Pharmacokinetic profile of oral mefloquine in clinically normal cats: Preliminary in vivo study of the potential treatment of feline infectious peritonitis (FIP).

Jane Yu, Benjamin Kimble, Jacqueline M. Norris and Merran Govendir
Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Sydney, NSW 2006, Australia; jane.yu@sydney.edu.au (JY); benjamin.kimble@sydney.edu.au (BK); jacqui.norris@sydney.edu.au (JN); merran.govendir@sydney.edu.au
Original article: Pharmacokinetic Profile of Oral Administration of Mefloquine to Clinically Normal Cats: A Preliminary In ‐ Vivo Study of a Potential Treatment for Feline Infectious Peritonitis (FIP)
8.6.2020

Brief summary:

In the search for antiviral agents against feline coronaviruses and feline caliciviruses, mefloquine, a human antimalarial drug, has been shown to reduce the viral load of feline coronaviruses and feline calicivirus in infected cells. In this study, mefloquine was administered orally to seven clinically healthy cats twice a week in four doses, and blood mefloquine concentrations were measured to determine the pharmacokinetic profile - drug movement in the body. The maximum blood concentration of mefloquine was 2.71 ug / ml and was reached 15 hours after a single oral dose. Side effects of mefloquine in some cats included vomiting after feeding without food and a slight increase in symmetric dimethylarginine (SDMA), an early renal biomarker. This study provides valuable information on the profile of mefloquine in cats as an initial step to its research as a possible treatment for feline coronavirus and feline calicivirus infection in cats.

Abstract: The pharmacokinetic profile of mefloquine was investigated to preliminary study the potential treatment of feline coronavirus infections (such as feline infectious peritonitis) or feline calicivirus infections. Mefloquine was administered orally at a dose of 62.5 mg to seven clinically healthy cats twice a week in four doses, and plasma mefloquine concentrations over 336 hours were measured by high performance liquid chromatography (HPLC). The maximum plasma concentration (Cmax) after a single oral dose of mefloquine was 2.71 ug / ml and the time to Cmax (Tmax) was 15 h. The elimination half-life was 224 h. Plasma concentrations reached a higher level of 4.06 ug / ml when mefloquine was fed. Dosage side effects in some cats included vomiting after administration without food. A slight increase in symmetric dimethylarginine (SDMA) but not creatinine serum concentrations was observed. Mefloquine can provide a safe and effective treatment for coronavirus and calicivirus infections in cats. 

Keywords: mefloquine; feline infectious peritonitis; pharmacokinetics; coronavirus; calicivirus 

Introduction

Feline coronavirus (FCoV) is an alpha coronavirus that occurs in two distinct pathotypes that can be distinguished by biological behavior but not by morphology [1]. Although the two pathotypes belong to the same type of virus, different names are used - feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV is highly contagious by the fecal-oral route. The infection is usually asymptomatic or can cause mild diarrhea [1,2]. FIP is a deadly, immune-mediated disease caused by virulent FCoV biotypes known as feline infectious peritonitis virus (FIPV) in domestic and wild cats. When cats with FIP show clinical signs, life expectancy ranges from a few days to a few weeks for the effusive form and from a few weeks to a few months for the non-fusive form [3-6]. However, there are a small number of cats that can survive for several years [7,8]. Treatment options are traditionally limited, but recent experimental treatments using protease inhibitors and nucleoside analogs have yielded promising results [9-13], although these treatment options have not yet been registered for the treatment of cats. The lack of available treatment options exacerbates the need to explore other available antiviral treatments. In addition, seeking treatment for various aspects of FCoV replication is of therapeutic value, as combination therapy for other viral infections is associated with higher rates of pathogen suppression and minimizing the development of antiviral resistance [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 emerging recently, leading to an outbreak of systemic disease, which is often fatal and, like FIP, has no effective antiviral treatment. [16] 

