SphK2 ( IC50 = 60 μM )

The antitumor activity of Opaganib (ABC294640)·HCl was tested in a syngeneic tumor model that uses the mouse JC mammary adenocarcimona cell line growing subcutaneously in immunocompetent BALB/c mice (Lee et al., 2003). Because of the excellent oral absorption described above, we determined the ability of orally delivered ABC294640·HCl to reduce tumor growth in vivo. The SK inhibitor was administered to fasted mice on odd days at doses ranging from 3.5 to 100 mg/kg. Body weights and tumor volumes were monitored daily. As demonstrated in Fig. 7, ABC294640·HCl caused dose-dependent reductions in the growth of the mammary adenocarcinoma xenografts. Body weights in each treatment group remained unchanged from vehicle-treated mice during the course of the study (data not shown). Comparison with the potencies in the tumor studies with the toxicity data described above reveals that ABC294640·HCl has a therapeutic index of greater than 7 (250 mg/kg nontoxic dose / 35 mg/kg antitumor activity). Thus, this SK2 inhibitor has an excellent therapeutic index.[1]

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To ensure that the antitumor effect of Opaganib (ABC294640)·HCl administration is mediated by the compound, its accumulation in tumors was quantified by LC/MS. In these experiments, mice bearing JC tumor xenografts were treated with 100 mg/kg ABC294640·HCl by intraperitoneal injection, and the concentrations of the compound in the plasma and the tumor were measured at 2 and 5 h. As indicated in Fig. 8A, approximately 75 μg/ml (197 μM) ABC294640 was present in the plasma at 2 h, and this decreased to 56 μg/ml (147 μM) at 5 h. The amounts of ABC294640 in the tumor at 2 and 5 h were determined to be 36 and 54 μg/g wet weight, respectively, corresponding to approximately 94 and 140 μM (assuming that 1 g approximately equals 1 ml). Therefore, amounts of ABC294640 well above those needed to block cell proliferation are accumulated in the tumors of intact mice.[1]

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Daily administration of 50 mg/kg Opaganib (ABC294640) led to a statistically significant delay in tumor growth over the 4 weeks of treatment. After 28 days, tumor tissues were excised, fixed, and sectioned, and slides were immunostained to assess the levels of beclin 1 and LC3 and stained by TUNEL to establish the number of apoptotic cells (Fig. 6B). As shown in Fig. 6C, the intensity of staining of both beclin 1 and LC3 was increased in the tumors from mice that were exposed to ABC294640 compared with the vehicle-treated mice. [2]

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ABC294640 (100 mg/kg, p.o.) significantly reduces tumor growth in mice with mammary adenocarcinoma xenografts; this effect is linked to S1P depletion. ABC294640 increases autophagy markers and delays tumor growth in mice with severe combined immunodeficiency carrying A-498 xenografts. ABC294640 enhances liver function and survival by preventing inflammation brought on by liver transplantation and the interaction between innate and adaptive immunity, two key processes that cause and exacerbate graft injury.

