ANTICANCER RESEARCH 35: 229-238 (2015)

Discovery of PAT-1102, a Novel, Potent and Orally Active Inhibitor with Antitumor Activity in Cancer Mouse Models

JAGADHESHAN HIRIYAN1, PRASAD SHIVARUDRAIAH1, GOVINDARAJULU GAVARA1, PAZHANIMUTHU ANNAMALAI2, SELVAKUMAR NATESAN3, GANESH SAMBASIVAM1 and SUNIL K. SUKUMARAN1

1Anthem Biosciences Pvt. Ltd., Bangalore, India; 2Micro Therapeutics Research Labs Pvt. Ltd., Chennai, India; 3DSK Innosciences, Kelamangalam, Hosur, India

Abstract. Aim: Histone deacetylase (HDAC) inhibitors are with antiproliferative activity against several human cancer a class of drugs that modulate transcriptional activity in cell lines and antitumor activity in mouse xenograft models. cells and are known to induce cell-cycle arrest and Based on the pre-clinical efficacy and safety profile of PAT- angiogenesis, the major components of tumor cell 1102, the compound demonstrates significant potential for proliferation. The aim of the present study was to evaluation as a novel drug candidate for cancer therapy. characterize a novel hydroxamic acid-based HDAC inhibitor, PAT-1102, and determine its efficacy and tolerability in pre- Histone acetylation/deacetylation is mediated by a class of clinical models. Materials and Methods: HDAC enzyme enzymes known as Histone acetyl transferase (HATs) and inhibition was measured using HeLa cell nuclear extracts, histone deacetylases (HDACs). HDACs are involved in many and recombinant HDAC enzymes. Antiproliferative activity biological processes, including development, cellular was assessed in a panel of cancer cell lines. Histone hyper- proliferation, differentiation, and apoptosis. Inhibition of acetylation status and p21 induction were assessed in HeLa HDACs is known to play an important role in epigenetic cells by immunoblotting. The effect on apoptosis was tested regulation by inducing cell death, apoptosis, and cell-cycle by caspase-3 activation and detection of cleaved poly-ADP arrest in cancer cells. The balance between histone ribose polymerase (PARP). Single-dose pharmacokinetics of acetylation and deacetylation, mediated by HATs and the compound were assessed in BALB/c mice following oral HDACs, respectively, is usually well-regulated, but the and intravenous administration. Antitumor efficacy was balance is often upset in diseases such as cancer (1). HDAC evaluated in tumor-bearing mice established from lung and inhibitors have emerged as an important class of anticancer colorectal cancer cells (A549 and HCT116, respectively). drugs and have proven to be efficacious in the clinic. The Results: PAT-1102 demonstrated potent HDAC-inhibitory mechanisms of action of HDAC inhibitors are thought to be activity and growth-inhibitory properties against a panel of related to altered gene expression and to changes in non- cancer cell lines. The optimized compound PAT-1102 exhibits histone proteins via regulation at the epigenetic and post- good aqueous solubility, metabolic stability and a favorable translational modification levels, respectively. In many tumor pharmacokinetic profile. Once-daily oral administration of cell lines, HDAC inhibitors cause up-regulation of the cell PAT-1102 resulted in significant antitumor activity and was cycle gene p21, blocking the cyclin–dependent kinase well-tolerated in mice. Conclusion: Our results indicate that complexes, leading to cell-cycle arrest and inhibition of PAT-1102 is a novel, potent, orally available HDAC inhibitor differentiation (2, 3). There exist 18 HDACs, which are classified according to functional and phylogenetic criteria (4). They are divided into Zn2+-dependent (class I, II and IV) and Zn2+-independent, adenine dinucleotide Correspondence to: Sunil K. Sukumaran, Ph.D., Discovery Biology, (NAD+)-dependent (class III) enzymes. Conventional Anthem Biosciences Pvt. Ltd, Hosur Road, Bangalore-560 099, HDACs are composed of 11 members which require Zn2+ as India. Tel: +91 8066724028, Fax: +91 8066724020, e-mail: [email protected] a co-factor for their deacetylase activity and are divided into four classes depending on their homology (5). The class III Key Words: Histone deacetylation, HDAC inhibitor, PAT-1102, HDACs (sirtuins) are structurally related to yeast SirT2, and . there is increasing evidence that they are critical