In the search for antiviral agents against feline coronavirus and feline calicivirus, mefloquine, a human antimalarial, has been shown to inhibit FCoV viral load in infected Crandell feline kidney cells without cytotoxic effects [17]. Its inhibition of cytopathic effects and viral replication at low concentrations supports further investigation of this drug as a potential antiviral therapeutic. In our previous project, we developed an in vitro model to determine the extent and rate of hepatic clearance (Cl) of mefloquine [18]. In cats, mefloquine undergoes phase I hepatic metabolism but not phase II conjugative metabolism [18]. There is no evidence that mefloquine delays elimination in clinically healthy cats. Plasma protein binding of mefloquine is approximately 99 % in the plasma of clinically normal cats as well as in the plasma of cats with FIP [19].

As mefloquine is currently used for malaria prophylaxis, information on its availability is available, including absorption, distribution and rate and extent of elimination of the drug in adults and infants, with some information also available in dogs [20], but not at this stage. no information is available on the pharmacokinetic profile (PK) of mefloquine in cats other than plasma protein binding [19]. Therefore, in order to assess whether the administration of mefloquine as an antiviral has any therapeutic advantage, it is necessary to determine the pharmacokinetic profile of mefloquine in clinically normal cats in order to develop the dosage and frequency of doses. Recognition of the pharmacokinetic profile of mefloquine in a clinically normal cat is a transitional step that bridges preclinical observations of mefloquine to feline medication in the future. The aim of this study was to examine the pharmacokinetic profile of mefloquine when administered 62.5 mg (10-12 mg / kg) orally twice weekly. The second objective was to document any changes in hematological and / or biochemical analytes and physiological responses to mefloquine in this dosing regimen.

Materials and methods 

The animals 

Eight adult cats (4 females, 4 males) were acquired by Invetus Pty Ltd. (Casino, New South Wales, Australia), an animal research facility, from its clinically normal animals. Body weight ranged from 5.0 to 5.8 kg (average 5.4 kg). The cats were 3 to 7 years old (average 5.5 years). Cats were selected based on clinically normal physical examination, normal fitness score, and body weight ≥ 5 kg. Exclusion criteria included cats with an abnormal physical examination, underweight cats, or cats that took medication. Seven cats were originally selected for the study. The cats were housed individually in boxes at the kennel and supplied with food and water ad libitum. The selection of clinically normal cats, their dosing with mefloquine, blood collection and shelter was carried out by the company Invetus. This study was approved by the Wongaburra Research Center's Animal Ethics Committee as a USY F 18120 W project on August 29, 2019 and by the University of Sydney Animal Ethics Committee as Protocol 2019/1662.

Drug administration and sampling

The cats were given a mask for blood collection and anesthesia with isoflurane in 100 % oxygen, and 2-4 ml of blood was collected from the jugular veins using a 22 mm diameter needle. Blood was collected into lithium heparin tubes, ethylenediaminetetraacetic acid (EDTA) tubes and serum tubes for mefloquine quantification, hematology and biochemistry. A 250 mg tablet of mefloquine (Lariam, Roche, Millers Point, New South Wales, Australia) was quartered along the line on the tablet. For each cat, the dose was a quarter of a tablet or 62.5 mg of mefloquine. The weight of each quarter of the tablet was recorded for consistency of dosing. Mefloquine (62.5 mg) was administered orally to each cat on days 0, 4 (corresponding to 96 h), 7 (168 h) and 10 (240 h). The cats were then monitored for any adverse reactions 2 hours after dosing. To determine mefloquine plasma concentrations, serial blood samples were taken into lithium heparin tubes at time 0 (before 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 day (t = 96, 168 and 240 h), blood samples were taken before the mefloquine dose, followed by wet or dry food for several minutes. Blood was also collected into EDTA and serum tubes for hematological and biochemical examination at 168 and 336 hours. The samples were centrifuged within 1 h after blood collection. Plasma and serum were immediately stored in a freezer (-20 ° C) within 90 minutes of blood collection. EDTA tubes were immediately shipped to Idexx East Brisbane, Qld. Australia, for hematological analysis. Serum tubes were sent to the Veterinary Pathology Diagnostic Services, The University of Sydney and the Idexx Reference Laboratory for biochemical analysis. Serum was also sent to the Idexx Reference Laboratory for analysis of serum symmetric dimethylarginine (SDMA) and creatinine. To determine plasma mefloquine concentrations, samples were analyzed at the Sydney School of Veterinary Science, The University of Sydney, within two months of blood collection. 

