The Aminosteroid Derivative RM-133 Shows In Vitro and In Vivo Antitumor Activity in Human Ovarian and Pancreatic Cancers

Ovarian and pancreatic cancers are two of the most aggressive and lethal cancers, whose management faces only limited therapeutic options. Typically, these tumors spread insidiously accompanied first with atypical symptoms, and usually shift to a drug resistance phenotype with the current pharmaceutical armamentarium. Thus, the development of new drugs acting via a different mechanism of action represents a clear priority. Herein, we are reporting for the first time that the aminosteroid derivative RM-133, developed in our laboratory, displays promising activity on two models of aggressive cancers, namely ovarian (OVCAR-3) and pancreatic (PANC-1) cancers. The IC50 value of RM-133 was 0.8 μM and 0.3 μM for OVCAR-3 and PANC-1 cell lines in culture, respectively. Based on pharmacokinetic studies on RM-133 using 11 different vehicles, we selected two main vehicles: aqueous 0.4% methylcellulose:ethanol (92:8) and sunflower oil:ethanol (92:8) for in vivo studies. Using subcutaneous injection of RM-133 with the methylcellulose-based vehicle, growth of PANC-1 tumors xenografted to nude mice was inhibited by 63%. Quite interestingly, RM-133 injected subcutaneously with the methylcellulose-based or sunflower-based vehicles reduced OVCAR-3 xenograft growth by 122% and 100%, respectively. After the end of RM-133 treatment using the methylcellulose-based vehicle, OVCAR-3 tumor growth inhibition was maintained for ≥ 1 week. RM-133 was also well tolerated in the whole animal, no apparent sign of toxicity having been detected in the xenograft studies.


Introduction
As a major public health concern worldwide, cancer is responsible for one in four deaths in the USA and Canada [1,2].Ovarian and pancreatic cancers are two aggressive cancers that share the characteristics of spreading insidiously while displaying atypical symptoms, and readily shifting to a drug resistance phenotype.Ovarian cancer is a heterogeneous disease that afflicts yearly 225,000 women worldwide [3][4][5].It is the most lethal among the gynecologic malignancies, due to its asymptomatic nature in its early etiology, and the lack of efficient diagnostic tools [2,5].As a result, 75% of women already present themselves with advanced stages of ovarian cancer (stages III-IV of the International Federation of Gynecology and Obstetrics classification) when first diagnosed [3,[6][7][8][9].For these women, the 5-year survival rate varies from 25% to 35% [3,10] and the gold standard treatment is surgical debulking and chemotherapy based on a combination of paclitaxel-and platinum-based regimens [6,[11][12][13].Although initial response to ovarian cancer treatment is favorable, the majority of patients become resistant to currently used treatments and more than 90% are subject to relapses after 18 months [14,15].On the other hand, pancreatic cancer, which is the fourth most lethal cancer, is an aggressive neoplasm presenting with a very poor prognosis [1,2,16].Its 5-year survival rate is of only 6% [1] due to late initial diagnosis, rapid progression of disease, and resistance to chemotherapy regimens in current use [17].As pancreatic tumors are characterized by extensive local invasion and early lymphatic as well as hematogenous metastases [18], only few patients are candidates to resection, and therefore, systemic gemcitabine-based chemotherapy is the most currently used form of treatment [19].Despite recent improvements in drug development, the length and quality of life of pancreatic cancer patients have not improved [20].Thus, there is an urgent need to develop new approaches for the management of these diseases, which are among most aggressive and lethal cancers and have limited therapeutic options, especially for patients with unresectable pancreatic cancer, whose general condition rapidly deteriorates [16,20,21].
The above considerations indicate that the development of novel therapeutic agents acting via different mechanisms of action is critically needed to overcome the major problem of drug resistance encountered with these two cancer types, and therefore, to improve the overall survival of patients.Compound RM-133 (Fig 1) is an aminosteroid that has been developed in our laboratory [22].It has shown antiproliferative activity on several cancer cell lines (HL60 human promyelocytic leukemia cells, T-47D human breast carcinoma cells, WEHI-3 mouse myelomonocytic leukemia cells, and LNCaP human prostate cancer cells) with IC 50 values ranging from 0.1 to 2 μM [23].RM-133 also showed a low to moderate risk of drug-drug interactions based on its weak inhibition of the liver enzymes CYP3A4 and CYP2D6 [23].In a foregoing in vivo assay, RM-133 blocked by 57% the growth of HL60 tumor xenografted in nude mice [22].In a preliminary study of the mechanism of action of this family of aminosteroids, an analog of RM-133 blocked HL60 cells in G0/G1 phase and induced apoptosis [24].
Herein, we report the promising anticancer activity of RM-133 on human adenocarcinoma OVCAR-3 cells, which is a model for drug resistance investigation in ovarian cancer [25], and on human pancreatic carcinoma (PANC-1) cells.We also report pharmacokinetic studies on RM-133 involving various vehicles, as well as its antitumor activity in models of ovarian and pancreatic cancers, namely the OVCAR-3 and PANC-1 tumors xenografted into nude mice.

