**Hydroxy-Propil-**β**-Cyclodextrin Inclusion Complexes of two Biphenylnicotinamide Derivatives: Formulation and Anti-Proliferative Activity Evaluation in Pancreatic Cancer Cell Models**

**Rosa Maria Iacobazzi 1,**† **, Annalisa Cutrignelli 2,**† **, Angela Stefanachi 2 , Letizia Porcelli 1 , Angela Assunta Lopedota 2 , Roberta Di Fonte 1 , Antonio Lopalco 2 , Simona Serratì 3 , Valentino Laquintana 2 , Nicola Silvestris 1,4 , Massimo Franco 2 , Saverio Cellamare 2 , Francesco Leonetti 2 , Amalia Azzariti 1,**‡ **and Nunzio Denora 2, \* ,**‡


Received: 20 July 2020; Accepted: 3 September 2020; Published: 7 September 2020

**Abstract:** Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with poor outcomes largely due to its unique microenvironment, which is responsible for the low response to drugs and drug-resistance phenomena. This clinical need led us to explore new therapeutic approaches for systemic PDAC treatment by the utilization of two newly synthesized biphenylnicotinamide derivatives, PTA73 and PTA34, with remarkable antitumor activity in an in vitro PDAC model. Given their poor water solubility, inclusion complexes of PTA34 and PTA73 in Hydroxy-Propil-β-Cyclodextrin (HP-β-CD) were prepared in solution and at the solid state. Complexation studies demonstrated that HP-β-CD is able to form stable host–guest inclusion complexes with PTA34 and PTA73, characterized by a 1:1 apparent formation constant of 503.9 M−<sup>1</sup> and 369.2 M−<sup>1</sup> , respectively (also demonstrated by the Job plot), and by an increase in aqueous solubility of about 150 times (from 1.95 µg/mL to 292.5 µg/mL) and 106 times (from 7.16 µg/mL to 762.5 µg/mL), in the presence of 45% *w*/*v* of HP-β-CD, respectively. In vitro studies confirmed the high antitumor activity of the complexed PTA34 and PTA73 towards PDAC cells, the strong G2/M phase arrest followed by induction of apoptosis, and thus their eligibility for PDAC therapy.

**Keywords:** pancreatic ductal adenocarcinoma; cyclodextrin inclusion complex; phase solubility studies; preformulation studies; biphenylnicotinamide derivatives

#### **1. Introduction**

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, which kills more patients every year than any other type of cancer excluding lung and colorectal. Although accounting for only 3% of new cancer cases in the United States, it is responsible for over 7% of all cancer deaths, with an overall five-year survival of less than 5% [1]. In 2019, in Italy, 13,500 new cases were expected (6800 in men and 6700 in women), about 3% of all male and female cancers [2]. The American Cancer Society estimates that in 2020 about 57,600 people (30,400 men and 27,200 women) will be diagnosed with pancreatic cancer, and that about 47,050 people (24,640 men and 22,410 women) will die of pancreatic cancer [1].

Since PDAC is generally diagnosed at an advanced stage, systemic therapy is the main strategy of treatment. Currently, the most successful chemotherapy regimens for this type of tumor are gemcitabine, FOLFIRINOX, and the combination gemcitabine/nabpaclitaxel. However, the clinical management of patients still remains an open challenge, because in most cases patients have inherent resistance to therapies. The poor outcome for PDAC patients is mainly due to the peculiarity of the desmoplastic stroma that represents up to 90% of the tumor mass, and is characterized by fibrosis, poor vascularization, high intratumoral pressure, immune infiltrates, and hypoxia, with consequent reduction of the bioavailability of the drugs also hindered by rapid elimination, metabolic inactivation, and not specific systemic toxicity [3,4].

The interaction between pancreatic cancer cells and the tumor microenvironment, including immune cells, endothelial cells, and fibroblasts, plays a crucial role in PDAC development and progression and in drug-resistance phenomena [5].

In this scenario, in order to identify new therapeutic strategies for PDAC, we planned to investigate, in a PDAC cells panel, the pharmacological efficacy of two newly synthesized N-biphenylnicotinamides, namely PTA34 and PTA73 [6,7], formulated as hydroxy-propil-β-cyclodextrin (HP-β-CD) inclusion complexes (Figure 1). β β

**Figure 1.** Chemical structures of PTA34, PTA73, hydroxy-propil-ββ-cyclodextrin (HP-ββ-CD) and graphical representation of the inclusion complex.

PTA34 and PTA73 molecules have been already classified as a novel, highly potent, and selective class of microtubule targeting agents (MTAs) and potential anti-angiogenic and vascular-disrupting agents in the Hodgkin lymphoma model [7]. Moreover, a remarkable antitumor activity of these molecules at low doses was assessed also in a PDAC model, MIA PaCa-2 cells [7], confirming that targeting microtubule dynamics could be effective against the abnormal proliferation of PDAC cancer cells [8–10].

However, preformulation studies conducted on PTA34 and PTA73 showed very low water solubility, which strongly limits the potential pharmaceutical development for these compounds due to the poor bioavailability of the drug. Therefore, the improvement of the aqueous solubility for the new PTA's formulations was an urgent need.

Different formulation strategies allow overcoming the limits of poorly soluble drugs, such as solid dispersions [11,12], addition of cosolvents [13], complexation, and size reduction [14,15], however the most studied and applied approach to improve the solubility and bioavailability of drugs is the complexation in cyclodextrins [16–18].

Cyclodextrins (CDs), are cyclic oligosaccharides containing at least 6 D - (+) glucopyranose units attached by α-(1,4) glucosidic bonds, with lipophilic inner cavities and hydrophilic outer surfaces. They are able to entrap hydrophobic drugs in their cavities, forming non-covalent inclusion complexes, thus allowing the dissolution in the aqueous phase of the drug included, making it suitable to diffuse in an aqueous medium, to come in contact with the membrane surface, and to permeate through the membrane. CDs are also able to interact with membrane components and to solubilize cholesterol, inducing perturbation in the lipid bilayer, and affect the membrane properties, such as fluidity and permeability. Moreover, the encapsulation in CDs protects the drug from chemical and enzymatic degradation [19–22].

Here, inclusion complexes of both PTA34 and PTA73 in hydroxy-propil-β-cyclodextrin (HP-β-CD), a semisynthetic cyclodextrin approved by the Food and Drug Administration (FDA) as an excipient for parenteral formulations, were developed (Figure 1) [23]. In detail, the inclusion complexes among the HP-β-CD and these biphenylnicotinamide derivatives were studied first in solution, by the analysis of the phase solubility diagram, according to Higuchi–Connors [24], and the construction of the Job plot [18] for the identification of the host–guest stoichiometric ratio. Next, the inclusion complexes were prepared at the solid state by freeze-drying and characterized in terms of incorporation degree and dissolution profiles.

Finally, in order to evaluate the effectiveness of the PTA's complexation in HP-β-CD, in terms of antitumor activity improvement, cytotoxicity studies, cell cycle analysis, and apoptosis determination were conducted in a panel of PDAC cell lines, AsPC-1, PANC-1, and MIA PaCa-2. The activities of the complexes PTA34/HP-β-CD and PTA73/HP-β-CD were compared to those of the corresponding pure molecules, showing a higher antiproliferative efficacy and an unaltered activity in terms of modulation of the cell cycle.

In conclusion, the two new cyclodextrin inclusion complexes have proven in vitro to be promising candidates for PDAC therapy, even if in vivo studies are needed in order to validate an actual clinical use of these formulations.

#### **2. Results and Discussion**

#### *2.1. Solubility and Phase-Solubility Studies of PTA73 and PTA34*

First of all, the solubility of PTA73 and PTA34 was determined at 37 ± 0.5 ◦C in ultra-pure water, and the results showed that the solubility is critical for the bioavailability of these compounds. In detail, the intrinsic solubility (S0), was equal to 1.95 µg/mL (5.78 × 10−<sup>6</sup> M) and 7.16 µg/mL (2.24 × 10−<sup>5</sup> M) for PTA 34 and PTA 73, respectively, thus complexation with a cyclodextrin could represent a valid solubilization strategy for these compounds. In Figure 2a,b the phase solubility diagrams of both the compounds are reported. It is evident that there is a linear correlation between solubility and HP-β-CD

concentration, therefore both diagrams are of the A<sup>L</sup> type, according to the classification proposed by Higuchi–Connors [24]. The solubility values obtained at 37 ± 0.5 ◦C in the presence of different HP-β-CD concentrations are shown in Table 1. The linear trend of the Higuchi–Connors diagram certifies the formation of an inclusion complex, with 1:1 host:guest stoichiometry, so that using the relative equation, as reported in the materials and methods section, it was possible to calculate the relative equilibrium constant (Ks). In particular, for PTA34 the presence of HP-β-CD at a maximum concentration of 45% *w*/*v* led to an increase in terms of aqueous solubility of about 150 times, bringing it from an initial solubility value of 1.95 µg/mL to a final solubility value of 292.5 µg/mL, and the calculated equilibrium constant was 503.9 M−<sup>1</sup> . For PTA73, instead, the presence of HP-β-CD at a maximum concentration of 45% *w*/*v* led to an increase in terms of aqueous solubility of about 106 times, bringing it from an initial solubility value of 7.16 µg/mL to a final solubility value of 762.5 µg/mL, and the calculated equilibrium constant was 369.2 M−<sup>1</sup> . − β −

β β **Figure 2.** Phase solubility diagrams. (**a**) PTA34/HP-β-CD; (**b**) PTA73/HP-β-CD.


β **Table 1.** PTA34 and PTA73 Water Solubility Values in Presence of HP-β-CD.

Data are the means of at least three determinations.

β The calculated Ks values indicated a good complexing capacity of the HP-β-CD towards these two new N-biphenylnicotinamide derivatives. Furthermore, PTA34 presented a higher complexation constant value, and this result is explained by the presence on aromatic ring of the para fluorine atom. Since the fluorine atom is a lipophilic substituent, PTA34 also showed a lower water solubility.

#### *2.2. Job's Plot Method*

In order to investigate the host-guest stoichiometric ratio and to confirm the linear behavior of the Higuchi–Connors diagrams, the construction of the Job plot was carried out. Since both compounds under analysis exhibited absorption in the visible spectrum, this determination was conducted via UV-VIS spectrophotometry and the graphs obtained are shown in Figure 3a,b. In both cases, a highly symmetrical trend, with a maximum value recorded at *r* = 0.5, is observed, highlighting the formation of a 1:1 inclusion complex. This result is fully in agreement with the A<sup>L</sup> trend of the solubility diagrams.

β β **Figure 3.** Job plots. (**a**) PTA 34/HP-β-CD; (**b**) PTA 73/HP-β-CD.

#### *β 2.3. Preparation of PTA34 or PTA73*/*HP-*β*-CD Inclusion Complexes at the Solid State and Determination of Their Incorporation Degree*

β β The inclusion complexes PTA34/HP-β-CD and PTA73/HP-β-CD were also prepared in the solid state by freeze-drying. A solid-state drug-cyclodextrin inclusion complex is certainly the most suitable tool to allow the administration of the drug, both orally and parenterally, overcoming the limit represented by its low solubility in water [17]. The lyophilized complexes were characterized through the determination of the incorporation degree, expressed as mg of PTA34 or PTA73 per 1 g of product, and were found to be 1.23 ± 0.42 and 2.91 ± 1.0 mg of drug per 1 g of lyophilized powder for PTA34 or PTA73, respectively.

#### *2.4. In Vitro Dissolution Studies*

In Figure 4 in vitro dissolution profiles, in 0.05 M phosphate buffer at pH 7.4, of PTA34 and PTA73, from their respective solid-state inclusion complexes, are shown. In the same graph the dissolution profiles of the two uncomplexed compounds are not reported because, due to their very low solubility, the dissolved quantity was well below the detection limit, and this prevented the quantitative determination via UV-VIS spectrophotometry in the dissolution medium. The lipophilic nature of both drugs limits their contact with the dissolution medium, causing them to float on the surface and hindering their dissolution. On the contrary, the freeze-dried complexes dissolve very

β

**100 125**

quickly once they are placed in the dissolution medium and, in both cases, 100% of the complexed drug was solubilized within the first 20 min of the dissolution process. **75 % PTA dissolved**

β **Figure 4.** Dissolution profiles of PTA/HP-β-CD solid complexes. β

Consequently, the complexation with HP-β-CD certainly represents a valid strategy for improving the solubility characteristics and the dissolution profile of these two biphenylnicotinamide derivatives. β β

β

PTA 34 PTA 73

#### *2.5. In Vitro Studies*

#### 2.5.1. Cytotoxicity

β β β β β The effectiveness of PTA34 and PTA73, both complexed in HP-β-CD and as pure compounds, was evaluated in three human pancreatic cancer cell lines AsPC-1, Panc-1, and MIA PaCa-2 cells, by MTT assay after 72 h of treatment. In order to demonstrate that the complexed drugs (PTA 34/HP-β-CD and PTA 73/HP-β-CD) did not lose their antiproliferative activity against tumor cells, in respect to uncomplexed ones (PTA34 and PTA73, respectively), the proliferation of all cells was determined after each drug exposure, and IC<sup>50</sup> was calculated. The dose/effect curves of PTA34/HP-β-CD vs. PTA34 (Figure 5, Panel (a)) and of PTA73/HP-β-CD vs. PTA73 (Figure 5, Panel (b)), as well as the IC<sup>50</sup> values reported in Table 2, show that both the complexes were even more active than the non-complexed ones, in AsPC- 1 and in PANC-1 cells, while in MIA PaCa-2 cells, where the lowest IC<sup>50</sup> values for PTA34 and PTA73 alone were recorded, the activity of the complexed compounds and pure molecules was comparable. β **β β**

(**a**)

0 0.2 0.4 0.6 0.8 1.0

Effect

**Effect**

0 2 4 6 8 10

Dose

0 0.2 0.4 0.6 0.8 1.0

Effect

**Effect**

**AsPC-1 PANC-1 Figure 5.** *Cont.*

0 2 4 6 8 10

0 2 4 6 8 10

**MIA Pa Ca-2**

β

Dose

0 0.2 0.4 0.6 0.8 1.0

Effect

**Effect**

Dose

**Dose Dose Dose**

**PTA34/HP- -CD PTA34 Ctrl HP- -CD**

(**b**)

β β β β β β β **Figure 5.** Dose/effect plots of the mean of three different cell proliferation experiments, conducted in PDAC cell lines incubated for 72 h with HP-β-CD (Ctrl HP-β-CD), PTA34, and PTA34/HP-β-CD (panel (**a**)) or with HP-β-CD (Ctrl HP-β-CD, PTA73, and PTA73/HP-β-CD (Panel (**b**)). Dose was expressed in each graph as 0.01–10 µM concentration range, in terms of PTA34 or PTA73, corresponding to 2.9–2900 µg/mL concentration range for HP-β-CD alone.

