New IMB16-4 Hot-Melt Extrusion Preparation Improved Oral Bioavailability and Enhanced Anti-Cholestatic Effect on Rats

Introduction

Cholestasis refers to hepatobiliary injury caused by excessive bile acid deposition in the liver, characterized by the disorder of bile production, secretion and excretion. The disease spectrum includes primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), genetic cholestatic liver disease, etc. Persistent cholestasis may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma.1–5 Unfortunately, effective treatment options for cholestasis are extremely limited. Ursodeoxycholic acid (UDCA) is the first-line drug for the treatment of cholestasis. In addition, nearly 50% patients are intolerant or unresponsive to it.6–8 Obeticholic acid, as a farnesoid X receptor agonist, was approved by the Food and Drug Administration (FDA) for the treatment of PBC in 2016. However, the side effect of pruritus seriously restricts its clinical application.9,10 Therefore, the researches of discovering effective and safe drugs for cholestasis are still very important and urgent.

N-(3,4-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide, abbreviated as IMB17-15, Ge has found that IMB17-15 could significantly enhance the mRNA and protein expression levels of peroxisome proliferator activated receptor α (PPARα) in vivo and vitro.11 PPARα, mainly expressed in the liver, which plays key roles in maintaining the homeostasis of lipid and bile acid. Activated PPARα inhibits the expression of CYP7A1, which is a crucial enzyme for the biosynthesis of bile acid.12–15 As the typical drugs of PPARα agonists, Fibrates were commonly recommended for the treatment of cholestasis in clinic.16–18 N-(3,4,5-trichlorophenyl)-2 (3-nitrobenzenesulfonamido) benzamide, also known as IMB16-4, was the sister compound of IMB17-15. It has been reported that IMB16-4 has amazing anti-fibrotic effect on the bile duct ligation (BDL)-induced liver fibrosis rats, and there is no any research of IMB16-4 on cholestasis by far. Based on the theory that analogous compounds should have similar bioactivity, IMB16-4 may be effective for the treatment of cholestasis through activating PPARα directly or indirectly.

However, IMB16-4 is nearly insoluble in water with poor oral bioavailability, which extremely obstruct the research programs.19–21 As an innovative high-technology in pharmaceutical industry, hot-melt extrusion (HME) technique was widely recommended for improving the solubility and oral bioavailability of insoluble drugs.22,23 In this study, a HME preparation was first applied to explore the effect of IMB16-4 on α-naphthy lisothiocyanate (ANIT) induced cholestasis in rats. Our data confirmed that the HME preparation significantly improved the oral bioavailability of pure IMB16-4. Furthermore, IMB16-4-HME at 100mg/kg (calculated by IMB16-4) exhibited stronger anti-cholestatic effect compared with pure IMB16-4 (400mg/kg), but caused liver injury as well, implying that a proper dose of IMB16-4-HME that balance the effect and safety should be explored in future research.

Materials and Methods Materials

IMB16-4 and IMB16-4-HME were provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Science (Beijing, China), ANIT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primers for quantitative real-time PCR were purchased from Sango Biotech Co., Ltd (Shanghai, China), HepG2 cells were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, China). All serum biochemical analyses were implemented by Automatic biochemical analyzer (Beckman Coulter, AU5800, USA), and other experimental supplies were purchased from commercial sources.

The Preparation of IMB16-4-HME

The preparation process of IMB16-4-HME was as follows. Briefly, 10g IMB16-4 was completely mixed with 40g of vinyl pyrrolidone/vinyl acetate copolymer (PVP-VA64). The mixture was transported to a hot-melt extruder (210 °C). Then a long yellow strip was obtained, which was manually pretreated into several short strips below 2 cm, then crushed by a pulverizer at 8000 rpm, and passed through an 80 mesh sieve, then IMB16-4-HME preparation were produced eventually.

Molecular Docking

The chemical structure of IMB16-4 and IMB17-15 were shown in Figure 1. To help rationalize the favorable potency of IMB16-4, a computational modeling study was performed to elucidate the binding mode of IMB16-4 as well as probe the possible interactions with PPARα. IMB16-4 was docked into the PPARα (PDB: 6KB2, 1.95 Å) of using Discovery StudioTM CDOCKER.24 6KB2 was optimized by removing water and adding hydrogens and charges, and then defined as a receptor protein. IMB16-4 was optimized with tool “Prepare Ligands” and given a CHARMM force field, followed by docking in the active cavity of protein 6KB2. The coordinate SBD_Site_Sphere is 13.9805, 7.00533, −8.45788, and the radius is 12Å. In order to get more poses, the parameter orientation: maximum bad orientations and orientation vdW energy threshold were increased to 999,999. The -CDOCKER ENERGY of best pose is 16.9015.