Drug analysis method and sample processing

The concentration of mefloquine in the samples was quantified by high pressure liquid chromatography (HPLC), and the plasma samples were adjusted according to a validated method [19]. 

Chemicals

Mefloquine, verapamil (as internal standard [IS]), sodium phosphate, trimethylamine and phosphoric acid were purchased from Sigma-Aldrich (Castle Hill, Sydney, New South Wales, Australia). Acetonitrile and HPLC grade methanol were purchased from Thermo Fisher Scientific (Macquarie Park, NSW, Australia).

HPLC conditions

The HPLC system consisted of a Shimadzu LC-20AT feed unit, a DGU-20A3 HT feed unit for solvent degassing, an SIL-20A automatic injector, a SPD-20A UV detector and a CTO-20A column furnace. Shimadzu LC Solution software (Kyoto, Japan) was used for chromatographic control, data collection and processing. Chromatographic separation was performed using a Polaris C18-A column (5 μm, 150 × 4.6 mm) with a 1.0 mm diameter Optic-guard C 18 precolumn (Choice Analytical, Thornleigh, NSW, Australia) while adjusting the column temperature. at 35 ° C. The isocratic mobile phase contained a mixture of 25 mM sodium phosphate with 0.5 L of TP 2 T triethylamine adjusted to pH 6.0 with phosphoric acid, 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 duration was 15 min. The diode array detector was set to a wavelength of 220 nm. 

Plasma mefloquine concentrations of 0.156, 0.313, 0.625, 1.25, 2.50 and 10.0 μg / ml were prepared by serial dilution for sample preparation. Solution IS was prepared in 100 % acetonitrile at a final concentration of 10 ug / ml. Feline plasma samples were used to prepare the standard curve. 

To extract proteins from plasma samples, 100 μl of acetonitrile containing 10 μg / ml IS was added to 100 μl of feline plasma samples. The samples were then vortexed and centrifuged at 14,000 g for 10 minutes. Ten microliters of supernatant was injected into the HPLC system for analysis. 

Pharmacokinetic analysis 

The data were evaluated by non-compartmental analysis, as the elimination phase was evident at only two time points, ie 48 and 96 h. The mean maximum plasma concentration (C max) and the time to C max (T max) of the first dose were determined by visual inspection of the plasma concentration-time curve of each cat over 96 hours. The difference in the natural logarithm of the plasma concentrations at 24 and 96 hours, ie the slope of the curve from 24 to 96 hours, gave. The elimination half-life was estimated using ln 2 / ke. Area under the curve (AUC_{0 ‐ t}) at 96 h was calculated to the last measurable concentration using the linear trapezoidal method. Apparent volume of distribution was calculated as: 

V / F = (Dose / AUC) × (1 / ke), (1)

where F is the oral bioavailability that cannot be determined because intravascular (intravenous) administration of mefloquine to cats has not occurred. Apparent clearance was calculated as: 

Cl / F = V × ke. (2)

Area under the torque curve (AUMC0_0-96h ) was calculated as the sum of the AUC when each of the concentration data was multiplied by time. The average residence time was calculated as 1 / ke.   

