animals
ArticlePharmacokinetic Profile of Oral Administration of
Mefloquine to Clinically Normal Cats: A Preliminary
In-Vivo Study of a Potential Treatment for Feline
Infectious Peritonitis (FIP)
Jane Yu *, Benjamin Kimble, Jacqueline M. Norrisand Merran Govendir

Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia;
benjamin.kimble@sydney.edu.au (B.K.); jacqui.norris@sydney.edu.au (J.M.N.);
merran.govendir@sydney.edu.au (M.G.)* Correspondence: jane.yu@sydney.edu.au
Received: 2 May 2020; Accepted: 5 June 2020; Published: 8 June 2020


Simple Summary: In searching for antiviral agents against feline coronaviruses and feline caliciviruses,
mefloquine, a human anti-malarial drug, has been demonstrated to reduce viral load of feline
coronaviruses and feline calicivirus in infected cells. In this study, mefloquine was administeredorally to seven clinically healthy cats twice weekly for four doses and mefloquine concentrations inblood were measured to investigate the pharmacokinetic profile—the movement of drug in the body.
The maximum blood concentration of mefloquine was 2.71 ug/mL and was reached 15 h after a single
oral dose was administered. Mefloquine side effects included vomiting following administration
without food in some cats, and mild increases in symmetric dimethylarginine (SDMA), an earlykidney biomarker. This study provides valuable information on mefloquine’s profile in cats as an
introductory step towards investigating it as a potential treatment for feline coronavirus and feline
calicivirus infection in cats.
Abstract: The pharmacokinetic profile of mefloquine was investigated as a preliminary study towards
a potential treatment for feline coronavirus infections (such as feline infectious peritonitis) or feline
calicivirus infections. Mefloquine was administered at 62.5 mg orally to seven clinically healthy cats
twice weekly for four doses and mefloquine plasma concentrations over 336 h were measured using
high pressure liquid chromatography (HPLC). The peak plasma concentration (Cmax) after a single
oral dose of mefloquine was 2.71 ug/mL and time to reach Cmax (Tmax) was 15 h. The elimination
half-life was 224 h. The plasma concentration reached a higher level at 4.06 ug/mL when mefloquine
was administered with food. Adverse effects of dosing included vomiting following administration
without food in some cats. Mild increases in serum symmetric dimethylarginine (SDMA), but notcreatinine, concentrations were observed. Mefloquine may provide a safe effective treatment for
feline coronavirus and feline calicivirus infections in cats.
Keywords: mefloquine; feline infectious peritonitis; pharmacokinetic; coronavirus; calicivirus

1. Introduction
Feline coronavirus (FCoV) is an alpha coronavirus which occurs in two distinct pathotypes thatcan be distinguished by their biological behaviour, but not by their morphology [1]. Although thetwo pathotypes belong to the same virus species, different names have been used—feline entericcoronavirus (FECV) and feline infectious peritonitis (FIP). FECV is highly contagious by the faecal
oral route. The infection is mostly asymptomatic or can cause mild diarrhoea [1,2]. FIP is a fatal,
Animals 2020, 10, 1000; doi:10.3390/ani10061000

www.mdpi.com/journal/animals

Animals 2020, 10, 1000

2 of 14immune-mediated disease induced by virulent biotypes of FCoV known as feline infectious peritonitis
virus (FIPV) in domestic and wild cats. Once cats with FIP show clinical signs, the median life
expectancy ranges from days to weeks for the effusive form, and weeks to months for the non-effusive
form [3–6]. However, there are a small number of cats who may survive for several years [7,8].
Traditionally, treatment options have been limited; however, recent experimental treatments using
protease inhibitors and nucleoside analogs have produced promising outcomes [9–13], although thesetreatment options are not yet registered for cats. The lack of available treatment options escalates theneed to investigate additional affordable antiviral treatments that are currently accessible. Additionally,
there is therapeutic value in finding treatments that target different aspects of FCoV replication, as
combination therapy in other viral infections is associated with higher rates of pathogen suppression
and minimisation of antiviral resistance development [14,15].
Feline calicivirus is an important and common cause of upper respiratory tract disease and oral
ulceration in cats, with more virulent forms of the virus recently emerging resulting in outbreaks of asystemic disease that is commonly fatal and like FIP, lacks effective antiviral treatments. [16].
In searching for antiviral agents against feline coronavirus and feline calicivirus, mefloquine, ahuman anti-malarial drug, was shown to inhibit viral load of FCoV in infected Crandell feline kidneycells without cytotoxic effects [17]. Its inhibition of cytopathic effects and viral replication at low
concentrations support further investigation of this drug as a potential antiviral therapeutic agent.
Our previous project developed an in vitro model to determine the extent and rate of hepatic clearance(Cl) of mefloquine [18]. Mefloquine undergoes phase I hepatic metabolism but not phase II conjugative
metabolism in the cat [18]. There is no evidence that mefloquine should have delayed elimination inclinically healthy cats. Mefloquine plasma protein binding is approximately 99% in both feline plasma
from clinically normal cats and plasma from cats with FIP [19].
As mefloquine is currently used for prophylaxis for malaria, information on its disposition,
including drug absorption, distribution and rate and extent of elimination, is available for humanadults and infants, with some information in dogs [20], but there is no information on mefloquine’s
pharmacokinetic (PK) profile in cats other than the plasma protein binding at this stage [19]. Therefore,
to assess whether mefloquine administration has any therapeutic advantage as an antiviral agent,
determining the pharmacokinetic profile of mefloquine in clinically normal cats is necessary so that adosage and dose frequency can be developed. Knowing the pharmacokinetic profile of mefloquinein the clinically normal cat is the transitional step that bridges preclinical mefloquine observations
to medicating cats with mefloquine in the future. The aim of this study was to investigate thepharmacokinetic profile of mefloquine when administered orally at 62.5 mg (10–12 mg/kg) twice
weekly. The second objective was to document any changes in haematological and/or biochemical
analytes and physiological responses to mefloquine at this dosage regimen.
2. Materials and Methods
2.1. Animals
Eight adult cats (4 females, 4 males) were recruited by Invetus Pty Ltd. (Casino, NSW, Australia),
an animal research facility, from their clinically normal stock animals. Body weights ranged from 5.0
to 5.8 kg (average 5.4 kg). The cats were aged 3–7 years (average 5.5 years). The cats were selectedbased on clinically normal physical examination, normal body condition score and body weight≥5 kg. Exclusion criteria included cats with abnormal physical examination findings, cats that were
underweight or those that were receiving medication. Seven cats were initially selected to enter the
study. The cats were housed individually in pens in a cattery and supplied with food and water ad
libitum. The selection of the clinically normal cats, their dosing with mefloquine, blood collection
and housing were performed by Invetus. This study was approved by Wongaburra Research Centre
Animal Ethics Committee as project USY F 18120 W on the 29th August 2019 and by The University ofSydney Animal Ethics Committee as protocol 2019/1662.

