key: cord-0700550-8lj7ylcr authors: Gao, Xiuge; Jing, Xian; Wang, Junqi; Zheng, Yuling; Qiu, Yawei; Ji, Hui; Peng, Lin; Jiang, Shanxiang; Wu, Wenda; Guo, Dawei title: Safety considerations of chloroquine in the treatment of patients with diabetes and COVID-19 date: 2022-04-22 journal: Chem Biol Interact DOI: 10.1016/j.cbi.2022.109954 sha: 44615ed6384a50530d3c435386125ec8f6ddd01e doc_id: 700550 cord_uid: 8lj7ylcr Patients with underlying diseases and coronavirus disease 2019 (COVID-19) are at increased risk of death. Using the recommended anti-COVID-19 drug, chloroquine phosphate (CQ), to treat patients with severe cases and type 2 diabetes (T2D) could potentially cause harm. We aimed to understand the safety of CQ in patients with T2D by administrating the recommended dose (63 mg/kg twice daily for 7 days) and a high dose (126 mg/kg twice daily for 7 days) of CQ in T2D rats. We found that CQ increased the total mortality of the T2D rats from 27.3% to 72.7% in the recommended and high-dose groups during the whole period. CQ also induced hematotoxicity of T2D rats in the high-dose group; the hepatic enzymes in T2D rats were significantly elevated. CQ also changed the electrocardiograms, prolonged the QTc intervals, and produced urinary leukocytes and proteins in the T2D rats. Histopathological observations revealed that CQ caused severe damage to the rats’ heart, jejunum, liver, kidneys, spleen, and retinas. Furthermore, CQ significantly decreased the serum IL-1β and IL-6 levels. In conclusion, the CQ dosage and regimen used to treat COVID-19 induced adverse effects in diabetic rats, suggesting the need to reevaluate the effective dose of CQ in humans. area (m 2 ). The remaining 12 healthy rats were used as negative controls and an equal 142 volume of saline water was administered during the entire experiment. The CQ 143 solution and saline water were administrated intragastrically every 12 h for 7 144 consecutive days. Regular food and clean water were provided for all the rats during 145 the CQ administration period. The schematic diagram of the animal experiment is 146 shown in Fig. 1 . The blood was analyzed using an automatic blood-counter system (Mindray, 191 Shenzhen, China) to determine the hematological changes of diabetic rats induced by In addition, to understand the effect of CQ on the urine of rats, the urinary 211 examination was carried out using a urine analyzer (Mindray, Shenzhen, China The rats' organs (hearts, livers, spleen, lungs, kidneys, testis, and thymuses) were 230 weighed, and the organ indexes were calculated in triplicate. Herein, organ index = 231 organ weight (g) * 100 / body weight (g). In addition, all the obtained organs from 232 each group were used for histopathological examination. Briefly, the fixed tissues 233 were cut into square sections (5 mm × 5 mm) and dehydrated in a series of alcohol 234 solutions (50 % ~ 100%) for 30 min at each concentration, followed by xylene twice 235 for transparency. The tissue blocks were then embedded into paraffin for at least 2 h. 236 Next, these paraffin blocks were cut into thin slices of about 3 μm using a slicer 237 (Leica, Germany). The slices were subsequently transferred to glass slides for drying 238 and dewaxing. These prepared tissue slices were stained using hematoxylin and eosin 239 (H&E) and appropriate washing to remove the free dyes. The stained tissue slices (Table S1 ). These clinical symptoms 254 were also observed in the high-dose CQ-treated diabetic rats, that exhibited more 255 severe symptoms, such as slow reaction, emaciation, and standing dorsal hair (Table 256 S1 ). As the time increased after CQ administration, most diabetic rats grew weak and 257 experienced sudden death, while the diabetic rats without CQ administration 258 experienced weight loss only. Starting on the fifth day after CQ treatment, even using 259 the recommended dose, the diabetic rats began to die. At the end of the CQ 260 administration period, the survival rate of the CQ-treated diabetic rats decreased to 261 72.7% (approved dose of CQ, P < 0.05) and 27.3% (high dose of CQ, P < 0.0001) 262 ( Fig. 2A) . However, all the diabetic rats receiving saline survived throughout the 263 entire animal experiment. In addition, compared to the healthy control group rats, the 264 bodyweight of the diabetic rats all decreased significantly among the control group (P 265 J o u r n a l P r e -p r o o f < 0.0001), approved-CQ dose group (P < 0.0001), and high-dose CQ group (P < 266 0.0001) (Fig. 2B) . Moreover, the doses of CQ (63 mg/kg and 126 mg/kg) decreased 267 the diabetic rats' blood glucose levels on day 3 (P < 0.01) and day 7 (P < 0.0001) ( Fig. 268 2C), and these rats showed decreased food and water intake on day 7 (P<0.0001), 269 compared to the diabetic and healthy rats administered saline ( Fig. 2D and 2E ). administration. *** , P<0.001 and **** , P<0.0001 compared to healthy rats; ## , P<0.01 and #### , Feed consumption of rats was recorded in each day. **** , P<0.0001, compared to healthy and 279 diabetic rats on day 7. (E) Water consumption of rats was recorded every day. Data are shown as 280 mean ± SD, n=3-11. **** , P<0.0001, compared to healthy rats on day 7; #### , P<0.0001, compared 281 to diabetic rats on day 7. 