A Soft-Drug Histone Deacetylase Inhibitor for Cutaneous T-Cell Lymphoma.

Abstract

Cutaneous T-Cell Lymphoma (CTCL) comprises a group of related lymphoproliferative disorders characterized by the presence of malignant lymphocytes in the skin. Most patients with CTCL enjoy a normal life expectancy, though all experience chronic morbidity due to the symptomatic and cosmetic manifestations of epidermotropism. There is no curative therapeutic option for patients with CTCL and consequently topical approaches have become a core component of standard care. Recently, activity against advanced CTCL has been demonstrated by a new class of targeted agents: histone deacetylase inhibitors (HDACi). One HDACi has been approved by the FDA for use in advanced disease (vorinostat; SAHA; Merck Research Laboratories, Rahway, NJ). Dose-limiting toxicities with parenteral HDAC inhibitors include fatigue, diarrhea, nausea and myelosuppression. The poor tolerability of the currently available, nonselective small molecules render most patients with this indolent disease unlikely to benefit from this pharmacologic class. We therefore conceived of reverse pro-drug HDAC inhibitors to allow high dose-intensity at the site of disease and presystemic metabolism to mitigate side effects and reduce exposure to genotoxic agents. Suberohydroxamic acid phenyl ester (SHAPE) is a first-in-class soft-drug HDACi. SHAPE is a soluble, scalable analog of SAHA possessing an isosteric, isoelectronic ester bond at a site predicted by molecular modeling not to impact target recognition features. Serum metabolism was confirmed using a real-time absorbance assay capable of monitoring SHAPE degradation to suberic acid hydroxamate and phenol. We have determined that the rapid hydrolysis of SHAPE in serum is due to the catalytic activities of butyrylcholinesterase and paraoxonase. Cellular inhibition of Class I and II HDACs was confirmed using high content imaging; effects on histone and tubulin acetylation comparable to SAHA are witnessed at concentrations above 20 micromolar. Active site binding to HDAC isoforms was determined using purified protein and a novel, miniaturized fluorescence polarization assay we designed to permit instantaneous kinetic measurements of labile compounds. Tolerability studies of topical SHAPE were performed on shaved immunocompetent mice and on immunodeficient animals bearing human skin xenografts. Histologic evidence of inflammation or damage was not visualized. We next performed experiments testing efficacy against CTCL in vivo. Using cell lines established from neoplastic, skin-homing lymphocytes arising from an interleukin-7 transgenic murine model of CTCL, we developed a transplantation model in syngeneic animals with rapid, homogeneous disease onset compatible with studies of investigational agents. Daily administration of SHAPE (50 mg/mL) for two weeks resulted in clearance of skin-homing lymphocytes, compared to vehicle treated controls. For use in murine model studies and anticipating human clinical trials, we identified acetylation response biomarkers using mass spectrometry and developed immunohistochemical protocols with acetylation state-specific antibodies. In sum, we report the design, characterization and translational studies supporting the clinical development of a novel, soft-drug HDAC inhibitor in CTCL.