Animals 2020, 10, 1000

3 of 142.2. Drug Administration and Sample Collection
To collect blood, cats were mask induced and anaesthetised with isoflurane in 100% oxygen and
2–4 mL of blood was obtained from the jugular veins using a 22-gauge needle. Blood was collected inlithium heparin tubes, ethylenediaminetetraacetic acid (EDTA) tubes and serum tubes for mefloquine
concentration quantification, haematology and biochemistry, respectively. A 250 mg mefloquine tablet(Lariam, Roche, Millers Point, NSW, Australia) was quartered along the tablet’s scored lines. A quarter
tablet, or 62.5 mg mefloquine, was the dose for each cat. The weight of each quarter tablet wasrecorded for dosing consistency. Mefloquine (62.5 mg) was administered to each cat orally on days 0,
4 (corresponding to 96 h), 7 (168 h) and 10 (240 h). The cats were then monitored for 2 h after dosing
for any adverse reactions. To determine mefloquine plasma concentration, serial blood samples werecollected into lithium heparin tubes at 0 (pre-treatment), 1, 2, 4, 8, 12, 24, 48, 96, 168, 240 and 336 h after
drug administration. On mefloquine administration days other than the first administration (t = 96,
168 and 240 h), blood samples were collected prior to dosing, and mefloquine was then administered,followed by wet or dry food administered within minutes after mefloquine dosing. Blood was alsocollected into EDTA and serum tubes for haematology and biochemistry at 168 and 336 h. Samples
were centrifuged within 1 h of blood collection. Plasma and serum were immediately stored in a freezer(−20 ◦ C) within 90 min of blood sampling. EDTA tubes were immediately sent to Idexx laboratory East
Brisbane, Qld. Australia, for haematological analysis. Serum tubes were sent to Veterinary Pathology
Diagnostic Services, The University of Sydney and the Idexx reference laboratory for biochemistry
analysis. Serum was also sent to the Idexx reference laboratory for serum symmetric dimethylarginine(SDMA) and creatinine. For the determination of mefloquine concentration in the plasma, samples
were batched and all samples were analysed by the veterinary pharmacology laboratory, Sydney
School of Veterinary Science, The University of Sydney, within two months of blood collection.
2.3. Drug Analysis Method and Sample Processing
Samples were quantified for mefloquine concentration by high pressure liquid chromatography(HPLC) with plasma samples modified from a validated method [19].
2.3.1. Chemicals
Mefloquine, verapamil (as the internal standard [IS]), sodium phosphate, trimethylamine,
phosphoric acid were purchased from Sigma-Aldrich (Castle Hill, Sydney, NSW, Australia).
HPLC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Macquarie
Park, NSW, Australia).
2.3.2. HPLC Conditions
The HPLC system consisted of a Shimadzu LC-20AT delivery unit, DGU-20A3 HT degassing
solvent delivery unit, SIL-20A auto injector, SPD-20A UV detector and CTO-20A column oven.
Shimadzu LC Solution software (Kyoto, Japan) was used for chromatographic control, data collection
and data processing. Chromatographic separation was performed with a Polaris C18-A column (5 µm,
150 × 4.6 mm) with a 1.0 mm Optic-guard C 18 pre-column (Choice Analytical, Thornleigh, NSW,
Australia), with the column oven temperature set at 35 ◦ C. The isocratic mobile phase contained a
mixture of 25 mM sodium phosphate with 0.5% triethylamine adjusted to pH 6.0 with phosphoricacid, acetonitrile and methanol (50:25:25) at a flow rate of 0.8 mL/min. For each sample, the injection
volume was 10 µL and the total run time was 15 min. The diode array detector was set at a wavelengthof 220 nm.
For sample preparation, mefloquine plasma concentrations of 0.156, 0.313, 0.625, 1.25, 2.50,
and 10.0 µg/mL were prepared by serial dilution. The IS solution was prepared in 100% acetonitrile ata final concentration of 10 ug/mL. Pre-treatment feline plasma samples were used for the preparationof the standard curve.