An HPLC-based SK activity assay that was recently developed is used to determine the IC50 values for ABC294640 and DMS. The test compounds are, in short, incubated with recombinant SK1 or SK2 and NBD-Sph in the isozyme-selective assay buffers described below, containing 400 μM MgCl2, 100 μM ATP, and 1 mg/ml fatty acid-free bovine serum albumin. The following is how HPLC separates the product, or NBD-S1P, from NBD-Sph: Utilizing a Waters 2495 fluorescence detector, a C8 Chromolith RP-8e column (100 × 4.6 mm) and a 1 ml/min mobile phase (pH 2.5 sodium phosphate buffer with acetonitrile/20 mM) at 45:55 make up the Waters 2795 HPLC system. Fluorescence is observed with excitation at 465 nm and emission at 531 nm. The NBD-S1P/(NBD-Sph + NBD-S1P) ratio is used to calculate the level of SK activity. 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors, and 1× phosphatase inhibitors were all present in each SK-isozyme selective assay buffer. 0.25% (final) Triton X-100 is added to the SK1 assay buffer, and 1 M (final) KCl is added to the SK2 buffer. The kinase reaction is stopped by adding 1.5 volumes of methanol after the assays have been running for two hours at room temperature. The samples are centrifuged at 20,000 g for 10 minutes to remove the precipitated protein, and the supernatants are then subjected to HPLC analysis. The ADP Quest assay system is used to measure kinase activity in the presence of different concentrations of sphingosine and ABC294640 in experiments to determine the Ki for inhibition of SK2 by ABC294640. In order to ascertain the impact of ABC294640 on cellular SK activity, near-confluent MDA-MB-231 cells undergo an overnight serum starvation protocol followed by exposure to different concentrations of ABC294640. Next, [3H]sphingosine is added to the cells at a final concentration of 1 μM. The cells take up the exogenous sphingosine, which is converted to S1P via SK activity, and [3H]S1P is separated from [3H]sphingosine by extraction and quantified by scintillation counting.

Cell Cycle, Apoptosis, and Mitochondrial Membrane Integrity Analyses.[2]
For cell cycle analyses, cells were exposed to various concentrations of Opaganib (ABC294640) for 24, 48, or 72 h, washed twice with PBS, and incubated in 0.5 ml of PI staining solution (50 μg/ml propidium iodide, 40 μg/ml RNase A in PBS) for 30 min at 37°C. Cell cycle distributions were analyzed in the Medical University of South Carolina Flow Cytometry Facility with a Becton Dickinson FACSCalibur Analytical Flow Cytometer. The activities of caspases 3 and 7 were measured by the caspase-Glo 3/7 Assay according to the manufacturer's instructions. In brief, A-498 cells were grown in white 96-well plates at a density of 10,000 cells per well. After incubation with Opaganib (ABC294640), 100 μl of the caspase reagent was added and plates were incubated at room temperature for 30 min. After incubation, luminescence levels were determined by using a SpectraMax M5 plate reader. Cells exposed to cisplatin were used as positive controls for apoptosis. For Annexin-V staining, after exposure to Opaganib (ABC294640), cells were trypsinized, resuspended in 10% fetal bovine serum-containing media, washed twice in PBS, and resuspended in Annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). To 100 μl of the cell suspension, 5 μl of Annexin-V solution was added and the mixture was kept at room temperature for 15 min. After incubation, 400 μl of Annexin buffer was added and cells were immediately analyzed by flow cytometry. For the analysis of mitochondrial membrane function, cells exposed to Opaganib (ABC294640) or cisplatin (positive control) were stained with 100 nM tetramethyrhodamine for 30 min in growth medium, and after washing with PBS, cells were immediately analyzed by flow cytometry. Both adherent and floating cells were collected for the apoptosis and flow cytometry analyses.

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In order to ascertain the impact of the test compounds on proliferation, 96-well microtiter plates are seeded with cells (1025LU, Hep-G2, A-498, MCF-7, Caco-2, MDA-MB-231, HT-29, Panc-1, DU145, T24, and SK-OV-3 cell lines) and left to adhere for a full day. Separate wells are filled with varying concentrations of ABC294640, and the cells are incubated for a further 72 hours. Using the sulforhodamine-binding assay, the number of viable cells is ascertained at the conclusion of this time. As a percentage of sulforhodamine-binding compared to control cultures, the percentage of cells killed is computed. GraphPad Prism is used to perform regression analyses of inhibition curves.

