Invest Clin 65(1): 83 - 98, 2024 https://doi.org/10.54817/IC.v65n1a08

Corresponding Author: Halis Suleyman, Department of Pharmacology, Institute of Health Sciences, Erzincan Bi-

nali Yildirim University, Erzincan, Turkey, Postcode: 24100; Phone: +90 530 9211909; Fax: +90 446 2261819.

E-mail: halis.suleyman@gmail.com; hsuleyman@erzincan.edu.tr

Pulmonary toxicity associated with

high-dose favipiravir and treatment

options: biochemical and histopathological

evaluation.

Bekir Elma 1, Bahadir Suleyman 2, Renad Mammadov 2, Bulent Yavuzer 3, Edhem Unver 4,

Durdu Altuner 2, Taha A. Coban 5, Behzad Mokhtare 6 and Halis Suleyman 3

1 Department of Thoracic Surgery, Faculty of Medicine, Division of Surgical Medical

Sciences, Gaziantep University, 27310, Gaziantep, Turkey.

2 Department of Pharmacology, Faculty of Medicine, Erzincan Binali Yildirim University,

24100, Erzincan, Turkey.

3 Department of Pharmacology, Institute of Health Sciences, Erzincan Binali Yıldırım

University, 24100, Erzincan, Turkey.

4 Department of Chest Diseases, Faculty of Medicine, Erzincan Binali Yıldırım University,

24100, Erzincan, Turkey.

5 Department of Medical Biochemistry, Faculty of Medicine, Erzincan Binali Yildirim

University, 24100, Erzincan, Turkey.

6 Department of Pathology, Faculty of Veterinary Medicine, Ataturk University, 25240,

Erzurum, Turkey.

Keywords: Favipiravir; lacidipine; thiamine pyrophosphate; adenosine triphosphate;

oxidative stress.

Abstract. Favipiravir is a broad-spectrum antiviral drug that is a viral RNA-

dependent RNA polymerase inhibitor. Favipiravir is used in high doses to treat

COVID-19 but has a side effect on humans at high doses. The side effects of

favipiravir have been associated with oxidative stress in the literature. In this

trial, we investigated the biochemical and histopathological effects of lacidip-

ine, thiamine pyrophosphate (TTP), and adenosine triphosphate (ATP), drugs

with antioxidant properties, on the lung toxicity caused by high-dose favipiravir

in rats. The rats were classified into five groups: healthy (HG), favipiravir alone

(Fav), lacidipine+favipiravir (LFav), TPP+favipiravir (TFav), and ATP+favipiravir

(AFav). Favipiravir (800 mg/kg) was administered twice daily for seven days. Laci-

dipine (4 mg/kg), TPP (20 mg/kg), and ATP (25 mg/kg) were administered once

daily for seven days. Oxidant (malondialdehyde), non-enzymatic (total glutathi-

one), and enzymatic (superoxide dismutase and catalase) antioxidant levels were

measured in the excised lung tissues. Furthermore, the tissues were histopatho-

logically examined. The systemic administration of high doses of favipiravir in-

creased oxidant levels and decreased antioxidant levels in the lung tissue of rats.

84 Elma et al.

Investigación Clínica 65(1): 2024

Toxicidad pulmonar asociada a altas dosis de favipiravir y

opciones de tratamiento: Evaluación bioquímica e histopatológica.

Invest Clin 2024; 65 (1): 83 – 98

Palabras clave: Favipiravir; lacidipino; pirofosfato de tiamina; trifosfato de adenosina;

estrés oxidativo.

Resumen. El Favipiravir es un fármaco antiviral de amplio espectro que es un

inhibidor de la ARN polimerasa viral dependiente de ARN. El Favipiravir se usa en do-

sis altas para tratar el COVID-19, pero tiene efectos secundarios en humanos a estas

dosis. Los efectos secundarios del favipiravir se han asociado con el estrés oxidativo

en la literatura. En este trabajo experimental, investigamos los efectos bioquímicos

e histopatológicos de lacidipina, pirofosfato de tiamina (TTP) y trifosfato de adeno-

sina (ATP), fármacos con propiedades antioxidantes, sobre la toxicidad pulmonar

causada por altas dosis de favipiravir en ratas. Las ratas se clasificaron en cinco gru-

pos: sanas (HG), favipiravir solo (Fav), lacidipina+favipiravir (LFav), TPP+favipiravir

