CRA-026440 is a novel, broad-spectrum, hydroxamic acid–based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of α-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity. [Mol Cancer Ther 2006;5(7):1693–701]

Chromatin is structurally complex, consisting of DNA, histone, and nonhistone proteins. Remodeling of the chromatin histone and nonhistone proteins around which the DNA is wrapped is a fundamental epigenetic mechanism regulating gene expression in vertebrates (1). It has been hypothesized that histone modification, achieved through histone acetyltransferase and histone deacetyltransferase (HDAC) enzymes, plays a key role in the regulation of gene transcription (24). The removal of acetyl groups from histones changes the accessibility of DNA to transcriptional factors, thus leading to transcriptional repression (24). In addition to histones, HDAC enzymes are known to deacetylate other proteins, including α-tubulin, suggesting complex, multifunctional roles for HDACs (5). In addition, HDACs have been reported to mediate the function of oncogenic translocation products in certain leukemias and lymphomas (610).

HDAC enzymes are divided into three major groups based on sequence, conformation, and function. Class I HDAC1, HDAC2, HDAC3, and HDAC8 are widely expressed in tissue and are largely restricted to the nucleus (11, 12). HDAC1 and HDAC2 form part of the transcription repression complex SIN3-HDAC and the nucleosome remodeling deacetylase complex NuRD-Mi2-NRD (13). Class II HDAC4, HDAC5, HDAC6, HDAC7, HDAC9a, HDAC9b, and HDAC10 are much larger in size, display limited tissue distribution, and can shuttle between the nucleus and the cytoplasm (11, 12). Class III HDACs consist of the sirtuins, proteins that have a unique enzymatic mechanism that is dependent on NAD+ (11, 12, 14, 15).

It is hypothesized that histone acetylation may play a role in tumor development and progression (6, 9, 16, 17). HDAC inhibitors have displayed selective growth inhibition against transformed cells (1821). Treatment of tumor cells with HDAC inhibitors induces growth arrest, differentiation, and the activation of caspase-dependent apoptosis (6, 11, 12). Moreover, HDAC inhibitors have exhibited inhibition of proliferation, invasion, migration, adhesion, and tube formation of endothelial cells (22). The expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), and antiangiogenic factors, such as von Hippel Lindau and neurofibromin 2, is also modulated by HDAC inhibitors (11).

Expression profiling studies have shown that treatment of HDAC inhibitors alters the expression of ∼2% to 10% of cellular genes (2325). Genes up-regulated by HDAC inhibitors include the tumor suppressor p21Cip1/WAF1, p16, gelsolin, histones, TBP-2, and caspases, and down-regulated genes include Her2/neu, VEGF, thioredoxin (26), and cyclin proteins (27). Other substrates of HDACs include p53 and BCL6 (6, 10).

A variety of structurally diverse compounds have been described to inhibit HDAC enzymes, including hydroxamic acids, benzamides, eletrophilic ketones, and cyclic peptides (11, 2830). Several hydroxamic acid–based inhibitors have been described (suberoylanilide hydroxamic acid, trichostatin A, oxamflatin, LAQ824, LBH589, PXD101, and CRA-024781), some of which are currently in clinical trials for cancer. We have focused on the characterization of a hydroxamic acid–based series of compounds optimized for in vivo efficacy and therapeutic index (31, 32). Herein, we describe the in vitro and in vivo characteristics of a novel HDAC inhibitor, CRA-026440.

Cell Lines

The human tumor cell lines were obtained from the American Type Culture Collection (Manassas, VA). They included human colorectal carcinomas (HCT116, HCT-15, and DLD-1), human breast carcinomas (BT-549 and MCF-7), human prostate carcinomas (CWR-22RV1 and NCI-PC3), human ovarian carcinomas (SKOV-3 and OVCAR-3), human lung carcinomas (NCI-H226 and A549), human pancreatic carcinoma (BxPC3), and human histiocytic carcinoma (U937). Human umbilical vein endothelial cells (HUVEC) were obtained from Cambrex (East Rutherford, NJ).

Histone Deacetylase Activity

HDAC activity was measured using a continuous trypsin-coupled assay that has been described in detail previously (33). For each isozyme, the HDAC protein in reaction buffer [50 mmol/L HEPES, 100 mmol/L KCl, 0.001% Tween 20, 5% DMSO (pH 7.4), supplemented with bovine serum albumin at concentrations of 0% (HDAC1), 0.01% (HDAC2, HDAC3, HDAC8, and HDAC10), or 0.05% (HDAC6)] was mixed with inhibitor at various concentrations and allowed to incubate for 15 minutes. Trypsin was added to a final concentration of 50 nmol/L, and acetyl-Gly-Ala-(N-acetyl-Lys)-AMC was added to a final concentration of 25 μmol/L (HDAC1, HDAC3, and HDAC6), 50 μmol/L (HDAC2 and HDAC10), or 100 μmol/L (HDAC8) to initiate the reaction. Negative control reactions were done in the absence of inhibitor in replicates of eight. After a 30-minute lag time, the fluorescence was measured over a 30-minute time frame using an excitation wavelength of 355 nm and a detection wavelength of 460 nm. The increase in fluorescence with time was used as the measure of the reaction rate. Inhibition constants Ki(app) were obtained using the program BatchKi (34).

