HDAC1 ( Ki = 7 nM ); HDAC3/SMRT ( Ki = 8.2 nM ); HDAC6 ( Ki = 17 nM ); HDAC2 ( Ki = 19 nM ); HDAC10 ( Ki = 24 nM ); HDAC8 ( Ki = 280 nM ); MBLAC2 ( Ki < 10 nM )

Abexinostat (CRA 024781) parenterally produced a statistically significant decrease in tumor growth in mice bearing colon tumor xenografts from DLD-1 or HCT116. Increased α-tubulin acetylation in peripheral blood mononuclear cells and altered expression of multiple genes, including those involved in apoptosis and cell growth, are observed in conjunction with tumor growth inhibition[1].

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Antitumor Activity and Induction of Acetylation by Abexinostat (PCI24781; CRA024781) In vivo [1] In order to assess the antitumor activity of CRA-024781 in vivo, mice bearing human colon tumor xenografts were dosed i.v. with the compound using various dosages and schedules. I.v. administration of CRA-024781 in a previous dose scheduling study in HCT116 xenografts identified two regimens with good therapeutic indices: (a) once daily every other day (q.o.d.) or (b) once daily for 4 consecutive days followed by 3 days without treatment each week (q.d. × 4 per week; dose scheduling data not shown). When CRA-024781 was evaluated in HCT116 or DLD-1 xenografts according to the first regimen at a dosage of 200 mg/kg i.v. q.o.d., statistically significant inhibition of tumor growth was observed (Fig. 4A and B). The inhibition of tumor growth was 69% (P < 0.000001) and 59% (P < 0.01) for HCT116 and DLD-1 models, respectively. Although some body weight loss approaching 13% relative to vehicle controls was observed in the DLD-1 study, no body weight loss was observed in the HCT116 study, suggesting that there was some tolerability variation across experiments, and that 200 mg/kg was near the maximum tolerated dose for q.o.d. administration (data not shown). CRA-024781 was then evaluated at multiple doses using the second regimen (q.d. × 4 per week). In the HCT116 model, inhibition of tumor growth was 48% (P < 0.05), 57% (P < 0.01), 82.2% (P < 0.0001), and 80.0% (P < 0.0001) for 20, 40, 80, and 160 mg/kg, respectively. None of these doses led to reductions in animal weight relative to vehicle by the end of the study (data not shown). In the DLD-1 model, CRA-024781 administered q.d. × 4 per week did not significantly inhibit tumor growth, although a marginally significant trend towards inhibition was observed with the highest dose of 160 mg/kg, showing 43% inhibition (P = 0.09) with no associated body weight loss (data not shown). In order to determine if the plasma concentrations of CRA-024781 attained in the studies were sufficient to inhibit HDAC enzymes in vivo, peripheral blood cells were examined ex vivo. As shown in Fig. 5, each treatment group had a measurable increase in tubulin acetylation at 2 and 6 hours after dosing. In summary, CRA-024781 exhibited statistically significant antitumor activity against both HCT116 and DLD-1 human colorectal tumor xenografts, although antitumor activity overall seemed to be more pronounced in the HCT116 xenograft model.

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Combining Abexinostat (PCI24781; CRA024781) with chemotherapy results in superior anti-STS effects in vivo. We next evaluated the impact of PCI-24781 alone and combined with chemotherapy in vivo. SKLMS1 growing i.m. and HT1080 growing s.c. or as experimental lung metastases were used in four-armed studies comparing the effects of low-dose doxorubicin, cisplatinum, PCI-24781, or PCI-24781 combined with either chemotherapy on human STS local and metastatic growth in SCID mice. Therapy was initiated after tumor establishment (100 mm3). Similarly, in the experimental lung metastasis model, treatment was initiated only after established metastases were identified by bioluminescence[3].

A linear mixed model was used to assess tumor growth (tabulated as log-transformed tumor volume) across treatment groups over time, and a linear regression model was used to assess tumor weights across treatment groups. Treatment with low-dose doxorubicin alone did not significantly affect SKLMS1 xenograft growth (Fig. 3A); Abexinostat (PCI24781; CRA024781) alone induced significant tumor growth inhibition (slope of tumor volume versus time for PCI-24781 treated mice versus control untreated tumors; P = 0.001). Most significantly, combined PCI-24781 and low-dose doxorubicin was markedly inhibitory compared with control, doxorubicin alone, or PCI-24781 alone treated tumor groups (P < 0.0001). Average group tumor weights at study termination were 1.62 g ± 0.47 for control, 1.34 g ± 0.43 for doxorubicin, 1.11 g ± 0.26 for PCI-24781, and 0.64 g ± 0.24 for combination (Fig. 3B). PCI-24781 alone significantly decreased tumor weight versus controls (P = 0.026). Moreover, tumor weight reduction due to combined PCI-24781-chemotherapy was markedly significant versus other treatments (P < 0.0001, = 0.0023, and = 0.047 for control, doxorubicin, and PCI-24781, respectively)[3].

