Aminosteroid RM-581 Decreases Cell Proliferation of All Breast Cancer Molecular Subtypes, Alone and in Combination with Breast Cancer Treatments.

Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.

An Aminosteroid Derivative Shows Higher In Vitro and In Vivo Potencies than Gold Standard Drugs in Androgen-Dependent Prostate Cancer Models.

The aminosteroid derivative RM-581 blocks with high potency the growth of androgen-dependent (AR+) prostate cancer VCaP, 22Rv1, and LAPC-4 cells. Notably, RM-581 demonstrated superior antiproliferative activity in LAPC-4 cells compared to enzalutamide and abiraterone, two drugs that exhibited a synergistic effect in combination with RM-581. These findings suggest that RM-581 may have an action that is not directly associated with the hormonal pathway of androgens. Furthermore, RM-581 completely blocks tumor growth in LAPC-4 xenografts when given orally at 3, 10, and 30 mg/kg in non-castrated (intact) nude mice. During this study, an accumulation of RM-581 was observed in tumors compared to plasma (3.3-10 folds). Additionally, the level of fatty acids (FA) increased in the tumors and livers of mice treated with RM-581 but not in plasma. The increase was greater in unsaturated FA (21-28%) than in saturated FA (7-11%). The most affected FA were saturated palmitic acid (+16%), monounsaturated oleic acid (+34%), and di-unsaturated linoleic acid (+56%), i.e., the 3 most abundant FA, with a total of 55% of the 56 FA measured. For cholesterol levels, there was no significant difference in the tumor, liver, or plasma of mice treated or not with RM-581. Another important result was the innocuity of RM-581 in mice during a 28-day xenograft experiment and a 7-week dose-escalation study, suggesting a favorable safety window for this new promising drug candidate when given orally.

6-Polyaminosteroid Squalamine Analogues Display Antibacterial Activity against Resistant Pathogens.

A series of 6-polyaminosteroid analogues of squalamine were synthesized with moderate to good yields and evaluated for their in vitro antimicrobial properties against both susceptible and resistant Gram-positive (vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus) and Gram-negative (carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa) bacterial strains. Minimum inhibitory concentrations against Gram-positive bacteria ranged from 4 to 16 µg/mL for the most effective compounds, 4k and 4n, and showed an additive or synergistic effect with vancomycin or oxacillin. On the other hand, the derivative 4f, which carries a spermine moiety like that of the natural trodusquemine molecule, was found to be the most active derivative against all the resistant Gram-negative bacteria tested, with an MIC value of 16 µg/mL. Our results suggest that 6-polyaminosteroid analogues of squalamine are interesting candidates for Gram-positive bacterial infection treatments, as well as potent adjuvants to fight Gram-negative bacterial resistance.

Antibiofilm and Antivirulence Properties of 6-Polyaminosteroid Derivatives against Antibiotic-Resistant Bacteria.

The emergence of multi-drug resistant pathogens is a major public health problem, leading us to rethink and innovate our bacterial control strategies. Here, we explore the antibiofilm and antivirulence activities of nineteen 6-polyaminosterol derivatives (squalamine-based), presenting a modulation of their polyamine side chain on four major pathogens, i.e., carbapenem-resistant A. baumannii (CRAB) and P. aeruginosa (CRPA), methicillin-resistant S. aureus (MRSA), and vancomycin-resistant E. faecium (VRE) strains. We screened the effect of these derivatives on biofilm formation and eradication. Derivatives 4e (for CRAB, VRE, and MRSA) and 4f (for all the strains) were the most potent ones and displayed activities as good as those of conventional antibiotics. We also identified 11 compounds able to decrease by more than 40% the production of pyocyanin, a major virulence factor of P. aeruginosa. We demonstrated that 4f treatment acts against bacterial infections in Galleria mellonella and significantly prolonged larvae survival (from 50% to 80%) after 24 h of CRAB, VRE, and MRSA infections. As shown by proteomic studies, 4f triggered distinct cellular responses depending on the bacterial species but essentially linked to cell envelope. Its interesting antibiofilm and antivirulence properties make it a promising a candidate for use in therapeutics.

Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel.

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.

Association Between Intermediate-Acting Neuromuscular-Blocking Agents and Short-Term Postoperative Outcomes in Patients with Gastric Cancer.

