*3.7. Biodistribution of FEN Solution and FEN-Loaded Soluplus*® *Micelles in Rats*

Figure 8 shows the total drug distribution in each organ measured 1 and 8 h after the intravenous injection of FEN solution and FEN-loaded Soluplus® micelles. After 1 h, the organ distribution of the drug was lower in micelles than in solution in all other organs except the spleen. After 8 h, both the solution and the micelles were present below the LOD in all organs. In particular, a significantly higher amount of drug was detected in the lungs 1 h after the administration of the solution than 1 h after the administration of the micelles. Additionally, when organs were obtained, some hemolysis was observed in the lungs (data not shown). *Pharmaceutics* **2020**, *12*, x 10 of 14

**Figure 8.** Total amount of fenbendazole (FEN) in each organ at 1 and 8 h after administration of FEN in 25% Cremophor EL®/EtOH solution and FEN-loaded Soluplus**®** micelles, \*\* *p* < 0.01, \*\*\* *p* < 0.001. **Figure 8.** Total amount of fenbendazole (FEN) in each organ at 1 and 8 h after administration of FEN in 25% Cremophor EL®/EtOH solution and FEN-loaded Soluplus® micelles, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### *3.8. In Vivo Toxicity Assay 3.8. In Vivo Toxicity Assay*

Figure 9 shows the total body weight changes and the survival rate after three injections of DPBS (control), FEN dissolved in 25% Cremophor EL**®**/EtOH, FEN dissolved in 25% DMA, FEN dissolved in 25% Tween 80**®**, or FEN-loaded Soluplus**®** micelles. Intravenous doses were administered on days 0, 4, and 8 of the experiment, and the survival rates and total body weights were measured. Compared with the control, the FEN-loaded Soluplus**®** micelle treatment group presented with 100% survival and higher body weight. One rat each in the Tween 80**®** and Cremophor EL**®**/EtOH groups died on day 6. In the DMA group, three rats died on day 10. After 2 weeks, half the rats in the DMA group, four rats in the Cremophor EL**®**/EtOH group, and one rat in the Tween 80**®** group survived. All animals in the micelle and control groups survived. Figure 9 shows the total body weight changes and the survival rate after three injections of DPBS (control), FEN dissolved in 25% Cremophor EL®/EtOH, FEN dissolved in 25% DMA, FEN dissolved in 25% Tween 80®, or FEN-loaded Soluplus® micelles. Intravenous doses were administered on days 0, 4, and 8 of the experiment, and the survival rates and total body weights were measured. Compared with the control, the FEN-loaded Soluplus® micelle treatment group presented with 100% survival and higher body weight. One rat each in the Tween 80® and Cremophor EL®/EtOH groups died on day 6. In the DMA group, three rats died on day 10. After 2 weeks, half the rats in the DMA group, four rats in the Cremophor EL®/EtOH group, and one rat in the Tween 80® group survived. All animals in the micelle and control groups survived.

(**a**)

(**b**)

**Figure 9.** Relative rat body weight changes and survival rates after three intravenous injections of Dulbecco's phosphate-buffered saline (DPBS) control, fenbendazole (FEN) dissolved in 25% Cremophor EL**®**/EtOH, FEN dissolved in 25% dimethylacetamide (DMA), FEN dissolved in 25%

*3.8. In Vivo Toxicity Assay* 

All animals in the micelle and control groups survived.

**Figure 8.** Total amount of fenbendazole (FEN) in each organ at 1 and 8 h after administration of FEN in 25% Cremophor EL®/EtOH solution and FEN-loaded Soluplus**®** micelles, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

Figure 9 shows the total body weight changes and the survival rate after three injections of DPBS (control), FEN dissolved in 25% Cremophor EL**®**/EtOH, FEN dissolved in 25% DMA, FEN dissolved in 25% Tween 80**®**, or FEN-loaded Soluplus**®** micelles. Intravenous doses were administered on days 0, 4, and 8 of the experiment, and the survival rates and total body weights were measured. Compared with the control, the FEN-loaded Soluplus**®** micelle treatment group presented with 100% survival and higher body weight. One rat each in the Tween 80**®** and Cremophor EL**®**/EtOH groups

**Figure 9.** Relative rat body weight changes and survival rates after three intravenous injections of Dulbecco's phosphate-buffered saline (DPBS) control, fenbendazole (FEN) dissolved in 25% Cremophor EL**®**/EtOH, FEN dissolved in 25% dimethylacetamide (DMA), FEN dissolved in 25% **Figure 9.** Relative rat body weight changes and survival rates after three intravenous injections of Dulbecco's phosphate-buffered saline (DPBS) control, fenbendazole (FEN) dissolved in 25% Cremophor EL®/EtOH, FEN dissolved in 25% dimethylacetamide (DMA), FEN dissolved in 25% Tween 80®, or FEN-loaded Soluplus® micelles on days 0, 4, and 8 (All doses are equal to 2 mgmL·kg−<sup>1</sup> ). (**a**) Relative daily body weight change. Missing data points indicate death of an animal. (**b**) Kaplan–Meier plot illustrating survival rates.