Figure 1 Chemical structure of IMB16-4 and IMB17-15, (A) means IMB16-4, (B) means IMB17-15.

Pharmacokinetics Study

12 male Sprague-Dawley (SD) rats weighting 220±20g were purchased from Hebei Yiweiwo Biotechnology company (Shijiazhuang, China, Permission No. SCXK (ji) 2020–002). The animal experiment was approved by the Ethics Committee of Hebei General Hospital (No. 202242) and were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals.25 SD rats were fasted overnight and divided into IMB16-4 and IMB16-4-HME groups with 6 rats in each group. Pure IMB16-4 and IMB16-4-HME were given at a single dose of 100mg/kg (calculated by IMB16-4). IMB16-4 and IMB16-4-HME were dispersed in the component solvent comprised 10% of dimethyl sulfoxide (DMSO) and 90% of 0.5% sodium carboxyl methyl cellulose aqueous solution (0.5% CMC-Na). The blood samples were collected from the eyes socket vein at the time points of 0.17, 0.5, 1, 2, 3, 4, 6, 8 and 12h after dosing of related drugs, and the plasma samples were obtained by centrifuging at 4500 revolutions per minute for 10 minutes. Then 50µL of plasma samples were mixed with 200µL internal standard (sulfamethoxazole) methanol solution, the mixture was vortex-mixed for 30s. After centrifugation at 13000 revolutions per minute for 10 minutes, the supernatant was obtained and transferred to vials, then 0.5 µL of liquor was subjected to HPLC-MS/MS analyzer (AB SCIEX, TRIPLE QUADTM 4500MD, USA). The mobile phase was comprised of 0.1% formic acid water (A) and methanol solution (B) in gradient elution (0~1.5 minutes, 40% B to 97% B; 1.5~2.2 minutes, 97% B; 2.2~3.2 minutes, 97% B to 40% B). Analysis was carried out on a Titank C18 column (2.1×100mm, 3µm, Phenomenex). The flow rate was 0.4 mL/min and column temperature was 30°C. The standard curve for IMB16-4 was the linear (R2>0.997) in the concentration range of 5–1000 ng/mL, and the plasma samples in IMB16-4-HME group were diluted 50-fold with blank rats plasma. The quantitative ion pairs of IMB16-4 and internal standard were m/z= 499.9/196.0 and m/z = 252.0/156.0. All pharmacokinetic parameters were calculated with DAS2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).

Pharmacodynamic Study

SD rats weighing 180–220g were purchased from Hebei Yiweiwo Biotechnology company, and all the rats had free access to water and food in a specific pathogen-free environment with a 12 h light/dark cycle. The animal experiment was approved by the Ethics Committee of Hebei General Hospital (No. 202238) and were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals.

All the rats were acclimated for one week before the experiment, and 32 rats were randomly divided into four groups: control group, no treatment (n=8); model group, treatment only with ANIT (n=8); IMB16-4-HME group, treatment with IMB16-4-HME (containing 100mg/kg of IMB16-4) and ANIT (n=8); IMB16-4 group, treatment with IMB16-4 (400mg/kg) and ANIT (n=8). IMB16-4 and IMB16-4-HME were dispersed in the component solvent comprised 10% of DMSO and 90% of 0.5% CMC-Na. The rats in IMB16-4 and IMB16-4-HME groups received daily gavage with related drugs for consecutive seven days. Meanwhile, the control and model groups were administrated intragastrically with the same volume of blank component solvent. On the fifth day (2 hours after the administration of IMB16-4 and IMB16-4-HME), the rats in model, IMB16-4 and IMB16-4-HME groups were received intragastrically with 100 mg/kg ANIT once (dissolved in olive oil, the dose was referenced to literature26). At the same time, the control group received the same volume of olive oil. 48 hours after the administration of ANIT, all the rats were sacrificed to collect the blood and liver, the blood samples were centrifuged at 4000 rpm for 10 min and then stored at −80°C. Liver samples were immediately collected and cut into two parts, one part was fixed with 4% paraformaldehyde solution for subsequent histopathological examination, the other part was snap-frozen in liquid nitrogen and stored at −80°C for qRT-PCR analysis.

Serum Biochemical Analyses

Serum levels of total bile acid (TBA), alkaline phosphatase (ALP), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured by the automatic biochemical analyzer with related kits.

Histopathological Assessment

The liver samples fixed in 4% paraformaldehyde solution were embedded in paraffin and sectioned to 5 μm slices, all slices were stained with hematoxylin and eosin according to the standard protocol. All pathological changes in the liver tissue were captured with a Leica microscope (Leica Microsystems CMS GmbH, Germany), the microscope analysis was performed by 200×.

