Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2

Abstract

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.

Fig. 1.

Structure of ABC294640 (CAS 915385-81-8).

Fig. 2.

Inhibition of sphingosine kinases. A, recombinant SK1 (circles) or SK2 (squares) was incubated with the indicated concentrations of ABC294640 (●, ■) or DMS (○, □), and the kinase activity was measured by use of the HPLC assay described under Materials and Methods. Values represent the mean ± S.E.M. for three experiments. B, recombinant SK2 was incubated with the indicated concentration of ABC294640, and kinase activity was determined in assays containing 2.5 (■), 5 (▴), 10 (▾), or 25 (♦) μM sphingosine by use of the ADP Quest assay as described under Materials and Methods. The reciprocal of the velocity is plotted against the ABC294640 concentration providing a Dixon plot of the results.

Fig. 3.

Time course of sphingolipid alterations by ABC294640. JC murine adenocarcinoma cells were exposed to 40 μM ABC294640 for 0 (blue), 12 (red), 24 (yellow), or 48 (green) h. Cells were harvested and the masses of the indicated sphingolipid species were quantified by mass spectrometry in the Lipidomics Core Facility as described under Materials and Methods. Labels indicate the chain length and saturation of molecular species of ceramide (Cer). Other labels refer to dihydro-C₁₆-ceramide (DHC16-Cer), dihydrosphingosine (DHSph), dihydrosphingosine 1-phosphate (DhSph-1P), sphingosine (Sph), and S1P.

Fig. 4.

Disruption of tumor cell migration and microfilament structure by ABC294640. A, A-498 cells were plated into a Boyden chamber and treated with the indicated concentration of ABC294640 for 4 h as described in Materials and Methods. The number of cells migrating to the opposite side of the filter was then quantified. B, A-498 cells were treated with the indicated concentration of ABC294640 for 24 h, and the amounts of G-actin and F-actin were determined as described in Materials and Methods. Cytochalasin D (cch) was used as a positive control at a concentration of 1 μM. C and D, A-498 cells were treated with 0 (C) or 50 μM ABC294640 (D) for 24 h, and then stress fibers were visualized with FITC-phalloidin as described under Materials and Methods.

Fig. 5.

Relationship between dose and plasma ABC294640 level. Mice were orally dosed with the indicated amounts of ABC294640·HCl in 0.375% Polysorbate-80 and bled at 30 min; the concentration of ABC294640 in the plasma was determined as described under Materials and Methods. Values represent mean ± S.E.M. (n = 3 mice per group).

Fig. 6.

Hematologic parameters in ABC294640-treated mice. Mice were orally dosed with 0 (Veh), 100 or 250 mg/kg ABC294640·HCl in 0.375% Polysorbate-80 daily for either 7 (A) or 28 (B) days. Blood was harvested by cardiac puncture and a complete blood count was performed. Values represent mean ± S.E.M. (n = 3 or 4 mice per group) for the red blood cells (RBC), hematocrit (HCT), total white blood cell (WBC), and neutrophil (Neutr) values.

Fig. 7.

Antitumor activity of orally administered ABC294640. Female BALB/c mice were injected subcutaneously with JC cells suspended in PBS. After palpable tumor growth, animals were treated every other day by oral gavage with 0 (□), 3.5 (♦), 10 (▾), 35 (▴), or 100 (■) mg/kg ABC294640·HCl in 0.375% Polysorbate-80. Values represent the mean ± S.E.M. (n = 5 mice per group) tumor volume normalized to treatment day 1 for each mouse. *, p < 0.05; **, p < 0.01.

Fig. 8.

Pharmacodynamic effects of ABC294640. A, accumulation of ABC294640 in tumors. Mice bearing JC tumor xenografts were treated by intraperitoneal injection of 100 mg/kg ABC294640·HCl, and tumors were harvested at the 2 or 5 h. The tumors were homogenized, and the amount of ABC294640 was quantified (n = 4 mice per group). Values represent the mean ± S.E.M. in micrograms per milliliter for plasma samples (filled bars) and micrograms per gram wet weight for tumor samples (open bars). B, induction of tumor apoptosis by ABC294640. Mice bearing JC tumor xenografts were treated by intraperitoneal injection of 100 mg/kg ABC294640·HCl. Tumors were harvested either 3 or 5 days after drug treatment, fixed, and sectioned, and the amount of apoptosis was quantified as TUNEL staining. Values represent the mean ± S.E.M. percentage of tumor cells that were TUNEL-positive (n = 4 mice per group). C, alteration of S1P levels by ABC294640. Tumors were harvested at the end of the experiment described in Fig. 7. The levels of sphingosine (filled bars) or S1P (open bars) were determined by liquid chromatography/tandem mass spectrometry. Values represent the mean ± S.D. levels compared with the vehicle-treatment group. **, p < 0.01 versus vehicle-treated mice.