H&E staining of different treatment arm tumors revealed marked tumor necrosis in Abexinostat (PCI24781; CRA024781) and combination treatment groups (Fig. 3C); viable tumor sections were immunohistochemically evaluated for the impact of different therapies on STS cell proliferation (PCNA) and apoptosis (TUNEL). Average PCNA and TUNEL-positive staining nuclei revealed 88 ± 2 and 20 ± 2.4 for control, 85 ± 1.2 and 16 ± 1.4 for doxorubicin, 71 ± 2.3 and 19 ± 1.1 for PCI-24781, and 57 ± 4.6 and 61 ± 18.4 for combination, respectively, suggesting that combination therapy had the strongest antiproliferation and apoptosis-inducing effects (P < 0.05). Sections were also stained for CD31 to examine different therapy effects on tumor-associated vasculature. No significant differences in CD31 counts were seen; however, markedly decreased numbers of large, patent blood vessels was discernable in the combination group[3].

Similarly, the effects of Abexinostat (PCI24781; CRA024781), cisplatinum, and their combination were tested in HT1080 xenografts (Fig. 4). No significant tumor growth differences over time were identified comparing cisplatinum- or PCI-24781–treated and control mice. However, significant tumor growth reduction was observed with combined therapy (P < 0.001 versus the other treatment groups; Fig. 4A). Average group tumor weights at study termination were 1.23 g ± 0.15 for control, 1.01 g ± 0.39 for cisplatinum, 1.09 g ± 0.54 for PCI-24781, and 0.36 g ± 0.22 for combination (Fig. 4B). No significant tumor weight reduction occurred due to PCI-24781 or cisplatinum alone treatments, whereas significant tumor weight reduction was observed with combined treatment (P = 0.0003, = 0.0038, and = 0.0011 versus control, cisplatinum, and PCI-24781 groups, respectively). Immunohistochemistry results were similar to those for the SKLMS1-treated tumors described above, showing increased necrosis in PCI-24781 and combination groups (Fig. 4C). Decreased proliferation and enhanced apoptosis were most pronounced in the combination-treated group (average PCNA and TUNEL-positive staining nuclei were 83 ± 7.1 and 18 ± 0.3 for control, 62 ± 2.8 and 17 ± 2.5 for cisplatinum, 50 ± 2.8 and 21 ± 5.9 for PCI-24781, and 38 ± 14.1 and 33 ± 8.6 for combination, respectively; P < 0.05). No significant difference in CD31 positivity was seen among groups, whereas a reduction in large blood vessels was observed in the combination group[3].

Lastly, the effect of the various therapies on STS lung metastases was evaluated. An experimental fibrosarcoma lung metastasis model was used; Abexinostat (PCI24781; CRA024781) combined with doxorubicin or cisplatinum was tested (Fig. 4D). Mice were followed by bioluminescence; representative sequential images are depicted in Fig. 4D, showing reduced bioluminescence in combination treatment mice. H&E staining revealed large metastatic deposits replacing much lung parenchyma in control- and chemotherapy-treated tumors; smaller lesions were seen in the PCI-24781 group, and small microscopic lesions were observed in combination treatment mice. Lung metastases weights were calculated by deducting the estimated average normal mouse lung weight from actual lung weight at study termination. Average lung metastases weight per group was 0.55 g ± 0.14 for control, 0.62 g ± 0.26 for cisplatinum, 0.48 g ± 0.33 for doxorubicin, 0.33 g ± 0.20 for PCI-24781 group, 0.15 g ± 0.19 for PCI-24781-cisplatinum, and 0.13 g ± 0.14 for PCI-2478-doxorubicin. A trend toward reduced metastatic load was seen in the PCI-24781–treated mice but it did not reach statistical significance, whereas PCI-24781 combined with either chemotherapy resulted in significant lung metastases weight reduction (P < 0.05) compared with control or cisplatinum treatment groups. Taken together, these data suggest that although PCI-24781 exhibits significant anti-STS effects in vitro, it is only marginally effective as monotherapy in vivo. However, combining PCI-24781 with low-dose conventional chemotherapy results in significant STS tumor and metastasis growth inhibition in vivo, an observation of potential clinical utility[3].