Cell viability assays
The cell proliferation assay was performed with a colorimetric method using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Cell Titer 96 Aqueous, Promega, Madison, WI).Microtiter 96-well plates (Becton-Dickinson Company, Lincoln Park, NJ) were seeded with 1 × 10 4 cells/well suspended in 100 μL of medium and pre-incubated for 24 h at 37°C in a 5% CO 2 atmosphere.A stock solution of RM-133 was prepared in EtOH (1×10 −2 M) and was diluted into experimental medium, which was added to each well at time zero.After a 72-h incubation, MTS (20 μL of the solution provided by the manufacturer) was added to each well and the plate incubated for 4 h.MTS is converted to water-soluble colored formazan by dehydrogenases present in metabolically active cells, and therefore, the MTS assay allows the immediate determination of absorbance of the soluble formazan directly in the cell medium and thus the measurement of viable cells.The A 490 of the medium was determined using a 96-well microplate reader INFINITE 200 PRO series (TECAN, Männedorf, Switzerland).The IC 50 value of RM-133 was determined for each of the two cell lines using the GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA).

Animals
All in vivo experiments were approved by our Institutional Animal Care and Use Committee (Comités de protection des animaux de l'Université Laval) and carried out according to the guidelines of the Canadian Council on Animal Care.For xenografts, homozygous ♀ nu/nu nude mice (24-42 days old) were purchased from Charles River Inc. (Saint-Constant, QC, Canada) and housed (four to five) in vinyl micro-isolated ventilated cages, equipped with air lids, which were kept in laminar airflow hoods and maintained under pathogen-limiting conditions.During the acclimatization and study period, the animals were housed under a controlled environment at 22 ± 3°C, with 50 ± 20% relative humidity and light set at 12 h/day (light on at 07:15).Rodent food (Rodent diet #T.2018.15,Harlan Teklad, Madison, WI) and water were provided ad libitum.For nu/nu nude mice, food and water were sterilized prior to dispensing to the animals.Standard Balb/c mice were used for pharmacokinetic studies.Animals were anesthetized with isoflurane and killed by cervical dislocation.

Effect of RM-133 on OVCAR-3 xenografts and drug plasma concentration in nude mice
First part.Twenty-two ♀ nu/nu nude mice (22-24 g) were inoculated s.c. with 5×10 6 OVCAR-3 cells (in 0.1 mL of growth medium containing 30% Matrigel (BD Biosciences, Bedford, MA)) into both flanks in each mouse via a 2.5-cm long 22-gauge needle.After 11 days, tumor-bearing mice were randomly assigned to two groups of 11 mice each according to tumor size, i.e. a control group (n = 12 tumors) and a treated group (n = 15 tumors).RM-133 was administered s.c.daily at 60 mg/kg in 0.1 mL of propylene glycol:EtOH (92:8).Animals in the control group received 0.1 mL of vehicle alone.Tumor size was measured twice weekly using a caliper.Two perpendicular diameters (L and W) were measured, and the tumor area (in mm 2 ) was calculated using the formula (L/2) x (W/2) x π.Animals were weighed at various intervals during the experiment.
Second part.At the end of the xenograft experiment, and in order to obtain preliminary pharmacological data, mice in treated groups were separated into 4 subgroups of 2-3 mice each, injected s.c. with RM-133 (60 mg/kg) and sacrificed after 3, 7, 12, or 24 h.In parallel, mice in the control group were also separated into 4 subgroups, treated with RM-133 (60 mg/kg) as for treated mice, and sacrificed at the same intervals.Blood was collected from mice by cardiac puncture, and the plasma concentration of RM-133 determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously reported [23].Tumors of the mice in the treated group were collected at necropsy, pooled, homogenized according to a known procedure [26] and the quantity of RM-133 was determined by LC-MS/MS.

Plasma concentration of RM-133 in mice at different injection doses
Eight ♀ Balb/c mice weighing approximately 20 g were separated in 4 groups.Mice in each group received a single s.c.injection of RM-133 (30, 60, 120, or 480 mg/kg).The vehicle used was propylene glycol:EtOH (92:8) and injection volume was 0.1 mL for all concentrations except at 480 mg/kg, where 0.2 mL was used.Blood was collected by cardiac puncture after 12 h, and the plasma concentration of RM-133 determined by LC-MS/MS as above.