Statistical analysis

Two cats were excluded from the statistical analysis due to vomiting. Mefloquine plasma concentration data for five cats (including cat E) at 24, 96, 168, 240 and 336 h were subjected to the 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. Mean plasma mefloquine concentrations were compared at 24, 96, 168, 240 and 336 h and were subjected to repeated on-way ANOVA measurements, as were mean SDMA and creatinine concentrations at 0, 168 and 336 h. Tukey's multiple comparison test was used to demonstrate whether the mean values at each time point differed significantly. Statistical analysis was accepted if p <0.05. Statistical analysis was performed using Graphpad Prism 8 (San Diego, CA, CA). 

The results

Following administration of 62.5 mg of mefloquine per cat, the mean dose was 11.8 mg / kg (median 12.3, range 10.8-12.5). The change in mefloquine plasma concentrations over 336 h (14 days) in seven cats is shown in Figure 1 and the actual mefloquine plasma concentrations of each cat at each time point are shown in Table 1. Figure 2 shows mefloquine plasma concentrations (ug / ml). during the first 24 hours. A single oral dose of mefloquine results in a Cmax of 2.71 μg / ml after the first dose, averaging 15 h (Tmax). Increases in mefloquine plasma concentrations were observed after 168, 240 and 336 h (Figure 1), after the second dose given just after 96 h, the third dose given just after 168 h and the fourth dose given just after 240 h, respectively when mefloquine was administered with food, with peak plasma concentrations reaching 4.06 μg / ml (average) after 240 h. One cat (cat C) vomited 15 minutes after dosing on day 0 (treatment 1). Mefloquine was re-administered to this cat on day 4 (treatment 2), but vomited approximately one hour after dosing and was therefore excluded from the study. Another cat (cat F) vomited 5 minutes after dosing on day 1 (treatment 1). This cat was successfully given a dose of mefloquine after feeding on the following day of treatment (96 h) and was re-enrolled in the study. This time, no vomiting was observed after dosing. Blood samples were taken from cat F at 168, 240 and 336 h as shown in Table 1. 

	Cat D	Cat A	Cat B	Cat G	Cat E	Cat C	Cat F
H	Castrated male	Castrated male	Castrated female	Castrated female	Castrated male	Castrated female	Castrated female
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	-	-
24	2.9	3.35	2.9	3.12	0.56	-	-
48	1.86	3.34	1.97	2.88	0.56	-	-
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	3.7	4.19	3.60	4.56	2.9	-	2.11

Pharmacokinetic (PK) parameters are shown in Table 2. Cats C and F were excluded from the PK analysis due to incomplete data. Because the PK profile of cat E distorts the data, indices for four cats (cat A, B, D, and G) with more consistent profiles were used for analysis. 

PK indices	Units	Average	SD	Median	Min	Max
ke (48-96 h)	1 / h	0.003	0.001	0.003	0.003	0.005
t1 / 2	h	224.18	51.60	233.94	153.24	275.60
Tmax	h	15.00	10.52	16.00	4.00	24.00
Cmax	μg / ml	2.71	0.66	2.71	2.09	3.35
AUC 0-96 h	μg / ml × h	228.30	62.23	228.18	166.59	290.25
AUMC 0-96 h	μg / ml × h 2	10737	2971.7	10576	7826.0	13968
MRT 0-96 h	h	326.50	13.60	337.46	221.17	397.47
V / Fobs (calculated 0-96 h)	L / kg	17.41	4.08	15.74	14.73	23.41
Cl / Fobs (calculated 0-96 h)	L / h / kg	0.06	0.02	0.052	0.04	0.085

Hematology and biochemistry of the serum of six cats (cats A, B, D, E, F and G) were performed before treatment (0 h), after 168 and 336 h. Cat C was excluded from the study after the first two checkpoints and therefore blood collection was not continued. Haematological results were unambiguous at all six time points. The results of biochemistry are shown in Table 3.