(TFav) y ATP+favipiravir (AFav). Se administró favipiravir (800 mg/kg) dos veces al

día durante siete días. Se administraron lacidipina (4 mg/kg), TPP (20 mg/kg) y

ATP (25 mg/kg) una vez al día durante siete días. Se midieron los niveles de antioxi-

dantes oxidantes (malondialdehído), no enzimáticos (glutatión total) y enzimáticos

(superóxido dismutasa y catalasa) en los tejidos pulmonares disecados. Además, los

tejidos fueron examinados histopatológicamente. La administración sistémica de

altas dosis de favipiravir aumentó los niveles de oxidantes y disminuyó los niveles

de antioxidantes en el tejido pulmonar de ratas. Paralelamente, el examen histopa-

tológico del tejido pulmonar reveló la presencia de graves infiltraciones de células

mononucleares en las zonas intersticiales y una pronunciada hiperplasia linfoide.

Lacidipina mostró una eficacia superior para mitigar el estrés oxidativo y prevenir

la disminución de antioxidantes inducida por favipiravir en comparación con TPP y

ATP. Histopatológicamente, la administración de lacidipina redujo significativamen-

te el daño oxidativo pulmonar. La TTP redujo moderadamente la lesión pulmonar

grave asociada al favipiravir. Sin embargo, el ATP fue ineficaz contra la lesión pul-

monar asociada al favipiravir. Lacidipina ofrece más beneficios terapéuticos que el

TPP en el tratamiento de la lesión pulmonar oxidativa causada por altas dosis de

favipiravir.

Received: 16-07-2023 Accepted: 28-10-2023

In parallel, the histopathological examination of the lung tissue revealed

the presence of severe mononuclear cell infiltrations in interstitial areas and

pronounced lymphoid hyperplasia. Lacidipine exhibited superior efficacy in mit-

igating oxidative stress and preventing the decline of antioxidants induced by

favipiravir compared with TPP and ATP. Histopathologically, the lacidipine admin-

istration significantly reduced lung oxidative damage. TTP moderately reduced

severe favipiravir-associated lung injury. However, ATP was ineffective against fa-

vipiravir-associated lung injury. Lacidipine offers more therapeutic benefits than

TPP in treating oxidative lung injury caused by high doses of favipiravir.

Pulmonary toxicity associated with high-dose favipiravir and treatment options 85

Vol. 65(1): 83 - 98, 2024

INTRODUCTION

Favipiravir is a nucleoside-derived pro-

drug with a wide range of antiviral activity.

