Antineoplastic activity of GP-2250 in-vitro and in mouse xenograft models.

This study examined the antineoplastic effects of GP-2250 (misetionamide), an oxathiazine derivative with broad activity, in multiple cancer cell lines and mouse xenograft models. Antineoplastic activity of GP-2250 was tested in >300 cancer cell lines using the OncoPanel cytotoxicity assay. GP-2250 activity was further tested in mouse xenograft models, in which GP-2250 or vehicle (10 ml/kg) was administered daily for 28 days by intraperitoneal injection in the lower right abdomen of CrTac:NCR-Foxn1nu mice with tumor volumes of 100 to 200 mm 3 . In the in-vitro models, GP-2250 increased cytotoxicity readings with IC50 and EC50 as well as indications of cell cycle blockage in pancreatic and ovarian cell lines. In mouse xenograft models, a reduction of 30-40% in tumor volume occurred in the GP-2250 group versus the vehicle group. On the final day of the study, tumor progression was significantly reduced in 4 tumor types: HT-29 in the GP-2250 500 and 1000 mg/kg groups, SKOV-3 in all GP-2250 treatment groups, Cal-27 in the GP-2250 1000 mg/kg group, and Hs-695T in the GP-2250 250 and 1000 mg/kg groups. Tumor regression in Cal-27 tumors was dose-dependent. GP-2250 demonstrated cytotoxic activity in vitro and reduced the tumor volume in a variety of human cancer cell lines in a xenograft mouse model. Given these results, as well as evidence of synergism with other anticancer drugs, GP-2250 shows promise as a new therapeutic agent for treating human cancers and is being evaluated in a phase 1 dose-escalation study (NCT03854100).

Felbamate-induced apoptosis of hematopoietic cells is mediated by redox-sensitive and redox-independent pathways.

Felbamate (FBM; 2-phenyl-1,3-propanediol dicarbamate) is an approved antiepileptic drug shown to be effective in a variety of seizure disorders refractory to other treatments. However, its use has been restricted because of association with occurrence of rare cases of aplastic anemia and hepatic failure. Since it was shown that FBM metabolism requires glutathione (GSH), we used two experimental protocols to determine if the effects of specific metabolites were sensitive to redox pathways. FBM and its metabolite W873 (2-phenyl-1,3-propanediol monocarbamate), at 0.1 mg/ml, induced increased apoptosis of bone marrow cells from B10.AKM mice as compared with B10.BR mice. Study of the effects of the drug on human promonocytic cell line U937 cells showed that FBM and the metabolite W2986 [2-(4-hydroxyphenyl)-1,3 propanediol dicarbamate], at higher concentrations (0.5 mg/ml), induced apoptosis in this cell line. We also observed that while FBM and its metabolites induced increased apoptosis of B cells with reduced intracellular GSH levels, addition of exogenous GSH decreased apoptosis induced by W873 but did not significantly affect apoptosis induced by FBM or W2986. Our results suggest that, at concentrations used during the present investigations, FBM metabolites induce apoptosis via redox-sensitive and redox-independent pathways.

Reactivity of atropaldehyde, a felbamate metabolite in human liver tissue in vitro.

Antiepileptic therapy with a broad spectrum drug felbamate (FBM) has been limited due to reports of hepatotoxicity and aplastic anemia associated with its use. It was proposed that a bioactivation of FBM leading to formation of alpha,beta-unsaturated aldehyde, atropaldehyde (ATPAL) could be responsible for toxicities associated with the parent drug. Other members of this class of compounds, acrolein and 4-hydroxynonenal (HNE), are known for their reactivity and toxicity. It has been proposed that the bioactivation of FBM to ATPAL proceeds though a more stable cyclized product, 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one (CCMF) whose formation has been shown recently. Aldehyde dehydrogenase (ALDH) and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes. This study examined effects of ATPAL and its precursor, CCMF on ALDH, GST and cell viability in liver, the target tissue for its metabolism and toxicity. A known toxin, HNE, which is also a substrate for ALDH and GST, was used for comparison. Interspecies difference in metabolism of FBM is well documented, therefore, human tissue was deemed most relevant and used for these studies. ATPAL inhibited ALDH and GST activities and led to a loss of hepatocyte viability. Several fold greater concentrations of CCMF were necessary to demonstrate a similar degree of ALDH inhibition or cytotoxicity as observed with ATPAL. This is consistent with CCMF requiring prior conversion to the more proximate toxin, ATPAL. GSH was shown to protect against ALDH inhibition by ATPAL. In this context, ALDH and GST are detoxifying pathways and their inhibition would lead to an accumulation of reactive species from FBM metabolism and/or metabolism of other endogenous or exogenous compounds and predisposing to or causing toxicity. Therefore, mechanisms of reactive aldehydes toxicity could include direct interaction with critical cellular macromolecules or indirect interference with cellular detoxification mechanisms.

