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Evaluation of Tacrolimus’ Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints

Wen-Wei Feng,Hsiu-Chao Chen,Gilbert Audira,Michael Edbert Suryanto,Ferry Saputra,Kevin Adi Kurnia,Ross D. Vasquez,Franelyne P. Casuga,Yu-Heng Lai,Chung-Der Hsiao,Chih-Hsin Hung

Simple SummaryDespite the clinical usefulness of tacrolimus in preventing organ transplant rejection, numerous studies have demonstrated its adverse effects in humans and animals. However, to date, no study has comprehensively addressed its toxicity toward zebrafish. Using various tests to assess the cardiovascular performance, various behaviors, and cognitive performance of the fish, we found that tacrolimus in relatively low concentrations altered the behaviors, social cohesion, and short-term memory of zebrafish in the larval and adult stages after acute and chronic exposures, respectively, with some alterations occurring in a dose-dependent manner. We hypothesize that tacrolimus may possess anxiotropic properties, which make it capable of modifying anxiety in the fish by affecting their nervous system. Nevertheless, considering the potential toxicity of tacrolimus, its usage, including in clinical applications, should be taken into account and monitored.AbstractTacrolimus (FK506) is a common immunosuppressant that is used in organ transplantation. However, despite its importance in medical applications, it is prone to adverse side effects. While some studies have demonstrated its toxicities to humans and various animal models, very few studies have addressed this issue in aquatic organisms, especially zebrafish. Here, we assessed the adverse effects of acute and chronic exposure to tacrolimus in relatively low doses in zebrafish in both larval and adult stages, respectively. Based on the results, although tacrolimus did not cause any cardiotoxicity and respiratory toxicity toward zebrafish larvae, it affected their locomotor activity performance in light–dark locomotion tests. Meanwhile, tacrolimus was also found to slightly affect the behavior performance, shoaling formation, circadian rhythm locomotor activity, and color preference of adult zebrafish in a dose-dependent manner. In addition, alterations in the cognitive performance of the fish were also displayed by the treated fish, indicated by a loss of short-term memory. To help elucidate the toxicity mechanism of tacrolimus, molecular docking was conducted to calculate the strength of the binding interaction between tacrolimus to human FKBP12. The results showed a relatively normal binding affinity, indicating that this interaction might only partly contribute to the observed alterations. Nevertheless, the current research could help clinicians and researchers to further understand the toxicology of tacrolimus, especially to zebrafish, thus highlighting the importance of considering the toxicity of tacrolimus prior to its usage.

Determination of tacrolimus in whole blood of pediatric liver transplant patients by UPLC–MS/MS: Informed its individualized dose

Saisai Ling,Ying Jin,Cuiyao He,Lisha Fu,Yuhua Deng,Xiaohui Qi,Zhenglei Wang,Yiying Wu

Tacrolimus is a potent macrolide immunosuppressant widely used in pediatric liver transplant patients. However, its narrow therapeutic window and significant inter-individual pharmacokinetic differences result in a poor correlation between blood concentrations and administered doses. To support its individualized dose, a sensitive, fast and robust ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to measure tacrolimus concentrations in whole blood of pediatric liver transplant patients. The method employed acetonitrile for protein precipitation, and sample separation was achieved using an Acquity UPLC CSH C 18 column (2.1 × 50 mm, 1.7 μm) with gradient elution. The mobile phase consisted of ammonium solution–water (0.5:1000, v /v) and methanol. The method demonstrated good linearity within a concentration range of 0.20–50.00 ng/mL. The intra- and inter-day precisions of tacrolimus in whole blood were less than 13.5 %, with the accuracy ranged from −7.2 % to 13.0 %. Selectivity, carryover, matrix effects, recovery, dilution reliability, and stability all met the standards set by relevant guidelines. The established UPLC–MS/MS method was successfully applied to measure the concentration of tacrolimus in the whole blood of pediatric liver transplant patients and support its individualized dose. Under the rapid UPLC–MS/MS method, 25 (65.8 %) patients required a dose increase, 3 (7.9 %) patients needed a dose reduction, and 10 (26.3 %) patients maintained their original dosage without adjustment.

