RAS; PPMTase (Ki = 2.6 μM)

Salirasib (40, 60, or 80 mg/kg, p.o.) significantly and dose-dependently inhibits the growth of tumors in vivo[1]. Salirasib (5 mg/kg, i.p.) causes an increase in Ras expression that is noticeably smaller than the increase seen in the dy^2J/dy^2J mice, and it also significantly reduces Ras expression in the dy^2J/dy^2J mice. In the dy^2J/dy^2J mice, salirasib treatment is linked to a significant inhibition of MMP-2 and MMP-9 activities[2]. In a model of subcutaneous xenograft mice, salirasib (10 mg/kg, i.p.) inhibits tumor growth without causing weight loss[3].

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FTS/Salirasib inhibits growth of ELT3 tumors in vivo [1]
Next, we examined the effects of FTS on ELT3 tumor growth in nude mice, i.e., on cell transformation in an in vivo model, as described in Material and Methods. In each of three independent experiments with different FTS doses, mice received 4 × 106 cells subcutaneously (s.c.) in the right flank, as described.21, 27 Treatment was started 5 days later, when the mice in the three experimental groups (n = 10 per group) received daily oral FTS (40, 60 or 80 mg/kg), whereas mice in a control group (n = 10) received only vehicle (carboxymethylcellulose). The mice were killed 54 days after the beginning of treatment. As shown in Figure 4a, from Day 33 onward, tumor growth was significantly inhibited by FTS at 80 mg/kg, and by Day 54 was inhibited by ∼ 85% compared to the control. This growth inhibition was dose dependent (Fig. 4b). At the end of the experiment (Day 54), when tumors were removed and weighed, FTS (80 mg/kg) had reduced tumor weight by 65.8 ± 12.5% (n = 10, p < 0.01; Fig. 4a). Notably, none of the mice, whether control or FTS-treated, had died even after 2 months. More detailed calibration may be needed to examine a possible effect of FTS on survival of the mice.

Parts of the tumors were also sectioned and stained with hematoxylin and eosin (Fig. 5A), which stains nucleic acids and cytoplasm, respectively. We also performed TUNEL staining for apoptotic cells (Fig. 5B). Pathological examination revealed much slower tumor cell proliferation in the Salirasib/FTS-treated groups than in the control. Shown in Figure 5A are typical sections (at magnifications 4×, 10× and 40×) of tumors from two FTS-treated mice; one tumor is relatively large (Figs. 5A-d–5A-f) and the other is a small tumor almost completely eradicated by FTS (Figs. 5A-g–5A-i). Also shown is a section of a tumor from a control mouse (Figs. 5A-d–5A-f). Four additional sections from each group yielded similar results (not shown). As shown in Figure 5A, FTS treatment (60 mg/kg, 40 days) caused a marked decrease in the number of tumor cells in the sections (white arrow), whereas, at the same time, the amounts of fibrotic connective tissue in those tumors were increased (black arrow). Statistical analysis of the stained sections from five mice in each group showed that the percentage of area stained was significantly smaller in the FTS-treated groups than in the control group (14.2% ± 3.6% compared to 25.7% ± 3.1%; n = 5, p < 0.05). Notably, TUNEL staining revealed no significant cell death and no difference between the control and the FTS-treated mice (n = 5; Fig. 5B). These results are consistent with the results of the in vitro experiments, which indicated that FTS inhibited the growth of ELT3 cells but did not induce cell death (Fig. 1).