It acts by inhibiting the viral RNA-dependent

RNA polymerase (RdRp) 1 and has been used

to treat viral infections, such as Ebola and

severe acute respiratory syndrome (SARS-

CoV-2) 2. Furthermore, favipiravir is approved

to be used as an antiviral medication in Ja-

pan to treat influenza virus infections 3. The

effectiveness of favipiravir against influenza

has been verified via cell cultures, animal

studies, and clinical trials 4. Additionally, fa-

vipiravir has been undergoing clinical trials

as an investigational drug owing to its poten-

tial application in treating the novel corona-

virus disease 2019 (COVID-19) 5. The results

of preclinical and clinical investigations in-

dicate that favipiravir shows promise as a

potential treatment option for severe infec-

tions caused by human rhinovirus, respira-

tory syncytial virus, metapneumovirus, para-

influenza viruses, and hantavirus pulmonary

syndrome 4. In addition, favipiravir has been

reported to be a promising and effective an-

tiviral medication for treating patients with

COVID-19 3. For COVID-19 treatment, a

loading dose of 2400–3000 mg (given in two

doses) every 12 h followed by a maintenance

dose of 1200–1800 mg every 12 h was rec-

ommended 6,7. Numerous studies have evalu-

ated favipiravir’s effectiveness and potential

side effects in treating patients with COV-

ID-19 8. High doses of favipiravir have been

associated with severe side effects in humans

9. Additionally, signs of toxicity have been

observed in animals administered with high

doses of favipiravir 10. Although the lethal

dose of favipiravir in animals is >2000 mg/

kg, it is administered to patients at higher

doses, such as 6000 mg/day on the first day

and 2400 mg/day on the second and subse-

quent days 11. Furthermore, the use of favipi-

ravir has been linked to toxic side effects in

humans, including diarrhea, nephrotoxicity,

elevated serum uric acid and transaminase

levels, and reduced white blood cell and neu-

trophil counts alongside symptoms such as

nausea, vomiting, abdominal pain, skin rash,

itching, delirium, hallucinations, convul-

sions, and potential teratogenicity 12-14. A re-

cent experimental study revealed that favipi-

ravir administration increased the levels of

malondialdehyde (MDA), a toxic byproduct

of lipid peroxidation (LPO), decreased the

levels of the non-enzymatic endogenous an-

tioxidant glutathione (GSH), and inhibited

activities of enzymatic antioxidants such as

superoxide dismutase (SOD), and catalase

(CAT), in liver tissue 15. Considering the

documented side effects of favipiravir, its

safety profile remains a concern based on a

pooled analysis of extensive studies 9. To the

best of our knowledge, no literature studies

have specifically investigated the impact of

high-dose favipiravir on lung function.

Herein, we investigated the therapeutic

effect of lacidipine, a drug derived from di-

hydropyridine and classified as an L-type cal-

cium (Ca2+) channel blocker, in mitigating

the potential pulmonary toxicity caused by

high-dose favipiravir 16. Lacidipine has been

primarily indicated for hypertension treat-

ment 17. Moreover, lacidipine exhibits anti-

oxidant activity by inhibiting the increase in

MDA levels and decrease in enzymatic and

non-enzymatic antioxidant levels in organs

and tissues 18. In addition, lacidipine re-

portedly inhibits acute and chronic inflam-

mation phases 19. Thiamine pyrophosphate

(TPP) is the active metabolite of thiamine,

and we investigated the therapeutic effect

of TPP against potential lung toxicity due to

high-dose favipiravir 20. Previous studies re-

port that TPP exerts a protective effect by

inhibiting elevated oxidant and proinflam-

matory parameters 21,22.

Moreover, we examined the therapeu-

tic effect of adenosine triphosphate (ATP)

against potential lung toxicity associated

with high-dose favipiravir administration.

ATP is a nucleoside triphosphate comprising

adenine, ribose sugar, and three phosphate

groups 23. ATP also synthesizes reactive

oxygen species (ROS)-scavenging antioxi-

86 Elma et al.

Investigación Clínica 65(1): 2024

dants24. In addition, ATP is an energy source

for synthesizing low molecular weight anti-

oxidants 25. This study aimed to biochemical-

ly investigate and histopathologically evalu-

ate the effects of lacidipine, TPP, and ATP on

the possible oxidative lung damage caused

by favipiravir in rats.

MATERIALS AND METHODS

Animals

In total, 36 albino Wistar-type male rats

weighing 285–297 g were obtained from Er-

zincan Binali Yıldırım University Experimen-

tal Animal Research and Application Center.

The experimental rats were subjected to

standard environmental conditions, main-

tained at an ambient temperature of 22°C

and a 12/12 h light–dark cycle. The rats had

ad libitum access to animal feed and tap wa-

ter. The study was approved by the local Ani-

mal Experiments Ethics Committee (Meet-

ing Date: 25.08.2022; Meeting Number:

2022/08; Decision Number: 37).

Chemicals

Thiopental sodium was obtained from

IE Ulagay (Turkey), favipiravir was obtained

from the Ministry of Health Training and

Research Hospital (Turkey), lacidipine was

obtained from GlaxoSmithKline (Turkey),

TPP was obtained from Biofarma (Russia),

and ATP was obtained from Zdorove Narodu

(Ukraine).

Experimental groups

The rats were classified into five

groups: healthy (HG), favipiravir alone (Fav),

lacidipine+favipiravir (LFav), TPP+favipiravir

(TFav), and ATP+favipiravir (AFav).

Experimental procedure

Lacidipine (4 mg/kg orally), TPP (20

mg/kg IP), and ATP (25 mg/kg IP) were ad-

ministered to the LFav (n = 6), TFav (n =

6), and AFav (n = 6) groups, respectively,

to initiate the experiment. The HG (n = 6)

and Fav (n = 6) groups were administered

with distilled water. Following the 1-h inter-

val following the administration of drugs and

distilled water, favipiravir was orally adminis-

tered at a dosage of 800 mg/kg twice daily

for seven days to all the animal groups ex-

cept the HG group. Favipiravir is known to

cause oxidative and inflammatory damage at

high doses 26. Lacidipine, TPP, and ATP were

administered once daily for seven days. TTP

and ATP have been investigated before in

these doses and found to be effective against

oxidative stress 27,28. Upon completion of this

timeframe, the animals were euthanized via

a high dose of thiopental sodium anesthesia

(50 mg/kg), following which their lung tis-

sues were extracted. MDA, tGSH, SOD, and

CAT levels were measured in the excised

lung tissues. Furthermore, the tissues were

histopathologically examined. The biochem-

ical and histopathological findings obtained

from all the animal groups were compared

and assessed for intergroup differences.

