Xanthine oxidase (E1263Ra) and aldehyde oxidase (ER0670) ELISA kits were purchased from FineTest Bioassay Technology Laboratory. Methanol, ethyl acetate, and other chemicals used in the study were obtained from Sigma-Aldrich.

The Institutional Animal Use and Care Committee of Firat University established rules for the humane treatment of all animals, and the same committee also approved the experiment's protocol (Ethical Approval Number: 2021/02). 25 female Sprague Dawley rats (12 weeks, 250-300 g) were obtained from the Laboratory of Experimental Animals of Firat University. The animals were provided with a standard pellet diet and ad libitum water. Rats were randomly divided into five experimental groups with five animals in each group. Group 1: Pioglitazone, Group 2: Citalopram, Group 3: Favipiravir, Group 4: Favipiravir+Pioglitazone, Group 5: Favipiravir+Citalopram.

Pioglitazone (Glifix tablet, Bilim İlaç Sanayii ve Ticaret A.Ş, Turkey) was dissolved in water and administered daily for 7 days by oral gavage at a dose of 1 mg/kg. Citalopram (Citol tablet, Abdi İbrahim İlaç Sanayii ve Ticaret A.Ş, Turkey) was dissolved in water and administered daily for 7 days by oral gavage at a dose of 1.5 mg/kg. Favipiravir (FAVIRA tablet, Novelfarma İlaç San. Ve Tic. Ltd. Şti., Turkey) was dissolved in 0.5% carboxymethyl cellulose (CMC) and administered to the rats by gavage at a loading dose of 50 mg/kg on the 6th day of the study, and a maintenance dose of 30 mg/kg was given on the 7th day. On the 7th day of the study, blood samples were collected at 0, 15, 30, and 45 min and 1, 2, 4, 6, and 8 h after ketamine (5 mg/kg)–xylazine (40 mg/kg) administration. Samples were collected into tubes containing EDTA from jugular veins (approximately 0.2 mL of blood was drawn each time) and were separated to sera by centrifugation at 4 °C for 30 min at 3.500 хg. After the end of the experiment; rats were sacrificed according to animal use guidelines. Liver tissues were obtained from decapitated animals at the end of the study. Tissues and plasma samples taken were stored in a deep freezer at -80 °C until analysis.

With the help of the IBM SPSS 22.0 package application, descriptive analysis done(SPSS Inc., Chicago, IL, United States). Descriptive statistics of the data are presented as Mean±SD. The Levene test and the Shapiro-Wilk test were used to test the homogeneity of variance and the assumption of normality, respectively. Mann-Whitney U test was used to compare pharmacokinetic parameters. The statistical analysis of enzyme activity was performed using one-way analysis of variance (ANOVA) and the post hoc Tukey HSD test, p<0.05 was considered statistically significant.

An arbitrary effect size of 0.25 (=0.05) was used to assess the statistical power of the sample size. The power was 0.998 with the sample size that was provided. The software G*Power 3.1 was used to calculate power.

Standard calibration lines subjected to linear regression analysis produced a range of Regression coefficient (R) values between 0.9986 and 0.9999. The lowest recovery value of the methods was determined as 85%. Chromatograms of favipiravir, pioglitazone, and citalopram concentrations in plasma are presented in Figure 1. The retention times for the favipiravir, pioglitazone and citalopram were 5.5, 7.5, and 8.5 min, respectively.

There was no significant difference in liver aldehyde oxidase (AO) enzyme activities between the groups in which pioglitazone and favipiravir were administered alone (p>0.05). However, when favipiravir and pioglitazone were administered together, AO enzyme activity was found to be statistically significantly decreased (p<0.008, Figure 2A). The xanthine oxidase (XO) enzyme activity was not significant difference between the favipiravir group and the favipiravir+pioglitazone group (p>0.05), but XO in these groups wassignificantly lower than the pioglitazone group (p<0.008, Figure 2B).

There was no significant difference in liver AO activity in the citalopram group and the favipiravir group (p>0.05), but the AO activity of the citalopram group was found to besignificantly higher than the favipiravir+citalopram group (p<0.008, Fig.2C). Liver XO enzyme activity was found to be significantly lower in the groups in which favipiravir was administered alone or in combination with citalopram compared to the group in which citalopram was administered alone (p<0.008, Figure 2D). There was no significant difference in XO activities in the groups in which favipiravir was administered alone and together with citalopram (p>0.05, Figure 2D).

The plasma concentration-time curve following oral administration of pioglitazone alone and in combination with favipiravir is presented in Figure 3A and Table 1. It was observed that administration of favipiravir did not affect the T_max, AUC, and Cl of pioglitazone. No statistically significant difference was observed in pharmacokinetic parameters between pioglitazone and favipiravir+pioglitazone groups (p>0.05).

