Lynn W. Maines

Pharmacology and Antitumor Activity of ABC294640, a Selective Inhibitor of Sphingosine Kinase-2

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 Ki of 9.8 μM, 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.

Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells.

Expression of the drug transport proteins, including P-glycoprotein (Pgp), in the brain vascular endothelium represents a challenge for the effective delivery of drugs for the treatment of several central nervous system (CNS) disorders including depression, schizophrenia and epilepsy. It has been hypothesized that Pgp plays a major role in drug efflux at the blood-brain barrier, and may be an underlying factor in the variable responses of patients to CNS drugs. However, the role of Pgp in the transport of many CNS drugs has not been directly demonstrated. To explore the role of Pgp in drug transport across an endothelial cell barrier derived from the central nervous system, the expression and activity of Pgp in bovine retinal endothelial cells (BRECs) and the effects of representative CNS drugs on Pgp activity were examined. Significant Pgp expression in BRECs was demonstrated by western analyses, and expression was increased by treatment of the cells with hydrocortisone. Intracellular accumulation of the well-characterized Pgp-substrate Taxol was markedly increased by the non-selective transporter inhibitor verapamil and the Pgp-selective antagonist PGP-4008, demonstrating that Pgp is active in these endothelial cells. In contrast, neither verapamil nor PGP-4008 affected the intracellular accumulation of [3H]paroxetine, [14C]phenytoin, [3H]clozapine or [14C]carbamazapine, indicating that these drugs are not substrates for Pgp. Paroxetine, clozapine and phenytoin were shown to be Pgp inhibitors, while carbamazapine did not inhibit Pgp at any concentration tested. These results indicate that Pgp is not likely to modulate patient responses to these drugs.

Efficacy of a novel sphingosine kinase inhibitor in experimental Crohn’s disease

AimActivation of sphingosine kinase (SK) is a key response to many inflammatory processes. The present studies test the hypothesis that an orally available SK inhibitor, ABC294640, would be effective in rodent models of Crohn’s disease.MethodsTrinitrobenzene sulfonic acid (TNBS) was administered rectally to mice and rats. Rats were treated with ABC294640 orally alone or in combination with olsalazine and disease progression was monitored.ResultsFor both rodent species, treatment with ABC294640 attenuated disease progression. Colon samples from the ABC294640-treated animals had improved histology and cytokine parameters when compared with vehicle-treated animals. The expression of SK was similarly increased in TNBS-treated animals and in human colon tissue specimens from inflammatory bowel disease patients relative to normal, control patients.ConclusionsSphingosine kinase may be a critical mediator of colonic damage during intestinal inflammation, and pharmacologic inhibitors of this enzyme may prove useful in the treatment of Crohn’s disease.

In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis

We examined the in vitro and in vivo effects of a probiotic, Escherichia coli strain M-17 (EC-M17), on NF-kappaB signalling, cytokine secretion and efficacy in dextran sulfate sodium (DSS)-induced murine colitis. NF-kappaB signalling was assessed using an NF-kappaB luciferase reporter cell line that was stimulated with TNF-alpha (100 ng/ml). p65 Nuclear binding and cytokine secretion (TNF-alpha, IL-1beta and IL-6) were evaluated using a RAW 264.7 macrophage cell line that was exposed to lipopolysaccharide (LPS; 5 microg/ml). Mice were administered vehicle, EC-M17, metronidazole, or EC-M17 plus metronidazole for 13 d. During the final 6 d, mice also received 2 % DSS. Parameters evaluated included disease activity index (DAI), histology, myeloperoxidase and NF-kappaB p65. EC-M17 dose dependently inhibited TNF-alpha-induced NF-kappaB signalling. At 5 x 109 colony-forming units/ml, EC-M17 inhibited NF-kappaB by >95 %. LPS-induced nuclear p65 binding was significantly inhibited (78 %; P 90 %) the LPS-induced secretion of TNF-alpha, IL-1beta and IL-6. In mice with DSS-induced colitis, EC-M17, metronidazole, and EC-M17 plus metronidazole significantly reduced DAI and colonic histology scores. Both EC-M17 and metronidazole reduced colonic IL-12, IL-6, IL-1beta and interferon-gamma. The combination of EC-M17 plus metronidazole resulted in more substantial cytokine reductions than were found with either treatment alone, and combination therapy significantly (P < 0.05 in both cases) reduced IL-1beta compared with EC-M17 and colonic histology scores compared with metronidazole. Alone, and in combination with metronidazole, EC-M17 improved murine colitis, probably due to an inhibitory effect on NF-kappaB signalling.