Effect of RM-133 on PANC-1 xenografts
Twenty female nu/nu nude mice (22-24 g) were inoculated with 5×10 6 PANC-1 cells per flank, in 30% matrigel-containing culture medium (0.1 mL).Ten days after inoculation, tumor-bearing mice were randomized in 2 groups of 10 mice each.Mice in the first group (n = 16 tumors) were treated s.c. with RM-133 (240 mg/kg, twice a day (AM and PM), every other day) in 0.2 mL of aqueous 0.4% methylcellulose:EtOH (92:8).Mice in the second (control) group (n = 18 tumors) received the vehicle only.As with previous xenografts, the tumor area was measured and mouse body weight monitored.

Statistics
The Duncan-Kramer test was used to analyze data, and statistical significance accepted at P < 0.05 [36].

RM-133 is cytotoxic towards human OVCAR-3 and PANC-1 cells
To evaluate the effect of RM-133 on the proliferation of OVCAR-3 and PANC-1 cancer cells, the latter were incubated with increasing concentrations of RM-133 for 72 h, and the IC 50 of the drug then measured.RM-133 clearly had an antiproliferative activity on the tested cell lines, with IC 50 values of 0.8 and 0.3 μM for OVCAR-3 and PANC-1, respectively (Fig 2).The IC 50 values in the 0.1-1 μM range obtained here were similar to those previously measured in a panel of human cancer cell lines with this family of aminosteroids [23].
The OVCAR-3 cell line was derived from a patient refractory to cytotoxic chemotherapy, and is therefore an interesting model to investigate drug resistance [25].The fact that RM-133 exhibited potent antiproliferative activity on a model (OVCAR-3) known for its chemoresistance, as well as on PANC-1 cells, prompted us to further investigate the in vivo activity of RM-133 in two tumor xenograft models using these two cancer cell lines.

RM-133 inhibits growth of OVCAR-3 tumor xenografts
Female nude mice were inoculated in both flanks with OVCAR-3 cells.Mice bearing tumors were randomized in 2 groups: one group was treated with RM-133 (60 mg/kg) and the other (control) received the vehicle only (propylene glycol:EtOH (92:8)).Tumors in the control group grew clearly more rapidly than those treated with the aminosteroid (Fig 3A).Starting from day 15 of drug therapy and until the end of the experimental period, RM-133-treated tumors became significantly smaller than in the control group, with a 60% difference between the two groups measured at the end of the treatment.Since the toxic potential of a given compound depends on its concentration and exposure time [37], and as mortality, clinical symptoms, and body weight changes are among the main indicators of its toxicity [38], it is noteworthy that over a 21-day treatment period with RM-133, there was no apparent effect on body weight (Fig 3B) nor apparent toxicity of the drug (e.g.abnormal behavior, death).
At the end of the OVCAR-3 xenograft experiment, both control and RM-133-treated groups separately received a single s.c.dose of RM-133 (60 mg/kg).Blood was collected after 3, 7, 12, and 24 h, and the drug plasma concentration measured by LC-MS/MS.The time course of RM-133 plasma concentration was similar in groups A and B. The drug exhibited a classic drug-releasing profile [39], with a bolus delivery of RM-133 followed by a sustained decrease in its plasma concentration (Fig 4).The maximum average plasma concentration (460 ng/mL) was observed 3 h following the bolus injection, and had decreased to 81 ng/mL after 24 h.These results demonstrate that RM-133 does not accumulate in blood during the 21 days-xenograft experiment.Tumors of the mice treated with RM-133 were also collected at necropsy in order to measure the quantity of RM-133.This later was present inside the tumor at a concentration of 1.2 μM (773 ng/g), which corresponds roughly to the concentration of RM-133 that inhibits 50% of OVCAR-3 cell proliferation (IC 50 = 0.8 μM).This result is in accordance with the 60% reduction of tumor size progression observed in the first OVCAR-3 xenograft experiment.Thus, RM-133 is not sufficiently concentrated inside the tumor to fully inhibit its growth.