The plasma concentration-time curve following oral administration of favipiravir alone and in combination with pioglitazone is presented in Figure 3 B and Table 1. When compared with the favipiravir group, the elimination t_1/2, AUC, AUMC, MRT, Vd (p<0.008), and Cl (p<0.004).values were found to be statistically significantly increased in the favipiravir+pioglitazone group.

Figure 3

Table 1

Parameters	Favipiravir	Favipiravir+Pioglitazone (Favipiravir)	Pioglitazone	Favipiravir+Pioglitazone (Pioglitazone)
t1/2(h)	16.17±0.18	17.14±0.54a	7.46±0.18	7.62±0.50
Tmax (h)	0.50	0.50	2.00±0.01	2.00±0.02
Cmax (μg/mL)	921.75±0.43	936.0±20.03	0.26±0.01	0.27±0.03
AUC 0-t (μg/mL*h)	5146.31±1.84	5589.88±36.74a	164.76±4.5	160.75±5.13
AUMC (h2*µg/mL)	423483.5±68.9	519976.26±384.59a	387.77±10.39	385.70±9.41
MRT (h)	23.47±0.25	24.97±0.65a	11.51±1.16	11.65±0.47
Vd (L/kg)	0.039±0.0009	0.0970±0.0110a	0.26±0.05	0.27±0.01
Cl (mL/h/kg)	0.0017±0.0001	0.0039±0.0003a	0.02	0.02

C_max: maximum plasma concentration reached, T_max: time to reach C_max, t_1/2=:elimination half-life, AUC: Area under the plasma concentration-time curve, AUMC: Area under the initial plasma concentration-time curve, MRT: Mean residence time, Vd: Volume of distribution at steady-state, Cl: clearance.

The plasma concentration-time curve following oral administration of citalopram alone and in combination with favipiravir is presented in Figure 4 A and Table 2. It was determined that the administration of favipiravir caused changes in some pharmacokinetic parameters of citalopram. While t_1/2, AUC, and MRT increased (p<0.008)significantly in the favipiravir+citalopram group compared to the citalopram group alone; it was determined that AUMC and Cl values decreased (p<0.008).

The plasma concentration-time curve following oral administration of favipiravir alone and in combination with citalopram is presented in Figure 4B and Table 2. t_1/2, C_max, AUMC, MRT, and Cl values were found to be statistically significantly increased (p<0.008)in the favipiravir+citalopram group compared to the group treated with favipiravir alone. AUC and Vd parameters were found to be significantly decreased (p<0.008).

Figure 4

Table 2

Parameters	Favipiravir	Favipiravir+Citalopram (Favipiravir)	Citalopram	Favipiravir+Citalopram (Citalopram)
t1/2(h)	16.17±0.18	17.03±0.34a	12.35±0.48	16.28±0.21b
Tmax (h)	0.50	0.50	1.00	1.00±0.01
Cmax (μg/mL)	921.75±0.43	932.0±0.90a	0.28±0.01	0.28±0.05
AUC 0-t (μg/mL*h)	5146.31±1.84	4791.88±230.53a	163.51±9.51	181.00±22.68b
AUMC (h2*µg/mL)	423483.5±68.9	426618.10±146.16a	821.58±12.09	155.78±2.19b
MRT (h)	23.47±0.25	24.59±0.18a	17.96±0.49	23.95±0.80b
Vd (L/kg)	0.039±0.0009	0.011±0.0061a	0.32	0.30±0.04
Cl (mL/h/kg)	0.0017±0.0001	0.0047±0.0011a	0.018	0.012b

C_max= maximum plasma concentration reached, T_max= time to reach C_max, t_1/2= elimination half-life, AUC= Area under the plasma concentration-time curve, AUMC= Area under the initial plasma concentration-time curve, MRT= Mean residence time, Vd= Volume of distribution at steady-state, Cl= clearance.

Orally administered favipiravir is metabolized in the liver mainly by AO and partially XO enzymes.7, 8 The AO enzyme metabolizes favipiravir and is also inhibited by favipiravir. 7 Therefore, drug-drug interactions become inevitable when favipiravir is co-administered with AO substrates or modulators. Citalopram is a drug used to treat depression and anxiety. In addition to cytochrome P450 enzymes, the AO enzyme also takes part in the metabolism of Citalopram. 11 It has been reported that pioglitazone, which is used in the treatment of type-2 diabetes, is an AO enzyme inhibitör.12, 14 Drug interactions are not known when favipiravir is co-administered with citalopram and pioglitazone. Therefore, in the current study, the possible drug interaction between favipiravir when used together with citalopram and pioglitazone was investigated.

Pioglitazone is mainly metabolized by CYP2C8, CYP3A4, and CYP2C9 enzymes. 19 Pioglitazone is an inhibitor of CYP2C8 and CYP3A4 in in vitro studies. However, it has not been reported to inhibit or induce CYP enzymes in in vivo studies. 19 In an in vitro study, it was found that pioglitazone inhibited the AO enzyme. 14 Therefore, the potential for drug interactions is increased when pioglitazone is co-administered with favipiravir.

