (or
10 mg/kg q 12 h) PO to achieve mean serum concentrations of
20  µg/ml. Cats weighing < 4 kg should have a 50 mg per day PO
(or 25 mg q 12 h) starting dose, and cats weighing > 4 kg should be
dosed 100 mg per day PO (or 50 mg q 12 h). Generic tablet formu-
lations may not be bioequivalent in dogs. Fluconazole serum con-
centrations were variable in dogs and cats, and therapeutic drug
monitoring should be considered in animals with treatment failure
or adverse effects.
AC K N OW L E D G M E N T S
The Phoenix® 8.0 software license was provided by Certara USA,
Inc. as a part of the company's Academic Centers of Excellence
program. Funded by a Mark Derrick Canine Research Fund and the
Department of Anatomy and Physiology, Kansas State University.
|
      9
C O N FL I C T O F I N T E R E S T
None.
AU T H O R C O N T R I B U T I O N S
KK contributed to study design, data collection and analysis, and
manuscript preparation. BK contributed to study design, data analy-
sis, and manuscript preparation. ZL contributed to data analysis and
manuscript preparation. AJR, ASH, JSP, JB, AKC, and AJ contributed
to data collection, and HJ contributed to data analysis. All authors
have read and approved the current manuscript.
ORCID
Kate KuKanich  https://orcid.org/0000-0002-0215-3284
Butch KuKanich  https://orcid.org/0000-0002-4037-3472
Zhoumeng Lin  https://orcid.org/0000-0002-8731-8366
REFERENCES
Arbona, N., Butkiewicz, C.D., Keyes, M., & Shubitz, L.F. (2020). Clinical
features of cats diagnosed with coccidioidomycosis in Arizona,
2004–2018. Journal of Feline Medicine and Surgery, 22, 129–137.
https://doi.org/10.1177/10986​12x19​829910
Askenazi, D.J., Moore, J.F., Fineberg, N., Koralkar, R., Clevenger, S., &
Sharer, J.D. (2014). Comparison of methods, storage conditions, and
time to analysis of serums and urine creatinine measured from micro-
samples by LC/MS vs. Jaffe. Journal of Clinical Laboratory Analysis, 28,
405–408. https://doi.org/10.1002/jcla.21701
Craig, A.J., Ramzan, I., & Malik, R. (1994). Pharmacokinetics of fluco-
nazole in cats after intravenous and oral administration. Research
in Veterinary Science, 57, 372–376. https://doi.org/10.1016/0034-
5288(94)90133​-3
Humphrey, M.J., Jevons, S., & Tarbit, M.H. (1985). Pharmacokinetic eval-
uation of UK-49,858, a metabolically stable triazole antifungal drug,
in animals and humans. Antimicrobial Agents and Chemotherapy, 28,
648–653. https://doi.org/10.1128/aac.28.5.648
KuKanich, B., KuKanich, K., Rankin, D., & Locuson, C.W. (2019). The effect
of fluconazole on oral methadone in dogs. Veterinary Anesthesia and
Analgesia, 46, 501–509. https://doi.org/10.1016/j.vaa.2019.02.003
Laporte, C.M., Cruz-Espindola, C., Thungrat, K., Schick, A.E., Lewis, T.P.
2nd, & Boothe, D.M. (2017). Quality assessment of fluconazole cap-
sules and oral suspensions compounded by pharmacies located in
the United States. American Journal of Veterinary Research, 78, 421–
432. https://doi.org/10.2460/ajvr.78.4.421
Lepak, A.J., & Andes, D.R. (2011). Antifungal PK/PD considerations in
fungal pulmonary infections. Seminars in Respiratory Critical Care
Medicine, 32, 783–794. https://doi.org/10.1055/s-0031-1295726
Liew, K.B., Loh, G.O.K., Tan, Y.T.F., & Peh, K.K. (2012). Development
and application of simple HPLC-UV method for fluconazole quan-
tification in human plasma. International Journal of Pharmacy and
Pharmaceutical Sciences, 4, 107–111. ISSN- 0975–1491.
Ludwig, H.C., Hanzlicek, A.S., KuKanich, K., & Payton, M.E. (2018).
