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]

Salirasib plasma pharmacokinetics [4]
Pharmacokinetic data were obtained and evaluable from 24 patients, involving 41 pharmacokinetic study periods (Table 4). The salirasib pharmacokinetic profile was characterized by slow absorption and a rapid elimination phase following oral administration (Fig. 1). Accumulation did not occur, as salirasib exposure (C max; day 1 AUCinf vs. day 15 AUC0–12 h) was similar between days 1 and 15 (P > 0.05). The T 1/2 (mean ± SD) was 3.6 ± 2.2 h on day 1. Salirasib had a ClS/F (mean ± SD) of 103.0 ± 112.5 and 73.3 ± 37.3 L/h on days 1 and 15, respectively, and showed extensive distribution in excess of blood volume with a V/F of 458.0 ± 466.1 and 255.7 ± 187.8 L on days 1 and 15, respectively (Table 4).
Despite the variability in the majority of the pharmacokinetic parameters, salirasib drug exposure (C max and AUC) and ClS/F were similar on days 1 and 15 (P > 0.05) according to matched pairs analysis. The lack of accumulation in C max or AUC is consistent with a dosing interval approximately four times longer (i.e., 12 h) than the T 1/2 (i.e., 3.6 h). C min concentrations appeared to reach a plateau at 8 days, whereas C 1 h values were consistent on days 1, 8, and 15. The systemic drug exposure (C max and AUC) increased in a manner proportionate with the increase in salirasib dose from 100 to 800 mg, as demonstrated by there being no significant difference between dose-normalized parameters by dose group (P > 0.05).

Toxicity [4]
To date, a total of 103 cycles of salirasib therapy have been administered. The median number of cycles per patient was 2 (range 1–13 cycles). All patients were treated at their initial dose level, except for one patient whose dose was escalated from 100 to 200 mg in the second cycle (total cycles, 2). The drug-related adverse events by dose are listed in Table 2. Only Grade 1–2 drug-related toxicities occurred. The most common toxic effect was diarrhea, which occurred in 79% of the patients (Grade 1 in 75% and Grade 2 in 4%). The duration of diarrhea increased with the salirasib dose. The frequency of diarrhea also was higher in patients treated with higher salirasib doses: 67% (2 of 3 patients), 67% (4 of 6), 100% (6 of 6), 67% (4 of 6), and 100% (3 of 3) in patients treated with 100, 200, 400, 600, and 800 mg of salirasib twice daily, respectively. Diarrhea did not result in abnormalities in clinical chemistry parameters, and it was usually reversible with oral antidiarrheal agents such as loperamide or diphenoxylate hydrochloride. Patients were instructed to use these medications if diarrhea occurred. Nineteen (79%) patients had no toxicities greater that Grade 1. Other toxicities included abdominal pain in 21% of patients and nausea, vomiting, and fatigue in 17% each. No classic DLT was reached at 800 mg twice daily, the highest dose tested. However, all three patients treated at this dose developed Grade 1–2 prolonged diarrhea, and it was decided that further dose escalation would not be tolerable. At 600 mg p.o. twice daily, no patients experienced a DLT or prolonged diarrhea, and therefore, it was felt that this dose would be optimal 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 sesquiterpenoid.
Salirasib has been used in trials studying the diagnostic of Carcinoma, Non-Small-Cell Lung.
Salirasib is a salicylic acid derivative with potential antineoplastic activity. Salirasib dislodges all Ras isoforms from their membrane-anchoring sites, thereby preventing activation of RAS signaling cascades that mediated cell proliferation, differentiation, and senescence. RAS signaling is believed to be abnormally activated in one-third of human cancers, including cancers of the pancreas, colon, lung and breast.

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The small GTPase proteins, Ras and Rheb, serve as molecular switches regulating cell proliferation, differentiation and apoptosis. Ras also regulates Rheb by inactivating the tuberous sclerosis complex (TSC), which includes products of the TSC1 and TSC2 genes encoding hamartin (TSC1) and tuberin (TSC2), respectively, and acts as a Rheb-specific GTPase-activating protein. Loss of function of TSC1 or TSC2 results in an increase in active Rheb.GTP with the consequent translational abnormalities and excessive cell proliferation characteristic of the genetic disorders, tuberous sclerosis and lymphangioleiomyomatosis (LAM). To determine whether inactivation of Rheb, Ras or both might be a potential treatment for LAM, we used TSC2-null ELT3 cells as a LAM model. The cells were treated with the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib), which mimics the C-terminal S-farnesyl cysteine common to Ras and Rheb. This C-terminus is critical for their attachment to cellular membranes and for their biological activities. Untreated, the ELT3 cells expressed significant amounts of Rheb but little Ras.GTP, and this phenotype was reversed by TSC2 reexpression. Treatment with FTS decreased Ras.GTP only slightly in the TSC2-null cells, but reduced their overactive Rheb as well as their proliferation, migration and tumor growth. Notably, TSC2 reexpression in these ELT3 cells rescued them from the inhibitory effect of FTS. Evidently, therefore, FTS blocks active Rheb in TSC2-null ELT3 cells and may have therapeutic potential for LAM.[1]

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The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy. [2]

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Background: Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.
Results: Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.
Conclusions: Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model. [3]

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Purpose: This phase I first-in-human trial evaluated salirasib, an S-prenyl derivative of thiosalicylic acid that competitively blocks RAS signaling.
Methods: Patients with advanced cancers received salirasib twice daily for 21 days every 4 weeks. Doses were escalated from 100 to 200, 400, 600, 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 toxicity was noted. Nineteen (79%) patients had no drug-related toxicity >Grade 1. Dose-limiting toxicity (DLT) was not reached, but all three patients treated with 800 mg experienced Grade 1-2 diarrhea, brogating dose escalation. Six patients were treated at a dose of 600 mg with no DLTs. Seven (29%) patients had stable disease on salirasib for ≥4 months (range 4-23+). The salirasib pharmacokinetic profile was characterized by slow absorption and a rapid elimination phase following oral administration. Salirasib exposure (C(max); day 1 AUC(inf) vs. day 15 AUC(0-12 h)) was similar between days 1 and 15 (P > 0.05). The T(1/2) (mean ± SD) was 3.6 ± 2.2 h on day 1.
Conclusions: Salirasib therapy was well tolerated. The recommended dose for phase II studies is 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.)