Corticosterone regulation of serotonin transporter and 5-HT1A receptor expression in the aging brain.

Hypercortisolemia is often observed in patients suffering from major depression. As the serotonergic (5‐hydroxytryptamine; 5‐HT) system plays a major role in the etiology of depression, a loss of endocrine and neurotransmitter system interactions, including corticosterone regulation of 5‐HT transporter (5‐HTT) and 5‐HT receptor expression, may underlie age‐related deficits in the regulation of the hypothalamic‐pituitary‐adrenal (HPA) axis and correlate with an increased incidence of depression with advancing age. In this study, female Fischer 344 rats, ages 3, 13, and 18 months, were bilaterally adrenalectomized and supplemented for 3 weeks with corticosterone (0, 200, or 600 mg; LC, MC, or HC, respectively) containing 21 day sustained‐release pellets implanted subcutaneously. Quantitative autoradiography of hippocampal and cortical regions using [3H]citalopram revealed a significant decrease in hippocampal 5‐HTT binding in the 3‐month HC treatment group compared to age‐matched MC and LC groups; this loss was not present in the 13‐ or 18‐month groups. Similarly, quantitative autoradiography using the radiolabeled 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐N‐propylamino) tetralin demonstrated a significant decline in receptor density in 3‐ and 13‐month MC and HC groups as compared to age‐matched LC groups in the hippocampus. These hormone treatments (MC or HC), however, failed to alter hippocampal 5‐HT1A binding site density in the 18‐month groups as compared to the age‐matched LC group. The 5‐HT2A receptor was also evaluated using [3H]ketanserin and showed no age‐ or corticosterone‐related changes in the cortex. Overall, an age‐associated deficit in the regulation of the hippocampal serotonergic system by varied corticosterone treatment was revealed in the present study, which may underlie the increased incidence of depression and hypercortisolemia found with advancing age. Synapse 32:58–66, 1999.

Experimental Osteoarthritis in Rats Is Attenuated by ABC294640, a Selective Inhibitor of Sphingosine Kinase-2

Background/Aims: Osteoarthritis (OA) is a progressive degenerative disease characterized by cartilage degradation and chondrocyte apoptosis, which may involve aberrant sphingolipid metabolism. ABC294640 is a compound that selectively inhibits sphingosine kinase-2, a key enzyme in the sphingolipid pathway. Our goal was to assess the pharmacological effects of ABC294640 in the monosodium iodoacetate (MIA) model of OA. Methods: MIA (3 mg) was injected into the right knee joint to induce osteoarthritis in rats. Subsequently, the rats were treated with vehicle, ABC294640 or tramadol over a 28-day period. To assess pain, incapacitance readings were obtained weekly. MIA-injected knee joints were evaluated for histological damage, cartilage degradation and chondrocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling histochemistry). Results: The percent weight bearing in vehicle/MIA rats significantly (p < 0.01) decreased from 48.8 ±0.8 (day 0) to 41.9 ±2.9 (day 28). In contrast, these values in ABC294640-treated rats were virtually the same on days 0 and 28. Knee joint histology scores were less severe in ABC294640-treated rats. Cartilage proteoglycan staining was more prominent in ABC294640/MIA animals than in vehicle/MIA rats. The percentage of apoptotic chondrocytes was decreased from 39.5% (vehicle treatment) to 25.8% (ABC294640 treatment). Conclusion: ABC294640 attenuated the knee joint histological damage and pain associated with MIA-induced OA in rats.