Animals 2020, 10, 1000

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Simple protein precipitation using 100 µL of acetonitrile containing 10 µg/mL of IS was added
to 100 µL of feline plasma samples to extract proteins from plasma samples. The samples were thenvortexed and centrifuged at 14,000× g for 10 min. Ten microlitres of supernatant was injected into the
HPLC system for analysis.
2.4. Pharmacokinetic Analysis
The data were evaluated using non-compartment analysis as the elimination phase was only
evident at two time-points, i.e., 48 and 96 h. The mean peak plasma concentration (C max) andtime to reach C max (T max) of the first dose were determined by visual inspection of the individual
cats’ plasma concentration vs time curve over 96 h. The difference in the natural log of the plasma
concentrations at 24 and 96 h, i.e., the slope of the curve from 24 to 96 h gave ke . The elimination
half-life was estimated by ln 2/ke . The area under the curve (AUC0-t ) at 96 h was calculated to the lastmeasurable concentration using linear trapezoidal method. The apparent volume of distribution was
calculated as:
V/F = (Dose/AUC) × (1/ke ),(1)where F is the bioavailability of the oral route and cannot be determined as the intravascular (IV)administration of mefloquine to cats has not been undertaken. The apparent clearance was calculated as:
Cl/F = V × ke .

(2)

Area under the moment curve (AUMC0–96h ) was calculated as the sum of the AUC when each of
the concentration data points was multiplied by time. The mean residence time was calculated as 1/ke.
2.5. Statistical Analysis
Two cats were excluded from statistical analysis due to vomiting. The mefloquine plasma
concentration data points for the five cats (including Cat E) at 24, 96, 168, 240 and 336 h underwent
Shapiro–Wilk normality test and all distributions were normal. However, SDMA concentrations were
not normal at t = 0 h, but were normal at 168 and 336 h. Creatinine concentrations were normal at 0,
168 and 336 h. The mefloquine plasma mean concentrations were compared at 24, 96, 168, 240 and
336 h and underwent a repeated measures on-way ANOVA as did the mean SDMA and creatinine
concentrations at 0, 168 and 336 h. Tukey’s multiple comparisons test was used to demonstrate whether
the means at each time point were significantly different. Statistical analysis was accepted if p < 0.05.
Statistical analysis was undertaken by Graphpad Prism 8 (San Diego, California, CA, USA).
3. Results
Following administration of 62.5 mg of mefloquine per cat, the mean dose per kg was 11.8 mg/kg(median 12.3, range 10.8–12.5). The change in mefloquine plasma concentrations over 336 h (14 days)
for seven cats are displayed in Figure 1 and the actual mefloquine plasma concentrations of each cat
at each time point are provided in Table 1. Figure 2 shows the mefloquine plasma concentrations(ug/mL) over first 24 h. A single oral dose of mefloquine results in a Cmax of 2.71 µg/mL after the
first dose, on average at 15 h (Tmax). Increases in mefloquine plasma concentrations were observed at
168, 240 and 336 h (Figure 1), after second dose administered just after 96 h, third dose administered
just after 168 h and fourth dose administered just after 240 h, respectively, when mefloquine wasadministered with food, with the peak plasma concentrations reaching 4.06 µg/mL (mean) at 240 h.
One cat (Cat C) vomited 15 min after dosing on day 0 (treatment 1). This cat was re-dosed withmefloquine on day 4 (treatment 2) but vomited approximately an hour after dosing, and was, therefore,
removed from the study. Another cat (Cat F) vomited 5 min after dosing on day 1 (treatment 1). This cat
was successfully dosed with mefloquine after feeding on the following treatment day (96 h) and was
re-introduced into the study. No vomiting was observed after dosing this time. Blood samples werecollected at 168, 240 and 336 h for Cat F, as shown in Table 1.