Biochemical analyzes

Preparation of samples

At this stage, 0.2 g of each removed tis-

sue was weighed for biochemical examina-

tion. Tissue samples were washed with cold

(+4°C) 0.15 M potassium chloride (KCl).

Tissue samples were homogenized in liquid

nitrogen. They were then passed into an ice-

cold phosphate buffer solution (50 mM, pH

7.4). The tissue homogenates were centri-

fuged at 5000 rpm for 20 min at +4°C, and

the supernatants were extracted to analyze

MDA, tGSH, SOD, and CAT.

Determination of MDA, GSH, SOD, CAT,

and protein

The levels of MDA, GSH, and SOD in

the supernatants derived from the lung tis-

sue samples from experimental animals were

measured using commercially available en-

zyme-linked immunosorbent assay (ELISA)

kits according to the manufacturer’s in-

structions (MDA catalogue no: 10009055;

tGSH catalogue no: 703002; SOD catalogue

no: 706002, Cayman Chemical Company).

Pulmonary toxicity associated with high-dose favipiravir and treatment options 87

Vol. 65(1): 83 - 98, 2024

CAT levels were determined according to

the method proposed by Goth 29. Protein

content was determined spectrophotometri-

cally at 595 nm according to the Bradford

method 30.

Histopathological examination

The rats underwent necropsy for his-

topathological assessment, and their lung

tissue samples were subsequently fixed in a

10% formalin solution. The tissues were sub-

jected to a series of alcohol–xylene solutions

and then embedded in paraffin blocks, from

which 5-µm thick sections were obtained for

histopathological evaluation. Then, these

sections were stained using hematoxylin–

eosin stain. The lung tissues were examined

using a light microscope (Olympus BX51,

Japan), and photographs were captured us-

ing a digital camera (Olympus DP 71) by a

pathologist blinded to the treatment proto-

col. Histopathological damage in each tissue

section was graded on a scale of 0–3 (0 =

normal, 1 = mild, 2 = moderate, and 3 = se-

vere) and assessed for mononuclear cell in-

filtrations in interstitial areas and lymphoid

hyperplasia.

Statistical analysis

All statistical analyses for the biochemi-

cal findings of the experiment were conduct-

ed using IBM SPSS® Statistics for Windows,

version 22.0 (IBM Corp, Armonk, NY, USA,

released in 2013). A significance level of p

<0.05 was considered statistically signifi-

cant. The biochemical results are expressed

as mean ± standard error ( ± SEM). The

normality of distribution for continuous

variables in the biochemical test results was

assessed using the Shapiro–Wilk test. The

significance level of the difference between

the groups was determined using a one-way

analysis of variance, as the distribution was

normal. The Levene’s test was performed to

determine whether the homogeneity of vari-

ances was ensured. Following the assump-

tion of homogeneity of variances, either the

Tukey Honest Significant Differences test or

the Games–Howell test was employed as a

post hoc test. The histopathological findings

were analyzed via the IBM SPSS® Statistics

program for Windows®, version 20.0 (IBM

Corp, Armonk, NY, USA, released in 2011). A

nonparametric Kruskal–Wallis test was per-

formed to assess between-group differences.

Subsequently, the group responsible for the

observed differences was identified using the

Mann–Whitney U test at a significance level

of p <0.05.

RESULTS

Biochemical results

MDA analysis results of lung tissue

The lung tissue of the high-dose favipi-

ravir–treated group exhibited higher MDA

levels than those in the lung tissue of the

HG, which was statistically significant (Table

1). Lacidipine, TPP, and ATP significantly

suppressed the increase in MDA levels in-

duced by high-dose favipiravir administra-

tion in lung tissue. However, ATP prevented

the increase of MDA in lung tissue weaker

than lacidipine and TPP. There was no signif-

icant difference in the MDA levels between

the HG, lacidipine, and TPP groups.

tGSH analysis results of lung tissue

The lung tissue of the high-dose favipi-

ravir–treated group exhibited lower tGSH

levels than the lung tissue of the HG, and

the difference was statistically significant

(Table 1). Lacidipine and TTP significantly

suppressed the decrease in tGSH levels in-

duced by high-dose favipiravir administra-

tion in lung tissue. However, ATP adminis-

tration did not exhibit a significant effect

on preventing a decrease in tGSH levels. A

statistically significant difference was de-

tected in the tGSH levels between the lung

tissue of the ATP-treated group and the HG.