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Ras expression and Ras-GTP [2]
WT and dy2J/dy2J mice were treated with either Salirasib/FTS or control for 12 weeks. Immunoblotting of skeletal muscle was carried out at the end of the study using anti-Ras antibody. Untreated dy2J/dy2J mice showed significantly higher Ras expression compared to the WT group (555.08±66.32 vs. 100±22.45 densitometry, percent of control; P<0.01; Figure 1A). FTS treatment was associated with significantly decreased Ras expression in the dy2J/dy2J mice (205.76±19.37; P<0.01). Treatment of WT mice with FTS caused an increase in Ras expression which was by far much lower than the increase observed in the dy2J/dy2J mice (206.76±29.37). In addition to Ras expression, Ras activity was measured at the end of the study by the Ras binding domain pull-down assay. In this assay the GTP-bound Ras is detected by its preferential binding to the RBD domain of Raf1 which is conjugated to sepharose beads [26]. We found that Ras-GTP levels were higher in dy2J/dy2J compared to the levels in WT mice (157.40±14.53 vs. 100±10.23; P<0.05. Figure 1B). The levels of Ras-GTP were significantly reduced in the FTS treated dy2J/dy2J group (97.16±10.54; P<0.01). Moreover, Ras-GTP in the treated dy2J/dy2J normalized and was comparable to the WT group. Treatment of the WT mice with FTS induced an increase in Ras-GTP (141.72±12.4) comparable to the increase in Ras expression in these mice. The nature of these increases observed in the WT mice is not known.
In addition, we measured the phosphorylation of ERK, a Ras downstream protein (Figure 1C). ERK phosphorylation was very high in the dy2J/dy2J mice compared to the WT group (424.97±63.85 vs. 100±21.56; P<0.02) while, Salirasib/FTS treatment significantly decreased this phosphorylation (175.62±21.53; P<0.02). Small increase in pERK was noted in the WT FTS (169.29±31.1) while, total ERK did not change at all.

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Muscle strength [2]
Total peak force was determined once a week using an electronic Grip Strength Meter. Significant difference in hind limb muscle strength was detected between the untreated dy2J/dy2J and the WT mice throughout the study (P<0.01; Figure 2). A significant increase in hind limb muscle strength was noted in the Salirasib/FTS treated compared to the untreated dy2J/dy2J mice (P<0.05). During the study period muscle strength increased from 3.01±0.27 to 4.79±0.22 (gram force/gram body weight) in the treated dy2J/dy2J mice, while remained unchanged from 2.67±0.22 to 2.72±0.19 in the untreated dy2J/dy2J group. Moreover, at the end of the trial the hind limb muscle strength of the dy2J/dy2J treated mice completely normalized to that of both WT groups (4.79±0.22 vs. WT: 4.64±0.39; WT+FTS: 4.77±0.25). Such a difference was not detected in the fore (stronger) limbs of the dy2J/dy2J mice (Figure S1), and in the fore and hind limbs of the treated and untreated WT groups.

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Muscle histology [2]
In comparison with the normal WT (Fig. 3A), Hematoxylin and eosin staining of untreated dy2J/dy2J mouse quadriceps muscle at the age of 18 weeks showed severe advanced dystrophic changes with abnormal variation of fiber size, internal nuclei and severe excessive fibrosis (Fig. 3B). The Salirasib/FTS treated dy2J/dy2J muscle showed considerable fibrosis attenuation (see next paragraph) but still abnormal myopathic changes with variation in fiber size, and increased number of central nuclei (Fig. 3C).

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Salirasib inhibits tumour growth in a subcutaneous xenograft model [3]
Finally, we assessed the in vivo antitumor activity of salirasib in a subcutaneous xenograft model of HepG2 cells in nude mice. From 5 days of treatment onwards, salirasib induced a statistically significant decrease in tumour volume (figure 8A). After 12 days of salirasib treatment, the mean tumour weight was 131.7 ± 18.9 mg compared with 297.5 ± 48.2 mg in the control group (vehicle), indicating that salirasib reduced tumour growth by 56 percent (figure 8B). Moreover, no overlap in tumour weight was observed between the control and the treatment groups, meaning that even the smallest tumour in the control group remained larger than the biggest tumour in the treatment group (figure 8C). Animals remained well throughout the entire experiment and no weight loss was observed upon treatment, suggesting that salirasib was well tolerated at this dose regimen (data not shown).

For methyltransferase assays in cell-free systems, total membranes of cultured cell lines (100,000 × g pellet) or synaptosomal membranes of the rat brain cerebellum are utilized. 50 μL of 50 mM Tris-HCl buffer, pH 7.4, 100 μg of protein, 25 μM [methyl-3H]AdoMet (300,000 cpm/nmol), and 50 μM AFC (prepared as a stock solution in DMSO) are used in methyltransferase assays. The assays are conducted at 37°C. 8% DMSO is used in all of the assays. As stated in the text, different AFC concentrations are used in a number of experiments. After 10 minutes, the reactions are stopped by adding 500 μL of a 1:1 chloroform:methanol mixture, followed by mixing and phase separation, and 250 μL of H2O. A 200 μl solution of 1 N NaOH and 1% SDS is added to a 125-μL portion of the chloroform phase that has been dried at 40°C. The vapor phase equilibrium method is used to count the methanol that is thus formed. When AFC is added, typical background counts (without AFC) range from 50 to 100 cpm, while typical reactions yield 500 to 6,000 cpm. Three duplicates of each assay are run, and background counts are deducted. Gel electrophoresis and methylation of endogenous substrates are carried out. In intact cells, protein carboxyl methylation is measured with 100 μCi/mL [methyl-3H]methionine. Nearly confluent cultures of the cells are cultivated in 10-cm plates with 5 mL of labeling medium before analysis.

Cells are harvested using 0.05% Trypsin-EDTA every day for one to seven days in time-dependent response studies. The Trypan blue exclusion method is then used under a microscope to count the cells. Cells are cultured in medium supplemented with salirasib or DMSO for three days in order to conduct dose response studies. As directed by the manufacturer, a colorimetric WST-1 assay is used to assess cell viability. Using GraphPad Prism software, nonlinear regression analysis is used to determine the IC50 value, or the point at which 50% of the cell growth is inhibited when compared to the DMSO control.

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Fluorescence-activated cell-sorter analysis [1]
For cell-death evaluation, cells were seeded at a density of 2 × 105 cells per 10-cm plate, treated for 72 hr with Salirasib/FTS, then collected and assayed by double staining with annexin-V/propidium iodide (PI) according to the manufacturer's instructions. The results obtained by FACSCalibur flow cytometry were analyzed with CBA software.

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Clonogenic assay [1]
ETL3 and TSC2-expressing ELT3 cells were plated at 2.5 × 104 cells per 24-well plate, grown for 24 hr and then treated with Salirasib/FTS or 0.1% Me2SO4 (control). After 3 days, the cells were detached and plated on 10-cm plates (1:50 dilution). Four days later, the cells were stained with crystal violet and colonies were counted.

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Immunoblot analysis [1]
ETL3 cells were plated at a density of 4 × 105 cells per 10-cm plate, grown for 24 hr and then treated with Salirasib/FTS or 0.1% Me2SO4 (control). Cells were lysed as described,14 and the lysates (100 μg protein) were immunoblotted with mouse anti-pan-Ras Ab, rabbit anti-Rheb Ab, rabbit anti-pS6K Ab, anti Flag Ab, rabbit anti-S6K Ab, mouse anti-pERK Ab, rabbit anti-ERK Ab and rabbit anti-β-tubulin Ab. Immunoblots were exposed to the appropriate secondary peroxidase-coupled IgG and subjected to enhanced chemiluminescence. Protein bands were quantified by densitometry with Image EZQuant-Gel Statistical Analysis Software.

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Total RNA purification and real-time PCR analysis [1]
Total RNA was isolated from untreated or 75 μM Salirasib/FTS-treated ELT3 cells using the RNeasy Plus Mini Kit. Purified RNA was used for real-time PCR as described.16 The following primers were used to target the Rheb gene: forward 5′-GCTATCGGTCTGTGGGAA-3′, reverse 5′-CTGCCCTGCTGTGTCTACA-3′ and for the housekeeping gene GAPDH: forward 5′-CCAGAACATCATC CCTGC-3′, reverse 5′-GGAAGGCCATGCCAGTGAGC-3′. mRNA expression of the target gene was normalized to the expression of the GAPDH reference gene.

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Measurement of Rheb.GTP [1]
The experiment was performed as described elsewhere.17 Briefly, ETL3 cells were plated at 4 × 105 cells per 10-cm plate, grown for 24 hr and then treated with Salirasib/FTS or 0.1% Me2SO4 (control). After 2 days, the cells were starved overnight and then switched to phosphate-free medium for 30 min before being labeled for 4 hr with 0.25 mCi/ml [32Pi] orthophosphate. Lysates from harvested cells were immunoprecipitated for 1 hr with protein-G sepharose beads and sc-6341 anti-Rheb Ab. Nucleotides were eluted at 65°C and subjected to thin-layer chromatography (TLC). GTP and GDP were used as markers and were detected with iodine. Signals were detected by exposure to X-ray film for 6 days at −80°C with intensifying screens. GDP and GTP were quantified by densitometry with Image EZ-Quant-Gel Statistical Analysis Software. The Rheb-bound GTP percentage was calculated as [GTP/(GDP × 1.5) + GTP] × 100%.

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Growth inhibition studies [3]
For time dependent response studies, cells were harvested with 0.05% Trypsin-EDTA daily for 1 to 7 days and counted under the microscope using the Trypan blue exclusion method. For dose response studies, cells were incubated in medium supplemented with Salirasib or DMSO for 3 days. Cell viability was determined using a colorimetric WST-1 assay according to the manufacturer's instructions.

Female athymic NMRI nu/nu mice, aged six weeks, are kept in cages with filters on top and are given unlimited access to food and drink. Twelve mice receive a subcutaneous injection of 5x10⁶ HepG2 cells suspended in 100 μL PBS to create tumors in their right lower flank. When palpable tumors have developed two weeks after cell inoculation, mice are divided into two groups: one for Salirasib treatment (n = 6) and the other for control (n = 4). Two of the animals had to be removed from the study because they do not develop tumors at that point. For 12 days, they are given daily intraperitoneal injections (10 mg/kg salirasib) or an equivalent volume of vehicle solution (PBS with 2.5% v/v ethanol, pH 8.0). A digital calliper is used to record tumor dimensions three times a week beginning on the first day of treatment. To estimate tumor volumes, use the formula V (mm³)=(length×width²)/2. To assess the effectiveness of treatment, tumour weights are noted at the moment of sacrifice.

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Athymic nude mice (6 weeks old) were housed in barrier facilities on a 12-hr light/dark cycle with unlimited food and water. Mice in the experimental group received orally administered Salirasib/FTS (0.1 ml) daily. Subcutaneous tumors were measured with a caliper, and animal weights were recorded every 4 days. Tumor volumes were calculated using the formula: [length × width] × [(length + width)/2]. [1]

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Following previous protocols in the rat model of liver cirrhosis, WT and dy2J/dy2J mice were injected intra-peritoneally 3 times a week with Salirasib/FTS 5 mg/kg or control solution (see below), for 12 weeks from the age of 6 weeks (n = 7/group, each group consisted of 4 males and 3 female mice). At the end of the study both hind limb muscles were dissected. Part of the muscle sample was frozen in liquid nitrogen and stored at −80°C for biochemical analysis. Quadriceps femoris muscle was rapidly frozen in isopentane pre-chilled by liquid nitrogen for cryostat sections and histology. [2]

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Six week old female athymic NMRI nu/nu mice were housed in filter-topped cages and received food and water ad libitum. Tumors were generated by subcutaneous injection into the right lower flank with 5 × 106 HepG2 cells suspended in 100 μl PBS in 12 mice. Two weeks after cell inoculation, when palpable tumours were established, mice were separated into Salirasib-treated (n = 6) and control group (n = 4). Two animals did not develop tumours at that time point and had to be excluded from the study. They received daily i.p. injections of 10 mg/kg Salirasib or a similar volume of vehicle solution (PBS containing 2.5% v/v ethanol, pH 8.0) for 12 days. Tumor dimensions were recorded three times per week with a digital calliper starting with the first day of treatment. Tumor volumes were estimated as follows: V (mm³) = (length × width²)/2. Tumour weights were recorded at the time of sacrifice in order to evaluate treatment response. [3]

Plasma pharmacokinetics of thaliracidib [4]
Evaluable pharmacokinetic data were obtained from 24 patients, involving 41 pharmacokinetic study periods (Table 4). The pharmacokinetic characteristics of thaliracidib were slow absorption and rapid elimination after oral administration (Figure 1). No accumulation occurred because the thaliracidib exposure (Cmax; AUCinf on day 1 and AUC0-12 h on day 15) was similar (P > 0.05). On day 1, T_1/2 (mean ± standard deviation) was 3.6 ± 2.2 hours. On days 1 and 15, the ClS/F (mean ± standard deviation) of salirasib were 103.0 ± 112.5 and 73.3 ± 37.3 L/h, respectively, and its distribution exceeded blood volume on both days 1 and 15, with V/F of 458.0 ± 466.1 and 255.7 ± 187.8 L, respectively (Table 4). Although most pharmacokinetic parameters were variable, paired analysis showed that salirasib drug exposure (C_max and AUC) and ClS/F were similar on days 1 and 15 (P > 0.05). No accumulation of Cmax or AUC was observed, consistent with a dosing interval (i.e., 12 hours) that was approximately four times the T1/2 (i.e., 3.6 hours). Cmin concentration appeared to plateau on day 8, while C1h values remained stable on days 1, 8, and 15. Systemic drug exposure (Cmax and AUC) increased proportionally with increasing salirasib dose from 100 mg to 800 mg, as confirmed by the lack of significant differences in dose-normalized parameters between dose groups (P > 0.05).

Toxicity[4]
To date, a total of 103 cycles of salilacillin treatment have been performed. The median number of treatment cycles per patient was 2 (range 1–13 cycles). Except for one patient who increased the dose from 100 mg to 200 mg in the second cycle, all patients were treated at the initial dose (total number of cycles 2). Table 2 lists the drug-related adverse events by dose. Only grade 1–2 drug-related toxicities occurred. The most common toxicity was diarrhea, with an incidence of 79% (75% grade 1, 4% grade 2). The duration of diarrhea increased with increasing salilacillin dose. The incidence of diarrhea was also higher in patients receiving higher doses of salilacillin: 67% (2 out of 3 patients), 67% (4 out of 6 patients), 100% (6 out of 6 patients), 67% (4 out of 6 patients), and 100% (3 out of 3 patients) in patients receiving 100, 200, 400, 600, and 800 mg salilacillin twice daily, respectively. Diarrhea did not cause abnormal clinical chemistry and was usually relieved by oral antidiarrheal medications such as loperamide or diphenoxylate hydrochloride. Patients were advised to take these medications if diarrhea occurred. No grade 1 or higher toxicities were observed in 19 patients (79%). Other toxicities included abdominal pain in 21% of patients and nausea, vomiting, and fatigue in 17% of patients each. At the dose of 800 mg twice daily (the highest dose tested), no typical dose-limiting toxicities (DLTs) were observed. However, all three patients receiving this dose developed grade 1-2 persistent diarrhea, making further dose increases intolerable. At a twice-daily oral dose of 600 mg, none of the patients experienced DLT or persistent diarrhea; therefore, this dose was considered the optimal dose for the Phase II study.

[1]. Farnesylthiosalicylic acid (salirasib) inhibits Rheb in TSC2-null ELT3 cells: a potential treatment for lymphangioleiomyomatosis. Int J Cancer. 2012 Mar 15;130(6):1420-9.

[2]. The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy. PLoS One. 2011 Mar 22;6(3):e18049.

[3]. Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition. Mol Cancer. 2010 Sep 22;9:256.

[4]. Phase 1 first-in-human clinical study of S-trans,trans-farnesylthiosalicylic acid (salirasib) in patients with solid tumors. Chemother Pharmacol. 2010 Jan;65(2):235-41.

Salirasib is a sesquiterpene compound. Thalirasib has been used in clinical trials for the diagnosis of non-small cell lung cancer. Thalirasib is a salicylic acid derivative with potential antitumor activity. Thalirasib disengages all Ras isoforms from their membrane anchoring sites, thereby preventing activation of the RAS signaling pathway and inhibiting cell proliferation, differentiation, and senescence. The RAS signaling pathway is believed to be aberrantly activated in one-third of human cancers, including pancreatic cancer, colon cancer, lung cancer, and breast cancer. The small GTPase proteins Ras and Rheb act as molecular switches, regulating cell proliferation, differentiation, and apoptosis. Ras also regulates Rheb by inactivating the tuberous sclerosis complex (TSC), which contains the products of the TSC1 and TSC2 genes, encoding hamartoma protein (TSC1) and nodule protein (TSC2), respectively. Ras functions as a Rheb-specific GTPase activator. Loss of TSC1 or TSC2 function leads to elevated levels of active Rheb-GTP, resulting in translational aberrations and excessive cell proliferation, characteristic of two hereditary diseases: tuberous sclerosis and lymphangioleiomyomatosis (LAM). To determine whether Rheb, Ras, or both inactivation might be a potential therapeutic approach for LAM, we used TSC2 knockout ELT3 cells as a LAM model. We treated these cells with the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib), which mimics the C-terminal S-farnesylcysteine residue shared by Ras and Rheb. This C-terminus is crucial for their binding to the cell membrane and their biological activity. Untreated ELT3 cells expressed high levels of Rheb but low levels of Ras-GTP, while re-expression of TSC2 reversed this phenotype. FTS treatment only slightly reduced Ras.GTP in TSC2-deficient cells, but reduced their overactive Rheb as well as cell proliferation, migration and tumor growth. Notably, re-expression of TSC2 in these ELT3 cells freed them from the inhibitory effect of FTS. Therefore, FTS can apparently block active Rheb in TSC2-deficient ELT3 cells and may have the potential to treat LAM. [1]

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The Ras superfamily is a class of guanosine triphosphate (GTP)-binding proteins that regulate a variety of intracellular processes involved in inflammation and fibrosis. Farnesylthiosalicylic acid (FTS) is a unique and potent Ras inhibitor that reduces inflammation and fibrosis in experimentally induced cirrhosis and improves inflammatory processes in animal models of systemic lupus erythematosus, neuritis and nephritis. This study evaluated the effects of free radical sclerotherapy (FTS) on Ras expression and activity, muscle strength, and fibrosis in a dy(2J)/dy(2J) mouse model of merosin-deficient congenital muscular dystrophy. Compared with wild-type mice, dy(2J)/dy(2J) mice showed significantly increased RAS expression and activity. FTS treatment significantly reduced RAS expression and activity. Furthermore, FTS treatment significantly reduced the phosphorylation level of ERK, a downstream protein of Ras, in dy(2J)/dy(2J) mice. Clinically, FTS-treated mice showed significantly enhanced hind limb muscle strength (measured by an electronic grip strength meter). Quantitative Sirius red staining and lower muscle collagen content indicated a significant reduction in fibrosis in the treatment group. The effects of FTS were associated with a significant inhibition of MMP-2 and MMP-9 activity. We concluded that the inhibition of RAS activity by FTS was associated with reduced fibrosis and enhanced muscle strength in a congenital muscular dystrophy dy(2J)/dy(2J) mouse model. [2]