Animals 2020, 10, 1000
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5 of 145 of 15

FigureFigure 1.1. Mefloquine
Mefloquine plasma
plasma concentrations
concentrations (ug/mL) ofof seven
seven cats
cats over
over 336
336 hh (14(14 daysdays after
after initial
initialdose).
Cats
weremedicated
with
62.5
mg
per
cat
(10–12mg/kg)
after
t
=
0,
96,
168
and
240
h.
Blood
was
dose). Cats were medicated with 62.5 mg per cat (10–12 mg/kg) after t = 0, 96, 168 and 240
h. Blood
taken
just prior
to treatment
as denotedby verticaldottedlines. lines.
was taken
just prior
to treatment
as denotedby verticaldottedTable 1. Mefloquine plasma concentrations of individual cats. Cat C and F were excluded frompharmacokinetic (PK) analysis due to incomplete data. Cat C was removed from the study due tovomiting, while Cat F vomited after the first treatment but was successfully re-introduced into thestudy on treatment day 2. Cat E was excluded from PK analysis because this cat’s profile was an outlier
and skewed the data.
Mefloquine Plasma Concentrations ug/mL
H

Cat D

Cat A

Cat B

Cat G

Cat E

Cat C

Cat F

Male
Neutered

Male
Neutered

FemaleNeutered

FemaleNeutered

Male
Neutered

FemaleNeutered

FemaleNeutered

0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1
0.54
1.58
0.31
1.97
0.22
0.86
2
0.95
1.95
0.89
2.79
0.40
2.19
4
1.13
2.10
1.84
3.23
0.49
8
1.59
2.23
2.20
2.89
0.64
12
1.77
2.97
2.14
3.22
0.72
Figure2. Mefloquine
plasmaconcentrations
h after cats were
24
2.09
3.35
2.09(ug/mL) over
3.12 the first 24
0.56- administered
48
1.86
3.34
1.97
2.88
0.56
62.5 mg (10–12 mg/kg) mefloquine at t = 0.
96
1.51
2.76
1.75
2.50
0.57
168
2.75
4.15
3.36
3.73
3.58
1.39
240
1.85
6.51
2.93
4.94
4.20
1.54
336
3.07
4.19
3.60
4.56
2.09
2.11

Figure 1. Mefloquine plasma concentrations (ug/mL) of seven cats over 336 h (14 days after initial
dose).
were medicated with 62.5 mg per cat (10–12 mg/kg) after t = 0, 96, 168 and 240 h. Blood 6 of 14
Animals
2020,Cats
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was taken just prior to treatment as denoted by vertical dotted lines.

FigureFigure 2.
2. Mefloquine
Mefloquineplasma
plasmaconcentrations
concentrations(ug/mL)(ug/mL)over
overthethefirst
first2424hhafter
aftercats
catswere
were administeredadministered62.5
mg(10–12mg/kg)
mefloquine
at
t
=
0.
62.5 mg (10–12 mg/kg) mefloquine at t = 0.

The pharmacokinetic (PK) indices are provided in Table 2. Cats C and F were excluded from thePK analysis due to incomplete data. As Cat E’s PK profile skewed the data, indices for four cats (Cat A,
B, D and G) with more consistent profiles were used for analysis.
Table 2. Pharmacokinetics indices of Cats A, B, D and G over the first 96 h.
PK Indices

Units

Mean

SD

MedianMinMaxke (48–96 h)
t1/2
Tmax
Cmax
AUC 0–96 h
AUMC 0–96 h
MRT 0–96 h

1/h
h
h
µg/mL
µg/mL × h
µg/mL × h2
h
L/kg
L/h/kg0.003
224.1815.00
2.71
228.3010737
326.50
17.41
0.06

0.001
51.60
10.52
0.66
62.23
2971.7
13.60
4.08
0.02

0.003
233.9416.00
2.71
228.1810576
337.46
15.74
0.052

0.003
153.244.00
2.09
166.597826.0221.17
14.73
0.04

0.005
275.6024.00
3.35
290.2513968
397.4723.41
0.085

V/Fobs (as calculated over 0–96 h)
Cl/Fobs (as calculated over 0–96 h)

SD—standard deviation; ke—elimination rate constant; t1/2—elimination half-life; Tmax—time to reach peakplasma concentration; Cmax—peak plasma concentration; AUC 0–96 h —area under the curve over 96 h; AUMC
0–96 h —area under the moment curve over 96 h; MRT 0–96 h —mean residence time; V/Fobs —apparent volume of
distribution; Cl/Fobs —apparent clearance.

Haematology and serum biochemistry of six cats (Cat A, B, D, E, F and G) were performed prior
to treatment (0 h), at 168 and 336 h. Cat C was removed from the study after the first two timepoints,
and therefore blood collection was not continued. Haematology results were unremarkable in all six
cats at all time points. Biochemistry results are illustrated in Table 3.
Biochemistry showed an increase trend in SDMA concentrations at 168 and 336 h in all six
cats. A repeated measure one- way ANOVA, comparing SDMA at all time-points, was statisticallysignificant p < 0.002. Tukey’s multiple comparisons tests show that mean SDMA at each time point
was statistically different: 0 vs 168 h p = 0.002; 168 vs 336 h p = 0.005. Figure 3 shows the median
SDMA concentrations at each time point with upper and lower range from six cats. The median SDMA
at 0 h was 8.0 g/dL (range 1.0–8.0), median SDMA at 168 h was 11.5 g/dL (range 8.0–13.0) and thatat 336 h was 14.0 g/dL (range 10.0–16.0). Creatinine concentrations did not differ significantly at 0,
168 and 336 h in six cats. Three cats had elevations in liver parameters including ALT, AST and ALP
pre-treatment. Among these three cats, one cat (Cat G) had an ALT value as high as 161 U/L and ALP
was 75.0 U/L pre-treatment, ALT and ALP remained elevated at 166 U/L and 87.0 U/L, respectively at
336 h. This cat had no adverse effects during the study. One cat with mildly increased ALT (142 U/L)