Quantification of Opaganib (ABC294640) in Plasma and Tumors. [1]
Plasma samples were prepared by centrifugation (5000g, 5 min at 4°C) of whole blood that was collected into syringes containing EDTA as an anticoagulant. Samples were spiked with 10 μg of an internal standard {3-(4-chlorophenyl)adamantane-1-carboxylic acid [2-(3,4-dihydroxyphenyl)ethyl]amide}, brought to 1 ml with water and extracted three times with 2 ml of ethyl acetate. Extracts were dried over nitrogen at 35°C, reconstituted in 0.2 ml of 0.1% formic acid in water/methanol (50:50, phase A), filtered, and transferred to vials. Analyses were performed by use of an Agilent 1100 binary pump HPLC system coupled to a Finnigan LCQ Classic ion trap quadrupole mass spectrometer running in electrospray ionization positive ion mode. Sample (10 μl) was injected and resolved by use of a Supelco Discovery C18 column (2.1 × 20 mm, 5-mm particle size) connected to a Zorbax precolumn with a mobile phase consisting of 0.1% formic acid in water/methanol (50:50). The flow rate was 0.3 ml/min, and samples were eluted by a linear gradient increasing from 50 to 100% methanol over 3 min. Opaganib (ABC294640) and the internal standard were detected at 5.1 min and 5.5 min, respectively, by use of a selected ion monitoring mode (m/z = 381 and 426, respectively). Peak areas were integrated by use of Xcalibur software, and Opaganib (ABC294640) concentrations were determined from a standard curve, which was linear in range of all plasma levels observed in these studies.

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Oral Bioavailability and Pharmacokinetic Studies.[1]
Formulations of Opaganib (ABC294640)·HCl were administered orally or intravenously to fasted female Swiss-Webster mice at a dose of 100 mg/kg in 0.1 ml of the indicated solvents. Blood samples were removed at 1 and 7 h after dosing, and the plasma concentration of Opaganib (ABC294640) was determined by reverse-phase LC/MS running in SIM mode as described above. For pharmacokinetic studies, female Swiss-Webster mice (6–8 weeks old) were fasted overnight and administered a bolus dose of 0.1 ml of Opaganib (ABC294640)·HCl either orally or intravenously. After dosing, mice were anesthetized with halothane, and blood was removed via intracardiac puncture at the indicated times. Plasma samples were processed, and Opaganib (ABC294640) levels were determined as described above. Noncompartmental pharmacokinetic analyses were performed with use of WinNolin software package.

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Toxicology Studies.[1]
Acute (7-day) and chronic (28-day) toxicology studies were conducted with Opaganib (ABC294640)·HCl. In the first study, Sprague-Dawley male rats (7–8 weeks old) were orally dosed with 0, 100, or 250 mg of Opaganib (ABC294640)·HCl/kg in 0.375% Polysorbate-80 in PBS daily for 7 days. The animals were observed daily for viability, signs of gross toxicity, and behavioral changes, and a battery of detailed observations were performed on study days 1 and 7. Blood was sampled from all animals on day 8 of the study for hematology, clinical biochemistry, and serology assessments, and the animals were sacrificed. Gross necropsies were performed on all study rats, and selected organs and tissues were evaluated in the control and high-dose level groups. In the second study, C57BL/6 mice were orally dosed with 0, 100, or 250 mg of Opaganib (ABC294640)·HCl/kg daily exactly as indicated above, and sacrificed at either day 7 or day 28 for hematology studies.

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Antitumor Evaluation.[1]
A syngeneic mouse tumor model that uses a transformed murine mammary adenocarcinoma cell line and BALB/c mice (Charles River, Wilmington, MA) was performed as described previously (Lee et al., 2003). Animal care and procedures were in accordance with guidelines and regulations of the Institutional Animal Care and Use Committee of the Penn State College of Medicine. Animals were housed under 12-h light/dark cycles, with food and water provided ad libitum. Tumor cells (1 × 106) were implanted subcutaneously, and tumor volume was calculated by use of the equation: (L × W2)/2. On detection of tumors, mice were randomly assigned to treatment groups. Treatment was then administered every other day thereafter, consisting of oral doses of 3.5, 10, 35, or 100 mg of Opaganib (ABC294640)·HCl/kg body weight or vehicle (0.375% Polysorbate-80). Whole body weights and tumor volume measurements were performed each day of treatment. On day 15, mice were dosed and euthanized 1 h later; tumors were excised and immediately frozen. p values were determined by use of one-way analysis of variance using GraphPad InStat.

