Abstract
Aim: Histone deacetylase (HDAC) inhibitors are a class of drugs that modulate transcriptional activity in cells and are known to induce cell-cycle arrest and angiogenesis, the major components of tumor cell proliferation. The aim of the present study was to characterize a novel hydroxamic acid-based HDAC inhibitor, PAT-1102, and determine its efficacy and tolerability in pre-clinical models. Materials and Methods: HDAC enzyme inhibition was measured using HeLa cell nuclear extracts, and recombinant HDAC enzymes. Antiproliferative activity was assessed in a panel of cancer cell lines. Histone hyper-acetylation status and p21 induction were assessed in HeLa cells by immunoblotting. The effect on apoptosis was tested by caspase-3 activation and detection of cleaved poly-ADP ribose polymerase (PARP). Single-dose pharmacokinetics of the compound were assessed in BALB/c mice following oral and intravenous administration. Antitumor efficacy was evaluated in tumor-bearing mice established from lung and colorectal cancer cells (A549 and HCT116, respectively). Results: PAT-1102 demonstrated potent HDAC-inhibitory activity and growth-inhibitory properties against a panel of cancer cell lines. The optimized compound PAT-1102 exhibits good aqueous solubility, metabolic stability and a favorable pharmacokinetic profile. Once-daily oral administration of PAT-1102 resulted in significant antitumor activity and was well-tolerated in mice. Conclusion: Our results indicate that PAT-1102 is a novel, potent, orally available HDAC inhibitor with antiproliferative activity against several human cancer cell lines and antitumor activity in mouse xenograft models. Based on the pre-clinical efficacy and safety profile of PAT-1102, the compound demonstrates significant potential for evaluation as a novel drug candidate for cancer therapy.
Histone acetylation/deacetylation is mediated by a class of enzymes known as Histone acetyl transferase (HATs) and histone deacetylases (HDACs). HDACs are involved in many biological processes, including development, cellular proliferation, differentiation, and apoptosis. Inhibition of HDACs is known to play an important role in epigenetic regulation by inducing cell death, apoptosis, and cell-cycle arrest in cancer cells. The balance between histone acetylation and deacetylation, mediated by HATs and HDACs, respectively, is usually well-regulated, but the balance is often upset in diseases such as cancer (1). HDAC inhibitors have emerged as an important class of anticancer drugs and have proven to be efficacious in the clinic. The mechanisms of action of HDAC inhibitors are thought to be related to altered gene expression and to changes in non-histone proteins via regulation at the epigenetic and post-translational modification levels, respectively. In many tumor cell lines, HDAC inhibitors cause up-regulation of the cell cycle gene p21, blocking the cyclin–dependent kinase complexes, leading to cell-cycle arrest and inhibition of 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, nicotinamide adenine dinucleotide (NAD+)-dependent (class III) enzymes. Conventional HDACs are composed of 11 members which require Zn2+ as a co-factor for their deacetylase activity and are divided into four classes depending on their homology (5). The class III HDACs (sirtuins) are structurally related to yeast SirT2, and there is increasing evidence that they are critical transcriptional regulators (6, 7). Class I comprises HDACs 1, 2, 3, and 8, which are located within the nucleus; class II comprises HDACs 4 to 7, 9, and 10, which are located in both the nucleus and the cytoplasm; and class IV comprises HDAC 11. Unlike conventional HDACs, class III HDACs are composed of seven mammalian sirtuins (SIRT1–7) (8). These are NAD+-dependent protein deacetylases localized in the nucleus (SIRT1, SIRT6, and SIRT7), mitochondria (SIRT3, SIRT4, and SIRT5), and cytoplasm (SIRT2).
Several structurally distinct classes of HDAC inhibitors have been purified from natural sources or synthetically developed, and some of them have advanced into phase I/II clinical trials in solid tumors and hematological malignancies (9). Although the mechanisms of action of HDAC inhibitors are still unclear, they are emerging therapeutic agents that have been clinically validated in patients with hematological malignancies, including cutaneous T-cell lymphoma (1) Two HDAC inhibitors, vorinostat (suberoylanilide hydroxamic acid; Merck & Co., Inc.) and depsipeptide (Romidepsin, FK-228; Gloucester Pharmaceutical Inc.), have been approved by the US food and drug administration (in 2006 and 2009, respectively).