β **Table 2.** Inhibitory Effect of PTA Compounds and PTA/HP-β-CD Complexes on Pancreatic Ductal Adenocarcinoma (PDAC) Cancer Cells.


β β β β β Furthermore, to exclude that the higher cytotoxic activity of the complexed compounds was to some extent attributable to the HP-β-CD, this was tested in the same concentration ranges utilized in the drug complexes. The dose/effect curves in Figure 5 for cyclodextrin (Ctrl HP-β-CD) clearly show that it was not toxic at the analyzed concentration range. These results evidenced a general improvement in the cytotoxic activity of these drugs following complexation, due to the increase in solubility in aqueous medium and to the enhancement of the plasmatic membrane permeability by HP-β-CD. As reported in the literature, this FDA approved excipient is characterized by a hydrophobic pocket capable of binding and solubilizing cholesterol, a critical component of the plasma membrane, thus having a role in the efflux and redistribution of cholesterol in mammalian cells [23,25–27]. Thus, the interaction of HP-β-CD with cholesterol in the plasma membrane can induce perturbation in the lipid bilayer, affecting the membrane properties such as fluidity and permeability. For these reasons, it is plausible to speculate that the cyclodextrin in the formulation of PTA compounds triggered the increase of intracellular uptake of the complexed drugs by enhancing the plasma membrane permeability. Ultimately, HP-β-CD could be considered a drug delivery enhancer for PTA 34 and PTA73.

#### 2.5.2. Cell Cycle Modulation

β β β β β β In order to investigate whether the PTA's complexation in HP-β-CD altered their mechanism of action, in terms of cell cycle modulation compared to uncomplexed drugs, we conducted the flow cytometry (FCM) analysis, after staining with propidium iodide, of cells previously treated for 24 h with compounds at 1µM in terms of PTA's concentration. In Figure 6, representative FCM histograms of cell cycle modulation by complexed drugs, or pure compounds in the three cell lines, are reported, as well as the quantification of three independent experiments. Results showed a strong arrest in the G2/M phase of AsPC-1 cells, induced by complexed and uncomplexed PTAs, with 93% vs. 95.50% and 94.1% vs. 95.58% for PTA34 vs. PTA34/HP-β-CD and PTA73 vs. PTA73/HP-β-CD, respectively. PANC-1 and MIA PaCa-2 cells were found to be less responsive to drugs with 57.9% vs. 66.9% and 64.9% vs. 67.3% for PTA34 vs. PTA34/HP-β-CD and PTA73 vs. PTA73/HP-β-CD, respectively in

β β

PANC-1 cells, and 36.7% vs..75.74% and 45.2% vs. 65.7% for PTA34 vs. PTA34/HP-β-CD and PTA73 vs. PTA73/HP-β-CD, respectively, in MIA PaCa-2 cells. However, in PANC-1 and MIA PaCa-2 cells, the complexed drugs induced an increase in the percentage of cells arrested in G2/M phase, compared to uncomplexed drugs, confirming an improvement of the drugs activity. In conclusion, PTA34/HP-β-CD resulted more active than PTA34 in inducing the arrest in G2/M phase in all PDAC cell lines while PTA73/HP-β-CD resulted more active than its pure counterpart only in MIA PaCa2 cells. Moreover, HP-β-CD alone (Ctrl HP-β-CD), tested at a concentration of 290 µg/mL, did not alter the cell cycle in all three investigated cell lines. These results allowed us to confirm that the complexation of these two biphenylnicotinamide derivatives with HP-β-CD, per se, did not modify the blocking activity in the G2/M phase of the cell cycle but may in some cases increase its effectiveness. β

β β **Figure 6.** Effects on cell cycle of PDAC cells by CD (Ctrl-CD), complexed PTA34 and PTA73 (PTA34/HP-β-CD and PTA73/HP-β-CD, respectively) and PTA34 and PTA73, alone. The modulation of cell cycle phases was evaluated by flow cytometry (FCM) analysis after staining cells with propidium iodide. In panel (**a**), a representative analysis of three independent experiments is reported for all cell lines investigated. In panel (**b**) the bar graph shows cell population percentage in each phase of the cell cycle.

#### 2.5.3. Apoptosis Assay

β β β The results of cell cycle analysis prompted us to investigate if the observed phase arrest in G2/M was followed by induction of apoptosis. Thus, the FITC Annexin V staining was carried out after 24 h of incubation of PDAC cells with PTA34 and PTA73 free and complexed in HP-β-CD at the same concentration used for cell cycle analysis (1 µM in terms of PTA's). Untreated cells and HP-β-CD treated ones, were used as references for PTAs and PTA's/HP-β-CD complexes, respectively. As representative of apoptosis analysis, the FCM dot plots of Annexin V/PI positive PDAC cells are reported in Figure 7a, whereas fold changes of Annexin V positive cells (early apoptosis plus late

apoptosis) in treated samples, versus their specific reference compound, are summarized in Figure 7b. The induction of apoptosis was more evident in AsPC-1 cells when treated with PTA34 complexed and not in respect to PTA73 and PTA73/HP-β-CD, conversely, in MIA PaCa-2 cells both the complexes showed a higher effectiveness than the uncomplexed counterpart. PANC-1 cells showed a completely different behavior, the arrest in G2/M phase after 24 h of treatment did not trigger the marked induction of apoptosis, rather, only a slowdown in cell growth, as observed in cell viability assay. Finally, the HP-β-cyclodextrin alone did not affect the basal condition of cells in terms of apoptotic events (Figure 7a). β

β β β β β **Figure 7.** (**a**) Representative apoptosis analysis of PDAC cells after 24 h treatment with HP-β-CD alone (Ctrl HP-β-CD), PTA34, PTA34/HP-β-CD, PTA73, and PTA73/HP-β-CD. Apoptosis was evaluated by using Annexin V/PI staining followed by FCM analysis. (**b**) Fold change of Annexin V positive cells (early plus late apoptosis) in treated samples versus their corresponding reference compounds (Ctrl cells and Ctrl HP-β-CD for PTAs and complexed-PTAs treated cells, respectively). The results are the mean ± SD of three independent experiments (\* *p* < 0.05; \*\*\* *p* < 0.001).

A possible explanation for the different sensitivity to these drugs, pure or complexed, of PDAC cell lines may lie in their cellular cholesterol levels. Li et al. documented an aberrant accumulation of cholesteryl ester (CEs) in human pancreatic cancer tissues and cell lines, but not in normal counterparts and, specifically, MIA PaCa-2 and PANC-1 cells had much higher levels of CEs than AsPC-1 [28]. Briefly, mammalian cells obtain cholesterol either from de novo synthesis or from the uptake of low-density lipoprotein (LDL). Overaccumulation of free (unesterified) cholesterol can be toxic to

cells, thus to prevent accumulation, cholesterol is converted primarily by the enzyme ACAT1, to CEs, which mainly exists as cytosolic lipid droplets. A different enzyme, the cholesteryl ester hydrolase (CEH), acts in the opposite direction by liberating cholesterol from CEs, which now can reach the plasma membrane. Biosynthesis and hydrolysis of CEs occur continuously, forming the cholesterol/CE cycle [29]. Thus, given the existence of the free cholesterol/esterified cholesterol cycle, it is plausible to hypothesize that low levels of esterified intracellular cholesterol correspond to low levels of cholesterol in the plasma membrane and vice versa. This might explain why AsPC-1 cells, having the lowest esterified-cholesterol levels compared to the other two PDAC cell lines, are more sensitive to the cyclodextrin-dependent solubilizing action of cholesterol, which in turn results in both increased intracellular uptake and antitumor activity of the delivered PTAs.

#### **3. Materials and Methods**

#### *3.1. Materials*

The synthesis of PTA34 (MW = 337.30) and PTA73 (MW = 319.30) was performed according to the already reported literature procedure [6,7]. HP-β-CD (hydroxypropil-β-cyclodextrin, MW = 1396 Dalton, substitution degree 0.65) was purchased from Farmalabor (Canosa, Italy). HCl and phosphate salts for the preparation of buffers were purchased from Fluka (Sigma Aldrich, Milan, Italy). Bidistilled water was bought from Carlo Erba (Milan, Italy). All other products and reagents used in this work were of analytical grade. Pancreatic cancer cell lines PANC-1, AsPC-1 and MIAPaCa-2 cells were purchased from ATCC. The MTT used for cytotoxicity studies and PI for cell cycle analysis were purchased from Sigma Aldrich (Milan, Italy). FITC Annexin V Apoptosis Detection Kit I was from BD PharmingenTM (San Jose, CA, USA, 556547).

#### *3.2. Quantitative Analysis of PTA34 and PTA73*

Quantitative analysis of PTA34 and PTA73 was carried out by spectrophotometry using a dual-beam UV-VIS Lambda 20 Bio spectrophotometer (Perkin Elmer, Milan, Italy) and quartz cuvettes, with a capacity of 1 mL, using methanol as solvent. In the case of PTA34 the reading was carried out at a wavelength of 320 nm, and a calibration straight line (r<sup>2</sup> = 0.9979), in the concentration range between 0.15 mg/mL (4.45 × 10−<sup>4</sup> mM) and 0.015 mg/mL (4.45 × 10−<sup>5</sup> mM), was obtained. For PTA73 the reading was carried out at a wavelength of 340 nm, and a calibration straight line (r<sup>2</sup> = 0.9999) in the concentration range between 0.16 mg/mL (5.01 × 10−<sup>4</sup> mM) and 0.016 mg/mL (5.01 × 10−<sup>5</sup> mM), was obtained.

#### *3.3. Solubility and Phase-Solubility Studies of PTA34 and PTA73*

The phase solubility study was conducted according with Higuchi and Connors. In detail, an excess of PTA34 or PTA73 was added to 1 mL of solutions containing HP-β-CD in the appropriate concentration (0–45% *w*/*v*) in 4 mL vials, with screw caps to avoid evaporation. The obtained mixtures were vigorously vortexed for about 5 min, and the suspensions thus obtained were placed under constant stirring in a thermostat bath at 37 ± 0.5 ◦C for about 48 h, until the saturation of the system was achieved. Then, each suspension was centrifuged at 13,200 rpm (MIKRO 22 R, Hettich Zentrifugen, Germany) in order to remove the uncomplexed drug and the supernatant was analyzed by spectrophotometry for the quantification of the drug, after appropriate dilution. All determinations were conducted at least in triplicate. The obtained data were used to determine the apparent stability 1:1 constant (K1:1) of the HP-β-CD inclusion complexes with the biphenylnicotinamide derivatives, using the Higuchi and Connors equation:

$$K\_{1:1} = \frac{slope}{S\_0(1 - slope)}$$

where *slope* is the slope of the obtained phase solubility diagrams straight line, and *S*<sup>0</sup> represents PTA34 or PTA73 intrinsic solubility determined in the same way.