0250-7005/2015 $2.00+.40 229 ANTICANCER RESEARCH 35: 229-238 (2015) transcriptional regulators (6, 7). Class I comprises HDACs profile, PAT-1102 has the potential to be evaluated further in 1, 2, 3, and 8, which are located within the nucleus; class II regulatory safety studies and the compound could have comprises HDACs 4 to 7, 9, and 10, which are located in advantages over other HDAC inhibitors with poor both the nucleus and the cytoplasm; and class IV comprises pharmacokinetic properties and dose-limiting side-effects. HDAC 11. Unlike conventional HDACs, class III HDACs are composed of seven mammalian sirtuins (SIRT1–7) (8). These Materials and Methods are NAD+-dependent protein deacetylases localized in the nucleus (SIRT1, SIRT6, and SIRT7), mitochondria (SIRT3, Cell lines, reagents, and animals. All the HDAC inhibitor compounds, including vorinostat and pracinostat , were synthesized SIRT4, and SIRT5), and cytoplasm (SIRT2). as per reported methods (21, 22). Human carcinoma cell lines were Several structurally distinct classes of HDAC inhibitors originally obtained from the American Type Culture Collection have been purified from natural sources or synthetically (ATCC; Manassas, VA, USA). Cell culture media was obtained from developed, and some of them have advanced into phase I/II Sigma-Aldrich (St. Louis, MO, USA) and fetal bovine serum (FBS) clinical trials in solid tumors and hematological malignancies was procured from Life Technologies (Carlsbad, CA, USA). Human (9). Although the mechanisms of action of HDAC inhibitors umblical vein endothelial cells (HUVEC) and endothelial growth are still unclear, they are emerging therapeutic agents that medium (EGM) were obtained from Lonza Inc. (San Diego, CA, USA). Antibodies against acetylated H3 and H4 were obtained from have been clinically validated in patients with hematological Merck Millipore (Billerica, MA, USA) and antibodies to malignancies, including cutaneous T-cell lymphoma (1) Two p21CIP/WAF1 were obtained from Sigma-Aldrich (St. Louis, MO, HDAC inhibitors, vorinostat (suberoylanilide hydroxamic USA). All other chemicals and solvents were obtained from Sigma- acid; Merck & Co., Inc.) and depsipeptide (, FK- Aldrich unless specified otherwise. 228; Gloucester Pharmaceutical Inc.), have been approved Athymic nude mice and severe combined immunodeficient by the US food and drug administration (in 2006 and 2009, (SCID) mice used for xenograft experiments were obtained from respectively). Harlan Laboratories (Indianapolis, IN, USA) and housed under pathogen-free conditions. All studies involving animals were Clinical trials with several HDAC inhibitors as single performed according to the protocols approved by the Institutional agents, in combination with conventional chemotherapies, or Animal Ethics Committee (IAEC) of Anthem Biosciences. as targeted drugs are under various stages of development. Hydroxamates exert nonspecific HDAC-inhibitory activity, In vitro HDAC enzyme assay. Nuclear fractions prepared from HeLa affecting all classes of HDACs (10). Other compounds cells as per established protocols were used as a source of HDAC specifically inhibit class I HDACs, e.g. the benzamide enzyme. Nuclear fractions were lysed with 0.5% Triton X-100 containing phosphate buffer (pH 8.0) and were centrifuged. HDAC (MS-275), or class I and IIa HDACs, as in the case inhibition screening of PAT-1102 and the reference inhibitor of the short-chain fatty acids valproic acid and butyrate (11). vorinostat was performed using a fluorescence-based assay with a Isotype-selective compounds are also increasingly becoming fluorescent substrate [Boc-Lys (Ac)-AMC Substrate] as reported available, e.g. tubacin, , and PC-34501 previously (19). Fluorescent, deacetylated substrate was detected selectively inhibit HDAC6, -1, and -8, respectively (12-15). with a microplate reader (Bio-Tek Instrument Inc., Winooski, VT, However, there has been an ongoing debate over whether USA). The 50% HDAC inhibitory concentration (IC50) was isoform and class-specific HDACIs are advantageous over determined by testing at concentrations of 0.001, 0.01, 0.1, 1 and 10 μM. Isoform selectivity was tested using recombinant HDAC1, broad-spectrum or pan HDAC inhibitors (16). Most of the HDAC2, HDAC3, HDAC6, and HDAC8 isoforms (Enzo Life HDAC inhibitors that have entered clinical trials have Sciences Inc., Farmingdale, NY, USA). limitations, including low bioavailability, low potency, cardiovascular safety issues, and potential for drug–drug Cell proliferation assays. Antiproliferative activity of the compounds interactions through cytochrome P450 inhibition (17). was tested against a panel of cancer cell lines including lung, cervical, Therefore, there is still a clinical opportunity for novel, colonic, brain, renal, leukemia, prostattic, pancreatic, skin, bone, orally available efficacious HDAC inhibitors with a wider breast, ovarian cancer by a cell viability assay using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent safety margin. (Sigma-Aldrich, St. Louis, MO, USA). Human cancer cell lines were We previously synthesized a novel series of hydroxamic cultured in complete media containing 10% heat-inactivated FBS and acid-based HDAC inhibitors with nanomolar potency against 100 U/ml penicillin, 100 μg/ml streptomycin in a humidified incubator HDAC (unpublished data). We had identified PAT- with 5% CO2 at 37˚C and subcultured twice weekly. Cells were seeded 1102(Figure 1) as a potent HDAC inhibitor which was active in 96-well plates at a density of 3×103 cells per well in 100 μl of against a wide range of cancer cell lines. In this report, we medium and were allowed to attach for 24 h. Stock concentrations of describe the preclinical profile of PAT-1102, a highly the compounds were made in dimethyl sulfoxide (DMSO). Media (100 μl/well) containing different concentrations of compounds (0.01, 0.1, efficacious HDAC inhibitor with excellent pharmacokinetic 1, 10 and 100 μM) were added to the cells and were incubated for 48 properties which has been shown to be orally efficacious and h. Vorinostat was tested as a reference compound in the assay. On the well-tolerated in murine cancer models. The study further day of termination, 50 μl of MTT reagent solution (5 mg/ml) was indicates that based on the preclinical efficacy and safety added to the medium and the cells were incubated for 3 h. The