Clinical trials with several HDAC inhibitors as single agents, in combination with conventional chemotherapies, or as targeted drugs are under various stages of development. Hydroxamates exert nonspecific HDAC-inhibitory activity, affecting all classes of HDACs (10). Other compounds specifically inhibit class I HDACs, e.g. the benzamide entinostat (MS-275), or class I and IIa HDACs, as in the case of the short-chain fatty acids valproic acid and butyrate (11). Isotype-selective compounds are also increasingly becoming available, e.g. tubacin, mocetinostat, and PC-34501 selectively inhibit HDAC6, -1, and -8, respectively (12-15). However, there has been an ongoing debate over whether isoform and class-specific HDACIs are advantageous over broad-spectrum or pan HDAC inhibitors (16). Most of the HDAC inhibitors that have entered clinical trials have limitations, including low bioavailability, low potency, cardiovascular safety issues, and potential for drug–drug interactions through cytochrome P450 inhibition (17). Therefore, there is still a clinical opportunity for novel, orally available efficacious HDAC inhibitors with a wider safety margin.
We previously synthesized a novel series of hydroxamic acid-based HDAC inhibitors with nanomolar potency against HDAC (unpublished data). We had identified PAT-1102(Figure 1) as a potent HDAC inhibitor which was active against a wide range of cancer cell lines. In this report, we describe the preclinical profile of PAT-1102, a highly efficacious HDAC inhibitor with excellent pharmacokinetic properties which has been shown to be orally efficacious and well-tolerated in murine cancer models. The study further indicates that based on the preclinical efficacy and safety profile, PAT-1102 has the potential to be evaluated further in regulatory safety studies and the compound could have advantages over other HDAC inhibitors with poor pharmacokinetic properties and dose-limiting side-effects.
Materials and Methods
Cell lines, reagents, and animals. All the HDAC inhibitor compounds, including vorinostat and pracinostat, were synthesized as per reported methods (21, 22). Human carcinoma cell lines were originally obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cell culture media was obtained from Sigma-Aldrich (St. Louis, MO, USA) and fetal bovine serum (FBS) was procured from Life Technologies (Carlsbad, CA, USA). Human umblical vein endothelial cells (HUVEC) and endothelial growth medium (EGM) were obtained from Lonza Inc. (San Diego, CA, USA). Antibodies against acetylated H3 and H4 were obtained from Merck Millipore (Billerica, MA, USA) and antibodies to p21CIP/WAF1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were obtained from Sigma-Aldrich unless specified otherwise.
Athymic nude mice and severe combined immunodeficient (SCID) mice used for xenograft experiments were obtained from Harlan Laboratories (Indianapolis, IN, USA) and housed under pathogen-free conditions. All studies involving animals were performed according to the protocols approved by the Institutional Animal Ethics Committee (IAEC) of Anthem Biosciences.
In vitro HDAC enzyme assay. Nuclear fractions prepared from HeLa cells as per established protocols were used as a source of HDAC enzyme. Nuclear fractions were lysed with 0.5% Triton X-100 containing phosphate buffer (pH 8.0) and were centrifuged. HDAC inhibition screening of PAT-1102 and the reference inhibitor vorinostat was performed using a fluorescence-based assay with a fluorescent substrate [Boc-Lys (Ac)-AMC Substrate] as reported previously (19). Fluorescent, deacetylated substrate was detected with a microplate reader (Bio-Tek Instrument Inc., Winooski, VT, USA). The 50% HDAC inhibitory concentration (IC50) was determined by testing at concentrations of 0.001, 0.01, 0.1, 1 and 10 μM. Isoform selectivity was tested using recombinant HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8 isoforms (Enzo Life Sciences Inc., Farmingdale, NY, USA).