		0h	0h	168h	168h	336h	336h
Biochemical analyte	Units	Average	Range	Average	Range	Average	Range	Reference interval (Idexx reference laboratory)
Glucose	mmol / 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
Phosphorus	mmol / l	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
Sodium	mmol / l	152	149-153	154	152-156	151	148-153	144-158
Potassium	mmol / l	5.10	4.50-5.20	4.50	4.10-5.20	4.40	4.10-4.70	3.70-5.40
Chloride	mmol / l	115	111-117	123	120-125	118	116-120	106-123
Bicarbonate	mmol / l	16.0	15.0-18.0	-	-	16.3 (4 cats)	15.0-18.0	12.0-24.0
Anion gap	mmol / l	25.8	25.2-27.1	-	-	20.6 (4 cats)	20.1-21.3	15.0-31.0
Total protein	g / L	75.3	67.0-86.0	71.0	65.2-80.7	71.3	65.0-83.7	60.0- 84.0
Albumin	g / l	31.7	29.0-36.0	28.9	27.8-30.0	30.0	28.0-32.0	25.0-38.0
Globulin	g / L	43.7	35.0-37.0	42.3	35.3-52.9	41.2	33.0-55.7	31.0-52.0
ALT	U / L	79.2	43.0-161	47.3	26.0-116	58.7	22.0-166	19.0-100.
AST	U / L	47.8	25.0-83.0	29.7	22.0-53.0	29.8	20.0-42.0	0.00-62.0
ALP	U / L	39.8	21.0-75.0	37.0	24.0-72.0	41.8	27.0-87.0	5.00-50.0
GGT	U / L	0.70	0.00-1.00	-	-	0.50	0.00-2.00	0.00-5.00
Total bilirubin	umol / l	3.00	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
CK	U / L	299	133-735	178	97.0-398	148	17.0-248	64.0-400
TT4	nmol / l	32.5	21.0-39.0	32.6	28.6-36.7		32.4-35.4	10.0-60.0

Biochemical examination showed a trend of increasing SDMA concentrations at 168 and 336 hours in all six cats. One-way repeated measures ANOVA comparing SDMA at all time points was statistically significant p <0.002. Tukey's multiple comparison tests show that the average SDMA value 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 ranges from six cats. The median SDMA at 0 h was 8.0 g / dl (range 1.0 - 8.0), the median SDMA at 168 h was 11.5 g / dl (range 8.0 - 13.0) and at 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 the six cats. Three cats had elevated liver parameters before treatment, including ALT, AST and ALP. Of these three cats, one cat (Cat G) had an ALT of up to 161 U / L and ALP was 75.0 U / L prior to treatment, and ALT and ALP remained elevated to 166 U / L and 87.0 U / L, respectively. at 336 h. This cat had no side effects during the study. One cat with slightly elevated ALT (142 U / L) was excluded from the study due to vomiting (cat C). Another cat with slightly elevated ALT (144 U / L) and AST (83.0 U / L) before treatment remained clinically healthy throughout the study and both liver parameters returned to normal after 168 and 336 h. Other biochemical analytical changes were unnoticed. Blood glucose was not provided at 0 h because artificial hypoglycemia was observed in all samples. This was suspected to be due to a problematic blood sampling method for glucose determination. 

Although cats C and F vomited on the first administration of mefloquine, all other cats tolerated the drug well on the next administration when mefloquine was administered with food. No other side effects were observed in any cat during the two weeks of treatment.  

Discussion

This is the first pharmacokinetic study of mefloquine in cats. The only reported use of mefloquine in animals is as an antimalarial for predators and penguins [21,22]. In addition to its use as an antimalarial, successful treatment with this drug has been reported in people with progressive multifocal leukoencephalopathy caused by John Cunningham virus (JCV) [23,24]. Its antiviral activity has been demonstrated in vitro with FCoV [17], feline calicivirus [25], dengue virus type 2 and Zika virus in humans [26] and more recently with pangolin coronavirus GX_P2X, which is a model for SARS-CoV-2, the causative agent COVID-19 in humans [27]. The exact mechanisms of its action as an antimalarial or antiviral agent are not known [17,28,29]. 