A Phase I Study of ABC294640, a First-in-Class Sphingosine Kinase-2 Inhibitor, in Patients with Advanced Solid Tumors

Purpose: Sphingosine kinases (SK1 and SK2) regulate tumor growth by generating the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). This phase I study investigated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of ABC294640, a first-in-class orally available inhibitor of SK2. Experimental Design: Escalating doses of ABC294640 were administered orally to patients with advanced solid tumors in sequential cohorts at the following dose levels: 250 mg qd, 250 mg bid, 500 mg bid, and 750 mg bid, continuously in cycles of 28 days. Serial blood samples were obtained to measure ABC294640 concentrations and sphingolipid profiles. Results: Twenty-two patients were enrolled, and 21 received ABC294640. The most common drug-related toxicities were nausea, vomiting, and fatigue. Among the 4 patients at 750 mg bid, one had dose-limiting grade 3 nausea and vomiting, and 2 were unable to complete cycle 1 due to diverse drug-related toxicities. The 500 mg bid dose level was established as the recommended phase II dose. ABC294640 administration resulted in decreases in S1P levels over the first 12 hours, with return to baseline at 24 hours. The best response was a partial response in a patient with cholangiocarcinoma at 250 mg qd, and stable disease was observed in 6 patients with various solid tumors across dose levels. Conclusions: At 500 mg bid, ABC294640 is well tolerated and achieves biologically relevant plasma concentrations. Changes in plasma sphingolipid levels may provide a useful pharmacodynamic biomarker for ABC294640. Clin Cancer Res; 23(16); 4642–50. ©2017 AACR.

Age-dependent loss of corticosterone modulation of central serotonin 5-HT1A receptor binding sites

A loss of endocrine and neurotransmitter system interactions, including corticosterone regulation of 5‐HT1Areceptors, may underlie the age‐related deficits in the hypothalamic‐pituitary‐adrenal (HPA) axis including adapting to stress. In this study, female Fischer 344 rats, (ages 3, 13, and 18 months), were bilaterally adrenalectomized and supplemented for 3 weeks with placebo or corticosterone (200 mg or 600 mg) containing 21 day sustained‐release pellets implanted subcutaneously (LC, MC, or HC, respectively). Scatchard analysis using the 5‐HT1A receptor agonist [3H]8‐hydroxy‐2‐(di‐N‐propylamino) tetralin (8‐OH‐DPAT) demonstrated a significant decrease in hippocampal receptor density in 3 month and 13 month MC groups (‐35.2 and ‐32.1%, respectively) as compared to age‐matched LC groups; a significant decline in 5‐HT1A receptor density in 3 month and 13 month HC groups was found compared to age‐matched MC groups (−16.7 and −22.0%, respectively). However, these hormone treatments (LC or HC) failed to alter hippocampal 5‐HT1A binding site density in the 18 month groups. Cortical 5‐HT1A receptor densities were altered in a similar age‐dependent manner. In contrast, the density of hypothalamic 5‐HT1A receptors in the 18 month LC group was significantly increased above that in the 3 month LC group. An additional indicator of the hippocampal response to corticosterone, the distribution of glial fibrillary acidic protein (GFAP), revealed an age‐related decline in responsiveness to hormone treatment in the oldest group. The present study has identified an age‐associated deficit in the regulation of hippocampal 5‐HT1A receptors by corticosterone which may underlie the diminished capacity of the aging HPA axis to cope with stress. J. Neurosci. Res. 53:86–98, 1998.

Compensatory responses in the aging hippocampal serotonergic system following neurodegenerative injury with 5,7‐dihydroxytryptamine