Cell Proliferation Assay

Ten tumor cell lines and HUVECs were cultured for at least two doubling times, and growth was monitored at the end of compound exposure using an Alamar Blue (Biosource, Camarillo, CA) fluorometric cell proliferation assay as described (35). CRA-026440 was assayed in triplicate wells at nine concentrations in half-log intervals from 0.0015 to 10 μmol/L. The concentration required to inhibit cell growth by 50% (GI50) and 95% confidence intervals were estimated from nonlinear regression using a four-variable logistic equation.

Histone and Tubulin Acetylation, p21Cip1/WAF1 Accumulation, Poly(ADP-Ribose) Polymerase Cleavage, and γ-H2AX

Tumor cells and subconfluent HUVECs were cultured for 18 hours in the presence of CRA-026440 concentrations ranging from 0.1 to 10 μmol/L. Cells were then collected and lysed in lysis buffer (M-Per; Pierce, Rockford, IL) containing protease inhibitors (Complete, Mini, EDTA-free; Roche, Indianapolis, IN) and phosphatase inhibitors (phosphatase inhibitor cocktail set II; Calbiochem, San Diego, CA). Lysates were electrophoresed in Novex Tris-glycine gels (Invitrogen, Carlsbad, CA), which were then blotted onto nitrocellulose and probed with either an anti-acetyl lysine antibody (Upstate, Lake Placid, NY) to detect acetylated histones, an anti-acetylated tubulin antibody (Sigma, St. Louis, MO), an anti-p21Cip1/WAF1 antibody (BD Biosciences, San Jose, CA), an anti-poly(ADP-ribose) polymerase antibody (Cell Signaling, Danvers, MA), or an anti-γ-H2AX antibody (Cell Signaling).

Ex vivo Angiogenesis Analysis

Aortic arch was isolated surgically from euthanized Fisher rats from Charles Rivers Laboratories (Wilmington, MA). It was sliced into 2-mm slices under aseptic conditions and placed in 48-well plates precoated with 150 μL of Matrigel (BD Biosciences). Aortic sproutings were observed about 3 days after incubation. The culture was then treated with CRA-026440 for an additional 5 days and was stained in crystal violet (0.5% crystal violet, 5% formaldehyde in 50% ethanol, and 50% PBS). The extent of angiogenesis was scored blindly on 0 to 3 scale (0, no sprouting; 1, some sprouting; 2, significant sprouting; and 3, extensive sprouting).

Pharmacokinetic Analysis

CRA-026440 was formulated in 20% SBE-β-cyclodextrin (CyDex, Lenexa, KS) in water and was given in i.v. bolus to female HCT 116 tumor-bearing BALB/c nu/nu mice. Plasma was prepared from each blood sample with lithium heparin. Tumor samples were collected and homogenized with 9 volumes of water, and the homogenate was collected. The plasma and tumor homogenate samples were processed using acetonitrile precipitation. The concentrations of CRA-026440 in the supernatants were determined by liquid chromatography tandem mass spectrometry. The limit of quantification was 1 nmol/L. Pharmacokinetic variables were determined with noncompartmental analysis using WinNonlin-Pro version 4.1 (Pharsight Corp., Mountain View, CA). Pharmacokinetic calculations were done using nominal doses and nominal collection times.

In vivo Efficacy Studies

Female BALB/c nu/nu mice were purchased from Charles River Laboratories. HCT116 at 3 × 106 and U937 at 4 × 106 were implanted s.c. Tumor-bearing mice were randomized based on tumor volume before the initiation of treatment. The treatment duration was 2 and 3 weeks for the U937 and the HCT 116 xenograft models, respectively. For combination studies, Avastin (Genentech, South San Francisco, CA) was given i.p. once a week, a day before the i.v. administration of CRA-026440. Tumor volume was calculated as follows: volume = 1/2 (X2Y), where X = tumor width and Y = tumor length. Inhibition of tumor growth was calculated as follows: 100 × [1 − (dT/dC)], where dT was the change in average tumor volume since the first dose in the treatment group, and dC was the change in average tumor volume since the first dose in the control group. Statistical analysis was done with one-way ANOVA, and P values were corrected for multiple comparisons to control by Dunnett's method.

Immunohistochemical Analysis

HCT 116 tumor-bearing nude mice were treated with CRA-026440 at 100 mg/kg daily for three consecutive days. Tumor xenograft samples were fixed overnight in 10% zinc-buffered formalin and embedded in paraffin. Immunohistochemical staining on Ki67 was done at BioPathology Sciences Medical Corp. (South San Francisco, CA).

Detection of Acetylated Tubulin in Whole Blood

Tumor xenograft and blood samples collected at various time points after dosing. Blood samples were processed in Red Cell Lysis Buffer (Roche) to isolate nucleated blood cells per manufacturer's instruction. Cells were pelleted and stored at −80°C until analysis. Acetylated tubulin was detected in blood lysates by Western blotting.