A trypsin-coupled assay that runs continuously is used to measure HDAC activity. Measurements in a reaction volume of 100 μL using 96-well assay plates are performed for inhibitor characterization. The HDAC protein is combined with PCI-24781 at different concentrations for each isozyme and incubated for 15 minutes. The reaction buffer is 50 mM HEPES, 100 mM KCl, 0.001% Tween 20, 5% DMSO (pH 7.4), supplemented with bovine serum albumin at concentrations of 0% (HDAC1), 0.01% (HDAC2, 3, 8, and 10), or 0.05% (HDAC6). The reaction is started by adding acetyl-Gly-Ala-(N-acetyl-Lys)-AMC at a final concentration of 25 μM (HDAC1, 3, and 6), 50 μM (HDAC2 and 10), or 100 μM (HDAC8). Trypsin is added to a final concentration of 50 nM. In duplicates of eight, negative control reactions are carried out without PCI-24781. A fluorescence plate reader is used to track reactions. The fluorescence is measured over a 30-minute period with an excitation wavelength of 355 nm and a detection wavelength of 460 nm, following a 30-minute lag time. Reaction rate is calculated using the fluorescence increase over time. The program BatchKi is used to obtain inhibition constants Ki(app).

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HDAC Activity [1]
HDAC activity was measured using a continuous trypsin-coupled assay that has been previously described in detail (11). For inhibitor characterization, measurements were done in a reaction volume of 100 μL using 96-well assay plates. 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, 3, 8, and 10), 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, 3, and 6), 50 μmol/L (HDAC2 and 10), or 100 μmol/L (HDAC8) to initiate the reaction. Negative control reactions were done in the absence of inhibitor in replicates of eight. Reactions were monitored in a fluorescence plate reader. 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.

Following at least two doubling times of culture for ten tumor cell lines and HUVEC, growth is assessed using an Alamar blue fluorometric cell proliferation assay to determine the end of compound exposure. The compound is assayed using half-log intervals ranging from 0.0015 to 10 μmol/L in triplicate wells in 96-well plates at nine concentrations. Each well has a final DMSO concentration of 0.15%. A four-parameter logistic equation is used to estimate the concentration needed to inhibit cell growth by 50% and 95% confidence intervals through nonlinear regression[1].

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Cell Proliferation Assay [1]
Ten tumor cell lines and HUVEC were cultured for at least two doubling times, and growth was monitored at the end of compound exposure using an Alamar blue fluorometric cell proliferation assay as previously described. The compound was assayed in triplicate wells in 96-well plates at nine concentrations using half-log intervals ranging from 0.0015 to 10 μmol/L. The final DMSO concentration in each well was 0.15%. The concentration required to inhibit cell growth by 50% (GI50%) and 95% confidence intervals were estimated from nonlinear regression using a four-parameter logistic equation.

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Histone and Tubulin Acetylation, p21Cip1/WAF1 Accumulation, Poly(ADP-Ribose) Polymerase Cleavage, and Phosphorylated Histone Variant H2AX [2]
Acetylated histone, acetylated tubulin, p21Cip1/WAF1, poly(ADP-ribose) polymerase (PARP) cleavage, and phosphorylated histone variant H2AX (γH2AX) proteins were detected by Western blotting in cells treated with Abexinostat (PCI24781; CRA024781). Tumor cells and subconfluent HUVEC were cultured for 18 hours in the presence of Abexinostat (PCI24781; CRA024781) concentrations ranging from 0.01 to 10 μmol/L. Cells were then collected and lysed in lysis buffer containing protease inhibitors and phosphatase inhibitors. Lysates were solubilized in SDS-PAGE reducing sample buffer, boiled, and electrophoresed in Novex Tris-glycine gels. The gels were blotted onto nitrocellulose and probed with either an antiacetyl lysine antibody to detect acetylated histone, an anti-acetylated tubulin antibody, an anti-p21Cip1/WAF1 antibody, an anti-PARP antibody, or an anti-γH2AX antibody. The blots were washed, incubated with an appropriate horseradish peroxidase–conjugated secondary antibody and the blots were developed for enhanced chemiluminescence.