This study utilized a multidisciplinary approach to examine injury‐induced compensatory responses in the aging hippocampal serotonin transporter (5‐HTT), a membrane protein implicated in a variety of neurodegenerative disorders. Age‐dependent cellular, anatomical, and physiological changes of the 5‐HTT were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents (fimbria‐fornix and cingulum bundle) to the dorsal hippocampus using the neurotoxicant 5,7‐dihydroxytryptamine (5,7‐DHT). Seven days following 5,7‐DHT administration, a uniform loss of the hippocampal 5‐HTT immunoreactivity was observed in both age groups. However, at 21 days 5‐HTT immunoreactivity in young 5,7‐DHT‐treated animals was similar to control levels, indicative of recovery, while older animals exposed to 5,7‐DHT did not show recovery of hippocampal 5‐HTT expression. 5‐HTT binding site density, as determined by quantitative autoradiography ([3H]citalopram), supported the immunohistochemical results by demonstrating a recovery of 5‐HTT binding sites in young, but not old animals, at 21 days following the lesion (P < 0.001). Furthermore, cellular electrophysiological function of hippocampal CA1 pyramidal neurons in 3‐ and 18‐month‐old F344 rats at 21 days following 5,7‐DHT or vehicle treatment were assessed using in vivo microiontophoretic application of serotonin (5‐HT). Independent of changes in sensitivity to the inhibitory effects of 5‐HT application, the time to recovery of cell firing following application of 5‐HT was significantly increased in the 18‐month 5,7‐DHT group compared to the 18‐month vehicle and 3‐month 5,7‐DHT groups (60 and 59% increases, respectively; P < 0.05). Overall, these series of studies comprise a model which can be used to identify cellular events underlying both the formation of injury‐induced compensatory processes in younger animals and the lack thereof with advancing age. Synapse 39:109–121, 2001.

Abstract C62: Phase I trial of ABC294640, a first-in-class sphingosine kinase-2 inhibitor.

Sphingosine kinases (SK1 and SK2) are new potential targets for anticancer drugs because SK inhibitors elevate ceramide levels thereby promoting apoptosis of tumor cells, and concomitantly reduce sphingosine 1-phosphate (S1P) levels thereby attenuating tumor cell proliferation and migration, as well as reducing host angiogenesis and inflammation. ABC294640 is a first-in-class orally-available inhibitor of SK2 that has activity both as a single-agent and in drug combinations in models of liver, kidney, pancreas, breast, ovary, colon and lung cancer, as well as leukemia and multiple myeloma. ABC294640 is currently in Phase I testing in patients with advanced solid tumors. The primary objectives are to identify the MTD, to determine the dose limiting toxicities (DLTs) and to evaluate the safety of ABC294640. Secondary objectives are to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of ABC294640, and to assess its antitumor activity. The study employs a standard 3+3 dose-escalation design. To date, this Phase I trial has enrolled 16 patients at the following dose levels: 250 mg po qd (6 patients), 250 mg po bid (4 patients), 500 mg po bid (4 patients) and 750 mg po bid (2 patients). In general, the drug has been well tolerated, with the most common side effects being low-grade fatigue and nausea. One pancreatic cancer patient at 250 mg po qd developed grade 4 DLT of hyperglycemia as disease progressed, which was possibly related to drug. Additionally, a patient at 750 mg po bid with a malignant neoplasm of the ovary experienced a grade 3 DLT of nausea and vomiting that was possibly drug-related. After 2 cycles of treatment (8 weeks), tumor imaging demonstrated that 6 of 10 patients had stable disease. Notably 2 patients have had prolonged stabilization of disease: 1 patient with metastatic cholangiocarcinoma (16 months), and 1 patient with metastatic/recurrent bladder cancer (11 months). The MTD has not yet been reached, and dose escalation is continuing. The t1/2 for ABC294640 was approximately 4 hours, and through at least the first 3 cohorts, PK parameters (Cmax and AUC) were proportional to the dose of ABC294640 received. Importantly, the average Cmax for patients receiving 500 mg ABC294640 (16.4 μM) is sufficient for anticancer efficacy in mouse models. This study includes the first-ever analysis of plasma S1P levels as a potential PD biomarker for activity of a sphingolipid-targeted drug, and will establish the degree of intra-and inter-patient variability needed for assessment of this PD marker in future clinical trials. Overall, this first-in-human study provides key information about the safety, toxicities, PK and PD of ABC294640 that are needed to support future Phase II and III clinical trials. (Supported by: P30 CA138313 from the National Cancer Institute to MUSC; R01 FD004102 from the Food and Drug Administration to Apogee; and SAP#4100061668 from the Pennsylvania Department of Health to Apogee.) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C62. Citation Format: Melanie B. Thomas, Carolyn D. Britten, Elizabeth Garrett-Mayer, Steve H. Chin, Tricia A. Bentz, Alan Brisendine, Terri L. Matson, Susan Cusack, Lynn W. Maines, Yan Zhuang, Charles D. Smith. Phase I trial of ABC294640, a first-in-class sphingosine kinase-2 inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C62.