Cell proliferation assays. Antiproliferative activity of the compounds was tested against a panel of cancer cell lines including lung, cervical, colonic, brain, renal, leukemia, prostattic, pancreatic, skin, bone, breast, ovarian cancer by a cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich, St. Louis, MO, USA). Human cancer cell lines were cultured in complete media containing 10% heat-inactivated FBS and 100 U/ml penicillin, 100 μg/ml streptomycin in a humidified incubator with 5% CO2 at 37°C and subcultured twice weekly. Cells were seeded in 96-well plates at a density of 3×103 cells per well in 100 μl of medium and were allowed to attach for 24 h. Stock concentrations of the compounds were made in dimethyl sulfoxide (DMSO). Media (100 μl/well) containing different concentrations of compounds (0.01, 0.1, 1, 10 and 100 μM) were added to the cells and were incubated for 48 h. Vorinostat was tested as a reference compound in the assay. On the day of termination, 50 μl of MTT reagent solution (5 mg/ml) was added to the medium and the cells were incubated for 3 h. The medium was then aspirated and 100% DMSO was added to solubilize the violet MTT-formazan product. The absorbance at 570 nm was measured on a Biotek Synergy HT 96-well plate reader by spectrophotometry. Assays were performed in duplicates for each concentration. Results are expressed as the percentage of growth inhibition with respect to that of the DMSO-treated control wells. A dose–response curve was generated for PAT-1102 and vorinostat and GI50 (concentration which inhibits 50% of cellular growth) values were interpolated from the growth curves using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).
Histone hyperacetylation and p21 induction by western blotting. Hyperacetylation of histones (H3 and H4) and p21CIP/WAF1 induction was measured by western blot of HeLa cell lysates as described elsewhere (2). Briefly, HeLa cells were cultured in 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 μM and incubated at 37°C overnight under 5% CO2. On the next day, the cells were washed in PBS and lysed using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration in the cell lysates was quantified using Bradford's method (20) and proteins were loaded on a 10% gel and subjected to immunoblotting for acetyl-histone H3 and acetyl-histone H4 using rabbit polyclonal antibodies to acetyl-histone H3 and H4.
Apoptosis assays [poly ADP ribose polymerase (PARP) cleavage and caspase-3 activation]. Caspase-3 activity was measured in HT-29 cells using a commercially available kit (Sigma- Aldrich, St. Louis, MO, USA). Briefly, HT-29 cells were cultured in McCoy's 5a medium containing 10% FBS and antibiotics. On the day of the study, 10,000 cells were seeded into each well of a 96-well plate and incubated for 12-16 h. PAT-1102 and vorinostat were added at concentrations ranging from 0.1 μM to 30 μM and cells were incubated for 48 h. The cells were then lysed in lysis buffer and the lysates were used to perform the assay according to the manufacturer's instruction. The assay is based on the hydrolysis of acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin by caspase-3, resulting in the release of the fluorescent 7-amino-4-methylcoumarin which is measured at an excitation and emission wavelength of 360 nm and 460 nm respectively. Apoptotic activity of PAT-1102 was also assessed in HeLa cells by detection of cleaved PARP, a substrate of caspase-3, in HeLa cell lysates by immunoblotting. Protein determinations were performed using Bradford method. Protein (10 mg) separated on sodium dodecyl sulfate 10% polyacrylamide gels was electroblotted onto Polyvinylidene fluoride membranes. Blots were probed with anti-human PARP (Cell Signaling Technology, Danvers, MA, USA) and visualized by enhanced chemiluminescence (Amersham GE Healthcare, Pittsburgh, PA, USA).
Matrigel angiogenesis assay. The in vitro angiogenesis assay was performed as described previously (18). HUVECs were cultured in EGM and plated in 24-well plates (5×104 cells per well) previously coated with Matrigel® (BD Biosciences, San Jose, CA, USA). The plates were then incubated overnight in the presence of PAT-1102 or vorinostat at 3.125, 6.25 and 12.5 μM in an incubator at 37°C under 5% CO2. The morphology of capillary-like structures formed were visualized using an inverted microscope and documented.