#### *3.4. Job's Plot Method*

The stoichiometric ratio between PTA34 or PTA73 and HP-β-CD in the inclusion complexes was determined by the continuous variation method or Job's plot method [18]. In detail, starting from CH3OH/H2O (55/45 *v*/*v*) equimolar solutions (1.0 × 10−<sup>4</sup> M) of PTA34/PTA73 and HP-β-CD, and keeping the total molar concentration of the species constant, a set of intermediate solutions with fixed volume was obtained by mixing them in the molar ratio ranged from 0 to 1. After stirring for 1 h, for each solution the absorbance (abs) was registered by UV–VIS spectroscopy at 320 nm for PTA34, and at 340 nm for PTA73. Then, ∆Abs × [PTA34 or PTA73] was plotted versus r, where

$$r = \frac{[PTA \text{34} or \text{PTA73}]}{[PTA \text{34} or \text{PTA73}] + [HP - \beta - CD]}$$

#### *3.5. Preparation of PTA34 or PTA73*/*HP-*β*-CD Inclusion Complexes at the Solid State and Determination of Their Incorporation Degree*

The PTA34 or PTA73 and HP-β-CD (PTA34/HP-β-CD or PTA73/HP-β-CD) inclusion complex was prepared in the solid state by freeze drying. Briefly, PTA34 or PTA73 and HP-β-CD were placed in water in a 1:1 molar ratio and the suspension was at first vigorously vortexed for about 5 min and then left under stirring for two days. At the end of the two days the samples were filtered through 0.22 µM cellulose acetate filters (Millipore), frozen, and freeze-dried (Lio 5P, Milan, Italy). The amount of PTA34 or PTA73 present in the PTA34 or PTA73/HP-β-CD solid complex was determined by solubilizing about 5 mg of each sample in 5 mL of deionized water. The obtained solution was filtered with 0.22 µM cellulose acetate filters (Millipore®), and the quantification of the drug was carried out via UV-VIS spectrophotometry, as described in Section 3.2.

The incorporation degree of PTA34 or PTA73 into the inclusion complexes was determined from the obtained absorbance, using the calibration straight line, and expressed as mg of PTA34 or PTA73 per 1 g of complex.

#### *3.6. In Vitro Dissolution Studies*

Dissolution experiments were performed at 37 ◦C according to the paddle method [30] and maintaining a rotational speed of 100 rpm during the test. Suitable quantities of PTAs/HP-β-CD solid complexes were suspended in the dissolution medium (0.05 M phosphate buffer at pH 7.4). At predetermined time intervals, 600 µL of suspension was collected and, in order to keep constant the initial volume, 600 µL of the same dissolution medium previously thermostated at the same temperature was added. Samples were subsequently filtered using a 0.22 µm membrane filter (Millipore® cellulose acetate), and the filtrates thus obtained were subjected to UV-VIS analysis after appropriate dilution. For quantitative analysis the calibration curve previously constructed was used, and the dissolution profiles shown correspond to the average of three determinations.

#### *3.7. Cell Culture*

Human pancreatic ductal adenocarcinoma (PDAC) PANC-1 cells, human pancreas adenocarcinoma ascites metastasis cells AsPC-1, and undifferentiated human pancreatic carcinoma MIAPaCa-2 cells, were purchased from ATCC®. PANC-1 and AsPC-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with fetal bovine serum (FBS), to a final concentration of 10%, L-glutamine 1% (*v*/*v*), and penicillin/streptomycin 1% (*v*/*v*). MIA PaCa-2 cells were grown in Roswell Park Memorial Institute (RPMI) medium, supplemented as above, and in addition with horse serum, to a final concentration of 2.5%. Cells were maintained in a humid atmosphere of 95% air and 5% CO<sup>2</sup> at 37 ◦C. All materials for cell culturing were purchased from EuroClone (Pero, Milan, Italy).

#### *3.8. Cell Proliferation Assay*

The 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide (MTT) assay was performed as previously described [5] to investigate the effect of hydroxy-propil-β-cyclodextrin-complexed PTA34 and PTA73 (PTA34/HP-β-CD and PTA73/HP-β-CD, respectively) on cell viability of PANC-1, AsPC-1, and MIA PaCa-2 cell lines. The same uncomplexed compounds and HP-β-CD alone were also tested as reference compounds. Untreated cells were used as a positive control. In particular, cells were seeded in 96 well plates at a density of 5000 cells/well, incubated for 24 h in culturing medium, and subsequently treated for 72 h, with compounds at 0.01–10 µM concentrations range, in terms of PTA34 or PTA73, included. HP-β-CD–cyclodextrin alone was tested in the concentrations range corresponding to that present in the complex, namely 2.9–2900 µg/mL. After the incubation period 10 µL of a 0.5% (*w*/*v*) MTT/PBS solution were added to each well, and the incubation was prolonged further for 2 h. After this period, medium was removed and replaced with 100 µL of DMSO. The absorbance in each well was measured by a microplate reader (MULTISKAN Sky, ThermoScientific). The results are shown as dose/effect plots of the mean of three different experiments. The IC<sup>50</sup> was defined as the drug concentration yielding a fraction of affected (no surviving) cells = 0.5, normalized with untreated cells, and was calculated utilizing CalcuSyn v.1.1.1 software (Biosoft, UK).

#### *3.9. Cell Cycle Analysis*

For the cell cycle analysis, human pancreatic cancer cells were seeded in 60 mm dishes at a density of 500,000/well and incubated for 24 h at 37 ◦C, with PTA34, PTA73 both complexed and not at concentration 1 µM in terms of PTA's, and with cyclodextrin alone at concentration equal to 290 µg/mL. Cells were then harvested, washed twice in ice-cold PBS pH 7.4, fixed in 4 mL of 70% ethanol, and stored at -20 ◦C until analysis. The cell cycle modulation induced by treatments was studied as previously described [7] by propidium iodide (PI) staining; the pellet was resuspended in PBS without Ca2<sup>+</sup> and Mg2+, containing 1 mg/mL RNase, 0.01% NP40, and 50 cgˆ /mL PI (Sigma), and then flow cytometry analysis was performed on Attune NxT acoustic focusing cytometer (Life Technologies). Data were interpreted using the CytExpert software v.1.2, provided by the manufacturer.

#### *3.10. Annexin V*/*PI Apoptosis Assay*

Annexin V/PI assay was conducted as previously described [31] to understand the mechanism of cell death, occurring after cell cycle arrest in phase G2/M. In particular after 24 h of treatment with compounds at 1 µM in terms of PTA's concentration, cells were subjected to Annexin-V FITC/propidium iodide staining, which allowed detection of early and late apoptosis as Annexin V positive cells, and late apoptosis as Annexin V/PI positive cells, and summarized in a graph as fold change of Annexin V positive cells (Annexin V plus Annexin V/PI) in treated samples versus their specific reference compound. FITC Annexin V Apoptosis Detection Kit I was from BD PharmingenTM (San Jose, CA, USA, 556547) and the analysis was performed on Attune NxT acoustic focusing cytometer (Life Technologies). Data were interpreted using the CytExpert software v. 1.2 provided by the manufacturer.

#### **4. Conclusions**

In conclusion, the complexation of PTA34 and PTA73 with hydroxy-propil-β-cyclodextrin was successful both in solution and in solid state, allowing an increase of the PTA's water solubility and a favorable dissolution profile with respect to the uncomplexed drug. In addition, the property of hydroxy-propil-β-cyclodextrin, which allows the enhancement of the plasma membrane permeability, was responsible for the increase of intracellular uptake of the complexed drugs, and consequently of their antitumor efficacy, as evidenced in studies conducted on PDAC cells model. Considering the promising results obtained, and that hydroxy-propil-β-cyclodextrin is an excipient already approved by the FDA for parenteral formulations, the inclusion complexes of PTA34 and PTA73, after an in vivo validation step, could be considered for parenteral administration in PDAC therapy.

**Author Contributions:** Conceptualization, N.D. and A.A.; methodology, A.C., R.M.I. and A.S.; software, R.D.F. and V.L.; validation, N.D., A.A., and F.L.; formal analysis, M.F., A.A.L., L.P. and S.S.; investigation, R.M.I., A.C., A.S., S.C., and A.L.; data curation, A.C., R.M.I.; writing—original draft preparation, R.M.I. and A.C.; writing—review and editing, A.C., R.M.I., A.S., A.A., N.S.; visualization, A.C., R.M.I., A.L., L.P.; supervision, A.A., F.L. and N.D.; funding acquisition, N.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Italian Ministry of Health, grant number RC 2018-2020: "Marcatori predittivo/prognostici tissutali e circolanti in pazienti con carcinoma del pancreas e delle vie biliari: dal nanostring alla biopsia liquida".

**Acknowledgments:** Authors acknowledge the University "Aldo Moro" of Bari for its finantial support.

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

#### **Abbreviations**


#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Aminopeptidase N Inhibitors as Pointers for Overcoming Antitumor Treatment Resistance**

**Oldˇrich Farsa <sup>1</sup> , Veronika Ballayová 1, \*, Radka Žáˇcková 1 , Peter Kollar 2 , Tereza Kauerová <sup>2</sup> and Peter Zubáˇc 1**


**Abstract:** Aminopeptidase N (APN), also known as CD13 antigen or membrane alanyl aminopeptidase, belongs to the M1 family of the MA clan of zinc metallopeptidases. In cancer cells, the inhibition of aminopeptidases including APN causes the phenomenon termed the amino acid deprivation response (AADR), a stress response characterized by the upregulation of amino acid transporters and synthetic enzymes and activation of stress-related pathways such as nuclear factor kB (NFkB) and other pro-apoptotic regulators, which leads to cancer cell death by apoptosis. Recently, APN inhibition has been shown to augment DR4-induced tumor cell death and thus overcome resistance to cancer treatment with DR4-ligand TRAIL, which is available as a recombinant soluble form dulanermin. This implies that APN inhibitors could serve as potential weapons for overcoming cancer treatment resistance. In this study, a series of basically substituted acetamidophenones and the semicarbazones and thiosemicarbazones derived from them were prepared, for which APN inhibitory activity was determined. In addition, a selective anti-proliferative activity against cancer cells expressing APN was demonstrated. Our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme.

**Keywords:** aminopeptidase N; acetamidophenones; Schiff bases; semicarbazones; thiosemicarbazones; inhibition of proliferation

#### **1. Introduction**

APN is sometimes called "a moonlighting enzyme". It is a widely expressed ectoenzyme with many functions that do not always depend on its enzymatic activity. The membrane-bound form of APN, which is expressed in the renal and intestinal epithelia, the nervous system, myeloid cells, and fibroblast-like cells, such as synoviocytes, is often referred to as hCD13, whereas the soluble form, which is present in human serum at a concentration of about 4.6 nM, is known as sCD13 [1]. There is a strong correlation between the expression and enzymatic activity of hCD13 and sCD13 and the invasive capacity of numerous tumor cell types. APN also serves as a receptor involved in endocytosis during viral infection such as in the human coronavirus HCoV-229E, among others [2]. As a signaling molecule, it takes part in adhesion, phagocytosis, and angiogenic processes [3]. The plasmatic concentration of sCD13 can be used as a prognostic marker for some cancers such as non-small cell lung cancer (NSCLC) including lung adenocarcinoma [4]. APN is a promising target for anticancer therapy. Newer research suggests that it serves as one of the molecular targets for the anticancer antibiotic actinomycin D and its simplified analogs [5]. Bestatin (3-Amino-2-hydroxy-4-phenylbutanoyl)-L-leucine or ubenimex in INN nomenclature, Figure 1, one of the most known APN inhibitors, was first isolated from the bacteria *Streptomyces olivoreticuli* in 1976. It was used as an anticancer agent for the treatment of lung cancer and acute myeloid leukemia in Japan for several years [6,7]. More recently, it has

**Citation:** Farsa, O.; Ballayová, V.; Žáˇcková, R.; Kollar, P.; Kauerová, T.; Zubáˇc, P. Aminopeptidase N Inhibitors as Pointers for Overcoming Antitumor Treatment Resistance. *Int. J. Mol. Sci.* **2022**, *23*, 9813. https:// doi.org/10.3390/ijms23179813

Academic Editor: Angela Stefanachi

Received: 26 July 2022 Accepted: 27 August 2022 Published: 29 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

also been demonstrated to inhibit cell proliferation, migration, and invasion in both renal cell carcinoma and prostate cancer [8,9]. Bestatin has also been shown to be capable of attenuating the acquired resistance of renal cell carcinoma to treatment with sorafenib, which is today a first-line therapy for this cancer [10]. Tosedostat, cyclopentyl (2S)-2-[[(2R)-2-[(1S)- 1-hydroxy-2-(hydroxyamino)-2-oxoethyl]-4-methylpentanoyl]amino]-2-phenylacetate, a synthetic dipeptide containing the carbohydroxamic group (Figure 1), is also a known APN inhibitor.