230 Hiriyan et al: PAT-1102, a Novel HDAC Inhibitor medium was then aspirated and 100% DMSO was added to solubilize Life Technologies. The final composition of the assay mixture the violet MTT-formazan product. The absorbance at 570 nm was included 100 μM of PAT-1102 (dissolved in DMSO), 0.5 mg/ml of measured on a Biotek Synergy HT 96-well plate reader by microsomal protein and cofactors (5.0 mM glucose-6-phosphate, spectrophotometry. Assays were performed in duplicates for each 0.06 U glucose-6-phosphate dehydrogenase, 2.0 mM MgCl2, 1.0mM concentration. Results are expressed as the percentage of growth NADP+, 0.5 mM uridine-diphosphate-glucuronic acid, 0.6 mM 3- inhibition with respect to that of the DMSO-treated control wells. A phosphoadenosine 5-phosphosulfate and 1 mM reduced glutathione). dose–response curve was generated for PAT-1102 and vorinostat and The compounds were incubated with liver microsomes in the GI50 (concentration which inhibits 50% of cellular growth) values presence of cofactors for 1 h at 37˚C. The reaction was stopped by were interpolated from the growth curves using GraphPad Prism the addition of ice-cold acetonitrile. The samples were then software (GraphPad Software Inc., La Jolla, CA, USA). centrifuged and supernatants were analyzed as per established protocols using liquid chromatography tandem mass spectrometry Histone hyperacetylation and p21 induction by western blotting. (API 3200 LC-MS/MS system; Applied Biosystems, Foster City, Hyperacetylation of histones (H3 and H4) and p21CIP/WAF1 CA, USA). The percentage of the parent compound remaining after induction was measured by western blot of HeLa cell lysates as 1 h of incubation was calculated with respect to the peak area of the described elsewhere (2). Briefly, HeLa cells were cultured in compound at time 0 using the system’s software. complete media and seeded into 6-well cell culture plates and on the following day, compounds were added at 0.03, 0.1, 0.3, 1 and 3 Pharmacokinetics of PAT-1102 in mice. Studies were conducted to μM and incubated at 37˚C overnight under 5% CO2. On the next determine the oral bioavailability and pharmacokinetics of PAT-1102 in day, the cells were washed in PBS and lysed using RIPA buffer male Balb/c mice. The study was performed after obtaining the (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration in the approval of the IAEC (Approval No. ABD/IAEC/PR/05R3-12-13). cell lysates was quantified using Bradford’s method (20) and Briefly, Mice aged 5-6 weeks weighing around 25-30 g were used for proteins were loaded on a 10% gel and subjected to immunoblotting the study. The animals were fasted overnight with free access to water. for acetyl-histone H3 and acetyl-histone H4 using rabbit polyclonal PAT-1102 or vorinostat was administered through oral gavage at a dose antibodies to acetyl-histone H3 and H4. of 50 mg/kg bodyweight in a formulation containing 0.5% methylcellulose in water and 0.1% Tween 80 at a dose volume of 10 Apoptosis assays [poly ADP ribose polymerase (PARP) cleavage and ml/kg bodyweight. For intravenous pharmacokinetics, the compounds caspase-3 activation]. Caspase-3 activity was measured in HT-29 were administered via tail vein at a dose of 10 mg/kg in a formulation cells using a commercially available kit (Sigma- Aldrich, St. Louis, containing 0.9% saline at a dose volume of 5 ml/kg. Blood samples MO, USA). Briefly, HT-29 cells were cultured in McCoy’s 5a were withdrawn at 15, 30, 60 min, 1 h, 2 h, 4 h and 6 h post dosing. medium containing 10% FBS and antibiotics. On the day of the The blood samples were centrifuged at 3,000 × g for 5 min at 4˚C for study, 10,000 cells were seeded into each