Anticonvulsant and antiepileptogenic effects of fluorofelbamate in experimental status epilepticus.

PURPOSE: To examine the seizure-protective properties of fluorofelbamate, a felbamate analog, on acute and chronic seizures in an experimental model of self-sustaining status epilepticus (SSSE). METHODS: SSSE was induced by stimulation of the perforant path for 30 min (PPS) through chronically implanted electrodes in free-running adult male Wistar rats. Fluorofelbamate was injected intravenously (i.v.) either 10 min, or 40 min after SSSE induction. Seizure and spike profiles were analyzed off-line. RESULTS: Fluorofelbamate injected during the early stages of SSSE (10 min after the end of PPS), shortened the duration of seizures in a dose-dependent manner. While a dose of 50 mg kg(-1) was ineffective, 100 and 200 mg kg(-1) reduced cumulative seizure time from 393 +/- 10 min to 15 +/- 8 min and 2.4 +/- 0.5 min respectively. Administration of fluorofelbamate (200 and 300 mg kg (-1)) at a late stage of SSSE, which is refractory to treatment with conventional anticonvulsants, also significantly attenuated seizures. Acute fluorofelbamate treatment (200 mg kg(-1) 10 min after PPS) significantly decreased the frequency of spontaneous seizures which follow SSSE after a 'latent' interval. Moreover, in contrast to control animals, fluorofelbamate-treated rats showed regression of spontaneous seizures, and an apparent remission of epilepsy within 2 months after SSSE. CONCLUSIONS: Acute treatment of SSSE with fluorofelbamate showed strong anticonvulsant effects even during the late stages of SSSE. In this model, it also displayed antiepileptogenic properties: it reduced the severity of chronic epilepsy after SSSE and lead to apparent remissions of that epilepsy.

Interaction between human serum albumin and the felbamate metabolites 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one and 2-phenylpropenal.

Felbamate is an anti-epileptic drug associated with hepatotoxicity and aplastic anemia. These toxicities are believed to be mediated by the formation of the reactive species 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one is a metabolic precursor for 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one exists in equilibrium with 3-oxo-2-phenylpropyl carbamate, which can undergo beta-elimination to form 2-phenylpropenal. The work presented here investigates the interaction between 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one and human serum albumin (HSA). HSA (40 mg/mL) was found to decrease the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one from 4.57 +/- 0.44 h to 1.07 +/- 0.10 h at pH 7.4. This decrease in the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was due to increased beta-elimination of 3-oxo-2-phenylpropyl carbamate, presumably through HSA-mediated general base catalysis. The k(cat) for HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was determined to be 12.04 min(-)(1) M(-)(1). Competitive binding assays using warfarin and ibuprofen showed that HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one is dependent on the subdomain IIA binding site of HSA. LC/MS/MS analyses of trypsin digests of HSA incubations with either 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one or 2-phenylpropenal identified HSA-2-phenylpropenal adducts formed specifically at residues His-242 and His-247. These HSA-2-phenylpropenal adducts were found to be slowly reversible, with a decrease in alkylation of 74.0 +/- 0.6% after extensive dialysis. Interestingly, only the bis-adduct (His-242 and His-247) could be identified after dialysis. These results demonstrate the first direct example of 2-phenylpropenal conjugation to a human protein in vitro and suggest the possibility that HSA may be involved in the development of felbamate toxicity either by antigen formation or as a route of detoxification of 2-phenylpropenal.