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Pharmacodynamic Studies and Tumor Accumulation of Opaganib (ABC294640).[1]
Apoptosis was measured in sections from tumors treated with Opaganib (ABC294640)·HCl using a TUNEL detection kit according to the manufacturer's instructions (In situ cell death detection kit; Roche Diagnostics). In brief, tumor sections were incubated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate, freshly prepared) for 8 min at room temperature and then washed twice with PBS. Sections were incubated with TUNEL reaction mixture in a humid atmosphere at 37°C for 60 min and mounted with crystal mounting medium. The amount of apoptosis was calculated for an average of 10 microscopic fields in each sample (magnification, 100×) and expressed as the percentage of cells that were TUNEL-positive. For the analyses of sphingolipids, frozen tumor slices were homogenized in ice-cold PBS to a final concentration of 10 mg/ml. A 0.5-ml aliquot of the homogenate was combined with 0.5 ml of methanol, 0.25 ml of chloroform, and 375 pmol each of internal standards C17-sphingosine and C17-S1P. Blank samples spiked with known amounts of sphingosine, S1P, and the internal standards were processed in parallel to provide a standard curve for quantification. After sonication, samples were incubated overnight at 48°C, followed by addition of 75 μl of 1 N potassium hydroxide in methanol. The samples were then sonicated and incubated at 37°C for 2 h. A portion (0.4 ml) of each sample was then transferred to a new tube, dried, reconstituted in 0.25 ml of phase A, filtered, and transferred to a vial. HPLC was performed as described above. Elution was performed at 0.45 ml/min with 65% phase B for 2 min followed by a linear gradient to 100% phase B over 5 min. Ions for C17-sphingosine, sphingosine, C17-S1P, and S1P were monitored at m/z 286 (parent ion) → 268 (daughter ion), 300 → 282, 366 → 250, and 380 → 264, respectively. Likewise, extracts of tumors from Opaganib (ABC294640)-treated mice were prepared and levels of Opaganib (ABC294640) were quantified by LC/MS as described above.

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SCID mice (6–8 weeks old) were injected with 3 × 106 A-498 cells per mouse, and after 3 to 4 weeks, mice bearing tumors of 100 to 150 mg were treated with either vehicle or 50 mg/kg Opaganib (ABC294640) 5 days/week for 4 weeks (eight mice per cohort). Tumors were measured twice per week, and volumes were calculated by using the following formula: volume = 1/2 × length × width2. At the end of the treatment, four animals from each cohort were sacrificed, and the paraffin-embedded tumor sections were stained with hematoxylin/eosin. Additional sections were deparaffinized and rehydrated in graded alcohols using standard procedures, and then subjected to TUNEL according to the manufacturer's instructions. The percentage of TUNEL-positive cells was determined by counting at least 100 cells each from at least three randomly selected fields. Additional sections were blocked with 10% normal goat serum in a humid chamber for 30 min, and then incubated antibodies for beclin 1 or LC3 overnight at 4°C, followed by secondary antibody for 60 min at room temperature.[2]

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Dissolved in 0.375% Polysorbate-80 in PBS; 100 mg/kg; oral givage