well of a 96-well plate and separation of plasma and the corresponding plasma samples were incubated for 12-16 h. PAT-1102 and vorinostat were added at collected into clean pre-labeled tubes. A liquid-liquid extraction concentrations ranging from 0.1 μM to 30 μM and cells were method was employed for obtaining PAT-1102 from plasma samples: incubated for 48 h. The cells were then lysed in lysis buffer and the 100 μl of plasma sample and 10 μl of internal standard solution of lysates were used to perform the assay according to the fluconazole were added to the centrifuge tube and vortexed for 2 min; manufacturer’s instruction. The assay is based on the hydrolysis of 2.5 ml of tertbutylmethyl ether were added and the mixture was acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin by caspase-3, vortexed for 15 min. The samples were centrifuged at 4300 × g for 15 resulting in the release of the fluorescent 7-amino-4-methylcoumarin min at 10˚C. The supernatant organic layer was separated and which is measured at an excitation and emission wavelength of 360 evaporated to dryness. The sample residues were reconstituted with nm and 460 nm respectively. Apoptotic activity of PAT-1102 was 200 μl of dilution solvent (methanol:water, 80:20 v/v). The also assessed in HeLa cells by detection of cleaved PARP, a substrate reconstituted samples were then transferred into auto sampler vials and of caspase-3, in HeLa cell lysates by immunoblotting. Protein analyzed using liquid chromatography tandem mass spectrometry (API determinations were performed using Bradford method. Protein (10 3200 LC-MS/MS system; Applied Biosystems, Foster City, CA, USA) mg) separated on sodium dodecyl sulfate 10% polyacrylamide gels as per methods established in-house. Data were analyzed using was electroblotted onto Polyvinylidene fluoride membranes. Blots WinNonlin version 5.2 (Pharsight Corporation, St. Louis, MO, USA). were probed with anti-human PARP (Cell Signaling Technology, Danvers, MA, USA) and visualized by enhanced chemiluminescence (Amersham GE Healthcare, Pittsburgh, PA, USA). In vivo anticancer activity in human tumor xenograft models. In vivo antitumor activity of PAT-1102 was assessed in 6-week-old athymic Matrigel angiogenesis assay. The in vitro angiogenesis assay was nude or SCID mice (C.B-17/IcrHsd-PrkdcscidLystbg-J) aged 5-6 performed as described previously (18). HUVECs were cultured in weeks and the study was performed after obtaining the approval of EGM and plated in 24-well plates (5×104 cells per well) previously the IAEC (Approval No.: ABD/IAEC/PR/25R2-12-13). Animals coated with Matrigel® (BD Biosciences, San Jose, CA, USA). The were purchased from Harlan Laboratories and housed in individually plates were then incubated overnight in the presence of PAT-1102 ventilated cages under controlled conditions and maintained on a 12- or vorinostat at 3.125, 6.25 and 12.5 μM in an incubator at 37˚C h light/12-h dark cycle, with food and water supplied ad libitum. under 5% CO2. The morphology of capillary-like structures formed A549 (lung adenocarcinoma) and HCT-116 (colorectal carcinoma) were visualized using an inverted microscope and documented. cells obtained from the ATCC were cultured in appropriate media containing 10% FBS and antibiotics. On the day of the implantation, Metabolic stability of compounds in human and mouse liver a 0.1 ml cell suspension was prepared containing 106 cells in hanks microsomes. The metabolic stability of test compounds was balanced salts solution mixed with Matrigel® (BD Bioscience, San determined using human and mouse liver microsomes obtained from Jose, CA, USA) in a 1:1 ratio. The cell suspension was injected

231 ANTICANCER RESEARCH 35: 229-238 (2015)

Table I. Histone deacetylase (HDAC) inhibitory activity of PAT-1102. Table II. Antiproliferative activity of PAT-1102 expressed as the mean HDAC inhibition by PAT-1102 was measured in HeLa cell nuclear concentration of the compound which inhibits 50% of cell growth, the extract as the enzyme source or against human recombinant HDAC 50% growth-inhibitory concentration (GI50). Antiproliferative effect of isoenzymes using a fluorescence-based assay as described in the PAT-1102 against a panel of cancer cell lines obtained from the Materials and Methods section. The inhibitory concentration IC50 was American Type Culture Collection using 3-(4,5-dimethylthiazol-2-yl)- calculated from curves of concentration used versus percentage 2,5-diphenyltetrazolium bromide (MTT) reagent as described under inhibition plotted using Graph Pad Prism software. Materials and Methods. GI50 was calculated using GraphPad Prism software. The mean GI50 was derived from individual assays performed Compound IC50 (nM) in triplicates.

HeLa HDAC1 HDAC2 HDAC3 HDAC6 HDAC8 GI50 (μM) nuclear extract Tissue Cell line PAT-1102 Vorinostat

PAT-1102 3 25 29 2 11 282 Lung A549 3.1±1.99 5.9±2.4 Vorinostat 78 83 78 32 128 1612 NCI-H23 0.9±0.25 3.2±1.0 NCI-H460 1.9±0.75 4.4±1.4 Calu-6 0.6±0.33 3.5±0.5 Cervix Ca Ski 1.1±0.49 6.0±5.3 Hela-229 1.1±0.23 4.7±3.2 Hela-S3 0.4±0.09 2.9±0.5 Colon Colo-205 0.3±0.05 1.6±0.1 HCT-15 19.3±3.8 5.2±1.9 HCT-116 0.1±0.04 2.2±0.0 HT-29 1.3±0.33 3.6±2.6 Brain IMR-32 0.4±0.09 1.8±0.2 U-87-MG 0.5±0.09 6.7±1.1 SH-SY-5Y 0.2±0.08 0.8±0.4 Renal ACHN 0.3±0.14 1.6±0.5 786-O 4.1±1.4 4.4±2.0 Leukemia RPMI-8226 0.4±0.3 2.2±2.3 K562 0.3±0.06 2.2±0.7 Prostate DU-145 0.2±0.06 1.3±0.0 PC-3 4.1±0.38 8.3±0.6 Figure 1. Structure of PAT-1102. Pancreas PANC-1 1.3 21.9 Skin A431 0.5±0.13 1.9±1.0 Bone KHOS 2.4±0.4 29.3±7.2 Breast MCF-7 2.6±1.26 5.5±1.2 Ovary SK-OV-3 1.6±0.10 4.4±1.5 subcutaneously into the flank of the animals under isoflurane PA-1 0.1±0.01 0.3±0.05 anesthesia. Tumor size was measured with a digital Vernier caliper and tumor-bearing mice were randomized into control and treatment groups (n=10) when the tumor volume reached approximately 100 mm3. Tumor volume was calculated using the formula, tumor volume=(length × width2)/2. Tumor-bearing mice were administered PAT-1102 once daily at 12.5, 25, 50 and 100 mg/kg doses by oral inhibitory activity of the compounds was tested against gavage. Vorinostat and pracinostat were administered once daily orally at 150 mg/kg and 75 mg/kg doses respectively. Tumor volume various HDAC isoforms, HDAC1, 2, 3, 6 and 8. (see Table I) and body weight were measured twice a week. Our results indicate that the compound is a pan-HDAC inhibitor similar to the reference compound vorinostat. Statistical analysis. The terminal tumor volumes from in vivo xenograft studies were subjected to one-way ANOVA analysis Effect on histone acetylation and p21 induction. The effect followed by Dunnett’s test when there were multiple treatment of PAT-1102 on histone acetylation in HeLa cells was groups. Results were considered statistically significant when p<0.05. assessed by treatment with the compound in culture. Acetylated histones (H3 and H4) were detected in cell Results lysates by western blotting using specific antibodies. H3 and H4 histone hyperacetylation was measured in HeLa cellular HDAC enzyme inhibition. The biological activity of the extract 24 h post-treatment with the compound as detected HDAC inhibitors was assessed in vitro using a cell-free by western blot. A dose-dependent accumulation of HDAC enzymatic assay. PAT-1102 exhibited potent HDAC- acetylated histones H3 and H4 was observed with PAT-1102, inhibitory activity, with an IC50 value of 3 nM. The the effect of which was comparable to that of vorinostat