Effects of tacrolimus on the TGF‑β1/SMAD signaling pathway in paraquat‑exposed rat alveolar type II epithelial cells

Yingli Ren,Xiangdong Jian,Zhongchen Zhang,Qiong Ning,Baotian Kan,Li Kong

Paraquat is a highly toxic pesticide, which often causes pulmonary interstitial fibrosis after poisoning, and there is no specific antidote. At present, limited studies have reported that tacrolimus, as an immunosuppressant, can inhibit pulmonary fibrosis, but the specific mechanism remains unknown. The aim of the present study was to demonstrate the effect of tacrolimus on the TGF‑β1 pathway associated with pulmonary fibrosis in paraquat exposed alveolar type II epithelial cells, and to identify the antipulmonary fibrosis mechanism of tacrolimus The rat alveolar epithelial type II RLE‑6TN cell line was exposed to paraquat and treated with or without tacrolimus for 24 h, or with a TGF‑β1 receptor type I/II inhibitor (LY2109761) for 1, 4, 8 or 16 h. MTT assays were used to detect the viability of rat alveolar type II epithelial cells under these different treatment conditions, while the concentrations of TGF‑β1, SMAD3, SMAD7 and connective tissue growth factor (CTGF) in the cell culture supernatant were determined using ELISAs. Additionally, reverse transcription‑quantitative PCR and immunofluorescence were used to analyze the mRNA and protein expression levels of TGF‑β1, SMAD3, CTGF and SMAD7. The results demonstrated that the inhibition of the proliferation of RLE‑6TN cells exposed to 200 nmol/l paraquat was 26.05±2.99%. The inhibition rate of 10 ng/ml tacrolimus on paraquat‑exposed alveolar type II epithelial cells was 18.40±3.49%. The inhibition rate caused by 5 µmol/l LY2109761 was 26.56±4.49%. The expression levels of TGF‑β1, SMAD3 and CTGF, as well as their concentrations in the culture supernatant, were significantly downregulated in the tacrolimus group compared with the paraquat group. However, both the concentration and expression levels of SMAD7 were significantly upregulated in the tacrolimus group compared with the paraquat group. In conclusion, tacrolimus can reduce the levels of TGF‑β1, SMAD3 and CTGF, increase the level of SMAD7 in TGF‑β1 signaling pathway and protect the development of pulmonary fibrosis in paraquat exposed alveolar epithelial cells.

Spherical carrier amplification strategy for electrochemical immunosensor based on polystyrene-gold nanorods @L-cysteine/MoS2 for determination of tacrolimus

Ze Zhang,Yingcong Zhang,Hongwei Yu,Shengzhong Rong,Hongmin Gao,Lingqiang Meng,Jianmin Dai,Hongzhi Pan,Dong Chang

A novel sensitive electrochemical immunosensor has been designed for determination of tacrolimus (Tac) based on spherical carrier amplification strategy. In this work, a spherical signal carrier was prepared by conjugating gold nanorods (AuNRs) functionalized L-cysteine ( [email protected] ) onto polystyrene nanoparticles (PS) to form a nanostructure, greatly increasing the amount of loaded capture antibodies. The PS was a stable spherical functional polymer with large specific surface area and was labeled with [email protected] via coupling reagent to increase active groups, biocompatibility and conductivity . The capture antibodies could be attached on the PS- [email protected] via linkage reagent glutaraldehyde (GA). Furthermore, single-layer molybdenum disulfide (MoS 2 ) fixed by chitosan were utilized to construct the base of this immunosensor, increasing the carrying capacity and stability of the electrode. After electrochemical reduction , the conductivity and electron transfer rate of MoS 2 were obviously improved, which offered a platform for this immunosensor and further amplified the signal. Under the optimized conditions, the proposed immunosensor revealed a linear Tac-concentration behavior from 1.0 to 30 ng mL −1 with a detection limit of 0.17 ng mL −1  (S/N = 3). The immunoassay was successfully applied to the quantification of Tac in serum samples with acceptable precision. The results indicated that a potentially analytical platform may be used to recognize the concentration of other drugs.

Physiologically-based pharmacokinetic modeling-guided rational combination of tacrolimus and voriconazole in patients with different CYP3A5 and CYP2C19 alleles

Fei Gong,Huihui Hu,Ying Ouyang,Zheng-Zheng Liao,Ying Kong,Jin-Fang Hu,Hua He,Ying Zhou