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Apoptosis Assay by Annexin V Staining. [2]
To determine potential synergy between Abexinostat (PCI24781; CRA024781) and the PARP inhibitor PJ34 (EMD Biosciences) in HCT116 cells, cytotoxicity was evaluated by assaying Annexin V–FITC (Biosource) binding after 96 h treatment with specified doses of the agents alone or in combination. The doses were adjusted to keep the ratio between the two drugs constant for the two combination treatments. Apoptosis was quantitated by using a FACSCalibur instrument (Becton Dickinson). The CalcuSyn program (Biosoft) was used to generate a median effects plot based on the CI calculated as described in ref. 29. A CI of >1 indicates antagonism, a CI of 1 indicates additivity, and a CI of <1 indicates synergy. To determine the percentage of apoptosis at 24 h in HCT116 cells, cells were treated with 0.2, 0.5, and 1.0 μM PCI-24781, and Annexin V binding was evaluated 24 h later.

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Immunofluorescence. [2]
HCT116 cells were grown on chamber slides and treated with 0.2 μM Abexinostat (PCI24781; CRA024781) for 24 h. Cells were then exposed to 10 Gy irradiation and incubated for 1 or 16 h after irradiation. Cells were then fixed and permeabilized in buffer containing 2% paraformaldehyde, 0.2% Triton X-100 in PBS. Cells were blocked in 2% BSA in PBS and probed with anti-RAD51 polyclonal antibody, followed by AlexaFluor594-conjugated secondary antibody. After staining, coverslips were mounted by using the Vecta shield mounting medium with DAPI and visualized by using fluorescence microscopy.

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TaqMan Gene Expression Assays. [2]
HCT116 cells were treated with Abexinostat (PCI24781; CRA024781) for various times, and total RNA was extracted by using the RNeasy kit. Total RNA was quantitated by using the Ribogreen RNA quantitation kit. One-step RT-PCR assays were set up in triplicate according to the instructions of the manufacturer by using the TaqMan master mix and 50 ng of total RNA as template. The amount of RNA in each reaction well was requantitated and used for normalizations. Gene expression assay probe sets for BRCA1, BRCA2, RAD51, and GADD45γ were purchased from Applied Biosystems, and assays were performed on an ABI PRISM 7300 instrument according to standard protocols.

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Western Blot Analysis. [2]
Cells were treated with Abexinostat (PCI24781; CRA024781) for the designated times and lysed, and total protein was quantitated by using the BCA protein assay kit. An equivalent amount of protein was loaded in each lane. The pan-caspase inhibitor Q-VD-OPh, which is used for blocking caspase cleavage, was purchased from xx Biomedicals. Total protein (30 μg) for each treatment was resolved on 4–15% gradient SDS/PAGE gels. Proteins were transferred to PVDF membrane and probed with the appropriate primary antibodies and secondary antibodies conjugated to AlexaFluor680 and IFdye800 were used. Imaging was performed by using an Odyssey scanner.

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Homologous Recombinational Repair Assay. [2]
Homologous recombinational repair activity was measured in DRAA8 Chinese hamster ovary cells as described in ref. 39. Briefly, DRAA8/CHO cells contained a nonfunctional GFP sequence and an internal GFP (iGFP) sequence integrated into the hprt locus. The mutant GFP sequence contained an 18-bp I-SceI recognition site, and DSBs were introduced by expression of the I-SceI endonuclease. The I-SceI expression vector (3.5 μg) was transfected by using nucleofection (Amaxa), and DSB-induced gene conversion by using the downstream iGFP repeat was quantitated by using FACS by measuring GFP signal. Abexinostat (PCI24781; CRA024781) was added at the appropriate concentrations 6 h after transfection, the live cells were gated based on 7-amino-actinomycin D (7-AAD) signal, and GFP expression in the gated live population was analyzed 36 h later.

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Clonogenic Survival Assays. [2]
To determine the effects of Abexinostat (PCI24781; CRA024781) treatment on colony formation in CHO-K1 and the NHEJ-deficient Ku80 mutant XRS5 cell lines, cells at the appropriate plating density were plated in a 10-cm dish and allowed to attach. Cells were exposed to 0.5, 1.0, or 2.0 μM concentrations of drug for 24 h, after which the cells were placed in fresh growth media without drug and allowed to incubate for 7–10 days. Colonies were fixed with 100% isopropyl alcohol and stained with 1% crystal violet. For each condition, the assay was performed in triplicate. In HCT116, NCI-H460, and A549 lung tumor cell lines, to determine the appropriate time of PCI-24781 treatment for synergy with radiation, cultures were pretreated with 1 μM PCI-24781 for various lengths of time, ranging from 2 to 24 h. Cells were exposed to 0, 2, 4, or 6 Gy irradiation (Gammacell 40, Atomic Energy of Canada, Ltd.), and clonogenic survival was assessed after 7–12 days. From the plating efficiencies at each dose, the percentage of colony-forming ability was calculated.