Microarray Analysis

Transcriptional profiles of HCT-116 cell line and tumor xenograft samples were obtained on Codelink Human Uniset 1 oligonucleotide microarrays (GE-Amersham, Piscataway, NJ) as described previously (31). For the cell line samples, total RNA was isolated from cells incubated with 0.31 μmol/L CRA-26440 for 2, 4, or 24 hours. For tumor samples, HCT-116 tumor-bearing mice received three consecutive daily i.v. doses of CRA-26440 or vehicle. Equal amounts of RNA from at least three animals per dose per time point were pooled together for analysis. Ten micrograms of biotinylated cRNA were fragmented and hybridized to the arrays. Arrays were hybridized, washed, and detected with Strepatavidin-Alexa 647 (Codelink Protocol v2.1). They were scanned with an Axon GenePix 4000B scanner (Axon Instruments, Sunnyvale, CA) and were processed with Codelink 4.0 Batch Processing software. The data were analyzed in Genespring (Agilent, Inc., Palo Alto, CA). Only genes flagged as Good by the Codelink quality control software were used in the analyses. Each treated sample was normalized to the corresponding vehicle control.

Taqman Analysis

Taqman Gene Expression Assays for selected genes were obtained from Applied Biosystems, Inc. (Foster City, CA). One-step reverse transcription-PCR was carried out in triplicate on 25 ng of total RNA from each sample on an ABI PRISM 7700 instrument according to standard protocols. The mRNA levels for each gene were normalized to the amount of RNA in the well as measured in parallel using Ribogreen (Molecular Probes, Carlsbad, CA). The treated samples were then normalized to the vehicle control for that time point.

Chemical Design and Synthesis

In the design of inhibitors of histone deacetylase, phenyl hydroxamic acids were employed as chelators of the active site Zinc(II). Further elaboration led to the aminopropynyl linking group attachment to the para-position of the phenyl hydroxamic acid. The aminopropynyl linking group was further modified by the addition of the 5-(2-dimethylaminoethoxy) indole capping group, which resulted in the potent, broad-spectrum histone deacetylase inhibitor CRA-026440 (Fig. 1). The Ki values against recombinant HDAC isoenzymes 1, 2, 3, 6, 8, and 10 were 4, 14, 11, 15, 7, and 20 nmol/L respectively.

Figure 1.

CRA-026440.

Effect on Tumor Cell Proliferation

To determine if HDAC inhibition by CRA-026440 affects the proliferation of tumor cells, a panel of human tumor cell lines was treated in vitro at various concentrations of inhibitor. Antitumor activity was observed in all 10 tumor cell lines tested, with GI50 values ranging from 0.12 to 9.95 μmol/L (Table 1). In addition, CRA-026440 had antiproliferative effect on HUVEC endothelial cells with a GI50 value of 1.41 μmol/L (Table 1; Fig. 2).

Table 1.

In vitro sensitivities of human cell lines to CRA-026440

Cell lineOriginTd (h)Assay (h)GI50% (μmol/L)* (95% confidence interval)
OVCAR-3 Ovary 46 120 0.1236 (0.11-0.14) 
SKOV-3 Ovary 44.4 120 1.033 (0.64-1.42) 
HCT116 Colon 14 48 0.3226 (0.25-0.40) 
DLD-1 Colon 22 48 2.8828 (1.77 3.99) 
HCT15 Colon 22 48 9.9514 (7.88-12.0) 
CWR-22RV1 Prostate 30.4 96 0.2781 (0.24-0.32) 
NCI-PC3 Prostate 18.8 48 0.6857 (0.39-0.98) 
MCF7 Breast 37 96 0.1575 (0.05-0.27) 
BT549 Breast 29 72 1.9032 (0.78-3.03) 
BxPC3 Pancreas 35.4 72 1.9588 (1.02-2.90) 
A549 Lung 23 48 1.2829 (0.88-1.69) 
NCI-H226 Lung 25.4 72 3.0154 (2.48-3.55) 
U937 Lymphoma 30 72 0.1956 (0.11-0.28) 
HUVEC Endothelium 15 48 1.4091 (0.56-2.23) 
Cell lineOriginTd (h)Assay (h)GI50% (μmol/L)* (95% confidence interval)
OVCAR-3 Ovary 46 120 0.1236 (0.11-0.14) 
SKOV-3 Ovary 44.4 120 1.033 (0.64-1.42) 
HCT116 Colon 14 48 0.3226 (0.25-0.40) 
DLD-1 Colon 22 48 2.8828 (1.77 3.99) 
HCT15 Colon 22 48 9.9514 (7.88-12.0) 
CWR-22RV1 Prostate 30.4 96 0.2781 (0.24-0.32) 
NCI-PC3 Prostate 18.8 48 0.6857 (0.39-0.98) 
MCF7 Breast 37 96 0.1575 (0.05-0.27) 
BT549 Breast 29 72 1.9032 (0.78-3.03) 
BxPC3 Pancreas 35.4 72 1.9588 (1.02-2.90) 
A549 Lung 23 48 1.2829 (0.88-1.69) 
NCI-H226 Lung 25.4 72 3.0154 (2.48-3.55) 
U937 Lymphoma 30 72 0.1956 (0.11-0.28) 
HUVEC Endothelium 15 48 1.4091 (0.56-2.23) 

Abbreviation: Td, doubling time.

