Evaluation of the Efficacy of OSU-2S in the Treatment of Non-Small-Cell Lung Cancer and Screening of Potential Targets of Action.

(1) Background: OSU-2S is a derivative of FTY720 and exhibits significant inhibitory effects on various cancer cells. There is currently no research on the mechanism of the impact of OSU-2S on NSCLC development. We analysed and validated the hub genes and pharmacodynamic effects of OSU-2S to treat NSCLC. (2) Methods: The hub genes of OSU-2S for the treatment of NSCLC were screened in PharmMapper, genecard, and KM Plotter database by survival and expression analysis. The effect of OSU-2S on hub gene expression was verified by Western blot analysis. The ex vivo and in vivo efficacy of OSU-2S on tumour growth was verified using A549 cells and a xenografted animal model. (3) Results: A total of 7 marker genes for OSU-2S treatment of NSCLC were obtained. AURKA and S1PR1 were screened as hub genes. Significant differences in the expression of AURKA and S1PR1 between normal and lung adenocarcinoma (LUAD) tissues were found in the GEPIA2 database; Western blot showed that OSU-2S could affect p-AURKA and S1PR1 protein expression. OSU-2S significantly inhibited tumour growth in A549 cells and xenografted animal models. (4) Conclusions: Our study confirms the inhibitory effect of OSU-2S on NSCLC, screens and demonstrates its potential targets AURKA(p-AURKA) and S1PR1, and provides a research basis for treating NSCLC with OSU-2S.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Design, synthesis and biological evaluation of new pyrrolidine carboxamide analogues as potential chemotherapeutic agents for hepatocellular carcinoma.

The successful targeting of different malignancies by OSU-2S, encouraged us to design and synthesize a novel series of pyrrolidine aryl carboxamide derivatives. In this context, we found that, the amide nature and tether length were found to be key determinant elements for the anticancer activity of these new and rigid analogues of OSU-2S. The most effective analogues induced apoptosis in cancer cells by a similar mechanism to that of OSU-2S, possibly via the activation of PKCδ in addition to their ability to induce cell cycle arrest and inhibition of cancer cell migration. Compound 10m, possesses anticancer potency comparable to that of OSU-2S when tested against cancer cell lines under study, and was found to be safer on normal cells. Furthermore, compound 10m, was found to be about 2-folds more potent than the anticancer drug Sorafenib in hepatocellular carcinoma (HCC). The newly developed compounds represent a therapeutically promising approach for the treatment of HCC.

Non-immunosuppressive FTY720-derivative OSU-2S mediates reactive oxygen species-mediated cytotoxicity in canine B-cell lymphoma.

OSU-2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti-tumour activity in several haematological and solid tumour models. We have recently shown OSU-2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre-clinical activity of OSU-2S in spontaneous B-cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU-2S mediated apoptosis in canine B-cell lines and primary B-cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU-2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU-2S-mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU-2S as small molecule for treating people and dogs with lymphoma.

OSU-2S/Sorafenib Synergistic Antitumor Combination against Hepatocellular Carcinoma: The Role of PKCδ/p53.

Background: Sorafenib (Nexavar®) is an FDA-approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, the low efficacy and adverse effects at high doses limit the clinical application of sorafenib and strongly recommend its combination with other agents aiming at ameliorating its drawbacks. OSU-2S, a PKCδ activator, was selected as a potential candidate anticancer agent to be combined with sorafenib to promote the anti-cancer activity through synergistic interaction. Methods: The antitumor effects of sorafenib, OSU-2S and their combination were assessed by MTT assay, caspase activation, Western blotting, migration/invasion assays in four different HCC cell lines. The synergistic interactions were determined by Calcusyn analysis. PKCδ knockdown was used to elucidate the role of PKCδ activation as a mechanism for the synergy. The knockdown/over-expression of p53 was used to explain the differential sensitivity of HCC cell lines to sorafenib and/or OSU-2S. Results: OSU-2S synergistically enhanced the anti-proliferative effects of sorafenib in the four used HCC cell lines with combination indices <1. This effect was accompanied by parallel increases in caspase 3/7 activity, PARP cleavage, PKCδ activation and inhibition of HCC cell migration/invasion. In addition, PKCδ knockdown abolished the synergy between sorafenib and OSU-2S. Furthermore, p53 restoration in Hep3B cells through the over-expression rendered them more sensitive to both agents while p53 knockdown from HepG2 cells increased their resistance to both agents. Conclusion: OSU-2S augments the anti-proliferative effect of sorafenib in HCC cell lines, in part, through the activation of PKCδ. The p53 status in HCC cells predicts their sensitivity toward both sorafenib and OSU-2S. The proposed combination represents a therapeutically relevant approach that can lead to a new HCC therapeutic protocol.

Quantification of OSU-2S, a novel derivative of FTY720, in mouse plasma by liquid chromatography-tandem mass spectrometry.

OSU-2S is a novel anti-cancer and immune modulatory agent designed specifically to avert the immunosuppressive effects and related toxicities observed in clinical studies with its predecessor analog, FTY720. To characterize its preclinical pharmacokinetics, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of OSU-2S in mouse plasma. Ethyl acetate extraction of samples containing OSU-2S and the internal standard, Sph-17, was followed by separation with a 6min gradient (water/0.1% formic acid and methanol/0.1% formic acid) on a reverse-phase C18 column at room temperature. Selected reaction monitoring was used for detection on a triple quadrupole mass spectrometer with positive ionization. The assay was linear over the concentration range 3-3000ng/mL with accuracy ranging from 103 to 111%, and both within- and between-run precision (CV%) ≤11%. All stability samples were within ±15% of nominal values, and replicates were within 15% CV. The assay was successfully applied to a mouse pharmacokinetic study of OSU-2S with intravenous and intraperitoneal administration. OSU-2S non-compartmental pharmacokinetic parameters, area under the concentration-time curve, clearance, and elimination half-life were estimated at 1522hµg/L, 3.06L/h/kg and 15.6h, respectively, for intravenous injection. Systemic availability after intraperitoneal injection was approximately 46%. These data demonstrate the OSU-2S compound displays acceptable pharmacokinetic properties for further in vivo pharmacologic evaluation, which can be facilitated by the validated LC-MS/MS assay.