There was a significant decrease in liver AO enzyme activities in the favipiravir + pioglitazone group compared to the groups treated with favipiravir and pioglitazone alone. However, AO enzyme activities were found to be similar in groups treated with favipiravir and pioglitazone alone. Our study results show that pioglitazone, like favipiravir, inhibits AO and inhibition is higher when both are used together(Fig.2A). 14 Possible interaction of pioglitazone with other drugs has been studied, but significant pharmacokinetic interactions have not been identified.13, 20 In the current study, we found that t_1/2, AUC, AUMC, MRT, Vd and Cl pharmacokinetic values of favipiravir were significantly increased when used with pioglitazone (Table 1). This difference in favipiravir pharmacokinetics is likely due to AO enzyme inhibition by pioglitazone (Figure 2 A).14 As a result of inhibition of the AO enzyme by pioglitazone, we found that the biological half-life and residence time of favipiravir were prolonged, and its plasma concentration increased.

In addition, we found that there was no significant difference in XO enzyme activities between the favipiravir+pioglitazone group and the favipiravir group, but these two groups had lower XO enzyme activities than the pioglitazone group (Figure 2B). Based on these results, it can be saidthat favipiravir partially occupies the enzyme as it is an XO substrate, as described in previous studies, whereas pioglitazone does not affect XO. 7, 19, 21

Clopidogrel inhibits pioglitazone metabolism and gemfibrozil increases the plasma concentration of pioglitazone. Itraconazole does not have a significant effect on the pharmacokinetics of pioglitazone. 22, 23 Clopidogrel and gemfibrozil increase pioglitazone plasma concentration, possibly by inhibiting CYP2C8. 22, 23 Similarly, in another study, it was reported that caffeine increased the C_max, T_max, AUC, and t_1/2 values of pioglitazone.24 In the current study, it was found that favipiravir did not affect pioglitazone pharmacokinetics. This is because pioglitazone is metabolized by cytochrome p450 enzymes and probably because favipiravir has no inhibition or induction effect on these enzymes. 7, 19

Citalopram is metabolized by cytochrome P450 enzymes10 and partially by AO enzyme. 11 Therefore, drug interactions may occur between favipiravir, which is both metabolized by AO and inhibits this enzyme, and citalopram. It has been reported that fluconazole, a CYP450 enzyme inhibitor, when used together with citalopram, changes the pharmacokinetics of citalopram. 25 In another study, cannabidiol was found to inhibit the CYP450-mediated metabolism of citalopram. 26

There has been an AO enzyme-related interaction between citalopram and favipiravir (Figure 2C).Since favipiravir is highly metabolized by AO, the simultaneous administration of citalopram, which is metabolized by AO, results in decreased metabolism by partially occupying the substrate-binding sites of the enzyme, reducing its association the enzyme.7, 11 Thus, the plasma concentration of favipiravir increased, and the time to stay in the body and the time to be eliminated from the body were prolonged (Table 2). In addition, as a result of inhibition of the AO enzyme by favipiravir, plasma residence time and the time taken for elimination from the body were prolonged for citalopram, which is partially metabolized by AO. The pharmacokinetic results and the data obtained from the liver AO enzyme activity support the study stating that although citalopram is mainly metabolized by CYP450 enzymes, the AO enzyme also has an important place in its metabolism. 11

Favipiravir may interact with pioglitazone and citalopram at the AO and XO enzyme level. As a result, the pharmacokinetics of these drugs may change. Therefore, concomitant use of favipiravir with pioglitazone and citalopram may increase the effects of these drugs and the risk of dose-related side effects.If the results obtained from this study are supported by human studies, it is thought that the concomitant use of these drugs in the clinic will be a guide in preventing the occurrence of adverse reactions.

Idea/Concept: Zeliha KESKİN ALKAÇ, Dilan AŞKIN ÖZEK, Songül ÜNÜVAR; Design:Zeliha KESKİN ALKAÇ, Dilan AŞKIN ÖZEK, Neşe BAŞAK TÜRKMEN; Data Collection and/or Processing: Zeliha KESKİN ALKAÇ, Dilan AŞKIN ÖZEK, Hande YÜCE, Fatih Ahmet KORKAK, Neşe BAŞAK TÜRKMEN, Sümeyye ASLAN; Analysis and/or Interpretation: Zeliha KESKİN ALKAÇ, Hande YÜCE, Fatih Ahmet KORKAK, Neşe BAŞAK TÜRKMEN; Literature Review: Zeliha KESKİN ALKAÇ; Writing the Article: Zeliha KESKİN ALKAÇ; Critical Review: Songül ÜNÜVAR, Dilan AŞKIN ÖZEK.