In vivo and in vitro antitumor activity of butyroyloxymethyl-diethyl phosphate (AN-7), a histone deacetylase inhibitor, in human prostate cancer Ada Rephaeli 1 * , Diana Blank-Porat 1,2 , Nataly Tarasenko 1 , Michal Entin-Meer 1 , Inesa Levovich 1,3 , Suzanne M. Cutts 4 , Don R. Phillips 4 , Zvi Malik 2 and Abraham Nudelman 3 * 1 Faculty of Medicine, Felsenstein Center for Medical Research, Tel Aviv University Beilinson Campus, Petach Tikva, Israel 2 Biology Department, Bar-Ilan University, Ramat Gan, Israel 3 Chemistry Department, Bar-Ilan University, Ramat Gan, Israel 4 Biochemistry, La Trobe University, Victoria, Australia AN-7, a prodrug of butyric acid, induced histone hyperacetylation and differentiation and inhibited proliferation of human prostate 22Rv1 cancer cells in vitro and in vivo. In nude mice implanted with these cells, 50 mg/kg AN-7 given orally thrice a week led to inhibition of tumor growth and metastasis, tumor regression in >25% of animals and increased survival. Median time to the experimental end point (tumor volume 2 cm 3 or death) in the untreated was 52 days, and average tumor volume was 0.8 6 0.18 cm 3 . At the same time, 94.4% of AN-7-treated mice survived and had average tumor volumes of 0.37 6 0.1 cm 3 . PSA expression was a useful marker for 22Rv1 lung metastasis detection. Sizeable metastases positively stained for PSA and limited air gaps were found in lungs of untreated mice. In animals treated with AN-7, lung morphology appeared normal. Primary tumors of treated animals were highly positive for PSA and had an elevated level of p21 and the proapoptotic protein Bax. Sections taken from AN-7- treated animals, examined under an electron microscope, exhib- ited condensed chromatin and apoptotic bodies. PSA serum levels were higher in untreated compared to treated animals and corre- lated with tumor volume. Since prolonged oral administration with 50 mg/kg or a single oral dose of 1.2 g/kg AN-7 did not cause adverse effects and the former exhibited significant anticancer activity, AN-7 is likely to display a high therapeutic index and may be beneficial for prostate cancer patients. ' 2005 Wiley-Liss, Inc. Key words: AN-7; histone deacetylase inhibitor; prostate-specific antigen; prodrug; lung metastasis; prostate carcinoma Modification of histones as molecular targets for disease ther- apy in general, and for cancer therapy in particular, is the subject of intense investigations. 1–4 The steady-state level of core histone acetylation, maintained by a dynamic balance between the activity of HATs and HDACs, provides a molecular communication link between chromatin and signal transduction. 5,6 In general, increased levels of histone acetylation lead to relaxation of the chromatin structure, allowing access of transcription factors and increased transcription, while decreased levels of acetylation are associated with repressed transcription. Considerable attention has been focused on developing HDA- CIs as potential anticancer agents. 1–4 In cancer cells, HDACIs affect the regulation of gene expression, cell growth, differentia- tion and apoptosis. A number of substances that display HDACI properties are in preclinical and clinical development, including AN-9 (a derivative of BA), the hydroxamic acid SAHA, trichosta- tin A, the benzamides MS-275 and CI-994 and the cyclic peptides trapoxin and depsipeptide. 7–12 For over a decade, we have studied prodrugs of low m.w. ali- phatic acids, primarily BA, as potential anticancer agents. 13–15 These compounds undergo esterase-dependent intracellular hydrolysis to release acids and aldehydes. The intracellular me- tabolic degradation is supported by the observation that the activ- ities of the compounds (including histone acetylation), modulation of gene expression, differentiation induction and proliferation inhibition are attenuated by esterase inhibitors. 16,17 In comparative studies, BA and its prodrugs elicited similar biologic effects; how- ever, the prodrugs affected cancer cells faster and at lower concen- trations than BA. The improved activity can be partially explained by the increased permeability of the prodrugs across cell mem- branes, leading to efficient delivery of BA to its cellular site of action. 18 HDACI esters induced transient hyperacetylation of his- tones and modulation of the early regulatory genes c-myc and c- jun, the tumor-suppressor gene RB as well as the antiapoptotic gene Bcl-2 in HL-60 and WEHI cells. 19–21 AN-9, the best-studied prodrug, effectively inhibited growth, proliferation and clonogenicity of a wide spectrum of cancer cells at concentrations 10- to 140-fold lower than BA. 13 When eval- uated in 76 primary solid human tumors (including colorectal, breast, lung, ovarian, renal and bladder), AN-9 was more active than BA in inhibiting solid-tumor colony-forming units. 22 Twenty-one primary samples of acute leukemia, including chemo- resistant cells, were also sensitive to the antiproliferative effects of AN-9, with an average IC 50 of 45.8 6 4.1 lM. While AN-9 dis- played synergy with DNA-interacting agents, BA itself did not. 23 The synergistic effects between AN-9 and DNA-disrupting agents have been observed in murine monocytic leukemia cells and the combination of AN-9 with daunorubicin led to a significant increase in survival of mice inoculated with acute monocytic leu- kemia cells. 24 AN-9 increased the level of doxorubicin- and mitox- antrone-DNA adducts and affected in a synergistic manner their antiproliferative activity in neuroblastoma and breast cell lines. The synergy can be attributed largely to the formaldehyde released from AN-9, which activates doxorubicin or mitoxantrone, result- ing in potentiation of anthracycline-DNA adduct formation. 25,26 The in vivo anticancer activity of AN-9 has been demonstrated in syngeneic murine models. 13,14,24 In a phase I clinical study, none of the 28 cancer patients treated with AN-9 at dosages rang- ing 0.047–3.3 g/m 2 /day every 3 weeks experienced dose-limiting toxicity. Dose escalation was limited by the maximum feasible volume of its intralipid formulation vehicle that could be adminis- tered safely in this schedule, resulting in a maximum deliverable daily dose of 3.3 g/m 2 . 7 A second generation of HDACIs designed Grant sponsor: Israel Science Foundation; Grant number: 542/00-4; Grant sponsor: Israel Cancer Research Fund; Grant sponsor: CaPCURE Israel; Grant sponsor: Marcus Center for Pharmaceutical and Medicinal Chemistry at Bar Ilan University; Grant sponsor: Australian Research Council. *Correspondence to: Faculty of Medicine, Felsenstein Center for Med- ical Research, Tel Aviv University Beilinson Campus, Petach Tikva, 49100, Israel. Fax: 1972-3-922-8096. E-mail: adarep@post.tau.ac.il Received 12 August 2004; Accepted after revision 14 January 2005 DOI 10.1002/ijc.21030 Published online 30 March 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: AN-1, methylidene dibutyrate; AN-7, butyroyloxy- methyl-diethyl phosphate; AN-9, pivaloyloxymethyl butyrate; AN-10, 1- butylidene dibutyrate; BA, butyric acid; DAB, diaminobenzidine; DDW, double-distilled water; DTT, dithiothreitol; HAT, histone acetyl transfer- ase; HDAC, histone deacetylase; HDACI, histone deacetylase inhibitor; HRP, horseradish peroxidase; IC 50 , concentration that inhibits 50%; IHC, immunohistochemistry; LD 50 , lethal dose for 50% of subjects; PSA, pros- tate-specific antigen; SAHA, suberoylanilide hydroxamic acid; TAE, TRIS-acetate EDTA; TE, TRIS-EDTA. Int. J. Cancer: 116, 226–235 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer