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. 2022 Jun 8:13:860598.
doi: 10.3389/fphar.2022.860598. eCollection 2022.

Cytotoxicity of Amphotericin B and AmBisome: In Silico and In Vivo Evaluation Employing the Chick Embryo Model

Ahmad Khosravi  1 , Iraj Sharifi  1 , Hadi Tavakkoli  2 , Elaheh Molaakbari  3 , Sina Bahraminegad  1 , Ehsan Salarkia  1 , Fatemeh Seyedi  4 , Alireza Keyhani  1 , Zohreh Salari  5 , Fatemeh Sharifi  6 , Mehdi Bamorovat  1 , Ali Afgar  7 , Shahriar Dabiri  8
Affiliations

Cytotoxicity of Amphotericin B and AmBisome: In Silico and In Vivo Evaluation Employing the Chick Embryo Model

Ahmad Khosravi et al. Front Pharmacol. .

Abstract

Leishmaniasis has been identified as a significant disease in tropical and subtropical regions of the world, with Iran being one of the disease-endemic areas. Various treatments have been applied for this disease, and amphotericin B (Amp B) is the second line of treatment. Side effects of this drug have been reported in various organs. The present study investigated the effects of different types of Amp B on fetal organs using in silico and in vivo assays (chicken embryos). In vivo analysis was done by checking pathological changes, angiogenesis, and apoptosis alterations on eggs treated by Amp B and AmBisome. In silico approach was employed to predict the affinity of Amp B and AmBisome to the vascular endothelial growth factor A (VEGF-A), its receptor (KDR1), apoptotic-regulator proteins (Bcl-2-associated X protein (Bax), B-cell lymphoma (Bcl-2), and Caspase-8. The ADME-toxicity prediction reveals that AmBisome possesses a superior pharmacological effect to Amp B. The best result of all the dockings in the Molegro Virtual Docker (MVD) was obtained between Bax, Bcl-2, Caspase-8, KDR1, and VEGF-A targets. Due to the lower Egap (HOMO-LUMO) of AmBisome, the chemical reactivity of AmBisome was higher than that of Amp B. In vivo analysis showed that embryos that received Amp B exhibited less vascular density than AmBisome. Amp B alone significantly increased the expression of apoptosis and decreased angiogenesis genes compared to AmBisome. The histopathology analysis of the treated embryos showed a reduction in the blood vessel collapse and an increase in degenerative and apoptotic-necrotic changes in the embryonic tissues. Overall, the results suggest the potential benefits of AmBisome over Amp B, which might be a better treatment strategy to treat leishmaniasis during pregnancy.

Keywords: amphotericin B; angiogenesis; apoptosis; chick embryo; in silico; in vivo; leishmaniasis; toxicity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Docking conformation and active sites. (A) Bax, (B) Bcl-2, (C) Caspase-8, (D) KDR1, and (E)VEGF-A target proteins (PDB, ID, 5W5X, 5JSN, 1I4E, 2QU5, and 5t89, respectively) using MVD studies. Detected cavities; green; carbon atom; grey; oxygen atoms; red; nitrogen atoms; blue.
FIGURE 2
FIGURE 2
(A) Amp B, (B) AmBisome molecular structure as stick and bond type in position for MMV 7.0.
FIGURE 3
FIGURE 3
Schematic of the best score docking solution of the Amp B ligands and (A) BAX, (B) Bcl-2 target and AmBisome, (C) BAX and (D) Bcl-2 target with the selected crystal structure of 5W5X and 5JSN, respectively, along with the pharmacophore and ligand map of MMV 7.0.
FIGURE 4
FIGURE 4
Schematic of the best score docking solution of the Amp B ligands and (A) Caspase-8, (B) KDR1, (C) VEGF-A target and AmBisome, (D) Caspase-8, (E) KDR1, and (F)VEGF-A target with the selected crystal structure of 1I4E, 2QU5, and 5t89, respectively, along with the pharmacophore and ligand map of MMV 7.0.
FIGURE 5
FIGURE 5
Amphotericin B and AmBisome affect the blood vessel system. (I) Control embryo with typical blood vessel system. (II and III) Amp B 1x and 2x, respectively. The blood vessel system is disrupted. (IV and V) AmBisome 1X and 2X, respectively. A smaller decrease in vascular density was exhibited compared to Amp B.
FIGURE 6
FIGURE 6
Vascular density following Amphotericin B and AmBisome. Control group, Amp B 1x and 2x, and L-Amp1 X and 2X, respectively. A significant reduction in vascular density is seen in both groups that received Amp B and AmBisome. The embryos that received the Amp B exhibited less vascular density than AmBisome (error bars show mean standard error; *p < 0.05 compared control group).
FIGURE 7
FIGURE 7
Amp B and AmBisome induced (I) apoptotic mediator and (II) angiogenesis genes in the chick’s extra-embryonic membrane vasculature. (I) The expression level of the apoptotic mediator (A) Caspase-8, (B) Apaf1, (C) AIF1, (D) Bax, and (E) Bcl2 (II) angiogenesis genes (A) VEGF and (B) KDR the Amp B -treated embryos compared to controls. The expression levels were normalized to GAPDH and HPRT and calibrated to controls (error bars show standard mean error; *p < 0.05).
FIGURE 8
FIGURE 8
Microscopy images of H&E staining of chick embryo (A) control group, (B) Amp B 1X,(C) Amp B 2X, (D) AmBisome 1X, (E) AmBisome 2X.
FIGURE 9
FIGURE 9
Microscopy images of IHC staining of CD34 from chick embryo (A) control group, (B) Amp B 1X, (C) Amp B 2X, (D) AmBisome 1X, (E) AmBisome 2X.
FIGURE 10
FIGURE 10
Microscopy images of IHC staining of Bax (up) and Bcl 2 (down) from chick embryo in (A) control group, (B) Amp B 1X, (C) Amp B 2X, (D) AmBisome 1X, (E) AmBisome 2X (40X).
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