**Figure 1. Figure 1.** The most known dipeptide APN inhibitors: bestatin (**a**) and tosedostat (**b**).

It has undergone more than ten clinical trials of phases 1 or 2 for the treatment of myeloid leukemias and solid tumors. Its anticancer activity is mainly attributed to the inhibition of the cleavage of proteins and peptides by M1 family aminopeptidases including APN. This disrupts normal cellular protein turnover, resulting in both peptide accumulation and a decrease in intracellular free amino acid content, a process that appears to preferentially affect metabolically active cells such as malignant cells. Such an inhibition triggers the phenomenon termed the amino acid deprivation response (AADR), a stress response comprising the upregulation of amino acid transporters and synthetic enzymes and the activation of stress-related pathways such as NFkB and other pro-apoptotic regulators [11]. Death receptor 4 (DR4 or TRAIL-R1), a member of the DR subgroup of the tumor necrosis factor (TNF) receptor superfamily, is overexpressed in various types of tumor cells. DR4 mediates extrinsic apoptotic cascades using binding to TNF-related apoptosis-inducing ligands (TRAIL or Apo2L). Unfortunately, resistance is often observed in the clinical application of TRAIL, which has undergone five clinical trials on its soluble recombinant form dulanermin, and another study for the treatment of peritoneal carcinomatosis continues [12]. In a recent study, bestatin markedly sensitized fibrosarcoma cells previously implanted in athymic nude mice to apoptosis induced by TRAIL [13]. Numerous further APN inhibitors were prepared as potential anti-cancer drugs. Recent progress in this field is summarized in a review article [1]. APN belongs among the zinc metallopeptidases. As far as a mechanism of inhibition is concerned, zinc chelation is frequently mentioned. Many reported inhibitors are attributed to this mechanism of action. Hydroxamic acids with the ureido fragment in their molecules [14–16] or without [17] use their carbohydroxamic group as the coordinating moiety for Zn 2+ . Vicinal cycloaliphatic amino ketones, specifically 3-amino-2-tetralone derivatives and analogs, use this complexation as their primary amino group together with the oxygen of the adjacent keto group [18]. Semicarbazones and thiosemicarbazones are known zinc chelators [19,20], although this fact has never been used in the design of metalloenzyme inhibitors. In this article, we describe the design, synthesis, and APN inhibition activity of a series of novel, basically substituted acetamidoacetophenone-semicarbazones and -thiosemicarbazones and their starting ketones, with either the dialkylamine group or a saturated nitrogenous heterocycle as a basic substituent in the acetamido part of the molecule.

#### **2. Results**

#### *2.1. Synthesis of Target Compounds*

Our target compounds were synthesized by a four-stage synthetic sequence that is depicted in Scheme 1.

**Scheme 1.** Synthesis of target compounds. Legend: a<sup>1</sup> to a<sup>11</sup> : a set of secondary amines; explanation of codes for ketones, thiosemicarbazides, and semicarbazides: XX-X-X: 1st Figure 1 for ketone, 2 for thiosemicarbazone, 3 for semicarbazone; 2nd Figure 2 for ortho substitution, 3 for meta and 4 for para; 3rd Figure 1 for Cl and then in the respective order: 2 diethylamine, 3 dipropyl amine, 4 dibutyl amine, 5 pyrrolidine, 6 piperidine, 7 azepane, 8 morpholine, 9 thiomorpholine, 10 N-benzylpiperazine, 11 N-methyl piperazine.

**Figure 2. Figure 2.** Effect of tested compounds on cell proliferation in THP-1, MCF-7, and DU-145 cell lines after 48 h of incubation. Proliferation was determined using WST-1 assay. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. Specific values, including statistical analysis, are given in the Supplementary Materials.

The starting 2-,3- or 4-aminoacetophenone **ao**, **am**, **ap** reacted with chloroacetyl chloride to give 2-, 3- or 4-(chloroacetamido)acetophenones **12-1**, **13-1**, **14-1**, which were then subjected to the reaction with individual secondary amines to give basically substituted ketones **12-2-1** to **14-11-1** (in case of dialkyl amines as reagents, 2-, 3- or 4-[2- (dialkylamino)acetamido]acetophenones were prepared). The reaction of such ketones with thiosemicarbazide in ethanol without the presence of any strong acid then led to the appropriate thiosemicarbazones as bases (**22-5-1–24-9-1**), whereas the analogous reaction with semicarbazide gave semicarbazones as bases, **32-2-1–34-10-1**. Analogous reactions with thiosemicarbazide in the presence of either perchloric or hydrochloric acid then led to appropriate thiosemicarbazones in the form of perchlorate or hydrochloride, **23-7-3–24-11-3.** The compounds were purified by a simple crystallization from the system ethanol/water with the addition of charcoal in the case of need and characterized with 1H- and 13-C-NMR, IR, and MS spectra. Two-dimensional NMR spectra (H-H-cosy, HMQC, HMBC) were used for the 1D NMR spectra interpretation. NOESY (NMR) spectra were used to determine the geometry of the Schiff bases. They revealed that the prepared products consisted of about equimolar amounts of *E* and *Z* (or *syn*/*anti*) isomers. The structural characteristics of the prepared compounds can be found in the Supplementary Materials as well as the procedure for the determination of their purity, and the yields and values of the purity of the target compounds and key intermediates are summarized in Table 1.


**Table 1.** Yields and values of purity, determined as chromatography homogeneity by HPLC, of the target compounds and their reaction intermediates.

#### *2.2. APN Inhibitory Activity and QSAR in It*

2.2.1. Determination of APN Inhibition

APN inhibitory activity was determined using a standardized spectrophotometry protocol using L-leucine-p-nitroanilide as a chromogenic substrate for APN. Measurements were performed in triplicate at 405 nm at a Cytation 3 well-plate reader. The results were processed into IC<sup>50</sup> values using GraFit 5 software and are listed in Table 2 below.

In some compounds, solubility problems occurred, which complicated the inhibition activity determination. We overcame these either by the use of cosolvents or by an alternative HPLC approach [21]. The details are summarized in the Supplementary Materials.


**Table 2.** Inhibitory activity of basically substituted acetamidoacetophenones and their semicarbazones and thiosemicarbazones expressed as IC<sup>50</sup> in µmol/L, and their logarithms and values of computed physical parameters used for construction of QSAR Equation (2). Optimized values of log IC<sup>50</sup> are listed only for compounds that were finally used for the construction of Equation (2) (see the text).

1 IC<sup>50</sup> values determined with the use of NMP as a cosolvent. <sup>2</sup> IC<sup>50</sup> values determined with the use of DMSO as a cosolvent.

#### 2.2.2. QSAR in APN Inhibitory Activity

The classical Hansch method of regression analysis was used to determine the dependence of the inhibitory expressed as IC<sup>50</sup> on the important structure descriptors. Typically, the activity of the members of a homologous series of biologically active compounds correlates with their lipophilicity. Furthermore, in our case, a hint of such a correlation was also found. This situation was expressed by Equation (1):

$$
\log \text{IC}\_{50} = 0.6925 \log \text{P} + 0.5684 \tag{1}
$$

where log P is calculated by an algorithm based on >12,000 experimental logP values using the principle of isolating carbons [22], as a parameter of lipophilicity was used. The low determination coefficient and F-statistic values (R<sup>2</sup> = 0.4033, F = 3.2615) indicated that the correlation was insufficient and a further structure parameter had to be added. An electronic parameter was then used because it was very probable that dissociation at various sites of the molecule could have an impact on coordination to Zn2+ cation as well as on interactions with the acidic or basic parts of the amino acid residues of the enzyme protein. Since our target compounds have two centers of acidity and at least two centers of basicity, it was more advantageous to express the electronic properties of a molecule with one electronic parameter, the isoelectric point pI, than with a set of dissociation constants. The set of pI values computed by the algorithm implemented in the Marvin software [23] was chosen for such a purpose. Ketones **14-2-1**, **14-6-1,** and **14-8-1** were preliminarily excluded because there was an assumption of a different Zn2+ complexation in them than in the thiosemicarbazones and semicarbazones. Further, **24-4-1** and **34-8-1** were excluded

as outliers during the regression analysis. Finally, a regression model with both parameters squared was found. It is expressed by Equation (2):

$$\begin{aligned} \text{logIC}\_{50} &= -0.4475(\text{logP})^2 - 0.1452(\text{pl})^2 + 1.8847(\text{logP})(\text{pl}) - 0.6101(\text{logP}) + \\ &0.6021(\text{pl}) + 1.1284 \end{aligned} \tag{2}$$

where the multiple correlation coefficient R was 0.9837, the determination coefficient R<sup>2</sup> was 0.9677, and the computed F-statistic was 29.9540. The IC<sup>50</sup> values for the prepared compounds, for which it was not possible to determine them experimentally due to their poor solubility or a precipitate formation, or those that have not yet been synthesized, were estimated by the calculation from Equation (2). They are listed in Table 3 below together with their calculated values of log P and pI.

**Table 3.** Calculated inhibitory activity of semicarbazides and thiosemicarbazides of basically substituted acetamidoacetophenones expressed as IC<sup>50</sup> in µmol/L together with the calculated values of log P and pI used for IC<sup>50</sup> calculation.


<sup>1</sup> Synthesized compounds in which IC<sup>50</sup> determination was not possible due to their poor solubility or precipitate formation. <sup>2</sup> Compounds that have not yet been synthesized.

The synthesis and testing of the above-mentioned unsensitized compounds, as well as other structurally related ones, which would serve as a test set for the confirmation of Equation (1), are planned as the continuation of this research.

#### *2.3. Proliferation Inhibitory Effects Induced by Thiosemicarbazides of Basically Substituted Acetamidoacetophenone Compounds in Human Cancer Cell Lines*

For the target compounds that effectively inhibited APN activity, the antiproliferative activity in the different human cancer cell lines THP-1, MCF-7, and DU-145 was evaluated as IC<sup>50</sup> values (50% inhibition concentration). The results are shown in Table 4, Figure 2, and the Supplementary Materials. All five tested compounds significantly decreased the proliferation of THP-1 and MCF-7 cell lines in a concentration-dependent manner. On the contrary, only compound 24-10-3 induced an antiproliferative effect in DU-145 cells as well.

**Table 4.** Cell proliferation of three human cancer cell lines THP-1, MCF-7, and DU-145 and IC<sup>50</sup> values were evaluated using WST-1 analysis after 48 h incubation with serial dilutions of tested compounds. The values shown are the mean ± SD from three independent experiments, each performed in triplicate.


#### *2.4. The Most Active Compounds and SAR*

#### 2.4.1. SAR in APN Inhibition

The compound **24-11-2**, which is 4-[2-(4-methyl piperazine-1-yl)acetamido]acetophenone thiosemicarbazone hydrochloride 34 with an IC<sup>50</sup> of 13.3 µmol/L, was found to be the most active compound, the activity of which was determined, whereas **22-4-1**, 2-[(diethylamino) acetamido]acetophenone thiosemicarbazone with an IC<sup>50</sup> of 3.4 µmol/L was the most active in the series of compounds with the activities predicted using Equation 2. The overall activity results suggest that there is no significant difference in the activity of semicarbazones and thiosemicarbazones. The terminal basic parts, which seem to improve the activity, are 4-methyl piperazine-1-yl, 4-benzyl piperazine-1-yl, piperidine-1-yl, azepane-1-yl, and pyrrolidine-1-yl in most bulkier substituents with rather greater basicity (except for pyrrolidine-1-yl). As far as the influence of the positional isomerism is concerned, that is, the position of a substituted 2-aminoacetamido substituent regarding a Schiff basecontaining group at the benzene ring, the results suggest that *p*-substituted compounds are more active than *m-* and *o-*derivatives.

#### 2.4.2. APN Inhibition vs. Antiproliferative Activity

The five compounds with the best values of IC<sup>50</sup> for APN (ranging between 13 and 23.5 µmol/L) underwent testing for inhibition of cell proliferation on the three different cell lines, which differ from one another in their levels of APN expression. All five compounds triggered a significant antiproliferative effect in the cell lines expressing APN, THP-1, and MCF-7, whereas in the cell line DU-145 with no APN expression, four out of these five compounds did not affect proliferation at all. The remaining compound also inhibited DU-145 cell proliferation but less than in APN-positive THP-1 or MCF-7 lines (compare Figure 2). These results could suggest that the antiproliferative activity is linked with APN inhibition although other mechanisms that can also participate in it.

#### **3. Discussion**

The antiproliferative effect was determined for five selected compounds with the most potent APN inhibitory activity. Proliferation inhibitory effect determination was performed in three human cancer lines that differed from each other in their levels of APN expression. Although APN expression has been proved in the human monocytic leukemia

cell line THP-1 [24] and breast carcinoma cell line MCF-7 [25], no APN expression was found in the DU 145 cells [26]. Our tested compounds showed a different effect in the cell lines used for analysis. All tested substances induced a significant antiproliferative effect in the cell lines expressing APN, THP-1, and MCF-7, whereas in the cell line DU-145 with no APN expression, the compounds **24-11-2**, **34-6-1**, **24-2-3**, and **24-5-3** did not affect proliferation at all. Compound 24-10-3 also decreased DU-145 cells but to a lesser extent than in APN-positive THP-1 or MCF-7 cells. In the case of this substance, we can consider another additional mechanism of antiproliferative action, but based on the data obtained, nothing closer can be stated. Although the obtained IC<sup>50</sup> values against the APN-positive cell lines are at the level of double-digit micromoles, the substances represent an interesting model structure for the development of potentially therapeutically useful APN inhibitors.

The APN inhibition activity results suggest that the complexation of Zn 2+ in the catalytic site of APN could be the mechanism underlying the inhibitory activity of basically substituted aminoacetophenones and their semicarbazones and thiosemicarbazones. A significant difference between the median inhibitory activity of the ketones (median IC<sup>50</sup> = 395.7 µmol/L) and thiosemicarbazones together with the semicarbazones (median IC<sup>50</sup> = 44.1 µmol/L) in compounds in which the IC<sup>50</sup> values were experimentally determined, further suggests that the mechanisms of the Zn 2+ complexation with ketones and the Schiff bases derived from them must be different. This assumption was taken into account in the construction of the QSAR dependence that led to Equation (2). Based on the structure of (T-4)-[2-[1-[5-Acetyl-2-(hydroxy-κO)-4-hydroxyphenyl]ethylidene]hydrazinecarbothioamidato (2-)-κN,κN2]aquazinc [20] (Figure 3a), we proposed a possible mode of the complexation of Zn 2+ cation with our basic thiosemicarbazones and semicarbazones. A tentative Zn 2+ complex of **24-8-1** is an example of such a coordination compound, as seen in Figure 3b.