232 Hiriyan et al: PAT-1102, a Novel HDAC Inhibitor

Figure 2. Effect on histone acetylation status and p21CIP/WAF1 induction by PAT-1102 in HeLa cells. Western blot analysis of acetylated histones, H3 and H4 in HeLa cell lysates. HeLa cells were treated with different concentrations of PAT-1102 or vorinostat. Cell lysates were prepared and subjected to immunoblotting. PAT-1102 treatment resulted in dose-dependent accumulation of acetylated H3 and H4. HeLa cells were cultured in media containing different concentrations of PAT-1102 or vorinostat for 24 h. p21CIP/WAF1 induction was measured in cell lysates using specific antibodies by western blotting. PAT-1102 treatment significantly induced P21 protein expression in a dose-dependent manner at the concentrations tested.

(Figure 2). We assessed the effect of PAT-1102 on the expression of p21CIP/WAF1, a cyclin-dependent kinase inhibitior, on HeLa cells by western blotting. The induction of p21CIP/WAF1 expression in HeLa cells by PAT-1102 was dose-dependent and the effect was comparable with that of vorinostat (Figure 2).

Induction of apoptosis of human cancer cells by PAT-1102. We investigated the ability of PAT-1102 to induce apoptosis of cancer cells and to determine if the apoptosis was caspase- dependent. In this regard, PAT-1102-induced caspase-3 activation in HT-29 cells was measured using a fluorescence- based assay. The compound significantly activated caspase-3 enzyme with an EC50 (Effective concentration at which shows 50% of pharmacological response) of 1.36 μM, which was superior to that of vorinostat (4.52 μM) (Table II). We analyzed PARP cleavage in HeLa cells using an antibody Figure 3. Induction of apoptosis of HeLa cells by PAT-1102, as measured by detection of cleaved poly ADP ribose polymerase (PARP). which specifically recognized a caspase_specific cleaved Apoptotic activity of PAT-1102 was assessed by detection of cleaved PARP fragment. As shown in the figure, we observed a dose- PARP in HeLa cell lysates by immunoblotting. As indicated in the dependent caspase-specific degradation of PARP by PAT- image, cleaved PARP was detected on treatment with 1, 3 and 10 μM 1102 in HeLa cells (Figure 3). concentrations of PAT-1102.