The drug-drug interactions (DDIs) between tacrolimus and voriconazole are highly variable among individuals. We aimed to develop a physiologically based pharmacokinetic (PBPK) model to predict the DDIs in people with different CYP3A5 and CYP2C19 alleles. First, pharmacokinetic data of humans receiving tacrolimus with or without voriconazole from the literature were used to construct and validate the PBPK model. Thereafter, we developed a model incorporating the metabolism of voriconazole mediated by CYP2C19 and the inhibitory effect of voriconazole on CYP3A4/5. Finally, the model was used to evaluate the dose adjustment of tacrolimus in people with different CYP3A5 and CYP2C19 alleles. When tacrolimus was administered alone (3 mg PO, single dose), the predicted AUC 0-∞ of tacrolimus in CYP3A5 nonexpressers (19.22) was 3.5-fold higher than that in expressers (5.48). Following voriconazole (200 mg PO, bid) administration in human with different CYP2C19 genotypes, the AUC 0-∞ of tacrolimus increased by 5.1- to 8.3-fold in CYP3A5 expressers and by 5.3- to 10.2-fold in CYP3A5 nonexpressers. The lower the gene expression level of CYP2C19 in the population, the higher the exposure to tacrolimus. When tacrolimus was combined with voriconazole (200 mg, bid; 400 mg, bid, on Day 1), the final model simulations suggested that the dose regimen of tacrolimus should be regulated to 0.15 mg/kg/day (qd) in CYP3A5 expressers with different CYP2C19 genotypes. For CYP3A5 nonexpressers, the dosing schedule of tacrolimus should be modified to 0.05 mg/kg/24 h for patients with 2C19 EM, 0.05 mg/kg/48 h for 2C19 IM and 0.05 mg/kg/72 h for 2C19 PM. In conclusion, a PBPK model with CYP3A5 and CYP2C19 polymorphisms was successfully established, providing more insights regarding the DDIs between tacrolimus and voriconazole to guide the clinical use of tacrolimus.

Tacrolimus-Loaded Cationic Nanoemulsion In-Situ Gel System: In-Vitro Characterization and Performance in a Dry-Eye Rabbit Model

Qiuhe Wang,Zheng Wu,Feifei Wang,Hua Zhang,Li Gan

Dry eye disease (DED) is a highly prevalent ocular surface disease that affects life quality and reduces productivity at work. The purpose of this study is to improve the efficacy of tacrolimus (FK506) in the treatment of DED using the special eye surface retention properties of cationic nanoemulsion (CNE) modified by thermosensitive in-situ gel (ISG) (CNE-ISG). The precorneal retention of CNE-ISG containing 0.05% FK506 (50 min) was longer than that of CNE containing 0.05% FK506 (25 min) and commercial suspension containing 0.1% FK506 (Talymus®) (10 min). Successfully modeled dry-eye rabbits were treated with 0.05% CNE-ISG (twice/day), 0.05% CNE and 0.1% suspension (Talymus®) (thrice/day). Schirmer's tear secretion test showed no significant difference between the CNE-ISG group and the healthy group after 5 days of treatment ( p  > 0.05). The results of a tear ferning test (TFT) showed that the tear-fern-like crystal branches in the CNE-ISG group returned to normal after 5 days of treatment. Histological analysis showed that the number of goblet cells in the CNE-ISG group significantly increased. HET-CAM stimulation test showed that the CNE-ISG group had no ocular irritation. The above results indicated that CNE-ISG might be a promising delivery system and as an effective dosage form was employed for FK506 in the treatment of DED.

Regenerating islet-derived protein 3 gamma (Reg3g) ameliorates tacrolimus-induced pancreatic β-cell dysfunction in mice by restoring mitochondrial function

Senlin Li,Hong Zhou,Mengyuan Xie,Zijun Zhang,Jing Gou,Jian Yang,Cheng Tian,Kun Ma,Cong-Yi Wang,Yi Lu,Qing Li,Wen Peng,Ming Xiang