There was no significant difference in the

tGSH levels between the HG, lacidipine, and

TPP groups.

88 Elma et al.

Investigación Clínica 65(1): 2024

SOD analysis results of lung tissue

The lung tissue of the high-dose favipi-

ravir–treated group exhibited lower SOD

activity compared with the lung tissue of

the HG, and the difference was statistically

significant (Table 1). Lacidipine and TPP

significantly alleviated the decrease in SOD

activity induced by high-dose favipiravir ad-

ministration in lung tissue; however, ATP

did not exhibit a similar effect. A statisti-

cally significant difference was found in the

SOD activities between the lung tissue of

the TPP and ATP groups and the HG. There

was no significant difference observed in

SOD activity between the HG and the laci-

dipine group.

Table 1

Effect of lacidipine, TPP, and ATP on oxidant and antioxidant levels in lung tissue

of rats administered with high doses of favipiravir.

Mean ± Standard error

MDA tGSH SOD CAT

Groups (nmol/mg protein) (nmol/mg protein) (U/mg protein) (U/mg protein)

HG 1.65±0.07 6.63±0.08 9.55±0.05 8.51±0.06

Fav 5.80±0.06 2.56±0.05 3.56±0.06 3.53±0.09

LFav 1.82±0.03 6.31±0.15 9.19±0.15 8.14±0.12

TFav 2.72±0.34 5.34±0.34 6.49±0.34 6.18±0.36

AFav 3.84±0.23 2.79±0.19 3.84±0.11 3.66±0.06

Comparison of p-values

Groups MDA tGSH SOD CAT

HG vs Fav <0.001 <0.001 <0.001 <0.001

HG vs LFav 0.215 0.386 0.302 0.116

HG vs TFav 0.118 0.056 0.001 0.006

HG vs AFav 0.001 <0.001 <0.001 <0.001

Fav vs LFav <0.001 <0.001 <0.001 <0.001

Fav vs TFav 0.001 0.002 0.001 0.003

Fav vs AFav 0.002 0.784 0.235 0.714

LFav vs TFav 0.201 0.163 0.001 0.011

LFav vs AFav 0.002 <0.001 <0.001 <0.001

TFav vs AFav 0.131 0.001 0.002 0.004

Abbreviations: TPP: thiamine pyrophosphate; ATP: adenosine triphosphate; HG: healthy group; Fav: alone favipiravir ad-

ministered group; LFav: lacidipine + favipiravir group; TFav: TPP + favipiravir group; AFav: ATP + favipiravir group; MDA:

malondialdehyde; tGSH: total glutathione; SOD: superoxide dismutase; CAT: catalase.

Footnotes: The Games-Howell test was applied as a post-hoc test after one-way ANOVA for all statistical evaluations.

Pulmonary toxicity associated with high-dose favipiravir and treatment options 89

Vol. 65(1): 83 - 98, 2024

CAT analysis results of lung tissue

CAT activity was lower in the lung tis-

sue of the high-dose favipiravir–treated group

than in the lung tissue of the HG, and the dif-

ference was statistically significant (Table 1).

Lacidipine and TPP significantly suppressed

the decrease in CAT activity induced by high-

dose favipiravir administration in lung tissue;

however, ATP did not suppress this decrease.

A statistically significant difference was de-

tected in the CAT activities between the lung

tissue of the TPP and ATP groups and those

of the HG. No significant difference was ob-

served in CAT activity between the HG and

the lacidipine group.

Histopathological results

Histopathologically significant differ-

ences were detected between the HG, Fav,

LFav, TFav, and AFav groups (Table 2; p<

0.05). The lung tissue samples of the HG ex-

hibited a normal histologic appearance (Fig.

1). Nevertheless, the rats in the high-dose fa-

vipiravir group exhibited severe mononucle-

ar cell infiltrations in interstitial areas (Fig.