**Figure 3.** κO) κN,κN2]aquazinc **Figure 3.** (**a**) Structure of (T-4)-[2-[1-[5-Acetyl-2-(hydroxy-κO)-4-hydroxyphenyl]ethylidene] hydrazinecarbothioamidato(2-)-κN,κN2]aquazinc according to [20], redrawn by ACD/ChemSketch; (**b**) Zn 2+ complex of **24-8-1**, i.e., 4-[(morpholine-4-yl)acetamido]acetophenone thiosemicarbazone as an example of the possibility of the coordination of the prepared Schiff bases with Zn 2+ .

The 2 + charge of such a complex cation must be compensated by the negatively charged carboxylates belonging to the dicarboxylic amino acid residues, i.e., Asp or Glu. Glu320, which together with His297 and His361 takes part in Zn 2+ complexation in the APN protein, and Glu264 or Glu298, which are nearby in the tertiary structure of the protein [27], can assume this role. Furthermore, when the whole group of Schiff bases with their determined anti-APN activities is separated into two groups—compounds with only one basic nitrogen atom (majority) and those with two basic nitrogens (piperazine derivatives)—the group of piperazines is markedly more active (median IC<sup>50</sup> = 22.3 µmol/L) than the rest (median IC<sup>50</sup> = 106.7 µmol/L). This fact could be caused by the possibility of the second nitrogen forming an ionic pair with a free carboxy group of another nearby dicarboxylic amino

acid and thus interacting with the protein with a greater affinity. The water molecule, the complexation of which is expected in this model, is also a ligand naturally coordinated to Zn<sup>2</sup> of APN [27] and we suppose that it remains coordinated when the spatial structure of the active site of the enzyme is changed by the binding of our ligand. A comparison of Figure 3a,b also suggests that the activity would benefit from the introduction of a chelating group into the *o*-position and an extension of the linker chain between the carbonyl of the acetamide group and the basic nitrogen to facilitate the formation of a donor–acceptor bond from the nitrogen to the zinc cation. This is the inspiration for our further synthesis of better APN inhibitors.

#### **4. Materials and Methods**

#### *4.1. Chemistry*

#### 4.1.1. General Information

All chemicals were purchased from commercial suppliers (Sigma-Aldrich, Darmstadt, Germany) and used as supplied without further purification. All reactions were monitored by TLC performed on precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany). For compounds 12-2-1 to 14-14-1, ethyl acetate:hexane:diethylamine = 3:2:1 was used as an eluent, UV light (254 nm). For compounds **22-2-1** to **24-14-1**, petroleum ether: diethylamine = 9:1 was used as eluent, UV light (254 nm), and iodine was used for the detection of spots. NMR spectra were recorded on an FT-NMR ECZR 400 (JEOL, Akishima, Tokyo, Japan) spectrometer using TMS as an internal standard. The FTIR spectra were obtained with a Smart MIRacle™ Nicolet™ Impact 410 FTIR Spectrometer (Thermo Scientific, West Palm. Beach, FL, USA) equipped with the ATR ZnSe module. MS spectra were measured on a Xevo TQ-S triple quadrupole MS spectrometer (Waters, Milford, MA, USA) and analyzed in the positive mode under the formation of [M-H]<sup>+</sup> ions. Melting points (uncorrected) were recorded on Kofler's block Büchi Labortechnik AG 535 (BUCHI Labortechnik AG, Flawil, Switzerland). Detailed spectral and other structural data of the prepared compounds can be found in the Supplementary Materials.

#### 4.1.2. General Procedure for the Preparation of **12-1**, **13-1**, and **14-1**

The 2-, 3-, or 4-aminoacetophenone 6.76 g (0.05 mol) was dissolved in 30 mL of acetone. Thereafter, 10 g (0.094 mol) of Na2CO<sup>3</sup> was added. Then, 7.18 mL (0.09 mol) of 2-chloroacetyl chloride was added to the reaction mixture dropwise. The reaction mixture was stirred for 4 h at room temperature. After the completion of the reaction, 50 mL of 2M, HCl was added to the reaction mixture. The mixture was cooled at 0–5 ◦C overnight. The precipitate was filtered off, washed with distilled water, and dried to a constant weight.

#### 4.1.3. General Procedure for the Preparation of **12-2-1** to **14-14-1**

The synthetized 2-, 3-, or 4-(chloroacetamido)acetophenone 0.847 g (0.004 mol) was dissolved in 30 mL of acetonitrile. Then, K2CO<sup>3</sup> 1.1 g (0.008 mol) was added to the mixture. Thereafter, 0.0044 mol of appropriate secondary amine was added to the suspension dropwise. The reaction mixture was stirred and refluxed for 4 to 8 h according to the secondary amine used. After the completion of the reaction (monitored by TLC), the reaction mixture was cooled at room temperature, potassium carbonate was filtered off, and the solvent was evaporated under reduced pressure to obtain the crude product. Synthetized 2-, 3-, or 4-[2-(dialkylamino)acetamido]acetophenones were washed with cooled distilled water and a small amount of ethanol and identified.

#### 4.1.4. General Procedure for the Preparation of **22-2-1** to **24-14-1** [28]

2-, 3-, or 4-[2-(dialkylamino)acetamido]acetophenone (0.004 mol) and thiosemicarbazide (0.008 mol) were added to 5 mL of 30% ethanol. The reaction mixture was refluxed for 3 h. After the completion of the reaction (monitored by TLC), the reaction mixture was kept at 0–5 ◦C overnight. The formed precipitate was then filtered off, washed with cold

distilled water, and dried to a constant weight. The final products were recrystallized from ethanol in case of need.

#### 4.1.5. General Procedure for the Preparation of **22-2-2** to **24-14-2** [29]

The thiosemicarbazide (0.01 mol) and the appropriate 2-, 3-, or 4-[2-(dialkylamino) acetamido]acetophenone (0.01 mol) were dissolved in 20 mL of methanol. The reaction mixture was stirred for 10 min at room temperature and then an equivalent amount of 35% hydrochloric acid was added. The reaction mixture was refluxed for 5 h. After the completion of the reaction, the reaction mixture was cooled at 0–5 ◦C overnight. The precipitate was filtered off and recrystallized from 96% ethanol.

#### 4.1.6. General Procedure for the Preparation of **22-2-3** to **24-14-3** [30]

Thiosemicarbazide (0.01 mol) was added to 15 mL of distilled water and the reaction mixture was stirred and heated at 70 ◦C for 50 min to dissolve the thiosemicarbazide. Then, 2 mL of 50% perchloric acid was added to the mixture and the mixture was stirred for another 5 min at the same temperature. The solution of 2-, 3-, or 4-[2- (dialkylamino)acetamido]acetophenone (0.011 mol) in 2 mL of ethanol and 13 mL of distilled water was added to the reaction mixture. The mixture was heated and stirred at 85 ◦C for 3 h. After the completion of the reaction, the reaction mixture was cooled at 0–5 ◦C overnight. The precipitate was filtered off, washed with a small amount of cooled distilled water, and dried to a constant weight.

#### 4.1.7. General Procedure for the Preparation of **32-2-1** to **34-14-1** [31]

Semicarbazide hydrochloride (0.0025 mol) and sodium acetate (0.0025 mol) were dissolved in 10 mL of 96% ethanol. The mixture was stirred at room temperature for 15 min. Then, 0.0025 mol of synthesized 2-, 3-, or 4-[2-(dialkylamino)acetamido]acetophenone was added to the reaction mixture and the stirring continued at room temperature for the next 12 to 48 h. The reaction progress was monitored by TLC. The final product was filtered off, washed with ethanol, and dried to a constant weight.

#### *4.2. Assessment of APN Inhibitory Activity*

IC<sup>50</sup> values against enzyme APN were determined using L-Leucine-*p*-nitroanilide (Sigma-Aldrich, Darmstadt, Germany) as the substrate and Microsomal Leucine aminopeptidase from porcine kidney EC 3.4.11.2, Type IV-S, ammonium sulfate suspension, 10–40 units/mg protein (Sigma-Aldrich, Darmstadt, Germany). The assay was performed by a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA) with appropriate Gen-5 software in 96-well plates. A 0.02 mol/L TRIS-HCl buffer solution at pH 7.5 was used as the assay buffer. All tested compounds were dissolved in TRIS-HCl buffer solution and compounds with poor solubility were dissolved in a small amount of DMSO or NMP used as a cosolvent. The assay mixture, which contained a variable amount of inhibitor solution (from 0 up to 80 µL), 10 µL of the enzyme solution, 5 µL of the substrate solution, and the assay buffer adjusted to 200 µL, was incubated at 37 ◦C for 40 min with short orbital shaking for 10 s. The hydrolysis of the substrate was monitored by the optical photometric method of absorbance in the visible and ultraviolet regions at a wavelength of 405 nm. The enzyme activity inhibitory rate was calculated from the measures of absorbance. The results of the 50% inhibitory activity of the enzyme (IC50) were determined through a regression analysis of the concentration/absorbance data by GraFit 5 software (Erithacus Software Ltd., East Grinstead, UK).

#### *4.3. QSAR Statistic and Parameters Calculations*

QCExpert 3.3 (TriloByte Statistical Software, Pardubice, Czech Republic) running onWindows 10 Education was used for the linear and multilinear regression calculations. ACD/ChemSketch was used for the log P values calculation by an algorithm based on >12,000 experimental logP values using the principle of isolating carbons [22]. MarvinS-

ketch 6.2.2 (ChemAxon Ltd., Budapest, Hungary) was the software used for the isoelectric point pI values calculation by the algorithm built into it [23].

#### *4.4. Evaluation of Proliferation Inhibitor Effects*

#### 4.4.1. Reagents

All tested compounds were dissolved in dimethyl sulfoxide (DMSO) from Sigma Aldrich. Their fresh solutions were prepared prior to each experiment, whereas the final concentration of DMSO in the assays never exceeded 0.1% (*v*/*v*). RPMI 1640 culture medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) were purchased from HyClone Laboratories, Inc. (GE Healthcare, Logan, UT, USA).

#### 4.4.2. Cell Culture

A human monocytic leukemia cell line (THP-1), human breast adenocarcinoma cells (MCF-7), and human prostate cancer (DU-145) cell line were obtained from ATCC. THP-1 and DU-145 cells were cultivated in an RPMI 1640 culture medium and MCF-7 cells were cultivated in a DMEM medium, both supplemented with the antibiotic solution (100 U/mL of penicillin, 100 µg/mL of streptomycin) and 10% FBS. Cells were maintained in a humidified incubator with 5% CO<sup>2</sup> at 37 ◦C and were regularly tested for the presence of mycoplasma contamination.

#### 4.4.3. WST-1 Analysis of Cell Proliferation

THP-1, MCF-7, and DU-145 cells were seeded in 96-well plates. Adherent cell lines were allowed to attach to the wells for 24 h. Cells were then treated with various concentrations of tested compounds to reach the final concentrations ranging between 1 µM and 100 µM and were incubated for 48 h. Cell proliferation was determined using Cell Proliferation Reagent WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium) (Roche Diagnostics, Mannheim, Germany), as previously described [32,33]. WST-1 analysis was performed in three independent experiments, with each condition tested in triplicate. The IC<sup>50</sup> values were determined using the nonlinear regression fourparameter logistic model using GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA). Statistical significance between the values was assessed by one-way analysis of variance (ANOVA) paired with Dunnett's post hoc test using GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA) at levels of \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

#### **5. Conclusions**

A series of 28 novel compounds based on the structure of 2-, 3-, or 4-[2-amino(acetamido)] acetophenone, where the amino group is a part of either a dialkylamino group or a saturated nitrogenous heterocycle, was synthesized. Twenty-two compounds from this series were tested for APN inhibitory activity. **24-11-2**, 4-[2-(4-methylpiperazine-1-yl)acetamido] acetophenone thiosemicarbazone hydrochloride with an IC<sup>50</sup> of 13.3 µmol/L was the most active of them. A QSAR study with the semicarbazones and thiosemicarbazones revealed the relationship between the activity on lipophilicity expressed as logP and the acido-basic behavior of the compounds expressed as isoelectric point pI. Equation (1) describing this relationship enabled them to predict the activities of 33 other members of the semicarbazone and thiosemicarbazone series, 6 of which had already been prepared, and the remaining 27 were only proposed for synthesis. The compound with the best-calculated activity was **22-4-1**, 2-[(diethylamino)acetamido]acetophenone thiosemicarbazone with an IC<sup>50</sup> of 3.4. The complexation of the Zn2+ cation of the active site of APN was proposed as the probable mechanism of activity, based on the similarity with other semicarbazones and thiosemicarbazones, which have served as ligands for the synthesis of transition metal complexes including those with Zn2+ as the central ion [16,17]. Five compounds with the best values of IC<sup>50</sup> for APN (ranging between

13 and 23.5 µmol/L) underwent testing for inhibition of cell proliferation on three different cell lines that differ from each other in their levels of APN expression. All five compounds triggered a significant antiproliferative effect in the cell lines expressing APN, THP-1, and MCF-7, whereas in the cell line DU-145 with no APN expression, four of these five compounds, **24-11-2**, 4-[2-(4-methylpiperazine-1-yl)acetamido]acetophenone thiosemicarbazone hydrochloride, **34-6-1**, 4-[2-(piperidine-1-yl)acetamido]acetophenone semicarbazone, **24-2-3**, 4-[2-(diethylamino)acetamido]acetophenone thiosemicarbazone perchlorate, and **24-5-3**, 4-[2-(pyrrolidine-1-yl)acetamido]acetophenone thiosemicarbazone perchlorate, did not affect proliferation at all. The remaining compound **24-10-3**, 4-[2-(4 benzylpiperazine-1-yl)acetamido]acetophenone thiosemicarbazone perchlorate also inhibited DU-145 cell proliferation but less than in the APN-positive THP-1 or MCF-7 lines. These results suggest that the antiproliferative activity is linked with APN inhibition, although other mechanisms can also participate in it. Furthermore, our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme. The results, including Equation 2, can enable the proposal and synthesis of highly active APN inhibitors, which could serve as potential anticancer or antiviral drugs, which could contribute to overcoming the resistance of cancers to contemporary treatments.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23179813/s1.