Animals 2020, 10, 1000

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was removed from the study due to vomiting (Cat C). Another cat with mildly increased ALT (144 U/L)
and AST (83.0 U/L) at pre-treatment remained clinically healthy throughout the study and both liver
parameters returned to normal range at 168 and 336 h. Other biochemical analytical changes wereunremarkable. Blood glucose at 0 h was not provided, as artifactual hypoglycaemia was seen in all
samples. This was suspected to result from a problematic collection method of blood glucose samples.
Table 3. Biochemistry results (mean and range) at 0, 168 and 336 h. Numbers outside reference intervals
are emboldened. Not all biochemical analytes were provided due to the availability of different
biochemical analytes at different laboratories, hence the blank cells.
Biochemical
Analyte

0h
Units

Mean

168 h
Range

Mean

336 h
Range

Mean

Range

Reference Interval (Idexx
Reference Laboratory)

Glucosemmol/L
5.10
3.90–6.30
4.50
3.40–5.40
3.20–7.50
SDMA
ug/dL
6.70
1.00–8.00
11.0
8.00–13.0
13.5
10.0–16.0
0.00–14.0
Creatinine
umol/L
115
90.0–140.
122
80.0–160.
120.
100.–140.
80.0–200.
Urea
mmol/L
8.00
6.80–10.2
7.70
6.90–9.10
8.08
6.90–9.20
5.00–15.0
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Phosphorus
1.70
1.40–2.00
1.40
1.17–1.63
1.32
1.20–1.60
0.00–2.30
Calcium
mmol/L
2.40
2.30–2.50
2.40
2.40–2.60
2.30
2.20–2.40
2.10–2.80
Sodiummmol/L
152
149–153154
152–156151
148–153
144–158
Biochemistry
increase trend
SDMA concentrationsat 168 and 336
h in all six cats.
Potassium
mmol/L showed
5.10 an 4.50–5.20
4.50 in 4.10–5.20
4.40
4.10–4.70
3.70–5.40
115
111–117123 comparing
120–125
118
AChloride
repeated mmol/Lmeasure oneway
ANOVA,
SDMA
at 116–120
all time-points,106–123
was statistically
16.3 (4 cats) 15.0–18.0
12.0–24.0
Bicarbonate
mmol/L
16.0
15.0–18.0
significant
p mmol/L< 0.002. Tukey’s
multiple comparisonsmean SDMA 15.0–31.0
at each time point
Anion gap
25.8
25.2–27.1- tests
20.6show(4 cats) that
20.1–21.3
Total
proteing/L
75.3
67.0–86.0
71.0
65.2–80.7
71.3
65.0–83.7
60.0–
84.0 the median
was statistically different: 0 vs 168 h p = 0.002; 168 vs 336 h p = 0.005. Figure 3 shows
Albuming/l
31.7
29.0–36.0
28.9
27.8–30.0
30.0
28.0–32.0
25.0–38.0
SDMA
at each35.0–37.0
time point
upper and41.2lower33.0–55.7
range from six 31.0–52.0
cats. The median
Globulinconcentrations
g/L
43.7
42.3 with35.3–52.9
ALT
U/L
79.2
43.0–161
47.3
26.0–116
58.7
22.0–166
19.0–100.
SDMA
at 0 h was
8.0 g/dL(range1.0–8.0),
medianSDMA at168 h 20.0–42.0
was 11.5 g/dL (range
8.0–13.0) and
AST
U/L
47.8
25.0–83.0
29.7
22.0–53.0
29.8
0.00–62.0
U/L 14.0 g/dL
39.8 (range
21.0–75.0
37.0
24.0–72.0
41.8
27.0–87.0did not differ
5.00–50.0
thatALP
at 336 h was
10.0–16.0).
Creatinine
concentrations
significantly at
GGT
U/L
0.70
0.00–1.00
0.50
0.00–2.00
0.00–5.00
0, 168 and 336
h in six3.00
cats. Threecats hadelevations
in liverparameters
including
ALT, AST and
Total bilirubinumol/L
3.00
2.10
0.40–2.70
2.60
2.00–3.00
0.00–7.00
Cholesterol
mmol/L
3.85
2.60–4.90
3.90
3.00–4.80
3.10
0.00–4.70
2.20–5.50
ALP pre-treatment. Among these three cats, one cat (Cat G) had an ALT value as high as 161 U/L and
CK
U/L
299
133–735178
97.0–398148
17.0–248
64.0–400
ALPTT4was 75.0
U/L pre-treatment,
ALP remained elevatedand 87.0 U/L,
nmol/L
32.5
21.0–39.0 ALT
32.6and 28.6–36.7
32.4–35.4 at 166 U/L
10.0–60.0