2A) and significant lymphoid hyperplasia in

the lung tissue (Fig. 2B). In contrast, mono-

nuclear cell infiltrations in interstitial areas

(Fig. 2C) and lymphoid hyperplasia (Fig. 2D)

were mild in the lacidipine group. In the TTP

group, mononuclear cell infiltrations in inter-

stitial areas (Fig. 3A) and lymphoid hyperpla-

sia (Fig. 3B) were moderate. However, mono-

nuclear cell infiltrations in interstitial areas

(Fig. 3C) and lymphoid hyperplasia (Fig. 3D)

were severe in the ATP group.

DISCUSSION

This study investigated the protective

effects of lacidipine, TPP, and ATP against

lung injury induced by high-dose favipira-

vir administration in rats. The investigation

involved biochemical and histopathological

analyses. Our biochemical experiments re-

vealed that the MDA levels increased in the

lung tissue of high-dose favipiravir–treated

animals, whereas the tGSH, SOD, and CAT

levels decreased significantly.

Our experimental results indicate that

high doses of favipiravir may lead to severe

oxidative damage. Herein, we measured MDA

levels as it is a toxic byproduct of LPO and a

significant indicator of oxidative stress 31. A re-

cent experimental study with results that align

with our biochemical findings reported that

favipiravir administration increased MDA levels

in liver tissue. Additionally, the drug decreased

endogenous antioxidant levels 15. Similarly, Bi-

lici et al. reported that rats treated with a high

dose of favipiravir exhibited increased oxidant

and decreased antioxidant levels 26.

Table 2

Effect of lacidipine, TPP, and ATP on histopathological scoring ndings

in lung tissue of rats administered with high doses of favipiravir.

Groups MNC infiltrations in interstitial areas Lymphoid hyperplasia

HG 0.00 ± 0.00a0.00 ± 0.00a

FAV 2.83 ± 0.40b2.83 ± 0.40b

LFav 0.16 ± 0.00a0.16 ± 0.00a

TFav 1.16 ± 0.40c1.16 ± 0.40c

AFav 2.66 ± 0.81b2.56 ± 0.80b

Abbreviations: TPP: thiamine pyrophosphate; ATP: adenosine triphosphate; MNC: mononuclear cell; HG: healthy group;

Fav: alone favipiravir administered group; LFav: lacidipine + favipiravir group; TFav: TPP+ favipiravir group; AFav: ATP+

favipiravir group.

Footnotes: The values given are mean ± standard deviation values. a, b, c: Groups marked with the same letter are statistically

similar, but there is a statistically signicant difference at the level of p < 0.05 among groups with different letters.

90 Elma et al.

Investigación Clínica 65(1): 2024

Fig. 1. Normal histological appearance of lung tissue belonging to the HG group (H&E x10).

Fig. 2. (A) Severe mononuclear cell infiltrations in interstitial areas (□) appearance in the lung tissue belon-

ging to the Fav group (H&E x20). (B) Severe lymphoid hyperplasia (arrowheads) appearance in the

lung tissue belonging to the Fav group (H&E x10). (C) Mild mononuclear cell infiltrations in inters-

titial areas (□) appearance in the lung tissue belonging to the LFav group (H&E x20). (D) Mild lym-

phoid hyperplasia (arrowheads) appearance in the lung tissue belonging to the LFav group (H&E x10).

Pulmonary toxicity associated with high-dose favipiravir and treatment options 91

Vol. 65(1): 83 - 98, 2024

Conversely, lacidipine, TPP, and ATP

administration significantly suppressed in-

creased MDA levels in the studied subjects.

In particular, it was observed that lacidipine

suppressed the increase in MDA levels to a

greater extent than TPP and ATP, bringing

them closer to the levels of the healthy con-

trol group. To the best of our knowledge,

there is no information in the literature

regarding the effect of lacidipine, TPP, and

ATP on lung injury induced by high-dose fa-

vipiravir. Previous studies have reported that

lacidipine significantly decreased MDA lev-

els and exerted a nephroprotective effect in

cyclosporine-induced nephrotoxicity 32. Pre-

vious research has also demonstrated that

lacidipine exhibits the highest potency as

an antioxidant among calcium channel an-

tagonists, effectively inhibiting membrane

LPO 33. In addition, TPP exerts an antioxi-

dant effect by significantly suppressing the

cyclophosphamide-induced increase in MDA

levels in female rats 21. Similarly, a previous

study reported that ATP protects kidney tis-

sue from bevacizumab-induced oxidative

damage by significantly inhibiting increased

MDA levels 34.