*

The GI50% data and 95% confidence interval were estimated from nonlinear regression using a four-variable logistic equation from single experiments.

Figure 2.

Growth curves of HCT 116 and HUVEC under different concentrations of CRA-026440. A, HCT 116; B, HUVEC.

Figure 2.

Growth curves of HCT 116 and HUVEC under different concentrations of CRA-026440. A, HCT 116; B, HUVEC.

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Effect on Protein Acetylation, p21Cip1/WAF1 Induction, and the Apoptosis Markers Poly(ADP-Ribose) Polymerase and γ-H2AX

We further characterized the antitumor mechanism of CRA-026440 by treating HCT116 colorectal tumor cells with increasing concentrations of CRA-026440 in vitro and measuring the accumulation of various mechanistic biomarkers proposed to be involved in the activity of HDAC inhibitors. Treating HCT116 cells with CRA-026440 resulted in the accumulation of both acetylated histones and acetylated tubulin (Fig. 3A). Acetylated tubulin was detectable at lower concentrations than acetylated histones, which might reflect differing sensitivities for specific HDACs or HDAC substrates, or differing sensitivities of the specific detection reagents used. In addition, CRA-026440 induced expression of the cyclin-dependent kinase inhibitor p21Cip1/WAF1, a protein postulated to play a role in the antitumor effect of HDAC inhibition (27, 36). This was confirmed by the expression profiling study in which p21 mRNA was induced by >2.5-fold in HCT 116 cells after treatment with CRA-026440 for 24 hours (data not shown). In addition, γ-H2AX formation, an early chromatin modification event following the initiation of DNA fragmentation during apoptosis (37) was detected along with poly(ADP-ribose) polymerase cleavage in HCT 116 in the low micromolar range. Although CRA-026440 was less potent in inhibiting HUVEC proliferation, pharmacodynamic analysis revealed similar extent of accumulation of acetylated tubulin in HUVEC upon CRA-026440 treatment (Fig. 3B).

Figure 3.

CRA-024460 activates biomarkers of HDAC inhibition. A, accumulation of acetylated histone, acetylated tubulin, p21Cip1/WAF1, cleaved poly(ADP-ribose) polymerase (PARP), and γ-H2AX are detected by Western blotting of lysates from treated HCT116. B, accumulation of acetylated histone in treated HUVECs.

Figure 3.

CRA-024460 activates biomarkers of HDAC inhibition. A, accumulation of acetylated histone, acetylated tubulin, p21Cip1/WAF1, cleaved poly(ADP-ribose) polymerase (PARP), and γ-H2AX are detected by Western blotting of lysates from treated HCT116. B, accumulation of acetylated histone in treated HUVECs.

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In vivo Pharmacokinetics

We assessed the pharmacokinetics of the compound to evaluate in vivo exposure. CRA-026440 was delivered i.v. to mice, and plasma concentrations were monitored over time (Fig. 4A). Based on these data, the clearance was calculated to be 38 mL/min/kg; the volume of distribution in the central compartment was 67 mL/kg. The steady-state volume of distribution was 454 mL/kg. The predominant plasma half-life was 7 minutes (44% of area under the curve); and the mean residence time was 12 minutes. One notable finding is that CRA-026440 had a different pharmacokinetic profile in tumor (Fig. 4B). It had a lower Cmax, but the compound concentration was maintained more steadily over time, probably due to the compound's large volume of distribution. These data suggested that CRA-026440 had sufficient in vivo exposure when dosed by the i.v. route of administration to be used to study the biological effects of HDAC inhibition in vivo.

Figure 4.

Pharmacokinetics of CRA-026440. A, plasma concentration versus time following i.v. administration at 10 mg/kg in female BALB/c mice. B, the concentrations of CRA-026440 in plasma and at HCT 116 tumor xenograft after a single i.v. administration at 50 mg/kg (plasma, ♦; tumor, ◊) in female nude BALB/c mice.

Figure 4.

Pharmacokinetics of CRA-026440. A, plasma concentration versus time following i.v. administration at 10 mg/kg in female BALB/c mice. B, the concentrations of CRA-026440 in plasma and at HCT 116 tumor xenograft after a single i.v. administration at 50 mg/kg (plasma, ♦; tumor, ◊) in female nude BALB/c mice.