RM-133 plasma concentration exhibits a logarithmic dependence on the amount injected
In previous experiments, RM-133 had demonstrated a maximal antitumor activity of 57% against HL60 xenografts [32], which is comparable to the 60% inhibition value measured in this report towards OVCAR-3 xenografts.At the end of the OVCAR-3 xenograft experiment, reached after the injection of 30 mg/kg represents only 17% of the injected amount, and this percentage decreased further with higher doses of RM-133.Thus, the trend of the dose-level relationship suggests that increasing the RM-133 dose leads to a plateau, likely as a result of low plasma solubility or of the poor bioavailability of RM-133 in the vehicle used (propylene glycol:EtOH (92:8)).However, the dose of 480 mg/kg in 0.2 mL of vehicle was not tolerated by mice (Fig 5), which manifested with motion discomfort.Following these observations, the vehicle alone (0.2 mL of propylene glycol:EtOH (92:8)) was administered to the mice.As suspected, reactions were the same in the animals, indicating that the vehicle itself (propylene glycol: EtOH (92:8), at 0.2 mL) was the culprit.The maximum volume tolerated was next determined as 0.1 mL.These observations led us to investigate alternative vehicles that would allow to yield higher RM-133 plasma levels with minimal adverse effects.

Vehicle optimization: screening of alternative vehicles for optimal RM-133 administration
Non-aqueous solvents such as dimethyl sulfoxide and polyethylene glycol, detergents (e.g.Tween, vegetable oils, etc.) and solubilizers (e.g.β-cyclodextrin, methylcellulose) offer strategic options for enhancing compound solubility in the drug discovery process [30,39].For this reason, 11 different vehicles previously used with steroidal derivatives were investigated.Firstly, based on the solubility of RM-133 in each vehicle (Table A in S1 File), eight vehicles were selected for a second round of investigation.In the latter experiments, mice received a single s.c. injection of each drug-free vehicle for tolerance and behavioral evaluation (Table B in S1 File).Based on our observations at different post-injection times (0.25-96 h), we eliminated the vehicle #3 (castor oil-based).In fact, following the administration of vehicle #3 alone, mice displayed many discomfort symptoms (itching, jumping, and microphtalmia), and during the following four days, mice presented wounds averaging ~24 mm 2 at the site of injection.
In a third experiment, the seven best tolerated vehicles containing RM-133 were injected s.c. in mice (120 mg/kg) and blood samples collected after 3 and 12 h for RM-133 plasma concentration measurement (Table 1).The highest RM-133 plasma concentration reached after 3 h was obtained with vehicle #6, followed (in that order) by vehicles #5, #2, #1, #7, #4, and #8.As expected, RM-133 plasma concentration had decreased after 12 h, but vehicle #6 still yielded the highest levels after the latter interval.In contrast, vehicle #8 yielded the lowest plasma concentration at both time intervals.Based on the results obtained from the three experiments described above (Tables A and B in S1 File, Table 1), we concluded that (i) RM-133 was poorly soluble in vehicle #5 (small lumps), (ii) that vehicles #1 and #7 led to skin lesions and vehicle #8 yielded the lowest plasma concentrations of the analyzed set.On the other hand, vehicle #6 led to the highest plasma concentrations of the set, whereas vehicles #2 and #4 did not show any adverse effect on mice.We thus selected vehicles #2, #4, and #6 to perform an additional tolerance test.

Effect of repeated subcutaneous injections of RM-133 in 3 selected vehicles (tolerance test)
RM-133 was administered to mice every other day using vehicles #2, #4, and #6.Animals were treated for one week with vehicles #2 and #4, but only once with vehicle #6.Mice behavior was monitored during the experiment, and macroscopic observation of organs was performed at necropsy (Table C in S1 File).Ten hours following the s.c.injection of the first dose of RM-133 (120 mg/kg/0.1 mL) in aqueous 25% β-cyclodextrin:EtOH (92:8) (vehicle #6), mouse mobility was considerably reduced.Twenty-four hours later, their general condition had further deteriorated, as seen with seclusion, dehydration, microphthalmia, bristling, accelerated breathing rate, and abdomen distension.Based on these observations, we abruptly terminated experiment with vehicle #6, 24 h post-injection.At necropsy, all mice in that group showed hemorrhagic lungs, loaded stomachs along with empty intestines, and 75% exhibited slightly enlarged kidneys.When correlated with the mouse deaths previously noted when administering vehicle #6 alone, these observations clearly indicated that the cyclodextrin-based vehicle was not tolerated by mice, and vehicle #6 was therefore eliminated from our screening studies.
Interestingly, and in accordance with the observations from the experiment performed with vehicles only, no adverse effect was noted when mice were treated with RM-133 using vehicles #2 and #4.Because the oily vehicle #4 (sunflower oil:EtOH (92:8) generated blisters at the injection site, we limited treatment to a single injection of RM-133 (240 mg/kg/0.2mL /every other day) for the xenograft experiments.However, the aqueous vehicle #2 (aqueous 0.4% methylcellulose:EtOH (92:8)) allowed to perform multiple injections (240 mg/kg/0.2mL, BID/every other day) without adverse effect on mice.At necropsy, organs did not show any detectable abnormality.Thus, both vehicles #2 and #4 were included for comparison for the in vivo studies on RM-133 antitumor activity.