**Author Contributions:** Conceptualization, O.F. and V.B.; methodology, O.F., T.K., P.K. and P.Z.; formal analysis, O.F. and T.K.; investigation, V.B., R.Ž., T.K., O.F. and P.Z.; data curation, T.K.; writing original draft preparation, O.F., P.K. and V.B.; writing—review and editing, O.F.; visualization, O.F.; supervision, O.F.; project administration, O.F.; funding acquisition, V.B. and O.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Masaryk University, grant numbers MUNI/IGA/0932/2021 and MUNI/A/1682/2020.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** No applicable.

**Data Availability Statement:** The data presented in this study are only available in this article and its Supplementary Materials.

**Acknowledgments:** Authors are grateful to Kristián Pršo from the Laboratory of Pharmacokinetics, Jessenius Medical Faculty in Martin, Comenius University in Bratislava, for the measurement and interpretation of MS spectra. The authors would like to thank Karel Souˇcek (Institute of Biophysics, the Czech Academy of Sciences, Brno) for his generous gift of the DU 145 cell lines. The authors are also grateful to Aleš Kroutil, Department of Chemical Drugs, Faculty of Pharmacy, Masaryk University, for consultations concerning metal chelates.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


### *Article* **Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells**

**Omar Prado-Carrillo 1,2 , Abner Arenas-Ramírez 1 , Monserrat Llaguno-Munive 1 , Rafael Jurado 1 , Jazmin Pérez-Rojas 1 , Eduardo Cervera-Ceballos <sup>3</sup> and Patricia Garcia-Lopez 1, \***


**Abstract:** Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20–30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinibresistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 µM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

**Keywords:** chronic myeloid leukemia; imatinib; tyrosine kinase; ketoconazole; P-glycoprotein; drug efflux transporter

#### **1. Introduction**

Chronic myeloid leukemia (CML), also known as chronic granulocytic leukemia, is a myeloproliferative disorder. It is characterized by neoplastic growth of myeloid cells in the bone marrow, leading to a significant increase of these cells in peripheral blood [1]. CML is traditionally described as a triphasic disease, beginning with the chronic phase and progressing to the accelerated phase, and finally to the blast phase. Chemotherapy given at the early chronic phase usually restores the patient to a normal-like state, which can be sustained for months or years. Nevertheless, without medical treatment or a lack of response to treatment, patients gradually progress to blast crisis [1,2].

The disease has its origins in the formation of the BCR-ABL1 gene, which results from the reciprocal translocation between chromosomes 9 and 22 and the fusion of the ABL1 and BCR genes to create a short chromosome called Philadelphia chromosome. The

**Citation:** Prado-Carrillo, O.; Arenas-Ramírez, A.; Llaguno-Munive, M.; Jurado, R.; Pérez-Rojas, J.; Cervera-Ceballos, E.; Garcia-Lopez, P. Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells. *Int. J. Mol. Sci.* **2022**, *23*, 7715. https://doi.org/ 10.3390/ijms23147715

Academic Editor: Angela Stefanachi

Received: 16 May 2022 Accepted: 6 July 2022 Published: 13 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

fused BCR-ABL1 gene codes for an abnormal oncoprotein with tyrosine kinase activity (Bcr-Abl) and with auto-phosphorylation capacity. Bcr-Abl activates multiple signaling pathways that cause the abnormal proliferation of hematopoietic stem cells (HSC) and thus the manifestation of the disease [3,4]. The BCR-ABL gene is present in all cases of CML. It provides a unique biomarker for diagnosis and is targeted during treatment with tyrosine kinase inhibitors (TKIs) to selectively inhibit Bcr-Abl. The first TKI, imatinib mesylate, became the basis of therapy for CML, transforming this disease from a fatal to a chronic one [5].

In 1996, Druker et al. [6] reported the in vitro effects of a specific inhibitor of the BCR-ABL tyrosine kinase on CML cell lines for the first time. This inhibitor was known as signal transduction inhibitor (STI571) but is now called imatinib (Gleevec®). In a phase 1 study of the advanced stage of the disease, STI571 not only controlled blood counts and restored the chronic phase, but 95% of patients achieved a complete hematologic response and a 60% greater cytogenetic response. Despite the short follow-up period existing at that time, imatinib was granted accelerated approval by the FDA in 2001 due to its exceptional efficacy and minimal toxicity [7]. It was established as the first-line treatment for CML [8,9].

The drug occupies the ATP-binding site on the BCR-ABL protein. The resulting conformational change in the tyrosine kinase quaternary structure inhibits autophosphorylation and phosphorylation of tyrosine residues on protein substrates. Thus, imatinib prevents the transduction of signals crucial for the abnormal and uncontrolled cell proliferation caused by the BCR-ABL gene in CML cells [10].

Since the bioavailability of imatinib is around 92% (86–99%) with a half-life of 18 h in healthy volunteers and patients [11], one dose/day seems to be appropriate. The drug is extensively metabolized by cytochrome P450 enzymes (CYP-P450). Additionally, it is a substrate of the drug transporter denominated P-glycoprotein (P-gp or MDR1 [multidrug resistance 1]), an ATP-dependent efflux pump that decreases intracellular drug concentrations [12]. Hence, P-gp influences drug absorption, distribution, metabolism, and excretion (ADME). Even with the high bioavailability of imatinib, its pharmacokinetics show a great variability in the responses of individuals, which are affected by both CYP-P450 and the P-gp efflux transporter [13].

The surprising efficacy of imatinib in CML is attenuated by resistance in a percentage of patients with advanced-stage CML [14–16]. The known mechanisms of resistance to imatinib can be divided into those BCR-ABL-dependent and BCR-ABL-independent. BCR-ABL-dependent mechanisms include mutations in the ABL kinase and/or mutations and amplification of the BCR-ABL gene. Among BCR-ABL-independent mechanisms is drug efflux mediated by ATP-binding cassette (ABC) transporters [17].

Specifically, it has been reported that the P-gp protein (encoded by the ABCB1 gene) may contribute considerably to resistance to imatinib. P-gp is capable of diminishing the intracellular concentration of imatinib by pumping it out of leukemia cells [17,18]. This efflux pump is located in normal human tissue in the liver, kidney, colon, adrenal gland, intestine, placenta, endothelial cells of the blood-brain barrier, and hematopoietic precursor cells. However, its expression is significantly elevated in drug-resistant tumors, making it an obstacle to successful chemotherapy. Therefore, a reduction of the level of P-gp by inhibitors could lead to the sensitization of CML resistant cells to imatinib and therefore the avoidance of resistance.

Numerous studies have identified various competitive substrates and inhibitors of P-gp, allowing for a greater understanding of the regulation of P-gp functions to overcome drug resistance. Several of these compounds are drugs originally approved for clinical indications unrelated to cancer [19], as is the case with ketoconazole. In 2004, Dutreix et al. described an increase in the plasma concentration of imatinib in healthy volunteers who had taken ketoconazole. The mechanisms involved were the blocking of imatinib metabolism by CYP3A4 and the inhibition of its extrusion from gastrointestinal cells by impeding P-gp activity [20]. To date, however, there have been no reports on the capacity of ketoconazole to reverse the resistance of CML cells to imatinib treatment. Thus, the purpose of the

current study was first to generate an in vitro model of CML cells resistant to imatinib, and secondly to explore the participation of the drug efflux transporter P-gp in such resistance. Subsequently, an evaluation was made of the capacity of ketoconazole to overcome resistance to imatinib through the inhibition of P-gp. Finally, the possible role of ketoconazole in triggering the apoptosis of imatinib-resistant cells was assessed.

#### **2. Results**

#### *2.1. The Development of CML K562 Cell Resistance to Imatinib*

By treating the K562 parent cell line with gradually increasing concentrations of imatinib (ranging from 1 to 2500 nM), the resistant phenotype was developed in about 8 months. Drug resistance was confirmed by a cell viability assay based on a 72 h exposure of sensitive (K562) and resistant (K562-RI) cells to imatinib. A clear difference between the two cell viability curves was observed, with the graph displaying a lesser effect of imatinib on the K562-RI cell line.

The IC50 of imatinib was determined for the K562-RI and K562 cell lines, finding the values of 2544 nM and 213 nM, respectively. Hence, there was an approximately 12-fold relative resistance, calculated as the ratio of the IC50 values of resistant and sensitive cells (Figure 1A).

#### *2.2. The Expression of P-Glycoprotein in Resistant and Susceptible CML Cells*

After confirming the resistant phenotype, the expression of P-gp was examined by Western blot, finding it to be around 4-fold greater in K562-RI than K562 cells (Figure 1B), correlating with the higher level of P-gp in resistant cells observed by immunofluorescence (Figure 1C). Also indicating a higher level of P-gp in resistant cells was the test with doxorubicin, which showed a lower intracellular accumulation of this compound in resistant than susceptible CML cells, as can be appreciated by the values of relative mean fluorescence intensity (RMFI) (Figure 1D).

#### *2.3. Effect of Ketoconazole on K562 Cells (Sensitive to Imatinib)*

The application of imatinib brought about a dose-dependent antiproliferative effect in K562 cells (Figure 2A), which was unmodified by the combination treatment with ketoconazole at 0.1 and 1.0 µM (Figure 2). However, significantly increased antiproliferative activity was detected with imatinib plus ketoconazole at 10 µM (Figure 2A). In this cell line, the IC50 of imatinib monotherapy was 232 nM, while that of imatinib co-incubated with ketoconazole at 10 µM was 150 nM, translating into a 0.65-fold reduction. According to the quantification of intracellular doxorubicin in sensitive cells, no change took place with ketoconazole at 10 or 20 µM (Figure 2B).

#### *2.4. Ketoconazole Induced a Reversal of the Resistance of K562-RI Cells to Imatinib*

As with K562 cells, the antiproliferative effect of imatinib on K562-RI cells was not improved by co-treatment with ketoconazole at 0.1 or 1.0 µM. However, a reversal of drug resistance in K562-RI cells was produced by applying imatinib plus ketoconazole at 10 µM (Figure 3A), resulting in an IC50 value of 186 nM. Considering the IC50 value of 1378 nM found after applying imatinib alone to K562-RI cells, the combination treatment afforded an approximately 7.5-fold reversal index.

#### *2.5. Effect of Ketoconazole on P-Glycoprotein Expression in Resistant Cells*

The Western blot (Figure 4A) and immunofluorescence assay (Figure 4B) performed with K562-RI cells demonstrated that imatinib alone (at 200 nM) and ketoconazole alone at 10 µM slightly reduced the expression of P-gp, while significantly diminished the level of P-gp, with imatinib plus ketoconazole causing a greater decrease (Figure 4).

β **Figure 1. The development of a CML cell line with resistance to imatinib treatment.** (**A**) Percentage of cell viability after applying imatinib alone (at distinct concentrations), considering CML cells sensitive (K562) and resistant (K562-RI) to this drug. The table denotes the IC50 of imatinib in each cell line and the relative resistance of K562-RI (calculated as the IC50 of K562-RI divided by that of K562). (**B**) P-gp expression levels in the K562 and K562-RI cell lines, based on Western blot and its densitometric analysis (β-actin was the load control); three Western blots from three independent experiments were used for densitometric analysis. (**C**) Analysis of P-gp evaluated by the immunofluorescence assay (the nuclei were visualized with DAPI). The mean fluorescence intensity was quantified by counting P-gp-positive cells from three independent experiment. Scale bars: 20 µm. (**D**) Representative histograms of the fluorescence of uptake of doxorubicin in the K562 and K562-RI cell lines. The empty histograms depict the control cells without doxorubicin. Data are expressed as the relative mean fluorescence intensity (RMFI) ± SEM of three independent experiments. The values of k562-RI were normalized with respect to K562. Statistical analysis was performed by comparing K562-RI to the parental cell line. \* *p* < 0.05; Student's *t*-test.

μ

μ

μ

μ **Figure 2. Effect of ketoconazole on K562 cells (sensitive to imatinib)**. (**A**) Cell viability of the K562 cell line exposed to imatinib in the absence or presence of ketoconazole (at 0.1, 1.0, and 10 µM). The table shows the IC50 of imatinib alone and imatinib plus ketoconazole. (**B**) Effect of ketoconazole (at 10 and 20 µM) on the fluorescence of doxorubicin in K562 cells, representative histograms with raw data and in a bar graph. Data are expressed as the relative mean fluorescence intensity (RMFI) ± SEM of three independent experiments. The cells treated with doxorubicin plus ketoconazole 10 and 20 µM were normalized against doxorubicin alone. Ktz, ketoconazole; Dox, doxorubicin. \* Significant difference (*p* < 0.05) by one-way analysis of variance (ANOVA) followed by Tukey's test.

#### *2.6. Effect of Ketoconazole on Apoptosis in Resistant Cells*

Since imatinib plus ketoconazole at 10 µM significantly inhibited the viability of K562- RI cells, apoptosis was explored as a possible mechanism of action. Externalization of phosphatidylserine to the outer surface of the plasma membrane is a clear sign of early apoptosis. K562-RI cells were stained with Annexin V-FITC (early apoptosis) and PI (late apoptosis) for flow cytometric analysis, which revealed the lack of any significant change in the percentage of apoptosis produced by imatinib alone or ketoconazole alone (versus the resistant control cells exposed to the vehicle only). However, there was indeed a significantly greater programmed cell death of resistant cells when using imatinib plus ketoconazole (~40%). These data suggest that exposure of cells to ketoconazole triggered apoptosis (Figure 5).

μ

μ

μ μ **Figure 3. Ketoconazole-induced reversal of K562-RI cell resistance to imatinib**. (**A**) Cell viability of the resistant cell line (K562-RI) exposed to imatinib in the absence and presence of ketoconazole (at 0.1, 1.0 and 10 µM). The table shows the IC50 of imatinib alone and imatinib plus ketoconazole. (**B**) Effect of ketoconazole on the fluorescence of doxorubicin in K562-RI cells. The bar graph portrays the relative fluorescence of doxorubicin in the presence of 10 or 20 µM of ketoconazole. Data are expressed as the relative mean fluorescence intensity (RMFI) ± SEM of three independent experiments. The cells treated with doxorubicin plus ketoconazole 10 and 20 µM were normalized against doxorubicin alone. Statistical analysis was performed by comparing the level of intracellular doxorubicin between the treatment with doxorubicin plus ketoconazole (10 or 20 µM) and doxorubicin alone. \* *p* < 0.05; determined with ANOVA followed by Tukey's test. Im, imatinib; Ktz, ketoconazole; Dox, doxorubicin.

μ

β μ μ **Figure 4. P-gp expression in resistant cells treated with imatinib and ketoconazole.** (**A**) P-gp expression levels in K562-RI cells exposed to imatinib in the absence and presence of ketoconazole based on Western blot and its densitometric analysis (β-actin was the load control); three Western blots from three independent experiments were used for densitometric analysis ± SEM. (**B**) analysis of P-gp evaluated by the immunofluorescence assay (DAPI was used to visualize the nuclei). The Mean Fluorescent Intensity was quantified by counting P-gp-positive cells (in 40–45 cells per group counted randomly) from three independent experiments. The cells K562-RI cells were exposed to imatinib alone (200 nM), ketoconazole alone (10 µM), or imatinib plus ketoconazole (200 nM/10 µM). \* *p* < 0.05; determined with ANOVA followed by Tukey's test. Scale bars: 20 µm. Ima, imatinib; Ktz, ketoconazole.

μ

μ

μ μ **Figure 5. Flow cytometric analysis of apoptosis in K562-RI cells at 96 h post-treatment.** Representative histograms depicting cells positive to Annexin V-FITC/IP (early and late apoptosis) by flow cytometric analysis; and percentage of total apoptosis. \* *p* < 0.05, examined with one-way analysis of variance (ANOVA) followed by Tukey's test. Data are expressed as the mean ± SEM of three independent experiments. Ima, imatinib (200 nM); Ktz, ketoconazole (10 µM); Ima + Ktz, the combination of imatinib plus ketoconazole (200 nM/10 µM).

μ

#### **3. Discussion**

~

Before 2001, the median survival of patients with CML was 5–7 years. Due to the introduction of TKI therapy, overall survival 5 years after treatment is now 92–95% (1). To date, the Food & Drug Administration (FDA) has approved four TKIs as first-line drugs for CML: imatinib, dasatinib, nilotinib and bosutinib. Imatinib is the first-generation drug, being the first to be approved in 2001 [21]. Whereas annual mortality for CML patients was 10–20% prior to the introduction of imatinib, it is now 1–2% [22].

There are several aspects to be considered in selecting one of the four inhibitors for CML therapy. Imatinib was the first TKI to receive approval by FDA for the treatment of patients with CML in chronic phase, is recommended for older patients with comorbidities, being the safest drug with the fewest side effects [23]. The use of dasatinib or nilotinib, TKI of the second generation, is justified in high-risk patients or young patients in need of a deep response with a first-line treatment. Whereas bosutinib, a third generation TKI, is intended for patients with chronic-, accelerated-, or blast-phase CML who cannot tolerate or are resistant to other therapies [24,25].

Most patients administered imatinib can carry on normal lives as long as they adhere to treatment. Since the dosing is carried out indefinitely, however, adherence is complicated, especially if patients experience side effects. In the latter case, adherence is likely to decline over time, leading a percentage of patients to develop resistance. The ADAGIO study showed that only 14.2% of patients took 100% of the prescribed imatinib doses. Furthermore, non-adherent patients constituted 23.2% of those with a suboptimal response and only 7.3% of those with an optimal response [26]. According to another study conducted on CML patients administered imatinib for a few years, poor adherence may be the predominant reason for a low level of response to imatinib and the development of resistance to the same [27].

Based on clinical trials, around 20–30% of patients eventually develop drug resistance [28], which consists of an absence of the initial desired response to the drug in a certain period of time (primary resistance), or the presence of the desired response followed by its loss (secondary resistance). Among the patients with primary or secondary resistance, many progress to accelerated phase and blast phase [15].

Researchers seeking new strategies to confront the emergence of resistance to imatinib have been obliged to gain new insights into the molecular mechanisms of resistance. Although there are several reports of resistance to imatinib being dependent on BCR-ABL1, the overexpression of P-gp (a drug efflux pump) could also be a crucial resistance mechanism [29–31], because it regulates the intracellular concentration of the drug.

In the current contribution, the first step was to establish drug resistance in the K562 cell line through its constant exposure to imatinib (for about eight months). The resistant cell line exhibited a drastic drop in its response to imatinib, evidenced by a significant increase in the IC<sup>50</sup> from 213 nM (K562) to 2544 nM. As can be appreciated, an approximately 12-fold greater dose is necessary to attain the same result with the K562-RI cells compared to the K562 cells.

The next step was to examine differences between the two cell lines in relation to certain parameters. In a recent clinical study on CML patients by Ammar et al. (2020), an association was found between elevated levels of P-gp and unresponsiveness to treatment, indicating that the overexpression of P-gp is probably a relevant mechanism in the development of resistance to imatinib [32]. According to the present data from Western blot analysis and immunofluorescence assays, P-gp levels were almost undetectable in the parent cells but were about 4-fold higher in K562-RI cells.

A high level of P-gp implies that its substrates can easily be removed from cells. Like imatinib, doxorubicin is a P-gp substrate and is efficiently pumped out of tumor cells when P-gp is highly expressed, leading to a reduced intracellular accumulation. To evaluate the activity of P-gp, the intracellular accumulation doxorubicin was herein analyzed after incubation of cells with this compound, finding a lower mean fluorescence intensity of intracellular doxorubicin in K562-RI versus K562 cells (Figure 1D). This low uptake of doxorubicin was accompanied by an elevated level of P-gp. Similar results have been reported by other researchers using in vitro models [30,31]. Moreover, K562-RI cells were incubated in doxorubicin only and doxorubicin plus ketoconazole, finding a greater intracellular fluorescence in the latter group with the P-gp inhibitor. Hence, an overexpression of the P-gp drug efflux pump appears to be an important mechanism of resistance of CML cells to imatinib.

The next question to be explored was the effect of applying a P-gp inhibitor to the K562 and K562-RI cell lines. P-gp, the best characterized molecule of the class of efflux pump transporters, is known to produce resistance to treatment by removing drugs from various kinds of cancer cells resistant to drug treatment. Consequently, several studies have evaluated the inhibition of this transporter to try to enhance the anti-proliferative activity of chemotherapy for distinct types of cancer. Some of these studies have tested compounds possibly capable of improving the pharmacokinetics of ITKs. However, there are few reports on the inhibition of P-gp to overcome the resistance of CML cells to imatinib.

Several researchers have demonstrated that ketoconazole is a strong inhibitor not only of CYP-P450 3A4 (CYP3A4) but also of P-gp [33]. On the other hand, imatinib is a substrate of P-gp and is metabolized by CYP-P450, mainly by the CYP3A4 isoenzyme. Thus, imatinib should be susceptible to drug interactions if administered concomitantly with potent inhibitors or inducers of CYP-P450 and/or P-gp. In this sense, the concomitant intake of imatinib and ketoconazole was found to increase the plasma concentration of imatinib in healthy subjects [20].

In the current contribution, imatinib plus 10 µM of ketoconazole was applied to resistant (K562-RI) CML cells with the aim of decreasing the overexpression of P-gp, enhancing the intracellular concentrations of imatinib, and accelerating the rate of programmed cell death. The result was the reversal of resistance to the standard drug. The IC50 of imatinib dropped from 1378 nM (without ketoconazole) to 186 nM (with ketoconazole), reflecting an approximately 7.5-fold reversal index. In K562 cells (with negligible levels of P-gp), this combination regimen only slightly improved the effect of imatinib. Contrarily, K562-RI cells subjected to the combination treatment showed a clear reduction in the expression of P-gp in Western blot and immunofluorescence assays (Figure 4), and a significantly greater rela-

tive fluorescence of intracellular doxorubicin after applying ketoconazole (10 and 20 µM) plus doxorubicin (versus doxorubicin alone) (Figure 3). Similarly, Siegsmund et al. found that applying ketoconazole in combination with vinblastine or doxorubicin to treat a highly resistant human cell line (KB-Vl) enhanced the intracellular accumulation of the latter two cytotoxic drugs. Both combinations reversed multidrug resistance with ketoconazole at 1, 3, and 10 µg/mL (∼2, 6, and 19 µM, respectively) [34], concentrations comparable to those used in the present work. In another study, it was also shown that ketoconazole was able to inhibit P-gp at a concentration of 6 µM in NIH-3T3-G185 cells that overexpressing human P-gp [35]. It has also been described that ketoconazole at 10 and 20 µM strongly enhanced cell growth inhibition and apoptosis of paclitaxel or cisplatin in ovarian cancer cells through its pregnane X receptor (PXR) antagonism. This nuclear receptor affects drug metabolism/efflux and drug-drug interaction through P-gp expression [36]. The lowest concentration of ketoconazole that presently demonstrated a positive effect was 10 µM, which is within the range of human plasma concentrations observed after a single oral dose of 200–400 mg [37]. Therefore, these non-toxic concentrations of ketoconazole can easily be achieved with clinical pharmacological doses of the drug and may be clinically well-tolerated if administered in combination with imatinib to treat patients with resistance to the standard drug.

Some additional mechanism by which ketoconazole is able to reverse resistance of imatinib could be related to regulation of P-gp/CYP3A4 by PXR, which regulates the expression of metabolic enzymes and transporters [38]. Ketoconazole has been reported as an inhibitor of PXR [39,40]. Therefore, it could regulate the transcription of the P-gp/CYP3A4 gene through disruption of PXR interaction with steroid receptor coactivator (SRC)-1.

A fundamental characteristic of cancer cells is their capacity to avoid apoptotic cell death. The cooperation between P-gp and molecules capable of inhibiting apoptosis-related proteins can generate a more robust drug resistance in cancer cells in general, and CML cells in particular. Among the numerous mechanisms utilized by CML cells to become resistant to imatinib, the avoidance of apoptosis is probably one of the most common.

The present study explored apoptosis as a possible mechanism of growth inhibition by the combination treatment. The Annexin V/PI assay, used for assessing early and late apoptosis, showed that imatinib plus ketoconazole increased apoptosis by 40%, whereas treatment with only imatinib or ketoconazole produced 25% greater apoptosis compared to control cells (Figure 5). Consequently, a plausible mechanism of ketoconazole for improving the efficacy of imatinib is its capacity to trigger apoptosis. Previous studies have reported that ketoconazole produce apoptosis inducing p53 levels and PARP cleavage in breast cancer cells [41], human colorectal and hepatocellular carcinoma cell lines [42]; therefore, a similar mechanism could be involved in the apoptosis of CML

On the other hand, Chen et al. recently reported that ketoconazole accelerates the process of apoptosis in hepatocarcinoma cells by exacerbating mitophagy, activating the PINK1/Parkin (PRKN) signaling pathway and downregulating cyclooxygenase-2 (COX-2), an inducible form of the enzyme that catalyzes the synthesis of prostanoids. The overexpression of COX-2 has been related with resistance to apoptosis [43]. In addition to these results, several reports reveled that the overexpression of COX-2 leads to increased P-gp expression [44]. Likewise, an overexpression of COX-2 and P-gp has been demonstrated in resistant K562 cells, with a decrease in apoptosis involving the Akt/p-Akt signaling pathway, which suggests the participation of COX-2 and P-gp in the development of resistance of CML cells [45]. We propose that ketoconazole stimulates apoptosis through COX-2 inhibition, in addition to P-gp inhibition, in imatinib-resistant cells (K562-RI).