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Antitumor Activity and Induction of Acetylation by CRA-026440 In vivo

Nude mice bearing human tumor xenografts were dosed i.v. with CRA-026440 once daily for 3 days followed by 4 days without treatment (qdx3). In the U937 histiocytic lymphoma model, statistically significant tumor growth inhibition of 58% (P < 0.05) was achieved after 2 weeks of treatment at 100 mg/kg (Fig. 5A). A trend toward efficacy, although statistically not significant, was noted at 50 and 25 mg/kg (Fig. 5A). Treatment at all dose levels was well tolerated with no mortality and 1.6% maximal body weight loss. In the HCT 116 colorectal tumor model, statistically significant tumor growth inhibition was observed after 3 weeks of treatment at 50, 75, 100, and 150 mg/kg (Fig. 5B). The inhibition of tumor growth was 42% (P < 0.05), 63% (P < 0.01), 77% (P < 0.01), and 93% (P < 0.01), respectively. The treatment was well tolerated at up to 100 mg/kg with a maximal weight loss of 6%. At 150 mg/kg, 9% weight loss and 30% mortality (3 of 10) were noted. The dose of 200 mg/kg qdx3 was not tolerated. Thus, the maximal tolerated dose under this schedule was ∼150 mg/kg (qdx3/wk for 3 weeks), as suggested by the mortality and weight loss. Additionally, a separate dose-scheduling study suggested that statistically significant tumor growth inhibition could be achieved at 100 mg/kg once daily for two consecutive days each week, or once daily on Monday and Friday each week (Fig. 5C). The tumor growth inhibition was 47% (P < 0.01) and 42% (P < 0.01), respectively. Because HDAC inhibition is known to affect the expression of genes involved in angiogenesis, we asked whether CRA-026440 would have greater efficacy in combination with an angiogenic inhibitor. To this end, CRA-026440 was tested in combination with Avastin in the HCT 116 tumor model. In this study, Avastin that was given at 25 mg/kg i.p. once a week yielded a tumor growth inhibition of 63% (P < 0.01). CRA-026440 at 25 and 50 mg/kg given qdx3 per week significantly inhibited tumor growth at 40% (P < 0.01) and 58% (P < 0.01), respectively. The combination of Avastin at 25 mg/kg once a week and CRA-026440 at 25 and 50 mg/kg for three consecutive days per week lead to more profound tumor growth inhibition of 84% (P < 0.01) and 85% (P < 0.01), respectively (Fig. 5D). Compared with Avastin alone, the combination yielded a marginally better result (P < 0.1). The combination of Avastin and CRA-026440 at 50 mg/kg was significantly better than CRA-026440 alone at 50 mg/kg (P < 0.05). Both combination treatments were well tolerated with maximal weight loss <3%.

Figure 5.

CRA-026440 inhibits the growth of human tumor xenografts in vivo. The growth of tumors over time is plotted in (A) U937 histiocytic tumor xenograft model (vehicle alone, ♦; 100 mg/kg, ×; 50 mg/kg, ▴; 25 mg/kg, ▪), (B) HCT 116 colon tumor xenograft model (vehicle alone, ♦; 150 mg/kg, ◊; 100 mg/kg, ×; 75 mg/kg, ▴; 50 mg/kg, ▪), (C) HCT116 colon tumor xenograft model (vehicle alone, ♦; 50 mg/kg qdx4/wk, ▪; 67 mg/kg qdx3/wk, □; 100 mg/kg qdx2/wk, ×; 100 mg/kg Monday + Friday/wk, ◊), (D) HCT 116 colon tumor xenograft model (vehicle alone, ♦; 25 mg/kg, ▴; 50 mg/kg, ▪; Avastin 25 mg/kg, ×; Avastin + CRA-026440 at 25 mg/kg, ▵; Avastin + CRA-026440 at 50 mg/kg, □). Ki67 immunohistochemical analysis: (E) HCT 116 xenograft tumors with three daily i.v. treatment of vehicle, (F) HCT 116 xenograft tumors with three daily i.v. treatment of CRA-026440 at 100 mg/kg. Error bars were calculated as described in Materials and Methods. *, P < 0.05; **, P < 0.01.

Figure 5.

CRA-026440 inhibits the growth of human tumor xenografts in vivo. The growth of tumors over time is plotted in (A) U937 histiocytic tumor xenograft model (vehicle alone, ♦; 100 mg/kg, ×; 50 mg/kg, ▴; 25 mg/kg, ▪), (B) HCT 116 colon tumor xenograft model (vehicle alone, ♦; 150 mg/kg, ◊; 100 mg/kg, ×; 75 mg/kg, ▴; 50 mg/kg, ▪), (C) HCT116 colon tumor xenograft model (vehicle alone, ♦; 50 mg/kg qdx4/wk, ▪; 67 mg/kg qdx3/wk, □; 100 mg/kg qdx2/wk, ×; 100 mg/kg Monday + Friday/wk, ◊), (D) HCT 116 colon tumor xenograft model (vehicle alone, ♦; 25 mg/kg, ▴; 50 mg/kg, ▪; Avastin 25 mg/kg, ×; Avastin + CRA-026440 at 25 mg/kg, ▵; Avastin + CRA-026440 at 50 mg/kg, □). Ki67 immunohistochemical analysis: (E) HCT 116 xenograft tumors with three daily i.v. treatment of vehicle, (F) HCT 116 xenograft tumors with three daily i.v. treatment of CRA-026440 at 100 mg/kg. Error bars were calculated as described in Materials and Methods. *, P < 0.05; **, P < 0.01.