respectively
at 336 dimethylarginine;h. This cat had ALT—alanine
no adverse transaminase;effects during
the study. transaminase;One cat withmildly increased
SDMA—symmetric
AST—aspartate
ALP—alkaline
phosphatase;
GGT—gamma-glutamyltranspeptidase;
CK—creatine
kinase; TT4—total
Numbers
ALT(142 U/L)
was removed fromthe study
due to vomiting(Cat thyroxine.
C). Anothercat outsidewith mildly
reference intervals are bolded.
increased ALT (144 U/L) and AST (83.0 U/L) at pre-treatment remained clinically healthy throughout
the study and both liver parameters returned to normal range at 168 and 336 h. Other biochemical
Although
Cat C andF vomitedon the firstadministration
all other cats
analyticalchanges
wereunremarkable.
Bloodglucose at of
0 mefloquine,
h was not provided,
as tolerated
artifactual
thehypoglycaemia
medication wellonsubsequentadministration
when
mefloquinewasadministered
with
food.
was seen in all samples. This was suspected to result from a problematiccollection
Nomethodother adverse
wereobserved in any cat over the two weeks of treatment.
of bloodeffects
glucosesamples.

Figure 3. SDMA results (ug/dL) of six cats at 0, 168 and 336 h. The median SDMA concentration of
Figure 3. SDMA results (ug/dL) of six cats at 0, 168 and 336 h. The median SDMA concentration ofeach time point is shown with the upper and lower range. The dotted line represents the referenceeach time point is shown with the upper and lower range. The dotted line represents the reference
interval (0.00–14.0).
interval (0.00–14.0).

Although Cat C and F vomited on the first administration of mefloquine, all other cats tolerated
the medication well on subsequent administration when mefloquine was administered with food. No
other adverse effects were observed in any cat over the two weeks of treatment.
4. Discussion

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4. Discussion
This is the first pharmacokinetic study of mefloquine in cats. Mefloquine’s only reported use
in animals is as an anti-malarial drug for raptors and penguins [21,22]. In addition to its use as ananti- malarial agent, successful treatment with this drug has been reported in people with progressive
multifocal leukoencephalopathy caused by John Cunningham virus (JCV) [23,24]. Its antiviral action
has been demonstrated in vitro in FCoV [17], feline calicivirus [25], dengue virus type 2 and Zika virus
in people [26], and recently, pangolin coronavirus GX_P2X, a model for SARS-CoV-2, the cause of
COVID-19 in people [27]. Its exact mechanisms of actions as an anti-malarial or antiviral agent are
unknown [17,28,29].
As mefloquine is an anti-malarial prophylactic and treatment for people, its pharmacokinetic
profile has been documented. In healthy human volunteers, mefloquine’s oral absorption half- life is
1–4 h (mean 2.1 h) [30]. The oral bioavailability of mefloquine in cats is unknown, as the required IV
AUC required for the calculation has not been undertaken in cats. However, the oral bioavailabilityof mefloquine was found to be about 67–90% (mean 78%) in dogs [20]. Mefloquine reaches peakplasma concentrations after around 6–24 h (median 17.6 h) in people [30]. When administered orally
to cats, the time to peak plasma concentrations (Tmax) is comparable to humans, on average 15 h.
The estimated total apparent volume of distribution in healthy people is approximately 19.2–22.1 L/kg
and systemic clearance is 0.026–0.042 L/h/kg [31], while the apparent mean ± SD volume of distribution
in cats is 17.4 ± 4.08 L/kg, and apparent clearance is 0.060 ± 0.020 L/h/kg when calculated over 0 to
96 h. Plasma protein binding was 98% in healthy human volunteers and patients with uncomplicatedfalciparum malaria [30] and also 99% in feline plasma from clinically normal cats and plasma from catswith FIP [19]. In humans, the elimination half-life of mefloquine is approximately 20 days in healthy
subjects, 10 to 14 days in patients with uncomplicated falciparum malaria [32,33] and 20 days in cases
involving severe malaria [33,34]. In humans, it is recommended that there is a loading dose and thenonce weekly medication [35,36]. Mefloquine is excreted slowly from the body through faeces andurine [31,37]. Our study estimated that the elimination half-life of mefloquine in clinically healthycats is approximately 224 h or approximately 9.3 days, similar to that of healthy people. The half-life
calculation was only based on the 24 to 96 h timepoints; further studies sampling a single dose
administration of mefloquine for longer than 96 h in cats may give a more definite result.
When mefloquine was administered with food, oral absorption was enhanced.
The pharmacokinetic analysis showed that the mean plasma concentration was higher (4.06 ug/mL)
at 240 h when mefloquine was given with food compared to plasma concentration of 2.71 ug/mL
at 15 h when mefloquine was given without feeding. Other considerations for the higher plasma
concentration at 240 h include the cumulative effect of the drug with multiple dosing and possible
enterohepatic circulation of the drug. In humans, the presence of food in the gastrointestinal tractaffects the pharmacokinetic properties of mefloquine by significantly increasing the rate and extent ofabsorption [38].
It has been demonstrated that mefloquine at a concentration of 10 µM showed marked inhibition
towards two biotypes of FCoV, FIPV WSU 79-1146 (FIPV1146) and FECV WSU 79-1683 (FECV1683),
acquired from the American Type Culture Collection (Virginia, USA), [17]. Given the molecularweight of mefloquine is 378 g/mol, a plasma concentration of 10 µM = 3.78 µg/mL is reached when
10 µM of mefloquine is used [17]. This study showed that when a single oral dose of mefloquineof ~12.5 mg/kg was administered, a peak plasma concentration (Cmax) of 2.71 µg/mL is reached.
A higher dose of mefloquine may be required for the drug to inhibit the FIP virus. However, it ispossible that when mefloquine is administered with food the Cmax will be much higher than thatreported at 12 to 24 h. To assess the efficacy of mefloquine towards FCoV virus, a clinical trial iswarranted. Additionally, mefloquine has been shown to inhibit cytopathic effects in cells infected by a
SARS-CoV-2a-related pangolin coronavirus (GX_P2X), making it a potential drug for use in cats with
SARS-CoV-2 infection [27].

