Acyclic NucleotideAnaloguewith Potent Antineoplastic Activity in Dogs with Spontaneous Non ^ Hodgkin ’ s Lymphoma

Purpose: GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objectivewas to characterize the antiproliferative activity, pharmacokinetics,pharmacodynamics, and safety of GS-9219. Experimental Design: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types.To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEGwere evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non ^ Hodgkin’s lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy. Results: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes andhematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events. Conclusion:GS-9219 may have utility for the treatment of NHL. Non–Hodgkin’s lymphoma (NHL) is the second fastest growing form of cancer and the fifth leading cause of cancer deaths in the United States. The American Cancer Society estimates that the annual incidence of all forms of NHL in the United States in 2006 was 58,870 cases. In 2006, the estimated number of deaths due to NHL in the United States was 18,840 (1). Despite the introduction of rituximab in 1997, the latest Surveillance Epidemiology and End Results 5-year relative survival data (1999-2003) show a 5-year survival rate of 63% in NHL (indolent NHL ranges from 71% to 89%, depending on subtype; aggressive NHL ranges from 34% to 54%; ref. 1). Therefore, there is still a major unmet medical need in NHL patients for novel agents with improved efficacy compared with existing treatment modalities, especially in NHL patients who have failed frontline therapy. The acyclic nucleotide 9-(2-phosphonylmethoxyethyl)guanine (PMEG) forms an active phosphorylated metabolite, PMEG diphosphate (PMEGpp), in cells and causes cytotoxicity in dividing cells due to potent inhibition of the nuclear DNA polymerases a, y, and q, resulting in inhibition of DNA Cancer Therapy: Preclinical Authors’ Affiliations: Department of Research and Development, Gilead Sciences, Inc., Foster City, California; Department of Experimental Therapeutics, M. D. Anderson Cancer Center, Houston, Texas; Center for Clinical Trials and Research, School of Veterinary Medicine and Paul P. Carbone Comprehensive Cancer Center, University of Wisconsin, Madison,Wisconsin; and College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado Received 8/21/07; revised11/16/07; accepted12/13/07. Grant support:Gilead Sciences, Inc. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Conflict of interest: H. Reiser, J.Wang,W.J.Watkins, A.S. Ray, R. Shibata, G. Birkus,T. Cihlar, S.Wu, B. Li, X. Liu, I.N. Henne, G.H.I.Wolfgang, M. Desai, G.R. Rhodes, A. Fridland,W.A. Lee, and D.B. Tumas are employees and/or shareholders of Gilead Sciences, Inc. Requests for reprints: Hans Reiser, Department of Research and Development, Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404. Phone: 650522-6327; E-mail: hans.reiser@gilead.com. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-2061 www.aacrjournals.org Clin Cancer Res 2008;14(9)May1, 2008 2824 Research. on August 29, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from synthesis and/or DNA repair (2). In rodent models, PMEG has activity against leukemia and melanoma. However, the utility of PMEG as an anticancer agent is limited by its poor cellular permeability and toxicity, especially for the kidney and gastrointestinal tract (3–5). We synthesized prodrug analogues of PMEG to improve permeability and selectivity, with the hypothesis that this would lead to better efficacy and a more favorable therapeutic window. As lymphoid malignancies were the primary target of this effort, our strategy was to identify a PMEG prodrug that effectively loaded peripheral blood mononuclear cells (PBMC) with PMEGpp and resulted in minimal plasma levels of PMEG. Compounds were analyzed for their antiproliferative activity in vitro and for their cell and tissue distribution in vivo following i.v. administration. As a starting point for prodrug design, we used the N-substituted prodrug of PMEG, 