Female BALB/c mice bearing JC tumors

Absorption of ABC294640.[1]
The HCl salt of ABC294640 (ABC294640·HCl) has been synthesized in multigram amounts for characterizations of its toxicity, pharmacokinetics, and in vivo efficacy. Formulation analyses were conducted to identify a suitable pharmaceutical composition for in vivo studies. We chose five different oral formulations from the Division of Drug Information Resources' Inactive Ingredient Guide, a compendium of all inactive ingredients in approved drug products currently marketed for human use, to assess their ability to support oral absorption of ABC294640. Solutions of ABC294640·HCl in water, 90% propylene glycol, 100% polyethylene glycol 400 (PEG400), 50% PEG400 or 0.375% Polysorbate-80 did not precipitate (measured as turbidity at 590 nm), and so were administered to fasted female Swiss-Webster mice at a dose of 100 mg/kg. Blood samples were removed at 1 and 7 h, and plasma levels of ABC294640 were determined by use of an internal standard and reverse-phase HPLC coupled to an ion-trap quadrupole mass spectrometer running in positive SIM detection mode. As shown in Table 3, substantial amounts of ABC294640 were detected in the blood 1 h after oral dosing, with the highest levels attained in the samples formulated in 90% propylene glycol. It should be noted that these ABC294640 concentrations are sufficient to inhibit SK activity and proliferation of tumor cells. By 7 h, the plasma concentrations decreased by approximately 50% in most cases. Effective absorption was observed in the sample formulated in 0.375% Polysorbate-80, and this solvent for ABC294640·HCl was used in further pharmacokinetic and efficacy analyses because of its low toxicity. [1]
To further understand the absorption properties of ABC294640·HCl, the relationship between plasma concentration and dose was examined. Mice were orally dosed with 10, 35, or 100 mg/kg ABC294640, and the plasma levels were determined at 30 min. As shown in Fig. 5, the plasma ABC294640 values demonstrated a good linear response with doses up to at least 100 mg/kg.

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Pharmacokinetics of ABC294640.[1]
Detailed pharmacokinetic studies were performed on ABC294640·HCl in 0.375% Polysorbate-80. Female Swiss-Webster mice were dosed with 50 mg/kg ABC294640 either intravenously or orally. Groups of mice (3 per group) were anesthetized, and blood was removed via cardiac puncture at time points ranging from 1 min to 7 h. Plasma concentrations of ABC294640 were determined by LC/MS, and pharmacokinetic parameters were calculated by use of the WinNolin software package (Table 4). Intravenous administration of ABC294640 resulted in high plasma concentrations that were eliminated with a half-time of clearance of 1.4 h. Although the peak plasma level of ABC294640 was lower when the compound was administered by oral gavage, the compound was much more persistent, probably reflecting continued absorption from the gastrointestinal tract, such that the calculated half-time for clearance was 4.5 h. It is noteworthy that comparison of the oral versus the intravenous pharmacokinetics of ABC294640 indicated an excellent oral bioavailability of 66% (F = AUCoral/AUCiv).

Toxicity of ABC294640.[1]
Preliminary toxicity studies were performed to determine the appropriate dose for in vivo efficacy testing. No immediate or delayed toxicity was observed in female Swiss-Webster mice treated with intraperitoneal doses of ABC294640·HCl up to at least 250 mg/kg. Repeated injections in the same mice every other day over 15 days showed a similar lack of toxicity at doses up to at least 250 mg/kg. Dose-escalation toxicity testing was performed via oral gavage with ABC294640·HCl dissolved in 0.375% Polysorbate-80, and no toxic effects were observed after administration of doses up to 1000 mg/kg. Therefore, the compound was considered to be suitable for more detailed in vivo studies.

Non-good laboratory practice acute toxicology studies were contracted to Eurofins|Product Safety Laboratories, in which ABC294640·HCl was given orally in 0.375% Polysorbate-80 to rats at doses of 0, 100, or 250 mg/kg daily for 7 days. There were no clinical observations or gross findings that were considered to be the result of ABC294640·HCl administration or otherwise. There were no significant changes in total body weight of the treated animals, although there was a slight decrease in body weight gain, consistent with a small decrease in food consumption, in the high-dose rats. Hematology studies (Table 5) indicated decreases in red blood cell number and hematocrit of approximately 20% in animals given either 100 or 250 mg/kg/day; and a slight increase in neutrophils and decrease in basophils in the treated rats. These changes would be scored as grade 0 toxicities on the standard National Cancer Institute scale for evaluating toxicity in clinical trials. It is noteworthy that no decreases in lymphocyte, platelet, or granulocyte counts were observed, indicating that the compound does not induce immunologic toxicities that are common with other anticancer drugs. Likewise, there were no drug-induced alterations of a broad panel of clinical chemistry or coagulation parameters. No gross abnormalities were noted for any of the euthanized animals when necropsied at the end of the 7-day observation period. Likewise, there were no treatment-related microscopic changes in any organ examined in the high-dose group, except for a slight decrease in the background level of extramedullary hematopoiesis in the spleen that may underlie the small decreases in the hematocrit.