Metabolic stability of compounds in human and mouse liver microsomes. The metabolic stability of test compounds was determined using human and mouse liver microsomes obtained from Life Technologies. The final composition of the assay mixture included 100 μM of PAT-1102 (dissolved in DMSO), 0.5 mg/ml of microsomal protein and cofactors (5.0 mM glucose-6-phosphate, 0.06 U glucose-6-phosphate dehydrogenase, 2.0 mM MgCl2, 1.0mM NADP+, 0.5 mM uridine-diphosphate-glucuronic acid, 0.6 mM 3-phosphoadenosine 5-phosphosulfate and 1 mM reduced glutathione). The compounds were incubated with liver microsomes in the presence of cofactors for 1 h at 37°C. The reaction was stopped by the addition of ice-cold acetonitrile. The samples were then centrifuged and supernatants were analyzed as per established protocols using liquid chromatography tandem mass spectrometry (API 3200 LC-MS/MS system; Applied Biosystems, Foster City, CA, USA). The percentage of the parent compound remaining after 1 h of incubation was calculated with respect to the peak area of the compound at time 0 using the system's software.
Pharmacokinetics of PAT-1102 in mice. Studies were conducted to determine the oral bioavailability and pharmacokinetics of PAT-1102 in male Balb/c mice. The study was performed after obtaining the approval of the IAEC (Approval No. ABD/IAEC/PR/05R3-12-13). Briefly, Mice aged 5-6 weeks weighing around 25-30 g were used for the study. The animals were fasted overnight with free access to water. PAT-1102 or vorinostat was administered through oral gavage at a dose 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 ml/kg bodyweight. For intravenous pharmacokinetics, the compounds were administered via tail vein at a dose of 10 mg/kg in a formulation containing 0.9% saline at a dose volume of 5 ml/kg. Blood samples were withdrawn at 15, 30, 60 min, 1 h, 2 h, 4 h and 6 h post dosing. The blood samples were centrifuged at 3,000 × g for 5 min at 4°C for separation of plasma and the corresponding plasma samples were collected into clean pre-labeled tubes. A liquid-liquid extraction method was employed for obtaining PAT-1102 from plasma samples: 100 μl of plasma sample and 10 μl of internal standard solution of fluconazole were added to the centrifuge tube and vortexed for 2 min; 2.5 ml of tertbutylmethyl ether were added and the mixture was vortexed for 15 min. The samples were centrifuged at 4300 × g for 15 min at 10°C. The supernatant organic layer was separated and evaporated to dryness. The sample residues were reconstituted with 200 μl of dilution solvent (methanol:water, 80:20 v/v). The reconstituted samples were then transferred into auto sampler vials and analyzed using liquid chromatography tandem mass spectrometry (API 3200 LC-MS/MS system; Applied Biosystems, Foster City, CA, USA) as per methods established in-house. Data were analyzed using WinNonlin version 5.2 (Pharsight Corporation, St. Louis, MO, USA).
In vivo anticancer activity in human tumor xenograft models. In vivo antitumor activity of PAT-1102 was assessed in 6-week-old athymic nude or SCID mice (C.B-17/IcrHsd-PrkdcscidLystbg-J) aged 5-6 weeks and the study was performed after obtaining the approval of the IAEC (Approval No.: ABD/IAEC/PR/25R2-12-13). Animals were purchased from Harlan Laboratories and housed in individually ventilated cages under controlled conditions and maintained on a 12-h light/12-h dark cycle, with food and water supplied ad libitum. A549 (lung adenocarcinoma) and HCT-116 (colorectal carcinoma) cells obtained from the ATCC were cultured in appropriate media containing 10% FBS and antibiotics. On the day of the implantation, a 0.1 ml cell suspension was prepared containing 106 cells in hanks balanced salts solution mixed with Matrigel® (BD Bioscience, San Jose, CA, USA) in a 1:1 ratio. The cell suspension was injected subcutaneously into the flank of the animals under isoflurane 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 gavage. Vorinostat and pracinostat were administered once daily orally at 150 mg/kg and 75 mg/kg doses respectively. Tumor volume and body weight were measured twice a week.