PURPOSE: This study examined whether different neuromuscular-blocking agents (NMBAs) work differently on the short-term outcomes of gastric cancer patients in terms of laboratory test results and severity of postoperative illness, and whether the effect is dose-related. PATIENTS AND METHODS: Data of 1643 adult patients receiving gastric cancer surgery were analyzed by employing generalized linear models (GLMs), to explore the effects of different NMBAs on neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR) at postoperative day 1 (POD1), POD3, POD7, and return to intended oncologic therapy (RIOT), among others. We adjusted multiple covariants, including patient-, anesthesia-, and surgical complexity-related risk factors. RESULTS: Without adjusting dosage of NMBAs, POD1NLR, POD1PLR (P < 0.05), POD3NLR, POD7NLR, POD3 lymphocytes, POD7LMR (P < 0.01) in gastric cancer patients administered with benzylisoquinoline NMBAs worsened, and the administration of aminosteroidal NMBAs was associated with less risk of transfer to ICU (P < 0.01); without adjusting the types of NMBAs, the highest dose of NMBAs postponed the RIOT (P < 0.05) and was negatively associated with POD3NLR, POD7NLR and POD7LMR (P < 0.01), and increased risk of postoperative transfer to ICU (P < 0.01). When patients given benzylisoquinolines were re-divided in terms of five equal quintiles, from low to high dose, RIOT was delayed and POD7LMR decreased significantly in the fourth and fifth quintile groups as compared to the first quintile group. A higher risk for postoperative transfer to ICU was found in the fifth quintile group as compared to the first quintile group. CONCLUSION: Patients with gastric cancer given benzylisoquinoline NMBAs had more unfavorable short-term outcomes, such as more severe inflammation and increased risk of transfer to ICU than their counterparts administered aminosteroidal NMBAs, and the effect of benzylisoquinolines was dose-related. The effect of aminosteroids on short-term outcomes was not dose-related in the dosage range we used.

Minor chemical modifications of the aminosteroid derivative RM-581 lead to major impact on its anticancer activity, metabolic stability and aqueous solubility.

The aminosteroid (AM) RM-581 is built around a mestranol backbone and has recently emerged as this family's lead candidate, showing in vitro and in vivo potency over different types of cancer, including high fatality pancreatic cancer. To extend the structure-activity relationships (SAR) to other estrane analogs, we synthesized a focused series of RM-581 derivatives at position C3 or C2 of its steroidal core. These new AM derivatives were first tested on a large selection of prostate, breast, pancreatic and ovarian cancer cell lines. The impact of these modifications on metabolic stability (human liver microsomes) was also measured. A SAR study revealed a fine regulation of anticancer activity related to the nature of the substituent. Indeed, the addition of potential prodrug groups like acetate, sulfamate or phosphate (compounds 8, 9 and 10) at C3 of the phenolic counterpart provided better antiproliferative activities than RM-581 in breast and pancreatic cancer cell types while maintaining activity in other cancer cell lines. Also, the phosphate group was highly beneficial on water solubility. However, the bulkier carbamate prodrugs 6 (N,N-dimethyl) and 7 (N,N-diethyl) were less active. Otherwise, carbon homologation (CH2) at C2 (compound 33) was beneficial to metabolic stability and, in the meantime, this AM conserved the same anticancer activity as RM-581. However, the replacement of the hydroxy or methoxy at C3 by a hydrogen or an acetyl (compound 17 or 21b) was detrimental for anticancer activity, pointing to a crucial molecular interaction of the aromatic oxygen atom at this position. Overall, this work provided a better knowledge of the structural requirements to maintain RM-581's anticancer activity, and also identified minor structural modifications to increase both metabolic stability and water solubility, three important parameters of pharmacological development.

Induction of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression in PANC-1 xenograft model.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC50 = 3.9 and 4.3 µM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60 mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

A 3D-QSAR Study on the Antitrypanosomal and Cytotoxic Activities of Steroid Alkaloids by Comparative Molecular Field Analysis.