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Background: Dysregulation of epidermal growth factor and insulin-like growth factor signaling plays an important role in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets ras/raf/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Thalilacimib is an S-isoprene cysteine analogue that has been shown to block ras and/or mTOR activation in a variety of non-hepatic tumor cell lines. We investigated the effects of thalilacimib on the growth of human hepatocellular carcinoma cell lines (HepG2, Huh7 and Hep3B) and its mechanism of action in vitro, and investigated its in vivo effects in a HepG2 cell subcutaneous xenograft model. Results: Thalirasib inhibited the growth of hepatocellular carcinoma cells in a time- and dose-dependent manner by suppressing cell proliferation and partially inducing apoptosis. In serum-containing medium, 150 μM thalirasib reduced the growth of all three cell lines by 50%. In contrast, under serum-free conditions, thalirasib showed better inhibitory effects on cell growth after stimulation with EGF or IGF2, with IC50 values ranging from 60 μM to 85 μM. The drug-induced antiproliferative effect was associated with the downregulation of cyclin A and the mild downregulation of cyclin D1, and the upregulation of p21 and p27. The induction of apoptosis was related to the overall pro-apoptotic balance, including caspase 3 activation, cytochrome c release, upregulation of death receptors, and decreased mRNA expression of apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, while ERK and Akt activation were not reduced. In vivo experiments showed that salirasib inhibited tumor growth from day 5. After 12 days of treatment, the average tumor weight of the treated animals was reduced by 56%. Conclusion: Our results are the first to show that salirasib inhibits the growth of human hepatocellular carcinoma cell lines by inhibiting proliferation and inducing apoptosis, which is related to the inhibition of ras and mTOR. The therapeutic potential of salirasib in human hepatocellular carcinoma (HCC) was further confirmed in a subcutaneous xenograft model. [3] Objective: This Phase I first-in-human trial evaluated salirasib, an S-isopentenyl derivative of thiosalicylic acid, which competitively blocks the RAS signaling pathway. Methods: Patients with advanced cancer were treated with salirasib twice daily for 21 days every 4 weeks. The dose was increased from 100 mg to 200 mg, 400 mg, 600 mg and 800 mg. Results: The most common toxicity was dose-related diarrhea (grade 1-2, 79% of 24 patients). Other toxicities included abdominal pain, nausea, and vomiting. No grade 3-4 toxicities were observed. Nineteen patients (79%) did not experience grade 1 or higher drug-related toxicities. Dose-limiting toxicities (DLTs) were not reached, but all three patients treated with 800 mg experienced grade 1-2 diarrhea, therefore no dose escalation was performed. Six patients treated with 600 mg did not experience DLTs. Seven patients (29%) remained stable for ≥4 months (range 4-23+ months) after treatment with salirasib. The pharmacokinetic profile of salirasib is slow absorption and rapid elimination after oral administration. Salirasib exposure (Cmax; AUC(inf) on day 1 and day 15) were similar (P > 0.05). The T1/2 (mean ± standard deviation) on day 1 was 3.6 ± 2.2 hours. Conclusion: Salirasib was well tolerated. The recommended dose in the phase II study was 600 mg twice daily. [4]

C22H30O2S

358.54

358.196

C, 73.70; H, 8.43; O, 8.92; S, 8.94

162520-00-5

5469318

White to light yellow solid powder

1.1±0.1 g/cm3

486.0±45.0 °C at 760 mmHg

64-66°C

247.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.559

8.53

1

3

10

25

498

0

S(C1=C([H])C([H])=C([H])C([H])=C1C(=O)O[H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H]

WUILNKCFCLNXOK-CFBAGHHKSA-N

InChI=1S/C22H30O2S/c1-17(2)9-7-10-18(3)11-8-12-19(4)15-16-25-21-14-6-5-13-20(21)22(23)24/h5-6,9,11,13-15H,7-8,10,12,16H2,1-4H3,(H,23,24)/b18-11+,19-15+

2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]sulfanylbenzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.97 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)