Quantitative Real-Time PCR

The total RNA from liver tissue was isolated with FastPure® Cell/Tissue Total RNA Isolation Kit (Nanjing Vazyme Biotech Co., Ltd, China) in accordance with the manufacturer’s instruction. The purity and concentration of the total RNA were detected with a spectrophotometer (Thermo Fisher Scientific, MA, USA) at 260 and 280 nanometre. Then 3 μg of total RNA was reverse transcribed to obtain the cDNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA). The mRNA expression levels of PPARα and CYP7A1 were detected with QuantStudio™ real time PCR System version 7500 (Applied Biosystems by Thermo Fisher Scientific). All data were calculated for comparison through 2−∆∆CT and the quantity of mRNA was normalized with the expression of GAPDH. The primers used in this research were listed in Table 1.

Table 1 Primers Sequences for RT-PCR

Cytotoxicity Assays

HepG2 cells were chosen for comparing the hepatotoxicity of IMB16-4 and IMB16-4-HME in vitro. Cultivation medium ingredient was as follows: Eagle minimal essential medium (MEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel), 1% sodium pyruvate and 1% nonessential amino acids (Solarbio, China). HepG2 cells were incubated at 37°C in an atmosphere of 5% CO2. In each test, the cell suspension was diluted with MEM and seeded into 96-well plates with 100 µL per well, incubated for 24 h. Subsequently, IMB16-4 and IMB16-4-HME (calculated by IMB16-4) suspensions containing different concentrations (0.1~25μg/mL) were added to 96-well plates with 100 µL per well and incubated for other 24 h, PVP-VA64 (0.4~100μg/mL) was set as control group. Then, 10 µL Cell Counting Kit-8 (CCK8) solution was added to 96-well plates and incubated for 2 h. Finally, the absorbance was determined with a Microplate Reader (Thermo Fisher Scientific, MA, USA) at 450 nm, and the cell survival rate was calculated according to the product instruction of CCK8. Cell survival rate (%)= (Absorbance of sample-Absorbance of blank)/(Absorbance of control - Absorbance of blank)×100. The half maximal inhibitory concentration (IC50) was calculated by GraphPad Prism (Version 8.2.0).

Statistical Analysis

All quantitative data were presented as the mean ± standard deviation (X ± SD) and analyzed by one-way analysis of variance (ANOVA) with SPSS software (Version 21.0), P<0.05 was considered as statistically significant. GraphPad Prism (Version 8.2.0) was used to visualize the final results.

Results Molecular Docking Results

As shown in Figure 2, the nitro group of IMB16-4 formed hydrogen bonding with Glu282. Furthermore, many hydrophobic binding interactions could be observed such as 3, 4, 5-trichlorophenyl group and His274, the sulfonyl moiety and Ala333, respectively, which indicated that IMB16-4 has great affinity with PPARα.

Figure 2 Molecular docking result between IMB16-4 and PPARα. (A) means 2D drawings, (B) means 3D drawings.

Pharmacokinetics Study

As shown in Figure 3, Table 2 and Supplement Tables 1-4. The HME preparation significantly improved the oral bioavailability of IMB16-4, the AUC0~12h of IMB16-4-HME after intragastric administration was increased 65-fold compared with that of pure IMB16-4. The Cmax value of IMB16-4 and IMB16-4-HME were 0.52±0.18 mg/L and 43.05±3.04 mg/L, respectively. In addition, the Tmax value of IMB16-4-HME was obviously earlier compared with that of pure IMB16-4, which suggested that the HME preparation greatly improved the absorption rate of IMB16-4.

Table 2 The Pharmacokinetic Parameters of IMB16-4 and IMB16-4-HME

Table 3 Effect of IMB16-4 and IMB16-4-HME on Serum Levels of TBA, ALP, TBIL and DBIL in ANIT-Induced Cholestasis in Rats. Mean ± SD, n=8

Table 4 The Results of Liver Histopathological Examination Grade

Figure 3 Plasma concentration-time profiles of pure IMB16-4 and IMB16-4-HME. Results are expressed as the mean with the bar showing SD values of six rats.

Serum Biochemical Analysis

As shown in Table 3 and Supplement Table 5, serum levels of TBA, ALP, TBIL and DBIL in model group were prominently elevated compared with that in control group, indicating serious cholestasis. After the treatment of pure IMB16-4, the level of TBA significantly decreased. However, the serum levels of ALP, TBIL and DBIL did not differ between IMB16-4 and model group, which suggested that pure IMB16-4 at 400mg/kg is not ideal for the treatment of cholestasis. On the other hand, IMB16-4-HME prominently decreased serum levels of TBA, ALP and the ratio of DBIL/TBIL, but elevated serum levels of TBIL and DBIL, which were sensitive indicators that reflected the liver function. It indicated that IMB16-4-HME plays a therapeutic role in resisting cholestasis, but it would cause liver injury at 100mg/kg (calculated by IMB16-4), the mechanism behind remains to be explored.