Matrigel angiogenesis assay. Angiogenesis involves the migration of endothelial cells and their organization into a human cancer cell lines by treating the cells with PAT-1102 network of tube-like structures. In HUVECs, PAT-1102 or vorinostat and the GI50 was determined. PAT-1102 inhibited endothelial tube formation at therapeutically treatment resulted in a dose-dependent inhibition of relevant concentrations in the micromolar range (Figure 4). proliferation of most of the cell lines tested at low The effect was comparable with that of vorinostat. micromolar concentrations (Table II). The inhibitory effect of PAT-1102 on proliferation of cancer cells was comparable Antiproliferative activity against cancer cells. The growth- or superior to that of vorinostat against several cell lines inhibitory activity of PAT-1102 was assessed in a panel of 29 under our experimental conditions.

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Figure 4. Effect of PAT-1102 and vorinostat on tube formation in human umblical vascular endothelial cells (HUVECs). PAT-1102 was tested for its effect on angiogenesis using the tube formation assay in HUVECs. Cells were cultured in complete media and plated in 24-well plates (5×104 cells per well) coated with Matrigel® (BD Biosciences) and treated with PAT-1102 or vorinostat at the concentrations mentioned, tube formation was assessed under a microscope. Treatment of HUVECs with PAT-1102 resulted in a dose-dependent inhibition of tube formation.

Figure 5. Single-dose oral and intravenous pharmacokinetics of PAT-1102 and vorinostat in BALB/c mice.

Metabolic stability in liver microsomes. To assess the vorinostat, BALB/c mice were orally dosed with 50 mg/kg metabolic stability of the compound, the compound was bodyweight, or injected with 10 mg/kg i.v. to calculate incubated in mouse liver microsomal fractions. PAT-1102 bioavailability. Mice were sacrificed at different time points was found to be more metabolically stable compared to between 15 min and 6 h, and plasma inhibitor concentrations vorinostat, with 100% of the compound remaining after 1 h were determined. The oral bioavailability of PAT-1102 was compared to 67% of that of vorinostat (data not shown). 10%, which was similar to that of vorinostat (11%) (Table III) but PAT-1102 yielded higher maximum plasma Bioavailability and pharmacokinetics in mice. To compare concentrations (1,313 ng/ml) compared to vorinostat (580 the pharmacokinetic properties of PAT-1102 to those of ng/ml) (Figure 5).

234 Hiriyan et al: PAT-1102, a Novel HDAC Inhibitor

Figure 6. Antitumor efficacy of PAT-1102 against xenografts in nude mice. a: Tumor growth kinetics in athymic nude mice subcutaneously implanted with 106 A549 cells and treated with PAT-1102 at 12.5, 25 and 50 mg/kg p.o. or vorinostat at 150 mg/kg p.o. (n=8 in each group) once daily for 21 days. PAT-1102 (50 mg/kg) vs. control: p<0.05, vorinostat vs. control: p<0.05 at day 21. b: Tumor growth kinetics in SCID mice subcutaneously implanted with 106 HCT-116 cells and treated with PAT-1102 at 25 and 50 and 100 mg/kg p.o. or pracinostat (SB-939) at 150 mg/kg, p.o, (n=8 in each group) once daily for 21 days. ***p<0.0001. Statistical analyses were performed by one-way ANOVA followed by Dunnett’s test. Data are represented as mean±SE.

Antitumor activity in human tumor xenograft models. To after 21 days (Figure 6a). TGI at 50 mg/kg of PAT-1102 was assess the ability of PAT-1102 to inhibit tumor growth in similar to that observed with vorinostat at 150 mg/kg. The vivo, we examined its effect in subcutaneous human efficacy was thus achieved at a third of the dose of the xenograft models in immunocompromised mice. The reference compound vorinostat. Furthermore, there was no antitumor efficacy was assessed in xenograft models significant reduction in bodyweight in the PAT-1102-treated established with A549 cells in athymic nude mice. Once- group compared to the vehicle control group. daily oral administration of PAT-1102 resulted in a We also evaluated the antitumor efficacy of PAT-1102 in a significant dose-dependent tumor growth inhibition (TGI) subcutaneous xenograft model established with HCT-116