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Histochemical analysis of HCT 116 tumor xenograft treated with CRA-026440 indicated a reduction of the number of Ki67-positive cells, confirming the compound's antiproliferative activity on tumors (Fig. 5E and F).

To analyze in vivo biomarkers that correlated with compound exposure and/or efficacy, peripheral blood cells and tumor xenograft samples were examined ex vivo. As shown in Fig. 6A, the peripheral blood samples in each treatment group had a measurable increase in tubulin acetylation at 2 and 6 hours after treatment. The level of tubulin acetylation returned to baseline after 24 hours, in agreement with the pharmacokinetic profile of CRA-026440 in circulation. Increased tubulin acetylation in the HCT 116 tumor xenograft was detectable at 24 hours after treatment (Fig. 6B), indicative of a more prolonged presence of CRA-026440 in tumor than in circulation, in agreement with the tumor pharmacokinetic profile (Fig. 4B).

Figure 6.

In vivo pharmacodynamics of CRA-026440. A, peripheral blood mononuclear cells were prepared from blood drawn at the time points indicated from mice following the final dose of 3 wks of qdx3 per week (three consecutive days of treatment each week) treatment of CRA-026440. B, HCT 116 tumor samples were collected at time points indicated from mice following the final dose of 3 wks at qdx3 per week treatment of CRA-026440. Acetylation of α-tubulin was detected by Western blot.

Figure 6.

In vivo pharmacodynamics of CRA-026440. A, peripheral blood mononuclear cells were prepared from blood drawn at the time points indicated from mice following the final dose of 3 wks of qdx3 per week (three consecutive days of treatment each week) treatment of CRA-026440. B, HCT 116 tumor samples were collected at time points indicated from mice following the final dose of 3 wks at qdx3 per week treatment of CRA-026440. Acetylation of α-tubulin was detected by Western blot.

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Transcriptional Profile of CRA-026440

The transcriptional profile of CRA-26440 in HCT-116 cells and tumors is shown in Fig. 7, where 100 mg/kg dose in vivo was compared with 0.31 μmol/L dose (the GI50 for CRA-026440 against HCT 116) in vitro. Genes in each sample were subjected to two-way hierarchical clustering using standard correlation on both the genes (along the vertical axis) and the samples (along the horizontal axis). There was a notable trend in the expression patterns with time that was conserved in both the in vivo and in vitro samples. In both cases, the initial response was composed of a rapid increase in expression of a set of genes (enclosed by the red box) within 2 hours followed by a more gradual but strong down-regulation of other genes at the mid and late time points (green boxes). The overall number of statistically significant, differentially expressed genes increased with time for both in vitro and in vivo experiments. The genes that were regulated in common between these conditions at each of the early (2 hours), mid (4–6 hours), or late (24 hours) time points include several that are known to be modulated by HDAC inhibition (31, 38, 39). The notable examples are the up-regulation of CTGF, DHRS2, FYN, RGL, SERPIN1, and PDE2A and the down-regulation of urokinase, NFkB p105, c-MYC, cyclin A1, and the tumor marker CA-9.

Figure 7.

Hierarchical clustering analysis of HCT116 tumors from mice treated with CRA-026440, along with the same cell line treated with CRA-026440 in vitro. Genes are colored red if they are up-regulated >1.8-fold and green if they are down-regulated >1.8-fold; otherwise, they are colored black. Each column corresponds to a CRA-026440 treated sample, which was normalized to its corresponding control (vehicle) treatment at the same time point. Boxed are genes that behave similarly across different treatments: red box, genes up-regulated; green boxes, genes that are down-regulated.

Figure 7.

Hierarchical clustering analysis of HCT116 tumors from mice treated with CRA-026440, along with the same cell line treated with CRA-026440 in vitro. Genes are colored red if they are up-regulated >1.8-fold and green if they are down-regulated >1.8-fold; otherwise, they are colored black. Each column corresponds to a CRA-026440 treated sample, which was normalized to its corresponding control (vehicle) treatment at the same time point. Boxed are genes that behave similarly across different treatments: red box, genes up-regulated; green boxes, genes that are down-regulated.

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Antiangiogenic Activity by CRA-026440

We observed that the expression levels of VEGF was only affected in vivo and not in vitro. In addition, it was also observed that the expression of several other angiogenic genes might have been down-regulated, especially at 24 hours. These included HIF-1a, EGFR, erbB2, angiopoietin-1, KDR (VEGFR2), FGFR3, and FGFR4 (Table 2). To examine whether CRA-026440 treatment affects the process of angiogenesis, CRA-026440 was tested in the rat aortic ring angiogenesis assay. The rat aortic ring culture was treated with CRA-026440 at 0.1 to 10 μmol/L after the vessels started to sprout. Inhibition by CRA-026440 on sprouting was detectable after 2 days of treatment. After 5 days of treatment, a dose-dependent inhibitory activity was noted in this assay with CRA-026440 at 10 and 1 μmol/L (Fig. 8A–F).