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Measurement of cell proliferation. [3]
Cell growth assays used CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega) per the manufacturer's instructions. Growth rates were analyzed 48 h after HDACi (Abexinostat (PCI24781; CRA024781)/SAHA) treatment and with doxorubicin or cisplatin, alone or combined with HDACi; several administration sequences were evaluated: 24-h pretreatment with PCI-24781 followed by the addition of chemotherapy for 24 h and conversely, pretreatment with chemotherapy followed by PCI-24781 as well as treatment with both compounds concomitantly for 24 h. Absorbance was measured at 490 nm wavelength; treated cell absorbance values are presented as a percentage of untreated cell absorbance.

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Clonogenic assay. [3]
STS cells were treated in culture dishes with DMSO (control) and varying concentrations of HDACi (Abexinostat (PCI24781; CRA024781)/SAHA) for 24 h. One hundred cells per well were replated, then allowed to grow in normal media for 10 d, then stained with a 6% glutaraldehyde, 0.5% crystal violet solution for 30 min. Staining solution was decanted from each well and cells washed with deionized H2O. Individual colonies retaining staining solution were counted.

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Cell cycle analysis. [3]
STS cell monolayers were treated with Abexinostat (PCI24781; CRA024781)/SAHA for varying periods. Propidium iodide/fluorescence-activated cell sorting (FACS) analysis was conducted as described.

Female BALB/c nu/nu mice are given 3×10⁶ tumor cells each, along with DLD-1 and HCT116 tumor cells, subcutaneously. When the tumor volume was on average -100 mm, treatment with Abexinostat (CRA 024781) began[1]. To measure the antitumor activity of Abexinostat (CRA 024781), mice with human colon tumor xenografts are given different doses and schedules of the drug intravenously [1].

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Pharmacokinetic Analysis [1]
Abexinostat (PCI24781; CRA024781) was formulated in 30% HP-cyclodextrin in water and a 10 mg/kg bolus dose was administered i.v. to female BALB/c nu/nu mice (16–22 g). Blood samples were collected over 24 hours. Plasma was prepared from each blood sample with lithium heparin and was assayed for Abexinostat (PCI24781; CRA024781) by liquid chromatography with tandem mass spectrometry. Mouse plasma samples were assayed for CRA-024781 by high-performance liquid chromatography with tandem mass spectrometric detection. Plasma samples were extracted by protein precipitation using acetonitrile (MeCN). A Hypersil C-18 column, 50 × 2.1 mm was used for sample separation. Samples were separated on reversed-phase high-performance liquid chromatography under linear gradient conditions using water/MeCN as mobile phases, at a flow rate at 0.5 mL/min. Samples were ionized under electrospray and quantified by multiple reactive monitoring, recording the transition from the molecular ion to the product ion: 398 → 200 m/z. Linearity (>0.25–1,000 ng/mL) was achieved with interday and intraday coefficient of variation (%) and deviation of the mean from theoretical (%) ±15%. The lower limit of quantitation was 0.25 ng/mL. Pharmacokinetic variables were estimated with compartmental methods using WinNonlin-Pro version 4.1 (Pharsight Corp., Mountain View, CA). Pharmacokinetic calculations were done using nominal doses and nominal collection times.

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In vivo Efficacy Studies [1]
Female BALB/c nu/nu mice were acclimated for 3 to 5 days prior to tumor implantation. All mice were maintained in sterilized translucent polycarbonate microisolator cages under specific pathogen–free conditions. HCT116 and DLD-1 tumor cells were implanted s.c. in nude mice at 3 × 106 per mouse. Tumor-bearing mice were randomized based on tumor volume prior to the initiation of treatment. Treatment with Abexinostat (PCI24781; CRA024781) started when the average tumor volume was ∼100 mm3. Tumor volume was calculated as follows: volume = 0.5 × X2 × Y, where X, tumor width; and Y, tumor length. The vehicle used in all studies was 20% HP-β-cyclodextrin in water. Dosages were given i.v. either every other day (q.o.d.) or for 4 consecutive days followed by 3 days without treatment for each week of the study (q.d. × 4 per week) as indicated. 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 on log-transformed data to meet the assumption of underlying normal distribution for the test to be valid, and P values were corrected for multiple comparisons to control by Dunnett's method. For single-group comparison, statistical analyses were done with t test on log-transformed data to meet the assumption of an underlying normal distribution for the test to be valid.