As mefloquine is an antimalarial prophylaxis and treatment of humans, its pharmacokinetic profile has been documented. In healthy volunteers, the oral half-life of mefloquine 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 calculation has not been performed in cats. However, the oral bioavailability of mefloquine in dogs has been found to be approximately 67-90 % (mean 78 %) [20]. Mefloquine reaches maximum plasma concentrations after approximately 6-24 h (median 17.6 h) in humans [30]. When administered orally to cats, the time to peak plasma concentrations (Tmax) is comparable to humans, averaging 15 hours. The estimated total apparent volume of distribution in healthy humans is approximately 19.2-22.1 l / kg and the 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 the apparent clearance is 0.060 ± 0.020 l / h / kg when calculated at 0 to 96 h. Plasma protein binding was 98 % in healthy human volunteers and patients with uncomplicated falciparum malaria [30] and also 99 % in cat plasma from clinically normal cats and FIP cat plasma [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, a loading dose and then treatment once a week is recommended [35,36]. Mefloquine is slowly excreted from the body via faeces and urine [31,37]. In our study, we estimated that the elimination half-life of mefloquine in clinically healthy cats is approximately 224 hours or approximately 9.3 days, similar to healthy humans. The elimination half-life calculation was based on 24 to 96 h time periods only; further studies, with sampling at a single dose of mefloquine longer than 96 hu in cats, may provide a clearer result.

Oral absorption was increased when mefloquine was administered with food. Pharmacokinetic analysis showed that the mean plasma concentration was higher (4.06 ug / ml) after 240 h when mefloquine was administered with food, compared to the plasma concentration of 2.71 ug / ml after 15 h when mefloquine was administered without food. . Other factors affecting the higher plasma concentrations after 240 hours include the cumulative effect of the drug when administered multiple times and the possible enterohepatic circulation of the drug. In humans, the presence of food in the gastrointestinal tract affects the pharmacokinetic properties of mefloquine by significantly increasing the rate and extent of absorption [38]. 

Mefloquine at 10 μM was shown to show significant inhibition against two FCoV biotypes, FIPV WSU 79-1146 (FIPV1146) and FECV WSU 79-1683 (FECV1683), obtained from the American Type Culture Collection (Virginia, USA), [17] . As the molecular weight of mefloquine is 378 g / mol, a plasma concentration of 10 μM = 3.78 μg / ml is achieved using 10 μM mefloquine [17]. This study showed that a single oral dose of mefloquine ~ 12.5 mg / kg achieved a maximum plasma concentration (Cmax) of 2.71 μg / ml. A higher dose of mefloquine may be required to inhibit FIP. However, it is possible that Cmax will be much higher when mefloquine is administered with food, as reported after 12 to 24 hours. The evaluation of the efficacy of mefloquine against FCoV deserves a clinical study. In addition, mefloquine has been shown to inhibit cytopathic effects in cells infected with SARS-CoV-2a coronavirus (GX_P2X), making it a potential drug for use in cats with SARS-CoV-2 infection [27].