In addition, enzymatic and non-enzy-

matic antioxidant parameters were mea-

sured to evaluate the possible oxidative

damage of favipiravir in lung tissue. It is

well-known that exposure to ROS from vari-

ous sources activates a cascade of defense

mechanisms in organs and tissues 35, such

as endogenous antioxidants 36. In case of in-

sufficient antioxidant levels to counteract

Fig. 3. (A) Moderate mononuclear cell infiltrations in interstitial areas (□) appearance in the lung tissue belon-

ging to the TFav group (H&E x20). (B) Moderate lymphoid hyperplasia (arrowheads) appearance in the

lung tissue belonging to the TFav group (H&E x10). (C) Severe mononuclear cell infiltrations in inters-

titial areas (□) appearance in the lung tissue belonging to the AFav group (H&E x20). (D) Severe lym-

phoid hyperplasia (arrowheads) appearance in the lung tissue belonging to the AFav group (H&E x10).

92 Elma et al.

Investigación Clínica 65(1): 2024

the oxidant accumulation, oxidative stress

occurs, leading to tissue damage 37,38. As re-

vealed by our experimental results, tGSH,

SOD, and CAT levels decreased in lung tis-

sue. It is widely recognized that GSH ex-

ists in two primary forms: the thiol-reduced

form and the disulfide-oxidized form known

as GSSG 39. GSH has several functions, in-

cluding antioxidant defense, detoxification,

maintenance of thiol status, and modulation

of cell proliferation 40. The protective effect

of GSH is attributed to its ability to react

with ROS and effectively perform detoxifi-

cation 41. SODs are enzymatic antioxidants

that play a role in the protection of cells

against oxygen toxicity 42. Likewise, CAT is

a significant antioxidant enzyme in various

cells that facilitates the breakdown of H2O2

into H2O and O2 42. Numerous prior studies

have assessed the levels of oxidants and anti-

oxidants above-mentioned to investigate the

occurrence of lung damage 43.

Our study revealed that lacidipine and

TPP administration effectively mitigated

the decline in tGSH, SOD, and CAT levels.

However, ATP did not demonstrate the same

suppressive effect on these antioxidant

markers. Moreover, lacidipine effectively

reduced the decline in these antioxidant

levels, restoring them to levels comparable

with those observed in the HG. To the best

of our knowledge, there is no data in the

literature regarding the antioxidant effect

of lacidipine against oxidative lung injury

due to favipiravir. Several studies have re-

ported that lacidipine protects heart tissue

from oxidant damage 44. In another study,

lacidipine treatment reportedly attenuated

the decrease in tGSH, SOD, and CAT lev-

els 45. It was determined that another drug,

TPP, suppressed the decrease in tGSH lev-

els and provided similar values as those of

the healthy control group; however, there

was a significant difference in SOD and CAT

activities between the healthy control and

TPP group. To the best of our knowledge,

there was no data in the literature regard-

ing the antioxidant effect of TTP against

favipiravir-induced oxidative lung injury. In

a previous study, TPP reportedly increased

tGSH levels in oxidative optic nerve damage

to levels comparable to those in the healthy

group 46. Demiryilmaz et al. reported that

SOD and CAT activities increased in rats

treated with TPP following oxidative liver

damage 47. Reportedly, ATP was ineffective

in preventing the decline of enzymatic and

non-enzymatic antioxidant levels and exhib-

ited a noticeable difference compared to

the values observed in the healthy control

group. To the best of our knowledge, there

were no reports in the literature investigat-

ing the antioxidant effect of ATP against

favipiravir-induced oxidative lung injury.

While Ozer et al. reported that ATP ad-

ministration increased the enzymatic and

non-enzymatic antioxidant levels in oxida-

tive ovarian damage, our study did not yield

similar findings 48.

Our study revealed a correlation be-

tween the biochemical results obtained from

the lung tissues of the animals and histo-

pathological findings. Severe mononuclear

cell infiltrations and lymphoid hyperplasia

in interstitial areas were observed in the

lung tissue of the favipiravir group, wherein

the oxidant levels were increased, and an-

tioxidant levels were decreased. However,

histopathological damage was observed to

be alleviated in the lacidipine group, which

best antagonized the effect of favipiravir on

oxidant and antioxidant parameters. While

histopathologic damage was moderate in

the TPP group, which prevented oxidant in-

crease and antioxidant decrease at a mod-

erate level, severe histopathologic damage

was detected in the ATP group, which could

not significantly prevent the decrease of an-

tioxidants caused by favipiravir. This is con-

sistent with the literature reporting that

histopathologic damage in lung tissue is as-

sociated with oxidant/antioxidant levels 49,50.