9-(2-phosphonylmethoxyethyl)-N-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), due to its specific intracellular activation and ability to limit plasma exposure to the nephrotoxic agent PMEG (6, 7). Phosphonoamidate prodrugmoieties were added to increase the efficiency of lymphoid cell and tissue loading. Here, we describe a novel compound, GS-9219 (diethyl N,N ¶-[({2-[2-amino-6(cyclopropylamino)-9H-purin-9-yl]ethoxy}methyl)phosphonoyl]di-L-alaninate), which met the pharmacokinetic and potency selection criteria. The cytotoxic activity of GS-9219 was evaluated in vitro against a panel of hematopoietic and nonhematopoietic cell lines. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo , the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with naturally occurring, advanced-stage NHL following a single i.v. administration of GS-9219 as monotherapy. Materials andMethods Compounds. GS-9219 (succinate salt), cPrPMEDAP, PMEG, PMEGp, PMEGpp, and 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP) were synthesized at Gilead Sciences, Inc. Cytarabine, cladribine, and fludarabine desphosphate were purchased from Sigma. Clofarabine was synthesized by Acme Biosciences. Deoxycoformycin was purchased from SuperGen. Cell culture and proliferation assays. All cell lines were purchased from the American Type Culture Collection and cultured in RPMI 1640 (Life Technologies/Invitrogen) supplemented with 15% fetal bovine serum (Hyclone), 2mmol/L L-glutamine (Life Technologies/Invitrogen), and antibiotics. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation using standard procedures. T and B cells were purified using antibody-conjugated magnetic beads. T lymphoblasts were generated by stimulation of CD3 T cells with 1 Ag/mL phytohemagglutinin (PHA-P; Sigma) and 10 units/mL interleukin-2 (Roche Applied Science; ref. 8). B lymphoblasts were generated by stimulation of CD19 B cells with 20 Ag/mL pokeweedmitogen (Sigma). Cell proliferation was quantified either by bromodeoxyuridine (BrdUrd) incorporation assay or by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Diagnostics). Metabolite analysis following in vitro cell loading with [C]GS9219. Twenty million PHA-stimulated T cells were incubated with 10 Amol/L [C]GS-9219 for 24 h. Cells were washed once with tissue culture medium and twice with PBS, incubated in 80% methanol overnight, and centrifuged at 14,000 rpm for 15 min to remove denatured proteins. The methanol extracts were lyophilized and dissolved in the high-performance liquid chromatography (HPLC) loading buffer. A portion of each sample was used for scintillation counting to calculate the total pmoles, and the rest was used for HPLC analysis to calculate the ratio of metabolites. HPLC analysis was done by gradient elution using a Phenomenex Prodigy column (5 Am, ODS3 150 4 mm), where buffer A is 25 mmol/L K2HPO4 (pH 6.0) and 5 mmol/L tetrabutylammonium bromide and buffer B is 25 mmol/L K2HPO4 (pH 6.0), 70% acetonitrile, and 5 mmol/L tetrabutylammonium bromide. Pharmacokinetic studies. The plasma and PBMC pharmacokinetic profiles of GS-9219 and select metabolites were determined following a 30-min i.v. infusion of 3 mg/kg GS-9219 (formulated as a 5% dextrose solution) to three male beagle dogs. At predefined time points, blood was drawn for plasma and PBMC isolation. Isolated PBMCs were resuspended in PBS. A small aliquot of cells was used for cell counting to determine the concentration of cells. The remaining cell suspension was centrifuged to pellet cells and the pellet was resuspended in 70% methanol lyses buffer. Plasma levels of GS-9219 and cPrPMEDAP, or PBMC levels of cPrPMEDAP were determined by reverse-phase liquid chromatography using 0.2% formic acid, an acetonitrile gradient, and a Synergi Fusion-RP 80A column (150 2.1 mm, 4 Am) or a Synergi Hydro-RP 80A column (50 2 mm, 4 Am), respectively (columns purchased from Phenomenex, Inc.). Detection of analytes was accomplished by mass spectrometry using an API 4000 triple quadruple instrument (Applied Biosystems/MDS Sciex) operating in multiple reaction monitoring an