Chloroquine, a 4-aminoquinoline with a similar mode of action to mefloquine [39], has shown an inhibitory effect against FIPV replication and an anti-inflammatory effect in vitro and improved the clinical evaluation of experimentally induced FIP in cats [40]. Although mefloquine has been shown to inhibit FIPV in vitro [17], its clinical efficacy in cats with FIP remains unknown. However, chloroquine caused an increase in ALT levels when used in FIP-infected cats. In this study, mefloquine did not increase ALT levels. Although some cats had elevated ALT levels prior to the mefloquine dose, no further increase was observed after four doses of mefloquine twice a week. Hydroxychloroquine has been studied in a clinical study for the treatment of COVID-19 in humans [41] and its antiviral properties against FIPV in vitro have also been recently investigated [42]. At when used with recombinant feline IFN-ω, hydroxychloroquine has shown increased antiviral activity against FIPV infection [42]. Further clinical studies are needed to verify its clinical efficacy and safety in cats with coronavirus or calicivirus infection. Side effects of mefloquine in humans are common, with 47-90 % people having some mild to moderate side effects [30,43,44]. The incidence of adverse events decreases with long-term use, from 44 % during the first 4 months to 19 % after one year [30,45]. The most common side effects include neuropsychiatric effects [46-48], gastrointestinal dysfunction [49], dermatological symptoms [50], haematological changes [51] or cardiovascular dysfunction [30,33]. In humans, nausea and vomiting are common side effects and may be dose and age dependent, with younger children being most at risk [49,52]. In this study, two cats vomited after the first administration of mefloquine without food. One cat (cat F) was successfully re-administered mefloquine after feeding and was therefore re-enrolled in the study. Cat C was also re-administered a second dose of mefloquine (day 4); however, this cat refused food before the second dose. Mefloquine was therefore dosed without food. This cat vomited again and was excluded from the study. No further vomiting was observed when mefloquine was administered with food. No other clinical side effects were observed in cats in our study. However, our cats were observed for only 14 days. Any delayed or long-term side effects of mefloquine in cats remain unknown. It is also possible that the incidence of side effects could be reduced with long-term administration, as observed in humans [30,45]. 

The cause of the lower plasma concentration curve of Cat E during the first dosing interval (0-96 h) remained unknown (Figures 1 and 2). The age difference may explain the lower plasma concentration curve of cats D and E, as these two cats are younger than the others (3 years versus 6-7 years). Decreased hepatic clearance, increased volume of lipid-soluble drugs with extended half-lives and increased oral bioavailability have been proposed to explain why older people have different pharmacokinetics compared to younger adults, and these causes could potentially contribute to differences in cat plasma concentrations. D and E [53,54]. However, no increase in volume of distribution and prolonged half-life were observed in older cats (cats A, B, C and G). Although human mefloquine blood levels during pregnancy are lower than in non-pregnant adults, there were no age-related differences in plasma mefloquine concentrations in the pharmacokinetic profiles [55,56]. Interestingly, maximum blood concentrations are 2-3 times higher in Asian adults compared to non-Asian volunteers, and the reason for this ethnic difference is unclear [30,57]. It is thought that a lower volume of distribution may have contributed to higher plasma concentrations due to lower body fat or differences in enterohepatic drug circulation in Asian volunteers [58]. In our study, it was not possible to identify differences in plasma concentrations between gender, castration, and body weight. Cat E also had normal biochemical analytes before treatment, at 168 and 336 h. Liver dysfunction causing altered drug metabolism is unlikely; however, it cannot be completely ruled out without determining bile acid levels before and after treatment to assess liver function. In humans, mefloquine is metabolised by cytochrome P450 3A (CYP 3A) in the liver [59]. In cats, CYP 3A activity was found to be lower in cats compared to cats [60]. However, the special plasma concentration curve of cat E cannot be explained. Another explanation could be that cat E could vomit without being observed after the first administration of mefloquine. 

The difference in pharmacokinetic profile is observed in healthy people compared to malaria-infected people. In humans, plasma mefloquine concentrations are 2-3 times higher in uncomplicated falciparum malaria compared to healthy volunteers. Uncomplicated malaria falciparum also has a shortened half-life [30,31,33,61]. The cause is not fully understood. One possible cause of the shortened half-life in patients with malaria is a decrease in enterohepatic circulation and greater fecal clearance. Another explanation for the differences in pharmacokinetic profile between the two groups is the difference in mefloquine binding to plasma proteins. Mefloquine is highly bound to plasma proteins, especially acute phase proteins such as alpha-1-acid glycoprotein (AGP) [62]. An increase in AGP in malaria is thought to lead to an increase in plasma protein binding of mefloquine, which affects the apparent volume of distribution [61]. High levels of AGP have been demonstrated in experimentally induced FIP [63] and naturally infected cats with FIP [64,65] and are commonly used in practice as a diagnostic tool for FIP [66]. Thus, it is possible that high levels of AGP and potentially other acute phase proteins in FIP-infected cats increase mefloquine binding to plasma proteins, altering the pharmacokinetic profile in these cats. Plasma protein binding of mefloquine in clinically normal and FIP-infected cats was investigated in vitro; however, the difference was ambiguous due to the unknown biological variability of the assay [19]. Further studies on the pharmacodynamics and pharmacokinetics of mefloquine are required in cats with FIP. 

During this study, a trend of increasing SDMA without a change in creatinine was observed in all cats (Figure 3). SDMA has been shown to be an early renal biomarker compared to creatinine [67-69] and increases in acute renal injury and chronic kidney disease [70]. Elevated serum SDMA concentrations in cats have been associated with decreased renal function as measured by glomerular filtration rate (GFR) [71]. Based on the results of our study, it is possible that mefloquine may cause decreased renal function in cats. Renal toxicity from antimalarial administration is rare in humans [72]. Another explanation for the elevated SDMA concentrations is the effect of general anesthesia and the cumulative effect of isoflurane during the study. Serum SDMA levels measured after induction of anesthesia (17.11 g / dl) have been shown to be significantly higher than levels measured before induction of anesthesia (12.39 g / dl) [73]. As cats were anesthetized with isoflurane during blood collection, this could potentially contribute to increased SDMA concentrations. Blood collection, mefloquine dosing, and cat recruitment were commissioned from an external designated animal research facility (Invetus Pty, Ltd., Casino, NSW, Australia) due to lack of subject availability, and this study was performed according to Invetus standard operating procedures. 

One limitation of this study was that not all cats had normal liver enzymes prior to treatment. Three cats had elevated ALT, AST and ALP levels before treatment. Because blood collection, pre-treatment blood tests, and treatment were performed at an external facility, investigators were unaware of elevated liver parameters prior to treatment. Investigators were also not involved in the recruitment process for these cats and the drug history and previous records of these cats were not known. Nevertheless, there was no significant increase in ALT and ALP after mefloquine treatment. ALT and ALP remained unchanged after 336 hours in one cat (cat G). The plasma mefloquine concentration curve in cat G did not differ significantly from the other cats (cats A, B and D). 

Another 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 (the low cat E concentration curve was omitted). Despite the small number of cats used in the analysis, an important description of the mefloquine drug profile in a clinically normal cat was provided. This preliminary information is crucial for all other research projects that involve the use of mefloquine in cats. 

Conclusions

The study provides preliminary data on the pharmacokinetic profile of mefloquine in cats and provides useful information for planning clinical trials of mefloquine for the treatment of cats with feline coronavirus (including FIP) and feline calicivirus infections and, if necessary, COVID-19 to potentially reduce virus shedding. . Further studies on its therapeutic effects are needed to determine the therapeutic benefit of mefloquine in cats with these diseases.

Author's contributions: Conceptualization, JMN and MG; methodology, MG and BK; software, BK and MG; validation, MG and BK; formal analysis, MG and BK; investigation, MG, BK and JY; sources, MG and BK; data curators, MG, JY and BK; preparation of the original proposal, JY; writing - review and editing, JY, MG and JMN; visualization, MG and JY; supervision, JMN and MG; project administration, MG; fundraising, JMN, MG and BK All authors have read and agreed to the published version of the manuscript.

Financing: This research was funded by the Winn Feline Foundation, grant number 2019_027, a donation from the estate of Christine Gai Atkins and a reference from Lesley Muir of the Sydney School of Veterinary Science.

Acknowledgments: The authors are grateful to Invetus for supporting the recruitment of cats, providing shelter, medication and taking the blood of cats.

Conflict of interests: The authors do not indicate any conflict of interest. The funders did not participate in the study design, data collection, analysis or interpretation, manuscript writing or decision to publish the results.

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