Histopathological Examination

As shown in Figure 4, Table 4 and Supplement 2, control group showed normal cellular structure of liver, in contrast, the liver in model group exhibited severe inflammatory cell infiltration, necrosis, tissue edema and bile duct epithelial cell damage. After the treatment of IMB16-4 or IMB16-4-HME, the liver histopathology showed less edema and necrosis, and the anti-cholestatic effect of IMB16-4-HME was prominently stronger compared with that of pure IMB16-4.

Figure 4 Effect of IMB16-4 and IMB16-4-HME on histological changes in the liver tissue of ANIT-induced cholestasis in rats (HE stained, 200×). The lesion locations were labelled with black arrows.

qRT-PCR

As shown in Figure 5 and Supplement Table 6, the mRNA expression level of PPARα in IMB16-4-HME group significantly increased compared with that in model group. Meanwhile, the CYP7A1 mRNA expression level prominently decreased after the treatment of IMB16-4 or IMB-16-4-HME. The qRT-PCR results confirmed that IMB16-4 protected against ANIT induced cholestasis by activating PPARα-CYP7A1 pathways, which is consistent with other PPARα agonists (such as fibrates).

Figure 5 Effect of IMB16-4 on the mRNA expression in rats, All data were expressed as mean ± SD (n=5). *** means P<0.001, **means P<0.01, *means P<0.05.

Cytotoxicity of IMB16-4 on HepG2 Cells

Figure 6, Table 5 and Supplement Table 7 showed the survival rate of HepG2 cells after 24h co-culture with PVP-VA64, IMB16-4 and IMB16-4-HME. When the concentration of IMB16-4 or IMB16-4-HME increased, the cell survival rate significantly decreased, and the IC50 of IMB16-4 and IMB16-4-HME on HepG2 cells is 12.06μg/mL, 8.289 μg/mL, respectively. In addition, PVP-VA64 has little negative effect on HepG2 cells viability at concentrations less than 100μg/mL, and in the concentrations range of 5–25μg/mL, the cell survival rate in IMB16-4-HME group is significantly lower compared with that in pure IMB16-4 group, suggesting that PVP-VA64 may enhance the drug load of IMB16-4 within HepG2 cells.

Table 5 The Hepatotoxicity of IMB16-4 and IMB16-4-HME on HepG2 Cells. Mean ± SD (n=3)

Figure 6 The cell survival rate of HepG2 cells at different concentrations of PVP-VA64 and IMB16-4.

Conclusion and Discussion

PPARα agonists have gained wide attention for the treatment of cholestasis in recent years. As the second-line drugs, fibrates were widely recommended for the treatment of cholestasis in various clinical guidelines due to its great efficiency and safety.27,29 It has been reported that IMB17-15 significantly enhanced the mRNA and protein expression levels of PPARα in vivo and vitro, which suggested that IMB17-15 may activate PPARα directly or activate some upstream signal pathways and then promote the PPARα expression. As the sister compound of IMB17-15, IMB16-4 could enhance the expression of PPARα as well. Molecular docking results demonstrated that IMB16-4 has great affinity with PPARα. Furthermore, the qRT-PCR analysis also confirmed that IMB16-4-HME prominently elevated the mRNA expression level of PPARα, and the mRNA level of CYP7A1 significantly decreased as well.

In this study, the oral bioavailability of IMB16-4-HME was improved 65-fold compared with that of pure IMB16-4. In addition, pharmacodynamics study demonstrated that IMB16-4-HME at lower dose exhibited stronger anti-cholestatic effect compared with pure IMB16-4. Cytotoxicity analysis suggested that the hepatotoxicity of IMB16-4-HME is absolutely attributed to IMB16-4 and that PVP-VA64 may increase the drug load within HepG2 cells. In conclusion, this study first reported the anti-cholestatic effect of IMB16-4 and its potential mechanism, our further research will be focused on the dosage form modification of IMB16-4 that balanced the pharmacokinetics, pharmacodynamics and safety.

Abbreviation

TBA, total bile acid; ALP, alkaline phosphatase; TBIL, total bilirubin; DBIL, direct bilirubin, ANIT, α-naphthy lisothiocyanate. UDCA, ursodeoxycholic acid; HME, Hot-Melt Extrusion.

Disclosure

The authors declare no conflicts of interest.

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