Histone deacetylase (HDAC) inhibitory activity of PAT-1102. HDAC inhibition by PAT-1102 was measured in HeLa cell nuclear extract as the enzyme source or against human recombinant HDAC isoenzymes using a fluorescence-based assay as described in the Materials and Methods section. The inhibitory concentration IC50 was calculated from curves of concentration used versus percentage inhibition plotted using Graph Pad Prism software.
Structure of PAT-1102.
Statistical analysis. The terminal tumor volumes from in vivo xenograft studies were subjected to one-way ANOVA analysis followed by Dunnett's test when there were multiple treatment groups. Results were considered statistically significant when p<0.05.
Results
HDAC enzyme inhibition. The biological activity of the HDAC inhibitors was assessed in vitro using a cell-free HDAC enzymatic assay. PAT-1102 exhibited potent HDAC-inhibitory activity, with an IC50 value of 3 nM. The inhibitory activity of the compounds was tested against various HDAC isoforms, HDAC1, 2, 3, 6 and 8. (see Table I) Our results indicate that the compound is a pan-HDAC inhibitor similar to the reference compound vorinostat.
Antiproliferative activity of PAT-1102 expressed as the mean concentration of the compound which inhibits 50% of cell growth, the 50% growth-inhibitory concentration (GI50). Antiproliferative effect of PAT-1102 against a panel of cancer cell lines obtained from the American Type Culture Collection using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent as described under Materials and Methods. GI50 was calculated using GraphPad Prism software. The mean GI50 was derived from individual assays performed in triplicates.
Effect on histone acetylation and p21 induction. The effect of PAT-1102 on histone acetylation in HeLa cells was assessed by treatment with the compound in culture. Acetylated histones (H3 and H4) were detected in cell lysates by western blotting using specific antibodies. H3 and H4 histone hyperacetylation was measured in HeLa cellular extract 24 h post-treatment with the compound as detected by western blot. A dose-dependent accumulation of acetylated histones H3 and H4 was observed with PAT-1102, the effect of which was comparable to that of vorinostat (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).
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.
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 which specifically recognized a caspase_specific cleaved PARP fragment. As shown in the figure, we observed a dose-dependent caspase-specific degradation of PARP by PAT-1102 in HeLa cells (Figure 3).
Matrigel angiogenesis assay. Angiogenesis involves the migration of endothelial cells and their organization into a network of tube-like structures. In HUVECs, PAT-1102 inhibited endothelial tube formation at therapeutically relevant concentrations in the micromolar range (Figure 4). The effect was comparable with that of vorinostat.
Antiproliferative activity against cancer cells. The growth-inhibitory activity of PAT-1102 was assessed in a panel of 29 human cancer cell lines by treating the cells with PAT-1102 or vorinostat and the GI 50 was determined. PAT-1102 treatment resulted in a dose-dependent inhibition of proliferation of most of the cell lines tested at low micromolar concentrations (Table II). The inhibitory effect of PAT-1102 on proliferation of cancer cells was comparable or superior to that of vorinostat against several cell lines under our experimental conditions.
Induction of apoptosis of HeLa cells by PAT-1102, as measured by detection of cleaved poly ADP ribose polymerase (PARP). Apoptotic activity of PAT-1102 was assessed by detection of cleaved PARP in HeLa cell lysates by immunoblotting. As indicated in the image, cleaved PARP was detected on treatment with 1, 3 and 10 μM concentrations of PAT-1102.
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.
Single-dose oral and intravenous pharmacokinetics of PAT-1102 and vorinostat in BALB/c mice.
Metabolic stability in liver microsomes. To assess the metabolic stability of the compound, the compound was incubated in mouse liver microsomal fractions. PAT-1102 was found to be more metabolically stable compared to vorinostat, with 100% of the compound remaining after 1 h compared to 67% of that of vorinostat (data not shown).