Background and Purpose Tacrolimus a first-line medication used after transplantation can induce β-cell dysfunction, causing new-onset diabetes mellitus (NODM). Regenerating islet-derived protein 3 gamma (Reg3g), a member of the pancreatic regenerative gene family, has been reported to improve type 1 diabetes by promoting β-cell regeneration. We aim to investigate the role of Reg3g in reversing tacrolimus-induced β-cell dysfunction and NODM in mice. Experimental Approach Circulating REG3A (the human homologue of mouse Reg3g) in heart transplantation patients treated with tacrolimus was detected. The glucose-stimulated insulin secretion and mitochondrial functions, including mitochondria membrane potential (MMP), mitochondria calcium, ATP production, oxygen consumption rate and mitochondrial morphology were investigated in β-cells. Additionally, effects of Reg3g on tacrolimus-induced NODM in mice were analysed. Key Results Circulating REG3A level in heart transplantation patients with NODM significantly decreased compared with those without diabetes. Tacrolimus down-regulated Reg3g via inhibiting STAT3-mediated transcription activation. Moreover, Reg3g restored glucose-stimulated insulin secretion suppressed by tacrolimus in β-cells by improving mitochondrial functions, including increased MMP, mitochondria calcium uptake, ATP production, oxygen consumption rate and contributing to an intact mitochondrial morphology. Mechanistically, Reg3g increased accumulation of pSTAT3(Ser727) in mitochondria by activating ERK1/2-STAT3 signalling pathway, leading to restoration of tacrolimus-induced mitochondrial impairment. Reg3g overexpression also effectively mitigated tacrolimus-induced NODM in mice. Conclusion and Implications Reg3g can significantly ameliorate tacrolimus-induced β-cell dysfunction by restoring mitochondrial function in a pSTAT3(Ser727)-dependent manner. Our observations identify a novel Reg3g-mediated mechanism that is involved in tacrolimus-induced NODM and establish the novel role of Reg3g in reversing tacrolimus-induced β-cell dysfunction. Graphical

Tacrolimus nanoparticles based on chitosan combined with nicotinamide: enhancing percutaneous delivery and treatment efficacy for atopic dermatitis and reducing dose

Kaiyue Yu,Yixuan Wang,Tao Wan,Yuanhao Zhai,Sisi Cao,Wenyi Ruan,Chuanbin Wu,Yuehong Xu

Integrating tacrolimus into eutectic oil-based microemulsion for atopic dermatitis: simultaneously enhancing percutaneous delivery and treatment efficacy with relieving side effects

Yixuan Wang,Sisi Cao,Kaiyue Yu,Fengdie Yang,Xiuming Yu,Yuanhao Zhai,Chuanbin Wu,Yuehong Xu

Background: Topical application of tacrolimus (FK506) was effective in treating atopic dermatitis (AD); however, the therapeutic efficiency is hampered by its poor penetration into the skin and local side effects of transient irritation symptoms with a burning sensation, a feeling of warmth or heat. Menthol and camphor have been widely used in topical compound formulations for adjunctive pharmacotherapy for antipruritics and analgesics owing to their cool nature, and both present skin penetration enhancing effects. Moreover, they can form a liquid eutectic oil to solubilize hydrophobic drugs.Purpose: Taking advantages of menthol/camphor eutectic (MCE), this work aims to integrate FK506 into MCE to construct a microemulsion system, i.e., FK506 MCE ME, which simultaneously enhances the percutaneous delivery and treatment efficacy, while reduces the side effects of FK506.Methods: The formulation of FK506 MCE ME was optimized and characterized. Different formulations containing FK506 were topically administered to treat 1–chloro–2, 4–dinitrobenzene (DNCB)-induced murine AD.Results: MCE solubilized FK506. FK506 in MCE ME penetrated skin in vitro more than in the commercial ointment, and MCE predominantly exerted the enhancing effects in MCE ME. FK506 MCE ME or FK506 MCE ME gel had greater effects on clinical symptoms, histological analysis, and IgE than did commercial FK506. The anti-pruritic and down-regulation of substance P effects of MCE ME vehicle mitigated the side effects of FK506 application.Conclusion: MCE ME presented the excellent properties of simultaneously enhancing the percutaneous delivery and treatment efficacy, while reducing the side effects of FK506 for AD. Therefore, MCE ME is a promising nanoscale system for FK506 to effectively treating AD with low irritation and high medication adherence.Chemical compounds studied in this article: Tacrolimus (PubChem CID: 445643); menthol (PubChem CID: 1254); camphor (PubChem CID: 2537)

Prediction of Drug–Drug Interaction between Tacrolimus and Principal Ingredients of Wuzhi Capsule in Chinese Healthy Volunteers Using Physiologically-Based Pharmacokinetic Modelling

Hongyan Zhang,Fengjiao Bu,Lei Li,Zheng Jiao,Guo Ma,Weimin Cai,Xiaomei Zhuang,Hai-Shu Lin,Jae-Gook Shin,Xiaoqiang Xiang