To further characterize the hematologic changes observed in the acute study, mice were treated with 0, 100, or 250 mg of ABC294640·HCl/kg daily for 28 days. As indicated in Fig. 6A, mice treated with 250 mg/kg experienced a 20% decrease in red blood cell count and hematocrit, and a modest increase in the number of circulating neutrophils on day 7, essentially identical to the previous study with rats. However, after 28 days of treatment (Fig. 6B), these parameters were restored to normal levels, indicating that the animals had fully recovered from any transient impairment of hematopoiesis. In addition, there were no changes in the brain or spleen weights of treated mice, whereas a slight decrease (12%) in liver weight was observed in mice treated with 250 mg/kg.

[1]. J Pharmacol Exp Ther. 2010 Apr;333(1):129-39.

[2]. J Pharmacol Exp Ther. 2010 May;333(2):454-64.

3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)-1-adamantanecarboxamide is an organochlorine compound.
Opaganib, also known as ABC294640, is a selective [sphingosine kinase-2 (SK2)](https://go.drugbank.com/polypeptides/Q9NRA0) inhibitor that is orally administered. This drug has potential anticancer, anti-inflammatory, and antiviral activities, with potential applications in oncology, inflammation, the gastrointestinal system, and COVID-19.
Opaganib is an orally available, aryladamantane compound and selective inhibitor of sphingosine kinase-2 (SK2) with potential antineoplastic activity. Upon administration, opaganib competitively binds to and inhibits SK2, thereby preventing the phosphorylation of the pro-apoptotic amino alcohol sphingosine to sphingosine 1-phosphate (S1P), the lipid mediator that is pro-survival and critical for immunomodulation. This may eventually lead to the induction of apoptosis and may result in an inhibition of cell proliferation in cancer cells overexpressing SK2. SK2 and its isoenzyme SK1 are overexpressed in numerous cancer cell types.

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Mechanism of Action
Opaganib selectively inhibits [sphingosine kinase-2 (SK2)](https://go.drugbank.com/polypeptides/Q9NRA0). This inhibition blocks the synthesis of sphingosine 1-phosphate (S1P) and its activities (which includes regulation of fundamental biological processes such as cell proliferation, migration, immune cell trafficking, angiogenesis, immune-modulation, and suppression of innate immune responses from T-cells). This drug has dual anti-inflammatory and antiviral activity targeting a host cell component and is unaffected by viral mutation, contributing to minimization of the likelihood of resistance. It is currently being investigated against COVID-19 as it has demonstrated anti-SARS-CoV-2 activity in studies.

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Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.[1]

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The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.[2]

C23H25CLN2O

380.91

380.165

915385-81-8

1185157-59-8 (HCl); 915385-81-8

15604015

White solid powder

1.3±0.1 g/cm3

589.5±50.0 °C at 760 mmHg

310.3±30.1 °C

0.0±1.7 mmHg at 25°C

1.632

4.16

1

2

4

27

551

0

C1C2CC3(CC(CC1C3)(C2)C1C=CC(=CC=1)Cl)C(NCC1C=CN=CC=1)=O

CAOTVXGYTWCKQE-UHFFFAOYSA-N

InChI=1S/C23H25ClN2O/c24-20-3-1-19(2-4-20)22-10-17-9-18(11-22)13-23(12-17,15-22)21(27)26-14-16-5-7-25-8-6-16/h1-8,17-18H,9-15H2,(H,26,27)

3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)adamantane-1-carboxamide

ABC294640; ABC 294640; ABC-294640; 3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)adamantane-1-carboxamide; CHEMBL2158685; Trade name Yeliva

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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