Ketoconazole is an antifungal drug known to impede fungal growth by preventing the synthesis of ergosterol (the fungal equivalent of cholesterol) [46,47]. According to recent reports and the current findings, this drug seems to have great potential for cancer therapy [48–53].

Since its approval by the FDA in 1982, the estimated number of prescriptions of ketoconazole in the United States has been increasing every year. The oral use of ketoconazole

is well tolerated in patients with limited toxicity. In the CALGB 9583 trial, 2% of patients who received 400 mg of ketoconazole three times per day, had grade 3 or 4 hepatotoxicity. However, the low ketoconazole dose (200 mg) was less toxic; thus, for patients who cannot tolerate high dose treatment, the low dose would be an option [54]. In another report, Outeiro et al. reported that only 1.7% of patients who received ketoconazole (400 mg/day for 28 days) experienced liver function abnormalities [55]. Several reports in the literature have also described that ketoconazole has mild toxicity and is rarely fatal in comparison with other azoles as voriconazole, fluconazole, itraconazole among others. Ketoconazole toxicity can be reversed upon drug discontinuation [56–58].

Thus, ketoconazole is a safe drug with a relatively low cost compared to the high price of new medications for cancer. The price of new anti-cancer drugs reflects the corresponding research and development costs, generally around a billion dollars or more [59,60]. One strategy now employed more and more frequently is the repositioning of drugs, which involves giving approved drugs new applications. Among the advantages is the known profile of safety and efficiency.

The literature describes a large number of P-gp substrates already approved by the FDA; nevertheless, to date there are few reported studies evaluating P-gp inhibition in CML patients. Cyclosporine was one of the first drugs studied concerning clinical P-gp modulations; despite the results in cells, it also showed high toxicity [61,62]. Recently several in vitro studies, using P-gp inhibitors, supported the role of efflux activity of this protein in CML resistance. Liu et. al, in 2018, demonstrated that non-toxic concentrations of nelfinavir, an anti-HIV drug, reverse the resistance of adriamycin (doxorubicin), colchicine, paclitaxel and imatinib in k562/ADR cells that overexpressed P-gp. Nelfinavir, in addition to being a protease inhibitor drug approved for the treatment of AIDS patients, it has also been proposed as a new antitumor drug for the treatment of CML [63]. Elacridar, a potent P-gp inhibitor, approved recently by FDA, has demonstrated interesting results about to promote a synergic effect with imatinib in resistant cells [64]. Azithromycin, antimicrobial drug, may be another interesting alternative to overcome imatinib resistance according to the results described of its ability to inhibit P-gp function and increase intracellular accumulation of imatinib [65]. Although all these drugs (including ketoconazole), show interesting results as potential drugs for the treatment of resistant CML, clinical trials are needed to demonstrate their effectiveness in reversing resistance by targeting P-gp and prove their low systemic toxicity in CML patients.

#### **4. Materials and Methods**

#### *4.1. Cell Lines*

Human CML K562 cells were acquired from the American Type Culture Collection (ATTC, Rockville, MD, USA) and they were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, at 37 ◦C in a humidified atmosphere with 5% CO2.

#### *4.2. Development of the CML Cell Line with Resistance to Imatinib Treatment*

The K562 cells were seeded in a culture flask with a surface area of 25 cm<sup>2</sup> , they were exposed to gradually increasing concentrations (ranging from 1 nM to 2500 nM) of imatinib over various months, establishing an imatinib-resistant culture. After about eight months, the resistant phenotype, designated as K562-RI, was confirmed with the cell viability assay. To maintain the resistance of the K562-RI cells, 250 nM of imatinib were added to the culture medium.

To evaluate the resistance of K562-RI to imatinib, these cells were seeded into 96-well plates (Costar, Cambridge, MA, USA) at a density of 12 × 10<sup>3</sup> viable cells per well in 150 µL of culture medium. They were exposed to increasing concentrations of imatinib for 72 h. Cell viability to test cell resistance to imatinib was assessed with the sodium 3 ′ -[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) assay (Roche Molecular Biochemicals), which is based on the cleavage of yellow tetrazolium salt XTT to form an orange formazan dye by metabolically active cells.

Briefly, 50 µL of XTT were added to each well with K562-RI cells (for a final concentration 0.3 mg/mL), followed by incubation at 37 ◦C for 2 h in a humidified atmosphere containing 5% CO2. The parent K562 cell line was also tested with the XTT assay for comparison. After quantifying the absorbance of the samples from both cell lines spectrophotometrically at 492 nm with an ELISA microplate reader (Thermo Scientific, Waltham, MA, USA), the percentage of viability and relative resistance was calculated. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate. For each experiment, the resistance of the K562-RI cell line was confirmed using the XTT assay to calculate cell viability and corroborate relative resistance.

Additionally, the level of P-gp was determined by Western blot; three independent experiments were performed, and the intracellular accumulation of doxorubicin was evaluated as an indirect measure of P-gp activity (given that doxorubicin is considered a substrate for P-gp transport). Data are expressed as the mean ± SEM of three independent experiments.

#### *4.3. Treatments with Imatinib and Ketoconazole*

K562 and K562-RI cells were seeded into 96-well plates (Costar, Cambridge, MA, USA) at a density of 12 × 10<sup>3</sup> viable cells per well in 150 µL culture medium, and then exposed for 72 h to various amounts of imatinib alone (0–2500 nM) or imatinib plus ketoconazole (0.1, 1.0, and 10 µM). Control cells were only in contact with the vehicle. At the end of the exposure period, cell viability was examined with the XTT assay, as aforementioned. The mean concentration in each set of four wells was determined in triplicate. The dose–response relationship for imatinib applied alone or in combination with ketoconazole was characterized with a sigmoidal function. The percentage of growth inhibition was calculated, and the IC50 values (the concentration of the drug required to afford 50% growth inhibition) were obtained from the survival curve fitted to a nonlinear regression using the GraphPad Software, Prism 7.0 (San Diego, CA, USA) The equations used were: "Dose-response curves-Inhibition" and "log(inhibitor) vs. normalized response"; with the following function: Y = 100/(1 + 10ˆ((LogIC50-X) × HillSlope))). Where the HillSlope describes the steepness of curves, and the IC50 is the concentration that provokes a response halfway between the minimum and maximum response [66].

#### *4.4. P-Glycoprotein Expression Analyzed by Western Blotting*

To evaluate the P-gp expression levels in resistant cells, K562 and K562-RI cells (1 × 10<sup>6</sup> cells) were seeded and incubated for 24 h, they were recollected and centrifuged. The resulting pellets were washed three times with PBS, then homogenized with a lysis buffer containing protease inhibitors. To evaluate the P-gp expression levels in K562- RI cells exposed to imatinib in the absence and presence of ketoconazole, K562-RI cells (1 × 10<sup>6</sup> cells) were cultured overnight, then treated with imatinib (200 nM), ketoconazole (10 µM), or imatinib plus ketoconazole (200 nM/10 µM) during 4 h, then the cells were recollected, washed with PBS and homogenized with a lysis buffer. The proteins were separated by centrifugation at 10,000× *g* and 4 ◦C, quantified by the BCA (bicinchoninic acid) assay, and separated electrophoretically on 4–20% gradient gel (Mini-Protean TGX 456-1094, Bio-Rad Laboratories, Inc., Burns, TN, USA). Markers (Bio-Rad, Hercules, CA, USA) were included to establish protein size. Subsequently, the proteins were transferred from the gel onto PVDF membranes (Amersham, UK), which were blocked with 5% non-fat dry milk at room temperature for 2 h. Membranes were incubated overnight at 4 ◦C with antibodies against P-gp (12683, 1:500, Cell Signaling Technology, Danvers, MA, USA) and β-actin (sc-69879, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were washed and incubated with IRDye® 800 CW goat anti-mouse or IRDye® 680RD goat anti-rabbit secondary antibodies (1:15,000; LI-COR, Biotechnology, Inc., Lincoln, NE, USA) for 1 h and then scanned on an Odyssey Imaging System. Their intensity of fluorescence

was calculated by using Image Studio software. In each figure, representative blot images were selected from the same gel.

#### *4.5. P-Glycoprotein by Immunofluorescence Assay*

K562-RI cells (1 × 10<sup>6</sup> cells) were cultured overnight in culture slides (CultureSlides, Falcon Corning, NY, USA), then treated with imatinib (200 nM), ketoconazole (10 µM), or imatinib plus ketoconazole during 4 h. Afterwards, the cells were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min, permeabilized with 0.1% Triton X-100 for another 15 min and blocked with Ultracruz Blocking reagent (sc-516214, Santa Cruz, CA, USA) for 1 h. At the end of this time, they were incubated with monoclonal antibody Mdr-1 conjugated to Alexa Fluor 488 (sc-55510 AF488, 1:200) at 4 ◦C overnight. Subsequently, the cells were washed three times, and DAPI (ENZ-53003) reagent was used to counterstain the nuclei. Finally, immunofluorescence images were examined through an inverted fluorescence microscope (Olympus XI51).

#### *4.6. Assessment of the Intracellular Accumulation of Doxorubicin*

The intracellular accumulation of doxorubicin, a P-gp substrate, was determined in K562 and K562-RI cells to appraise P-gp activity. Briefly, the cells (1 × 10<sup>6</sup> cells) were seeded in triplicate in 25 cm<sup>2</sup> plates and exposed to doxorubicin (10 µg/mL) in the presence or absence of ketoconazole (10 µM) for 1 h. Control cells were only in contact with the vehicle. The cells were centrifuged after incubation, and the pellets were washed three times with ice-cold phosphate buffer solution (PBS). Subsequently, the cells were analyzed by flow cytometry (Guava® easyCyte, Merck Millipore), obtaining data from 10,000 acquired events with InCyte software (Merck Millipore, Darmstadt, DE, USA). The fluorescence of doxorubicin was quantified at 488 nm excitation and 575 nm emission wavelength.

#### *4.7. Determination of Apoptosis by Flow Cytometry*

Externalization of phosphatidylserine was evaluated with the Annexin-V-FLUOS Staining Kit. Cells (1 × 10<sup>6</sup> ) were seeded in Costar® 6-well Clear TC-treated Plates and exposed with imatinib alone (200 nM), ketoconazole alone (10 µM), or imatinib plus ketoconazole for 96 h. At the end of the incubation period, the cells were harvested, centrifuged at 2000 rpm for 5 min, washed once with PBS, and centrifuged again. Then they were resuspended in Annexin-V-FLUOS labeling solution, Annexin V-FITC (FITC), and propidium iodide (PI), to be incubated for 15 min at room temperature in the dark, according to the Annexin-V-FLUOS Staining Kit protocol. Flow cytometry was carried out to obtain 5 × 10<sup>3</sup> cells. The analysis of annexin was conducted with the BD FACS Canto II BD flow cytometry system, and BD FACSDiva software 6.0. The results were expressed as the total percentage of cells undergoing apoptosis. At least three independent experiments were performed for each assay.

#### **5. Conclusions**

The current study provides evidence that P-gp, a drug efflux pump, plays an important role in the development of resistance to imatinib in CML cells. In vitro testing presently showed the capacity of an inhibitor of P-gp, ketoconazole, to reverse CML cell resistance to imatinib treatment. In resistant CML cells, the antifungal ketoconazole inhibited the overexpression and efflux function of P-gp. Additionally, it increased the intracellular concentration of doxorubicin (a P-gp substrate) when resistant cells were incubated with doxorubicin plus ketoconazole. Thus, a possible corresponding increment in the intracellular concentration of imatinib may have taken place when resistant cells were incubated with this drug plus ketoconazole. Finally, ketoconazole triggered greater apoptosis of resistant CML cells. According to these findings on the mechanisms of action of ketoconazole in CML cells resistant to imatinib, the administration of imatinib plus ketoconazole is likely to favor reversal of resistance in CML patients treated unsuccessfully with imatinib

alone; therefore, we propose the repositioning of ketoconazole for the treatment of CML in patients resistant to imatinib.

**Author Contributions:** Conceptualization, P.G.-L.; methodology, O.P.-C., A.A.-R., R.J., M.L.-M. and J.P.-R.; software, R.J.; investigation, E.C.-C., O.P.-C. and A.A.-R.; writing, P.G.-L.; supervision, P.G.-L.; funding acquisition, P.G.-L. and E.C.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially financed by CONACYT (Mexico City, Mexico) through grant number: 142062.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available on request to the corresponding author.

**Acknowledgments:** We thank Bruce Allan Larsen for proofreading the manuscript.

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

#### **References**