Table 2.

Modulation of expression of select genes in angiogenesis by CRA-026440

Tumors (h)
Cells (h)
Differentially expressed angiogenesis-related genes
26242424SymbolGene NameGenbank
−3.3 −4.7 −5.5 1.2 −1.3 1.3 VEGF Vascular endothelial growth factor AF022375 
−1.4 −5.3 1.2 1.7 1.7 KDR Kinase insert domain receptor (VEGFR2) NM_002253 
1.8 1.4 −2.1 −1.0 1.2 −3.1 FZD3 Frizzled homologue 3 NM_017412 
−1.7 −2.2 −1.8 −1.2 −1.3 −4.3 FGFR4 Fibroblast growth factor receptor 4 NM_022963 
1.2 −1.6 −1.7 1.5 −2.0 1.9 ERBB3 Erythroblastic leukemia viral oncogene homologue 3 NM_001982 
−1.0 1.1 −1.7 1.1 −1.2 −2.4 HIF1A Hypoxia-inducible factor 1, α subunit NM_001530 
1.0 1.0 −1.6 1.0 −1.2 −1.4 NF2 Neurofibromin 2 NM_016418 
−1.2 1.1 −1.5 −1.6 1.1 −2.2 FGFR3 Fibroblast growth factor receptor 3 NM_022965 
1.0 −1.6 −1.2 1.1 −1.4 −4.3 TNFAIP2 TNF α-induced protein 2 NM_006291 
1.0 −1.6 −1.2 1.0 −1.7 1.0 EGFR Epidermal growth factor receptor NM_005228 
−1.2 −1.6 −1.1 1.0 −1.4 −1.2 ERBB2 Erythroblastic leukemia viral oncogene homolog 2 NM_004448 
1.4 1.2 1.1 −1.1 −2.4 SPRY2 Sprouty homologue 2 (DrosophilaNM_005842 
1.6 1.2 1.4 1.4 −1.0 −1.6 FGF2 Fibroblast growth factor 2 (basic FGF) NM_002006 
1.5 1.1 1.6 2.9 2.5 −2.3 ANGPT1 Angiopoietin 1 NM_001146 
1.4 1.4 3.9 −4.3 −1.7 −2.7 VEGFC Vascular endothelial growth factor C NM_005429 
Tumors (h)
Cells (h)
Differentially expressed angiogenesis-related genes
26242424SymbolGene NameGenbank
−3.3 −4.7 −5.5 1.2 −1.3 1.3 VEGF Vascular endothelial growth factor AF022375 
−1.4 −5.3 1.2 1.7 1.7 KDR Kinase insert domain receptor (VEGFR2) NM_002253 
1.8 1.4 −2.1 −1.0 1.2 −3.1 FZD3 Frizzled homologue 3 NM_017412 
−1.7 −2.2 −1.8 −1.2 −1.3 −4.3 FGFR4 Fibroblast growth factor receptor 4 NM_022963 
1.2 −1.6 −1.7 1.5 −2.0 1.9 ERBB3 Erythroblastic leukemia viral oncogene homologue 3 NM_001982 
−1.0 1.1 −1.7 1.1 −1.2 −2.4 HIF1A Hypoxia-inducible factor 1, α subunit NM_001530 
1.0 1.0 −1.6 1.0 −1.2 −1.4 NF2 Neurofibromin 2 NM_016418 
−1.2 1.1 −1.5 −1.6 1.1 −2.2 FGFR3 Fibroblast growth factor receptor 3 NM_022965 
1.0 −1.6 −1.2 1.1 −1.4 −4.3 TNFAIP2 TNF α-induced protein 2 NM_006291 
1.0 −1.6 −1.2 1.0 −1.7 1.0 EGFR Epidermal growth factor receptor NM_005228 
−1.2 −1.6 −1.1 1.0 −1.4 −1.2 ERBB2 Erythroblastic leukemia viral oncogene homolog 2 NM_004448 
1.4 1.2 1.1 −1.1 −2.4 SPRY2 Sprouty homologue 2 (DrosophilaNM_005842 
1.6 1.2 1.4 1.4 −1.0 −1.6 FGF2 Fibroblast growth factor 2 (basic FGF) NM_002006 
1.5 1.1 1.6 2.9 2.5 −2.3 ANGPT1 Angiopoietin 1 NM_001146 
1.4 1.4 3.9 −4.3 −1.7 −2.7 VEGFC Vascular endothelial growth factor C NM_005429 

NOTE: Selected genes involved in angiogenic processes, which are regulated by HDAC inhibition with CRA-026440 in vivo and in vitro. Genes that are down-regulated <1.5-fold are in boldface. Ratios marked with an asterisk could not be calculated due to lack of expression control.

Figure 8.