Animals 2020, 10, 1000

9 of 14Chloroquine, a 4-aminoquinoline with similar mode of action to mefloquine [39], has been
demonstrated to have inhibitory effect against FIPV replication and anti-inflammatory effect in vitro
and improved clinical score of experimentally induced FIP cats [40]. While mefloquine has been shownto inhibit FIPV in vitro [17], its clinical efficacy in cats with FIP remains unknown. Chloroquine has,
however, caused an increase in ALT levels when used in FIP infected cats. In this study, mefloquine
did not cause an increase in ALT levels. Although some cats had elevated ALT levels prior tomefloquine dosing, a further increase after four doses of mefloquine twice weekly was not seen.
Hydroxychloroquine has been investigated in a clinical trial for COVID-19 treatment in people [41]and recently, its antiviral property against FIPV has also been studied in vitro [42]. When usedwith recombinant feline IFN-ω, hydroxychloroquine showed increased antiviral activity against FIPV
infection [42]. Further clinical studies are needed to verify its clinical efficacy and safety in catswith coronavirus or calicivrus infections. The incidence of adverse effects following mefloquine
administration in people is common with 47–90% of people experience some mild or moderate adverse
effects [30,43,44]. The incidence of adverse effects decreases with prolonged use, from 44% during thefirst 4 months to 19% after one year [30,45]. The most common adverse effects include neuropsychiatriceffects [46–48], gastrointestinal dysfunction [49], dermatological signs [50], haematological changes [51]or cardiovascular dysfunction [30,33]. In humans, nausea and vomiting are both common adverse
effects and can be dose-dependent and age-related, with the highest risk in younger children [49,52].
In this study, two cats vomited following the first mefloquine administration without food. Mefloquine
was successfully re-administered to one cat (Cat F) after feeding and this cat was therefore re-introduced
into the study. Cat C was also re-dosed at the second mefloquine dosing (day 4); however, this cat
refused food before the second dosing. Mefloquine was therefore dosed without food. This cat vomited
again and was removed from the study. When mefloquine was administered with food, no further
vomiting was observed. No other adverse effects were seen clinically in the cats in our study. However,
our cats were only observed for 14 days. Any delayed or long-term adverse effects of mefloquine incats remain unknown. It is also possible that the incidence of adverse effects might reduce with longterm administration, as observed in people [30,45].
The cause of the lower plasma concentration curve of Cat E over the first dosing interval (0–96 h)
was unknown (Figures 1 and 2). An age difference may explain the lower plasma concentration
curve of Cat D and E as these two cats are younger than the others (3 years old vs. 6–7 years old).
Reduction in hepatic clearance, increase in volume of distribution of lipid soluble drugs with prolonged
half-life and increased oral bioavailability were proposed to explain why elderly people have different
pharmacokinetics compared to younger adults and these causes could potentially contribute to thedifferences in plasma concentration in Cats D and E [53,54]. However, an increase in volume of
distribution and prolonged half-life were not observed in the older cats (Cats A, B, C and G). In humans,
while mefloquine blood concentrations during pregnancy are lower than those in non-pregnant
adults, no age-related differences in mefloquine plasma concentrations were found in pharmacokinetic
profiles [55,56]. Interestingly, the maximum blood concentrations are 2–3 times higher in Asian adults
compared to non-Asian volunteers, the reason for this ethnic difference is unclear [30,57]. It has beensuggested that the smaller volume of distribution secondary to a lower body fat content or differencesin enterohepatic circulation of the drug in Asian volunteers could have contributed to the higher
plasma concentrations [58]. In our study, the differences in plasma concentrations between gender,
neutering status and body weight could not be identified. Cat E also had normal biochemical analytes
pre-treatment, at 168 and 336 h. Liver dysfunction causing altered metabolism of the drug is unlikely;
however, it cannot be completely excluded without the pre- and post-prandial bile acid level to assessliver function. In people, mefloquine is metabolised by cytochrome P450 3A (CYP 3A) in the liver [59].
In cats, CYP 3A activities have been found to be lower in female cats compared to male cats [60].
However, the odd plasma concentration curve of Cat E cannot be explained. Another explanation could
be that Cat E might have vomited after the first mefloquine administration without being observed.