As part of our research for new leads against human African trypanosomiasis (HAT), we report on a 3D-QSAR study for antitrypanosomal activity and cytotoxicity of aminosteroid-type alkaloids recently isolated from the African medicinal plant Holarrhena africana A. DC. (Apocynaceae), some of which are strong trypanocides against Trypanosoma brucei rhodesiense (Tbr), with low toxicity against mammalian cells. Fully optimized 3D molecular models of seventeen congeneric Holarrhena alkaloids were subjected to a comparative molecular field analysis (CoMFA). CoMFA models were obtained for both, the anti-Tbr and cytotoxic activity data. Model performance was assessed in terms of statistical characteristics (R², Q², and P² for partial least squares (PLS) regression, internal cross-validation (leave-one-out), and external predictions (test set), respectively, as well as the corresponding standard deviation error in prediction (SDEP) and F-values). With R² = 0.99, Q² = 0.83 and P² = 0.79 for anti-Tbr activity and R² = 0.94, Q² = 0.64, P² = 0.59 for cytotoxicity against L6 rat skeletal myoblasts, both models were of good internal and external predictive power. The regression coefficients of the models representing the most prominent steric and electrostatic effects on anti-Tbr and for L6 cytotoxic activity were translated into contour maps and analyzed visually, allowing suggestions for possible modification of the aminosteroids to further increase the antitrypanosomal potency and selectivity. Very interestingly, the 3D-QSAR model established with the Holarrhena alkaloids also applied to the antitrypanosomal activity of two aminocycloartane-type compounds recently isolated by our group from Buxus sempervirens L. (Buxaceae), which indicates that these structurally similar natural products share a common structure⁻activity relationship (SAR) and, possibly, mechanism of action with the Holarrhena steroids. This 3D-QSAR study has thus resulted in plausible structural explanations of the antitrypanosomal activity and selectivity of aminosteroid- and aminocycloartane-type alkaloids as an interesting new class of trypanocides and may represent a starting point for lead optimization.

Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133.

The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.

UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.

Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 µL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.

Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstane-3α,17ß-diol derivatives.

Aminosteroid derivatives represent a new family of compounds with promising antiproliferative activity over different cancer cell lines. Among all the aminosteroid derivatives synthesised in our laboratory, we have identified E-37P as one of the more potent when tested in vitro. Unfortunately, the pharmacokinetic properties of E-37P decrease its effectiveness when tested in vivo. To improve the bioavailability and increase the efficiency of aminosteroid E-37P, two series of analog compounds were synthesised by classic chemical synthesis, they were then characterized, and the concentration that inhibits 50% of cell proliferation (IC50) was determined on different cell lines. RM-133, a 5α-androstane-3α,17ß-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2ß and an ethinyl at position 17α, showed very good antiproliferative activity among the five cancer cell lines studied (IC50=0.1, 0.1, 0.1, 2.0 and 1.1 µM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively). Moreover, the plasmatic concentration of RM-133 at 3h, when injected subcutaneously in rats, was 2.3-fold higher than that of E-37P (151 vs 64.8 ng/mL). Furthermore, RM-133 weakly inhibited the two representative liver enzymes, CYP3A4 and CYP2D6, indicating a very low risk of drug-drug interactions. The cytotoxicity of RM-133 against normal cells was tested on peripheral blood lymphocytes (PBL) obtained from different donors and previously activated with phytohemagglutinin-L. PBL responded differently to treatment with RM-133, we observed a stimulation of cell proliferation and/or cytotoxicity in a dose-dependent manner. Based on these results, additional studies are currently underway to evaluate the selectivity of our lead compound against normal cell lines in a more detailed fashion.

Effect of 21-aminosteroid U74389G on the Extent of Brain Damage and Edema in the Newborn Rats with Hypoxic-ischemic Injury

PURPOSE: To investigate the effect of 21-aminosteroid U74389G (U) on the extent of brain damage and edema formation in the newborn rats with hypoxic ischemic (HI) brain injury. METHODS: This is a randomized, placebo-controlled, experimental study. The subjects were 113 seven-days-old rats with HI injury. Pups were treated with 3, 10, or 20 mg/ kg of U intraperitoneally 30 minutes before hypoxia (Group 1, 2, 3: n=10, 13, 11), 10 mg/kg of U immediately after hypoxia (n=11) (Group 4), 10 mg/kg of U 30 minutes before and after hypoxia (n=n=13) (Group 5), or vehicle (n=12) (Group C). We expressed the degree of brain infarction and brain edema in % atrophy (Left hemisphere-Right hemisphere/Left hemispherex100) and water content % (wet weight-dry weight/wet weightx100) RESULTS: There were significant reductions in the diameters of right hemisphere compared with those of left hemisphere in vehicle and U treated animals (P<0.05). As to the cortical thickness, group 2, 3 and 5 pups showed no significant reductions in the right side compared with the left side implicating that U treatment in these groups was of benefit in attenuating HI cortical injury, while there was significant difference between the right and left side in group 1, 4 and C animals (P<0.001). There was a significant difference (P< 0.01) in % atrophy of group 2, 3, 5 versus group C, but the mean % atrophy was similar in groups 1, 4 and C. There was a significant (P<0.05) increase of water content in right hemisphere compared with left hemisphere both in U and vehicle treated groups. CONCLUSION: Pre-treatment and prepost-treatment at moderate doses (10 or more mg/kg) of 21-aminosteroid U74389G reduced the extent of perinatal hypoxic-ischemic brain damages, especially in the cortex, but do not affect the extent of brain edema.