Antitumor activity of RM-133 on OVCAR-3 xenografts using two different vehicles
With the sunflower-based vehicle (Fig 6A ), RM-133 started to inhibit the tumor growth after 20 days, but the effect became significant only at day 28.By the end of treatment (40 days),

Antitumor activity of RM-133 on PANC-1 xenografts
As with ovarian cancer, pancreatic cancer typically shows resistance to currently available therapies and presents a very poor prognosis.Based on the results obtained with the OVCAR-3 xenografts, we used vehicle #2 (aqueous 0.4% methylcellulose:EtOH (92:8) to assess the effect

Conclusion
The cytotoxic activity of the aminosteroid RM-133 has been first evaluated in vitro in two human cancer cell lines, and the IC 50 values in the low micromolar range that were obtained (0.8 μM for OVCAR-3 (ovary) and 0.3 μM for PANC-1 (pancreas)) prompted us to assess the potential of RM-133 in tumor xenograft models.RM-133 (60 mg/kg/day) reduced OVCAR-3 tumor xenograft growth by 60% in nude mice, using propylene glycol:EtOH (92:8) as vehicle and s.c.injections.The failure to achieve a stronger tumor growth inhibition pointed towards a poor bioavailability of RM-133 with the vehicle used.Upon investigating the solubility, plasma concentration, and tolerance of RM-133 delivered with 11 different vehicles, two alternative vehicles (aqueous 0.4% methylcellulose:EtOH (92:8) and sunflower oil:EtOH (92:8)) were selected for further xenograft experiments.Thus, long-term treatment with RM-133 reduced PANC-1 pancreatic tumor growth by 63% in methylcellulose-based vehicle.Interestingly, RM-133 completely inhibited OVCAR-3 tumor growth using either sunflower-based and methylcellulose vehicles; and using the latter vehicle only, RM-133 further reduced tumor size down to 78% of its initial size.However, the antitumor response observed using the oily, sunflowerbased vehicle was delayed and less efficient compared to the methylcellulose-based vehicle.Furthermore, as observed with the methylcellulose-based vehicle, RM-133 maintained its antitumor action against the OVCAR-3 model, even one week after cessation of treatment.RM-133 was well tolerated by mice during the whole experimental period, and no weight loss was recorded.These promising results that have been obtained with ovarian and pancreatic cancer models, which are known as highly refractory to current therapies and as having a very poor prognosis, urge us to pursue our studies with the aminosteroid RM-133 and optimize its dosage, schedule, and mode of delivery, especially regarding its effect towards pancreatic cancer.

Fig 4 .
Fig 4. Time course of RM-133 plasma concentration following a single injection.RM-133 (60 mg/kg) in 0.1 mL of propylene glycol:EtOH (92:8) was injected s.c.into nu/nu nude mice.Group A: mice were previously treated daily for 21 days with RM-133 at 60 mg/kg; group B: mice not previously exposed to the drug.doi:10.1371/journal.pone.0144890.g004

Fig 5 .
Fig 5. Plasma concentration of RM-133 as a function of the dose (30, 60, 120, and 480 mg/kg) injected.RM-133 was measured by LC-MS/MS 12 h following a single s.c.injection in Balb/c mice, using propylene glycol:EtOH (92:8) as vehicle.The volume of injection is 0.1 mL except for the highest dose used (480 mg/kg), where 0.2 mL were used.The volume of 0.2 mL, with and without RM-133, produced some motion discomfort.doi:10.1371/journal.pone.0144890.g005

a
Single injection of RM-133 (120 mg/kg) in 0.1 mL of vehicle.b NT: this vehicle was not tested in this experiment based on tolerance and behavioral evaluation in mice.doi:10.1371/journal.pone.0144890.t001RM-133 had completely inhibited (i.e. by 100%) OVCAR-3 tumor growth.However, the oily nature of vehicle #4 generated big blisters, which also hampered tumor measurements.When RM-133 treatment was stopped at day 40 due to vehicle-induced effects, tumor growth resumed and no significant difference subsisted between the two groups seven days after treatment termination (Fig 6A).The latter results show that the aminosteroid RM-133 was responsible for tumor growth inhibition.No significant difference in body weight was observed between the untreated (control) and treated (RM-133) group (Fig 6B).Furthermore, no toxicity symptom was either observed during the study or at necropsy.

Table 1 .
Time course of RM-133 plasma concentrations with various vehicles.