Candidate prognostic indicators in cats with histoplasmosis treated
with antifungal therapy. Journal of Feline Medicine and Surgery, 20,
985–996. https://doi.org/10.1177/10986​12X17​746523
Malik, R., Wigney, D.I., Muir, D.B., Gregory, D.J., & Love, D.N. (1992).
Cryptococcosis in cats: Clinical and mycological assessment of 29
cases and evaluation of treatment using orally administered flu-
conazole. Journal of Medical and Veterinary Mycology, 30, 133–144.
https://doi.org/10.1080/02681​21928​0 000181
Mazepa, A.S., Trepanier, L.A., & Foy, D.S. (2011). Retrospective compari-
son of the efficacy of fluconazole or itraconazole for the treatment of
 |
10       KUKANICH et al.

systemic blastomycosis in dogs. Journal of Veterinary Internal Medicine,
25, 440–445. https://doi.org/10.1111/j.1939-1676.2011.0710.x
Mould, D.R., & Upton, R.N. (2012). Basic concepts in population
modeling, simulation, and model-based drug development. CPT:
Pharmacometrics & Systems Pharmacology, 1, e6. https://doi.
org/10.1038/psp.2012.4
Muñana, K.R., Otamendi, A.J., Nettifee, J.A., & Papich, M.G. (2018).
Population pharmacokinetics of extended release levetiracetam in
epileptic dogs when administered alone, with phenobarbital or zonis-
amide. Journal of Veterinary Internal Medicine, 32, 1677–1683. https://
doi.org/10.1111/jvim.15298
Papich, M.G. (2011). Fluconazole. In Saunders Handbook of Veterinary
Drugs (3rd ed., pp. 316–318). St. Louis, MO: Elsevier.
Papich, M.G. (2017). Ciprofloxacin pharmacokinetics in Clinical Canine
Cases. Journal of Veterinary Internal Medicine, 31, 1508–1513. https://
doi.org/10.1111/jvim.14788
Reinhart, J.M., KuKanich, K., Jackson, T., & Harkin, K.R. (2012). Feline
histoplasmosis: Fluconazole therapy and identification of potential
sources of Histoplasma species exposure. Journal of Feline Medicine
and Surgery, 14, 841–848. https://doi.org/10.1177/10986​ 12X12​
452494
Riviere, J.E. (1999). Study design and data analysis. In Comparative phar-
macokinetics: Principles, techniques and applications, (239–258). Ames,
IA: Iowa State Press.
Sikina, E.R., Bach, J.F., Lin, Z., Gehring, R., & KuKanich, B. (2018).
Bioavailability of suppository acetaminophen in healthy and hospi-
talized ill dogs. Journal of Veterinary Pharmacology and Therapeutics,
41, 652–658. https://doi.org/10.1111/jvp.12664
Thoresen, S.I., Tverdal, A., Havre, G., & Morberg, H. (1995). Effects of
storage time and freezing temperature on clinical chemical param-
eters from canine serum and heparinized plasma. Veterinary Clinical
Pathology, 24, 129–133. https://doi.org/10.1111/j.1939-165x.1995.
tb009​54.x
Vaden, S.L., Heit, M.C., Hawkins, E.C., Manaugh, C., & Riviere, J.E.
(1997). Fluconazole in cats: Pharmacokinetics following intrave-
nous and oral administration and penetration into cerebrospinal
fluid, aqueous humor and pulmonary epithelial lining fluid. Journal of
Veterinary Pharmacology and Therapeutics, 20, 181–186. https://doi.
org/10.1111/j.1365-2885.1997.tb000​93.x
Wilson, A., KuKanich, K., Hanzlicek, A., & Payton, M. (2018). Clinical
presentation, treatment, and prognostic indicators in dogs with his-
toplasmosis. Journal of American Veterinary Medical Association, 252,
201–209. https://doi.org/10.2460/javma.252.2.201
S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: KuKanich K, KuKanich B, Lin Z, et al.
Clinical pharmacokinetics and outcomes of oral fluconazole
therapy in dogs and cats with naturally occurring fungal
disease. J vet Pharmacol Therap. 2020;00:1–10. https://doi.
org/10.1111/jvp.12888