CRA-026440 inhibits vessel sprouting in the rat aortic ring assay. Treatment started when sprouting was visible on day 5. Treatment went from days 5 to 10. A, cremophor/ethanol/saline, (B) Taxol at 100 nmol/L, (C) SBE-β-CD, (D) CRA-026440 at 10 μmol/L, (E) CRA-026440 at 1 μmol/L, (F) CRA-026440 at 100 nmol/L, (G) composite angiogenesis scores of two independent rat aortic ring assays.

Figure 8.

CRA-026440 inhibits vessel sprouting in the rat aortic ring assay. Treatment started when sprouting was visible on day 5. Treatment went from days 5 to 10. A, cremophor/ethanol/saline, (B) Taxol at 100 nmol/L, (C) SBE-β-CD, (D) CRA-026440 at 10 μmol/L, (E) CRA-026440 at 1 μmol/L, (F) CRA-026440 at 100 nmol/L, (G) composite angiogenesis scores of two independent rat aortic ring assays.

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In summary, CRA-026440 exhibited statistically significant antitumor activity against both HCT116 and U937 human tumor xenografts. Evidence suggesting possible antiangiogenic activity of CRA-026440 was obtained. CRA-026440, when given in combination with Avastin, exhibited additional antitumor activity in the HCT116 xenograft model.

A number of HDAC inhibitors have been characterized both in vitro and in vivo (1, 11, 31), and some are being developed clinically to treat cancer (1, 11, 31, 40). Different HDAC inhibitors have displayed different selectivity against HDAC isoenzymes; for example, FK228 inhibits HDAC1 and HDAC2 but not HDAC4 or HDAC6 (41), and MS-275 is potent against HDAC1 and HDAC3 but not HDAC8 (42). Herein, we have described CRA-026440, a novel hydroxamic acid–based, broad-spectrum inhibitor of HDAC enzymes with potent antitumor activity in vitro and in vivo. CRA-026440 inhibited potently all of the HDACs tested with Ki values ranging from 4.1 to 20 nmol/L.

CRA-026440 inhibited the growth of a number of human tumor cell lines and nonneoplastic HUVEC in vitro. Growth inhibition was accompanied by changes in several known biomarkers of HDAC response. These biomarkers included the accumulation of acetylated histones and acetylated tubulin, the accumulation of the tumor suppressor protein p21Cip1/WAF1, and the generation of cleaved poly(ADP-ribose) polymerase fragment and phosphorylated H2AX, two known markers of apoptosis.

CRA-026440 had an in vivo pharmacokinetic profile that suggested adequate exposure to enable HDAC inhibition. One noteworthy finding is that the plasma pharmacokinetic profile of CRA-026440 be different from the tumor pharmacokinetic profile. CRA-026440 was present for much longer duration at HCT 116 tumor xenograft than in circulation. The results suggest of the need to monitor drug exposure at tumors to have a more relevant correlation between exposure and effects. The 50 mg/kg i.v. dose achieved significant tumor growth inhibition in the HCT 116 tumor xenograft model. The in situ concentration of CRA-026440 was maintained above the measured cellular GI50 for HCT116 tumor cells for ∼6 hours. The data suggest that in situ CRA-026440 concentration need to be maintained above a threshold for around 6 hours to achieve antitumor efficacy. This is consistent with our observation with another HDAC inhibitor CRA-024781 (31). Additionally, the preclinical efficacy of CRA-026440 was manifested primarily as tumor growth inhibition and not sustained tumor regression. Thus, it is inferred that CRA-026440 may need to be given on regular basis to achieve sustained antitumor effect.

The expression profiling analysis of HCT 116 treated by CRA-026440 has suggested a possible role of CRA-026440 in angiogenesis. This is supported by the finding in the chronic rat aortic ring assay in which CRA-026440 did exhibit clear, dose-dependent inhibition on vessel sprouting. However, the effect of CRA-026440 on overnight HUVEC tubule formation assay was marginal (data not shown). It must be noted that the potency of CRA-026440 against rat HDAC enzymes is unknown. Thus, it is difficult to directly compare the outcomes of the HUVEC Matrigel and rat aortic ring assays. However, the results may suggest that CRA-026440 would have a more chronic antiangiogenic activity, as was manifested in the aortic ring assay (4–5 days) rather than an acute activity as was in the Matrigel assay (overnight).

Some HDAC inhibitors have been shown to work synergistically or additively with an array of anticancer agents (6, 10, 11). Here, the notion that HDAC inhibition could potentiate antiangiogenic activity prompted us to examine the combination of Avastin and CRA-026440 in the HCT 116 tumor model. We observed additional efficacy with this combination. Such additive benefit could be achieved at doses of CRA-026440 much below its maximal tolerable dose, without manifesting any noticeable signs of toxicity. This finding suggests that CRA-026440 may be used in combination with a standard angiogenic inhibitor for cancer therapy.

In summary, we have identified a novel inhibitor of HDAC (CRA-026440) with antitumor activity in vitro and in vivo. The results presented here show that CRA-026440 inhibits HDAC enzymes and has antitumor properties in vivo at doses that are tolerated and may therefore be active in a clinical setting in cancer patients. Based on these results, efforts are continuing on further assessing CRA-026440 as an antitumor agent.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

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