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Table III. Comparative pharmacokinetic parameters for PAT-1102 and Table IV. Tumor growth inhibition (TGI) in subcutaneous A549 and vorinostat after intravenous (I.V.; 10 mg/kg) and oral administration (50 HCT116 tumor xenograft models established in mice. mg/kg) in BALB/c mice. The pharmacokinetic parameters shown were calculated by a non-compartmental method using the WinNonlin 4.0 % TGI software. Data shown are averages derived from three individual animals. Compound Dose A549 HCT-116 (mg/kg, p.o.) xenograft xenograft Parameters PAT-1102 Vorinostat PAT-1102 12.5 27 NA Oral I.V. Oral I.V. 25 37 38 50 52 48 Dose (mg/kg) 50 10 50 10 100 NA 72 Tmax (h) 0.25 − 0.08 − Vorinostat 150 54 NA Cmax (ng/ml) 1313 − 580 − Pracinostat 75 NA 77 T1/2 (h) 0.4 − 0.8 − AUClast (h ng/ml) 586 1194 347 625 TGI: Tumor growth inhibition was calculated with respect to the growth Vss (l/kg) − 12.5 − 4.3 of tumor in the vehicle-treated control group after 21 days treatment Cl (ml/min/kg) − 681 − 247 with compound; NA: Not available. MRTlast (h) − 0.25 − 0.21 F% (oral bioavailability) 9.8 11.1

−: Not detected; AUC: area under the plasma concentration–time curve from 0 h to last time point; Cmax: peak plasma concentration; Tmax: time to reach Cmax; T1/2: terminal half-life; MRT: mean retention time, preclinical xenograft models of solid tumors, such as lung CL: clearance; F%: fractional bioavailability. and colorectal cancer, comparable with other HDAC inhibitors such as vorinostat, and pracinostat currently in clinical development. PAT-1102 has increased plasma drug concentrations compared to vorinostat. These favorable colorectal carcinoma cells in SCID mice. We compared the pharmacokinetic properties translate into a dose-dependent efficacy with that of another HDAC inhibitor, pracinostat and enhanced antitumor efficacy in cancer models. The which is in clinical development. PAT-1102 was administered reduced toxicity of PAT-1102 could facilitate the use of orally at three doses, 25, 50 and 100 mg/kg/day in a 21-day higher doses of the drug in the clinic in order to maximize study and compared to pracinostat (75 mg/kg/day). Once- antitumor activity. Therefore, PAT-1102 has the potential to daily oral administration of PAT-1102 resulted in a dose- be more effective in targeted clinical trials than other dependent TGI (Figure 6b). Under our experimental HDAC inhibitors with poor pharmacokinetic properties and conditions, PAT-1102 was found to be better tolerated dose-limiting side-effects. We believe the improved compared to pracinostat as evidenced by reduction in bioavailability and safety profile of this compound could bodyweight and greater mortality with pracinostat, indicating help achieve better efficacy in clinical trials. that PAT-1102 has a better safety profile compared to pracinostat. Acknowledgements

Discussion The Authors sincerely thank the management of Anthem Biosciences for their constant support and encouragement in carrying out this study. We have identified PAT-1102 as an orally bioavailable, small molecule designed to inhibit HDAC, resulting in References anticancer activity. In vitro mechanism of action studies demonstrate that PAT-1102 is able to inhibit HDAC 1 Kim HJ and Bae SC: Histone deacetylase inhibitors: molecular isoforms and up-regulate molecules involved in cancer cell mechanisms of action and clinical trials as anticancer drugs. Am death. PAT-1102, similarly to vorinostat, is a pan inhibitor J Transl Res 3(2): 166-179, 2011. of class I, II, and IV HDACs, but with a higher potency 2 Richon VM, Sandhoff TW, Rifkind RA and Marks PA: Histone against HDAC isoenzymes. PAT-1102 displays a favorable deacetylase inhibitor selectively induces p21WAF1 expression and pharmacokinetic profile after oral administration and is gene-associated histone acetylation. Proc Natl Acad Sci USA 97: orally bioavailable in rodent models. PAT-1102 exhibits 10014-10019, 2000. 3 Sandor V, Senderowicz A, Mertins S, Sackett D, Sausville E, antiproliferative activity against a broad range of cancer Blagosklonny MV and Bates SE: p21-dependent G(1) arrest with cell types in in vitro studies (Table II), with greater than or down-regulation of cyclin D1 and up-regulation of cyclin E by similar potency to that of leading HDAC inhibitors in the histone deacetylase inhibitor FR901228. Br J Cancer 83: development. PAT-1102 also inhibits tumor growth in 817-825, 2000.

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