Bioavailability and pharmacokinetics in mice. To compare the pharmacokinetic properties of PAT-1102 to those of vorinostat, BALB/c mice were orally dosed with 50 mg/kg bodyweight, or injected with 10 mg/kg i.v. to calculate bioavailability. Mice were sacrificed at different time points between 15 min and 6 h, and plasma inhibitor concentrations were determined. The oral bioavailability of PAT-1102 was 10%, which was similar to that of vorinostat (11%) (Table III) but PAT-1102 yielded higher maximum plasma concentrations (1,313 ng/ml) compared to vorinostat (580 ng/ml) (Figure 5).
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 assess the ability of PAT-1102 to inhibit tumor growth in vivo, we examined its effect in subcutaneous human xenograft models in immunocompromised mice. The antitumor efficacy was assessed in xenograft models established with A549 cells in athymic nude mice. Once-daily oral administration of PAT-1102 resulted in a significant dose-dependent tumor growth inhibition (TGI) after 21 days (Figure 6a). TGI at 50 mg/kg of PAT-1102 was similar to that observed with vorinostat at 150 mg/kg. The efficacy was thus achieved at a third of the dose of the reference compound vorinostat. Furthermore, there was no significant reduction in bodyweight in the PAT-1102-treated group compared to the vehicle control group.
We also evaluated the antitumor efficacy of PAT-1102 in a subcutaneous xenograft model established with HCT-116 colorectal carcinoma cells in SCID mice. We compared the efficacy with that of another HDAC inhibitor, pracinostat which is in clinical development. PAT-1102 was administered orally at three doses, 25, 50 and 100 mg/kg/day in a 21-day study and compared to pracinostat (75 mg/kg/day). Once-daily oral administration of PAT-1102 resulted in a dose-dependent TGI (Figure 6b). Under our experimental conditions, PAT-1102 was found to be better tolerated compared to pracinostat as evidenced by reduction in bodyweight and greater mortality with pracinostat, indicating that PAT-1102 has a better safety profile compared to pracinostat.
Comparative pharmacokinetic parameters for PAT-1102 and vorinostat after intravenous (I.V.; 10 mg/kg) and oral administration (50 mg/kg) in BALB/c mice. The pharmacokinetic parameters shown were calculated by a non-compartmental method using the WinNonlin 4.0 software. Data shown are averages derived from three individual animals.
Discussion
We have identified PAT-1102 as an orally bioavailable, small molecule designed to inhibit HDAC, resulting in anticancer activity. In vitro mechanism of action studies demonstrate that PAT-1102 is able to inhibit HDAC isoforms and up-regulate molecules involved in cancer cell death. PAT-1102, similarly to vorinostat, is a pan inhibitor of class I, II, and IV HDACs, but with a higher potency against HDAC isoenzymes. PAT-1102 displays a favorable pharmacokinetic profile after oral administration and is orally bioavailable in rodent models. PAT-1102 exhibits antiproliferative activity against a broad range of cancer cell types in in vitro studies (Table II), with greater than or similar potency to that of leading HDAC inhibitors in development. PAT-1102 also inhibits tumor growth in preclinical xenograft models of solid tumors, such as lung 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 pharmacokinetic properties translate into a dose-dependent and enhanced antitumor efficacy in cancer models. The reduced toxicity of PAT-1102 could facilitate the use of higher doses of the drug in the clinic in order to maximize antitumor activity. Therefore, PAT-1102 has the potential to be more effective in targeted clinical trials than other HDAC inhibitors with poor pharmacokinetic properties and dose-limiting side-effects. We believe the improved bioavailability and safety profile of this compound could help achieve better efficacy in clinical trials.
Tumor growth inhibition (TGI) in subcutaneous A549 and HCT116 tumor xenograft models established in mice.
Acknowledgements
The Authors sincerely thank the management of Anthem Biosciences for their constant support and encouragement in carrying out this study.
- Received September 20, 2014.
- Revision received October 21, 2014.
- Accepted October 27, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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