282 As shown in Table 1 , compared with healthy rats and diabetic rats, high-dose CQ As shown in Table 2 , the recommended and high doses of CQ significantly 296 elevated the levels of serum alanine aminotransferase (ALT), aspartate 297 aminotransferase (AST), and the ratio of ALT/AST compared to the healthy rats and 298 diabetic rats (P < 0.05 and P < 0.01, respectively). Furthermore, the diabetic rats and 299 the CQ-treated diabetic rats (both doses) all had significantly increased levels of 300 lactate dehydrogenase (LDH) and creatine kinase (CK). The urea levels of T2D rats 301 were significantly higher than those of the healthy rats (P < 0.01); however, CQ 302 effectively decreased urea to normal levels. The amylase (AMY) levels were 303 significantly reduced in the diabetic rats and CQ-treated diabetic rats (P < 0.01). As shown in Table 3 , high-dose CQ significantly prolonged the QRS interval of 309 the T2D rats (P < 0.01). Of note, the diabetic rats and CQ-treated diabetic rats showed 310 a significant increase in QT intervals (P < 0.01) compared to healthy rats. Compared 311 to the diabetic rats, the recommended-dose and high-dose CQ groups had significantly 312 prolonged QT intervals (P < 0.01). Compared to the recommended dose of CQ, the high dose of CQ induced more severe QT prolongation in the diabetic rats (P < 0.01). In addition, compared to the healthy and diabetic rats, the recommended dose (63 315 mg/kg) of CQ treatment decreased the heart rate (HR) of the diabetic rats (P < 0.05). The relevant electrocardiographs of rats are presented in Fig. 3 and show CQ 317 markedly changed the peak profile, especially QT-interval prolongation. 318 Table 3 319 Electrocardiogram changes of T2D rats induced by CQ. Table 4 , compared to healthy rats in the control group, the diabetic 330 rats had high nitrite and glucose urine levels. In addition, the diabetic rat urine levels Next, the CQ-treated rat organ index changes are shown in Table 5 . They indicate the diabetic rats and CQ-treated diabetic rats had significantly increased 340 organ indexes for the livers, lungs, and testis (P < 0.05 and P < 0.01) compared to 341 those of the healthy rats. Similarly, 63 mg/kg CQ administration induced a significant 342 increase in the kidney index (P < 0.05) compared with the control group of rats. In 343 contrast, the thymus indices of the diabetic and CQ-treated diabetic rats significantly 344 decreased (P < 0.05 and P < 0.01, respectively) compared to the control group. However, there was no significant difference between the diabetic rats and CQ-treated 346 diabetic rats (P > 0.05). Moreover, during the CQ administration period, the diabetic 347 rat heart index did not significantly change (P > 0.05). Organ indexes of rats on day 7 post-administration of CQ. We further evaluated the histopathological changes of the CQ-treated rats for 350 seven days. The results showed that several organic rat tissues were damaged to 351 varying degrees. These organs and tissues include the heart, jejunum, liver, kidney, 352 retina, and spleen. In detail, compared with healthy and diabetic rats, the approved 353 dose of CQ induced vacuolar degeneration and lipid deposition of the heart (Fig. 4) . In contrast, a high dose of CQ caused more severe myocardial damage such as focal 355 necrosis, myocardial fiber swelling, and inflammatory cell infiltration (Fig. 4) . In 356 addition, the CQ-treated T2D rat jejunum were severely damaged in a dose-dependent 357 manner; the histopathologic structure changes included necrosis and shedding of 358 intestinal villus, disruption of intestinal crypt cells structure, and attenuation of the 359 serosal layer (Fig. 4) . However, other intestinal segments of the CQ-treated T2D rats 360 had no apparent lesions compared with healthy rats (supplemental Fig. S1 ). For the 361 liver, CQ aggravated the cytoplasm vacuolization and granular degeneration induced 362 by STZ pretreatment (Fig. 4) compared with healthy rats. Furthermore, the approved 363 dose of CQ induced T2D rat glomerular cell swelling, and the high dose of CQ caused 364 renal tubules necrosis, extreme vacuolization, and the loss of cellular integrity (Fig. 4) . However, we observed focal vacuolar degeneration of the renal tubular epithelial cells 366 in T2D rats with no CQ (Fig. 4) . For the eyes of T2D rats, the approved dose of CQ 367 triggered congestion of the retinal optic nerve layer vessels (Fig. 4) , and the high dose 368 of CQ induced hemorrhage in the inner granular layer of the retina (Fig. 4) . CQ 369 treatment caused diffuse vacuolization of macrophages in the medullary regions of the 370 T2D spleen. However, this phenomenon was not observed in the healthy rat spleen 371 (Fig. 4) . STZ-induced rat pancreas injury during T2D model development was 372 alleviated by CQ treatment in a dose-dependent manner, including reduced vacuolar 373 degeneration (Fig. 4) . However, other organs and tissues showed no apparent 374 pathologic injury. These organs were the lungs, caecum, colon, rectum, thymus, testis, 375 bladder, stomach, and local tissues of the eye (supplemental Fig. S1 ). 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