#### **4. Discussion**

FEN is a drug used as an anthelmintic. However, in recent years, the potential of the drug as an anticancer drug through various mechanisms and its few side effects are being revealed [5,6]. However, the low solubility and bioavailability poses an obstacle to its use [10]. To overcome this deficiency and obtain the advantages of micelles, we formulated, optimized, and tested polymeric FEN micelles. Here, we tested four different polymers and sought the optimal formulation. We compared their physicochemical properties, including particle size, poly-dispersity index, zeta-potential, and encapsulation efficiency. The present study showed that Soluplus® had the highest EE (%) and DL (%), the most appropriate size, and the lowest PDI. Nanoparticles < 200 nm have numerous drug delivery benefits and excellent micelle-forming ability. Small nanoparticles reduce the incidence of nonspecific interactions, including those that occur in the reticuloendothelial system. Secondly, the intravenous injection of micelles obviates the need for kidney excision and mitigates cancer accumulation via the EPR effect [40]. Also, by obtaining TEM data, we clearly showed that the nanoscale micelles we wanted were formed. All four polymers had negative zeta-potentials. Hence, they created electrostatic repulsion and remained safely within the physiological environment [41]. In vitro release profiles disclosed that the micelle formulation had 3-fold and 1.5-fold slower FEN release rates than the solution at 6 and 72 h, respectively. Micelle formulations can solubilize hydrophobic drugs and impede their rapid drug release, possibly because intermolecular interactions occur between the drug and the hydrophobic micelle core. The lipophilic moiety of Soluplus® consists of polyvinyl caprolactam-polyvinyl acetate, which should hydrophobically interact and form hydrogen bonds with FEN. The MTT assay confirmed the short-term (48 h) cytotoxicity of FEN. The difference between the IC<sup>50</sup> of free FEN and FEN-loaded Soluplus® micelles was <sup>≈</sup>10% according to the MTT assay. The IC<sup>50</sup> was slightly higher for the micelles than for the free drug as the former had a 48-h release rate of only 11.8%. Nevertheless, the micelles and their drug load were internalized, possibly through endocytosis [30,42]. Therefore, despite the low release rate, it is expected that the difference in effect was not large. The IC<sup>50</sup> for free FEN drug was 3070 nM (3.07 µM). Curcumin has an IC<sup>50</sup> range of 5.43–108.69 µM, and its anticancer action is downregulation of the BCL-2 family. 17-AAG has an IC<sup>50</sup> range of 0.1–2.37 µM, and its anticancer mechanism is the suppression of heat shock protein 90. Hence, the IC<sup>50</sup> of FEN indicates that the drug has sufficient anticancer efficacy [43–45]. The clonogenic assay confirmed the long-term inhibition of cell reproduction. It revealed that the FEN micellar formulation entirely inhibited colony formation at 1740 µM but was also effective at only 174 µM. Furthermore, both the size and PDI of the FEN-loaded Soluplus® micelles remained stable for two weeks at three temperatures. Therefore, this product is appropriate for long-term transport and storage in various situations. The FEN-loaded Soluplus® micelles also maintained stability at 37 ◦C. This is the temperature inside the body, and in terms of temperature, we may expect stability inside the body. The FEN-loaded Soluplus® micelles had superior pharmacokinetic parameters compared to the solution, including higher AUC and C0. This is expected to be owing to the decrease in the volume of distribution and total clearance of FEN. Thus, FEN-loaded Soluplus® micelles may exhibit better bioavailability than the free FEN drug solution at equal doses. The biodistribution study indicated that lung damage could be responsible for the observed high pulmonary drug accumulation in the solution group. Cremophor EL® increased the total numbers of cells and macrophages in the lungs, which might suggest inflammation [46]. Therefore, the observed unknown lung injury may have induced localized FEN accumulation. For the in vivo toxicity assay, three commercially applied or extensively tested solubilizing agents were selected and compared against FEN-loaded Soluplus® micelles. However, all the products may induce side effects and could be hepatotoxic and/or neurotoxic. Here, the toxicity of these agents was adjudged by determining the reduction in body weight and survival rate in rats. In the solubilizing agent groups, <sup>≥</sup> 2 rats died. The tween80® group had the largest number of deaths with five. Three were killed in the DMA group and two in the Cremophor EL®/EtOH group. It is inferred that this death was caused by the toxicity of solubilizing agent. In contrast, none of the rats in the control and FEN-loaded Soluplus® micelles groups died and they presented with consistent weight gain. Therefore, the FEN-loaded Soluplus® micelles may be considered relatively low toxicity.

#### **5. Conclusions**

Here, we tested various excipients to solubilize the veterinary anthelmintic FEN. It was recently discovered that this drug also has anticancer efficacy. We selected the Soluplus® micelle by optimization experiments and evaluated its physicochemical properties including particle diameter, zeta-potential, encapsulation efficiency, and drug release. We also conducted pharmacokinetic and biodistribution studies on rats intravenously injected with FEN-loaded Soluplus® micelles. The micellar formulation had superior bioavailability compared to that of free FEN. The biodistribution results indicate that FEN solution is mainly distributed to the lungs and liver compared to other organs. In addition, FEN solution was more specifically distributed to the lungs than micelles. In vivo toxicity tests showed that the FEN-loaded Soluplus® micelle formulation was less toxic than FEN solubilized with other excipients. As FEN has already demonstrated anticancer efficacy, the nanoparticle formulation developed here merits further preclinical research. The ultimate objective is to conduct human clinical safety and efficacy trials on FEN-loaded Soluplus® micelles.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/10/1000/s1, Figure S1: Representative chromatograms of fenbendazole (FEN) and genistein (internal standard [IS]) in stock solution and biological plasma sample.

**Author Contributions:** Conceptualization, I.S.J. and D.H.S.; methodology, I.S.J. and M.J.J.; software, I.S.J., M.J.J.; validation, I.S.J. and D.H.S.; formal analysis, I.S.J.; investigation, I.S.J.; resources, I.S.J.; data curation, I.S.J.; writing—original draft preparation, I.S.J.; writing—review and editing, I.S.J., M.J.J. and D.H.S. visualization, I.S.J. and M.J.J.; supervision, C.-W.P., Y.B.C., J.-S.K.; project administration, I.S.J. and D.H.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1C1C1009996).

**Acknowledgments:** The authors would like to thank Yu Jin lee, Moon Sup Yoon, Hee Ji Shin for their technical assistances.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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