Effect of U74389G on Neurological Deficit and the Formation of Brain Edema after Focal Ischemia in Rats

In the early stage of the development of ischemic brain injury, oxygen free radical-mediated lipid peroxidation is suspected to play an important role. U74389G is considered as one of the effective scavengers of free radicals and inhibitors of lipid peroxidation. However, the cytoprotective effect of U74389G in rodent models of neocortical infarction still remains controversial. Forty-four adult Sprague-Dawley rats weighing 250-300gm were subjected to permanent or transient middle cerebral artery occlusion(MCAO) using modified Longa's method while anesthetized with ketamine and xylazine. In the permanent MCAO groups, the animals were subjected to 4-hours of occlusion and were treated with vehicle(control, n=9), 3mg/kg U74389G(low-dose treatment, n=9) or 7mg/kg U74389G(high-dose treatment, n=10) intravenously at the time of occlusion and then half of the initial dose was administered 30 minuites after occlusion respectively. In the transient MCAO groups, the animals were subjected to 0.5 hours of occlusion and 3.5-hours of reperfusion and were treated with vehicle(control, n=8) or 3mg/kg U74389G(treatment, n=8) intravenously at the time of occlusion and then half of the initial dose was administered at the time of reperfusion respectively. Four hours after occlusion neurological evaluation was performed and then the animals were sacrificed immediately to determine brain water content. In the rats of permanent MCAO, treatment with both low and high-dose U74389G did not protect them against neurological deficit;however, treatment with high-dose U74389G was found to reduce brain edema in the ischemic core(19%, p<0.05) and intermediate regions(23%, p<0.05). In the rats of transient MCAO, treatment with U74389G was found to protect them against neurological deficit(p<0.05) and reduce brain edema in the ischemic core region(58%, p<0.05). These results suggest that free radical-mediated lipid peroxidation plays a significant role in focal ischemic reperfusion brain injury and that 21-aminosteroids might have the potential to be a useful supplementary drug as a cerebral protective agent for the thrombolytic therapy of acute cerebral infarction.

The Effects of 21-Aminosteroid U74389F on Posttraumatic Spinal Cord Blood Flow and Somatosensory Evoked Potential

A growing body of biochemical, physiological, and pharmacological evidence has suggested that oxygen free radical-induced lipid peroxidation plays a key role in progressive posttraumatic spinal cord ischemia. Recently, it has been reported that the newly developed compound, U74006 F, a non-glucocorticoid 21-aminosteroid, is extremely potent as an inhibitor of lipid peroxidation and effective preventor from the posttraumatic ischemia. In this investigation, the effects of 21-aminosteroid U74389F, analog of U74006F on posttraumatic spinal cord blood flow and somatosensory evoked potential have been studied in cats. The results of this study are summarized as follows:(1) U74389F, given 3 mg/kg, blood flow decreased significantly and somatosensory response returned to none of five cats. (2) U74389F, given 10 mg/kg or 20 mg/kg, the blood flow did not decreased and maintained near the preinjury level. The recovery rate of somatosensory evoked potential ranged from 40 to 60%. (3) There was no statistical difference of blood flow change between the cats treated with 10 mg/kg and those treated with 20 mg/kg of U74389F. It is thought that the adequate dosage of U74389F to prevent posttraumatic spinal cord ischemia is more than 10 mg/kg or 20 mg/kg of U74389F, was statistically significant compared to much more decrease in naloxone-treated cats. The reason is thought to be much longer half life of U74389F in serum. From the above results, it is speculated that U74389Fhas beneficial effect on posttraumatic spinal cord ischemia and functional recovery. The effects last longer than those of naloxone.