Pneumonia is a widely recognized condition

characterized by inflammation of the lungs,

typically due to an infection 51. There is cur-

rently no information available in the litera-

Pulmonary toxicity associated with high-dose favipiravir and treatment options 93

Vol. 65(1): 83 - 98, 2024

ture suggesting that favipiravir induces in-

terstitial inflammation via oxidative stress in

the lungs. However, Tomoda Y et al. reported

the development of drug-induced intersti-

tial pneumonia in a patient receiving clopi-

dogrel 52. Jo T et al. reported that class III

antiarrhythmic drugs, epidermal growth fac-

tor receptor inhibitors, and numerous other

drugs can cause interstitial pneumonia 53.

Reportedly, the broncho-alveolar lavage fluid

of a patient with drug-associated pneumo-

nia contained abundant lymphocytes, mac-

rophages, neutrophils, and eosinophils 52. A

study reported that ROS-induced oxidative

stress may be one of the underlying factors

in interstitial pneumonia development 54,

supporting our biochemical and histopatho-

logical findings.

In conclusion, the systemic adminis-

tration of high doses of favipiravir increased

oxidant levels and decreased antioxidant

levels in the lung tissue of rats. In parallel,

the histopathological examination of the

lung tissue revealed the presence of severe

mononuclear cell infiltrations in interstitial

areas and pronounced lymphoid hyperpla-

sia. To the best of our knowledge, this was

the first study focusing on the impact of

high-dose systemic administration of favipi-

ravir on lung tissue in terms of biochemical

alterations and histopathological changes.

Lacidipine exhibited superior efficacy in

mitigating oxidative stress and preventing

the decline of antioxidants induced by fa-

vipiravir compared with TPP and ATP. His-

topathologically, lacidipine administration

significantly reduced lung oxidative dam-

age. TTP moderately reduced severe favip-

iravir-associated lung injury. However, ATP

was ineffective against favipiravir-associat-

ed lung injury. Based on our findings, it can

be concluded that lacidipine offers more

therapeutic benefits than TPP in treating

oxidative lung injury caused by high doses

of favipiravir. We believe investigating pro-

inflammatory and inflammatory cytokines

can provide valuable insights into further

understanding this issue.

ACKNOWLEDGMENTS

The authors thank Enago – https://

www.enago.com.tr/ceviri/ for their manu-

script translation and editing assistance.

Conflict of interest

There are no conflicts of interest be-

tween the authors or between family mem-

bers of the scientific and medical com-

mittees. The authors do not have any

consultancy, expertise, working conditions,

shareholdings or similar situations that

could lead to potential conflicts of interest

in any company.

ORCID authors

• Bekir Elma(BE):

0000-0001-5083-8088

• Bahadir Suleyman (BS):

0000-0001-5795-3177

• Renad Mammadov (RM):

0000-0002-5785-1960

• Bulent Yavuzer (BY):

0000-0001-7576-0678

• Edhem Unver(EU):

0000-0002-0322-8102

• Durdu Altuner(DA):

0000-0002-5756-3459

• Taha A. Coban (TAC):

0000-0003-1711-5499

• Behzad Mokhtare(BM):

0000-0002-9075-7239

• Halis Suleyman(HS):

0000-0002-9239-4099

Authorships contribution statement

BE: conceptualization, methodology,

software, formal analysis, resources, data

curation, writing original draft preparation,

writing-review & editing, supervision, proj-

ect administration; BS: conceptualization,

94 Elma et al.

Investigación Clínica 65(1): 2024

methodology, validation, formal analysis,

investigation, resources, writing original

draft preparation, writing-review & editing;

RM: methodology, validation, formal analy-

sis, investigation, resources, writing-review

& editing; BY: methodology, software, for-

mal analysis, investigation, resources, data

curation, writing-review & editing, visual-

ization; EU: methodology, validation, inves-

tigation, resources, writing-review & edit-

ing; DA: methodology, validation, formal

analysis, writing original draft preparation,

writing-review & editing; TAC: conceptu-

alization, methodology, validation, formal

analysis, resources, writing original draft

preparation, writing-review & editing; BM:

conceptualization, methodology, validation,

formal analysis, resources, writing original

draft preparation, writing-review & edit-

ing; HS: conceptualization, methodology,

validation, formal analysis, investigation,

writing original draft preparation, writing-

review & editing, visualization, supervision,

project administration.

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