Schisantherin A and schisandrin A, the most abundant active ingredients of Wuzhi capsule, are known to inhibit tacrolimus metabolism by inhibiting CYP3A4/5. We aimed to predict the contribution of schisantherin A and schisandrin A to drug–drug interaction (DDI) between Wuzhi capsule and tacrolimus using physiologically-based pharmacokinetic (PBPK) modelling. Firstly, the inhibition mechanism of schisantherin A and schisandrin A on CYP3A4/5 was investigated. Thereafter, PBPK models of schisantherin A, schisandrin A and tacrolimus were established. Finally, tacrolimus pharmacokinetics were evaluated after the combined use with schisantherin A or schisandrin A. The blood area under the curve (AUC) of tacrolimus increased 1.77- and 2.61-fold after a single dose and multiple doses of schisantherin A, respectively. Meanwhile, schisandrin A inhibited tacrolimus metabolism to a smaller extent. Also, it showed that mechanism-based inhibition (MBI) played a more important role in DDI than reversible inhibition after long-term administration, while reversible inhibition was comparable to MBI after single-dose administration. In conclusion, we utilized PBPK modelling to quantify the contribution of schisantherin A and schisandrin A to DDI between tacrolimus and Wuzhi capsule. This may provide more insights for the rational use of this drug combination.

Au/Ag@ZIF-8 nanocomposite as solid phase extraction adsorbent and SERS substrate for tacrolimus label-free therapeutic drug monitoring in human serum

Yaqin Zhan,Jinru Cao,Ying Wang,Xuesong Li,Yixing Li,Huihui Zeng,Wenyi Huang,Hao Cheng,Si Gao,Lijun Li,Jun Feng

Surface Enhanced Raman Scattering (SERS) has been extensively utilized in therapeutic drug monitoring (TDM) due to its rapid detection speed, high sensitivity and straightforward sample pretreatment. In this study, Au/AgNPs were obtained through the reduction of AgNO 3 on the surface of AuNPs. Subsequently, Au/AgNPs were embedded into the tetrahedral lattice of ZIF-8 MOFs, resulting in the formation of Au/Ag@ZIF-8 nanocomposites. The Au/Ag@ZIF-8 nanocomposites exhibit a robust electromagnetic enhancement of Au/Ag bimetallic nanoparticles and a considerable adsorption capacity of ZIF-8 MOFs. This enables the pre-enrichment of target molecules in the vicinity of the electromagnetic field of the Au/AgNPs, thereby enhancing the sensitivity of SERS detection. The SERS substrate also exhibits high stability and reproducibility, as well as molecular sieving effects, due to the fact that Au/AgNPs are embedded into the tetrahedral lattice of ZIF-8. A TDM method for tacrolimus (FK506) in human serum was developed by using Au/Ag@ZIF-8 nanocomposites as solid phase extraction (SPE) adsorbent and SERS substrates. The results showed that under the optimized conditions, tacrolimus exhibited satisfactory linearity within the concentration range of 10 −5 -10 −11  mol L −1 , with a correlation coefficient (R 2 ) of 0.9944, and the limit of detection (LOD) was as low as 6.4 pg mL −1 . The recoveries were observed to range between 92 % and 105 %, with an RSD of below 8 %. The method is highly sensitive, exhibiting a sensitivity that is 3-6 orders of magnitude higher than that of existing analytical techniques. It has the potential to be applied in a clinical setting to biological samples.

Solid dispersion-based pellet for colon delivery of tacrolimus through time- and pH-dependent layer coating: preparation, in vitro and in vivo studies

Guo Jian,Fang Huihui,Gui Shuangying,Huang Yuzhe

The intent of the present investigation is to develop and evaluate colon-specific coated tacrolimus solid dispersion pellet (SDP) that retards drug release in the stomach and small intestine but progressively releases in the colon. Tacrolimus-SDP was prepared by extrusion-spheronization technology and optimized by the micromeritic properties including flowability, friability, yields and dissolution rate. Subsequently, the pH-dependent layer (Eudragit L30D55) and time-dependent layer (Eudragit NE30D and L30D55) were coated on the SDP to form tacrolimus colon-specific pellets (CSP) using a fluidized bed coater. Under in vitro gradient pH environment, tacrolimus only released from CSP after changing pH to 6.8 and then quickly released in the phosphate buffer solution of pH 7.2. The Cmax of CSP was 195.68 ± 3.14 ng/mL at Tmax 4.5 ± 0.24 h where as in case of SDP, the Cmax was 646.16 ± 8.15 ng/mL at Tmax 0.5 ± 0.03 h, indicating the ability of CSP targeted to colon. The highest area under the curve was achieved 2479.58 ± 183.33 ng·h/mL for SDP, which was 2.27-fold higher than tacrolimus suspension. However, the best biodistribution performance was achieved from CSP. In conclusion, SDP combining of pH- and time-dependent approaches was suitable for targeted delivery of tacrolimus to colon.