Animals 2020, 10, 1000

10 of 14

A difference in pharmacokinetic profile is observed in healthy vs humans infected with malaria.
In humans, mefloquine plasma concentrations are 2–3 times higher in uncomplicated falciparum
malaria when compared to healthy volunteers. Uncomplicated falciparum malaria also has a reduced
half-life [30,31,33,61]. The cause is not entirely understood. One possible cause of the reduced half-life
in malaria patients is the decrease in enterohepatic circulation and greater faecal clearance. Anotherexplanation of the variation in pharmacokinetic profile between the two groups is the difference in theplasma protein binding of mefloquine. Mefloquine extensively binds to plasma proteins, particularly
acute phase proteins such as alpha-1-acid glycoprotein (AGP) [62]. The increase in AGP in malaria
is thought to result in increased mefloquine plasma protein binding, thus affecting the apparent
volume of distribution [61]. High levels of AGP have been demonstrated in experimentally induced
FIP [63] and naturally infected FIP cats [64,65], and is commonly used as a diagnostic tool for FIP inpractice [66]. Thus, it is possible that the high level of AGP and potentially other acute phase proteinsin FIP infected cats increases mefloquine plasma protein binding, altering the pharmacokinetic profilein these cats. The plasma protein binding of mefloquine in clinically normal and FIP infected cats has
been explored in vitro; however, the difference was equivocal due to the unknown biological variabilityof the assay [19]. Further studies on pharmacodynamics and pharmacokinetics of mefloquine areneeded in cats with FIP.
An increase trend in SDMA with no change in creatinine was observed in all cats (Figure 3) during
this study. SDMA has been shown to be an early renal biomarker compared to creatinine [67–69]
and increases in acute kidney injury and chronic kidney disease [70]. Increased serum SDMA
concentrations were associated with reduced renal function measured by glomerular filtration rate
(GFR) in cats [71]. Based on the result of our study, it is possible that mefloquine may cause reduced
renal function in cats. Renal toxicity from anti-malarial drug administration is rare in people [72].
The other explanation of increased SDMA concentrations is the influence of general anaesthesia andthe cumulative effect of isoflurane during the study. Serum SDMA levels measured after anaesthesia
induction (17.11 g/dL) have been shown to be significantly higher than those measured before
anaesthesia induction (12.39 g/dL) [73]. As the cats were anaesthetised with isoflurane during bloodcollections, this could potentially contribute to the increase in SDMA concentrations. The bloodcollection method, mefloquine dosing and recruitment of cats was outsourced to an external designatedanimal research facility (Invetus Pty, Ltd.,Casino, NSW, Australia) because of lack of subject availability
and this study was conducted according to Invetus’ standard operating procedures.
One of the limitations of this study was that not all cats had normal liver enzymes pre-treatment.
Three cats had elevated ALT, AST and ALP levels pre-treatment. As the blood collection, pre-treatment
blood tests and treatment were performed at an external facility, the investigators did not know about
the elevated liver parameters prior to treatment. The investigators were also not involved in the
recruitment process of these cats, and the drug histories and previous disease records of these catswere unknown. Despite this, a significant increase in ALT and ALP after mefloquine treatment did
not occur. ALT and ALP remained unchanged at 336 h in one cat (Cat G). The mefloquine plasma
concentration curve of Cat G did not differ substantially from the other cats (Cat A, B and D).
An additional limitation is the small number of cats in our study. Only five cats had complete
mefloquine plasma concentrations and only four cats were included in the pharmacokinetic analysis(low concentration curve of cat E was omitted). Despite the small number of cats used in the analysis,
an important description of mefloquine’s drug profile in the clinically normal cat was provided.
This preliminary information is crucial for any further research projects that involve mefloquine use
in cats.
5. Conclusions
The study provides preliminary data for the pharmacokinetic profile of mefloquine in catsand provides useful information for the planning of clinical trials of mefloquine to treat cats withfeline coronavirus (including FIP) and feline calicivirus infections and possibly, if the need arises,

Animals 2020, 10, 1000

11 of 14for COVID-19 to potentially reduce viral shedding. Further studies on the therapeutic effects of
mefloquine in cats with these diseases are needed to determine its therapeutic advantage.
Author Contributions: Conceptualization, J.M.N. and M.G.; methodology, M.G. and B.K.; software, B.K. and
M.G.; validation, M.G. and B.K.; formal analysis, M.G. and B.K.; investigation, M.G., B.K. and J.Y.; resources,
M.G. and B.K.; data curation, M.G., J.Y. and B.K.; writing—original draft preparation, J.Y.; writing—review andediting, J.Y., M.G. and J.M.N.; visualization, M.G. and J.Y.; supervision, J.M.N. and M.G.; project administration,
M.G.; funding acquisition, J.M.N., M.G. and B.K. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Winn Feline Foundation, grant number 2019_027, donation from the Estateof Christine Gai Atkins and from the Lesley Muir Bequest from the Sydney School of Veterinary Science.
Acknowledgments: The authors are grateful to Invetus for their support in recruiting the cats, providing housing,
medicating and blood collection of the cats.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of thestudy; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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