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In Vivo Studies. [2]
BALB/c female nude mice were implanted s.c. with 3 × 106 HCT116 cells in a 1:1 ratio with Matrigel into the right-hand flank 24 h after administering 4.5 Gy (whole-body irradiation). Mice were entered into study ≈10 days after implantation or when tumor volumes reached a minimum of 75–100 mm3. Four treatment regimens were followed with three animals in each group, each receiving a dose of 200 mg/kg: (i) vehicle; (ii) animals received a single oral dose 4 h before the end of the study (1×); (iii) animals received one oral dose 28 h before the end of the study and a second dose 6 h later (2×); and (iv) animals were dosed as in the 2× group but also received a third dose the following morning, 24 h after the first dose was administered and 4 h before the end of the study (3×). Animals were humanely killed, tumors were extracted, and total protein was isolated and quantitated by using the BCA protein assay kit. Total protein (30 μg) was loaded for each tumor, and Western blotting was performed to determine RAD51, acetylated tubulin, and actin levels.

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In vivo therapeutic studies. [3]
Trypan blue staining confirmed viable STS cells (SKLMS1 or HT1080 cells 1 × 106/0.1 mL HBSS/mouse) were injected into the flank (SKLMS1 i.m., HT1080 s.c.) of 6-wk-old female SCID mice (n = 40/experiment), growth was measured twice weekly; HT1080GL cells (stably expressing green fluorescent protein/Luciferase) were tail vein injected, resulting in experimental lung metastases that could be followed by bioluminescence. When average tumor volumes reached 100 mm3, the mice were assigned to four treatment groups (7-8 mice/group): (a) control (vehicles only); (b) doxorubicin/cisplatinum (1.2 mg/kg/biweekly and 2 mg/kg/biweekly, respectively, i.p.); (c) Abexinostat (PCI24781; CRA024781) (50 mg/kg/d ×5 d/wk, i.p.); and (d) Abexinostat (PCI24781; CRA024781) plus chemotherapy (PCI-24781 initiated 24 h before chemotherapy in all cases). PCI-24781 dose and treatment schedules were determined per company recommendations. A similar experimental design was used for lung metastasis treatment, initiated when bioluminescence showed established lung metastases; the study included six arms so that Abexinostat (PCI24781; CRA024781) combined with either chemotherapeutic agent could be evaluated. The mice were followed for tumor size and body weight and sacrificed when control group tumors reached 1.5 cm average largest dimension or when bioluminescence suggested control group had significant pulmonary tumor load. Tumors were resected, weighed, and frozen or fixed in formalin and paraffin-embedded for immunohistochemical studies. Similarly, lungs were resected, weighed, and fixed in formalin.

In vivo Pharmacokinetics [1]
Having established that Abexinostat (PCI24781; CRA024781) displayed HDAC inhibitory and antitumor properties in vitro, we assessed the pharmacokinetics of the compound to evaluate exposure in order to enable further testing in efficacy models. CRA-024781 was delivered i.v. to female mice and plasma concentrations were monitored over time (Fig. 3). Based on these data, the clearance was calculated to be 297 mL/min/kg, the volume of distribution in the central compartment was 3,750 mL/kg, the steady-state volume of distribution was 9,230 mL/kg, the predominant plasma half-life was 6.7 minutes (73% area under the curve), and the mean residence time was 31 minutes. These data suggest that CRA-024781 had sufficient in vivo exposure when given by the i.v. route of administration for use in the study of the biological effects of HDAC inhibition in vivo.

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CRA-024781 had an in vivo pharmacokinetic profile that suggested adequate drug exposure to allow HDAC inhibition in vivo. The 10 mg/kg i.v. dose achieved a plasma concentration above the measured cellular GI50% for HCT116 tumor cells for 15 minutes. However, it is difficult to predict a priori the in vivo drug exposure (area under the curve) that will be adequate for efficacy based on GI50% or on the appearance of mechanistic biomarkers (protein acetylation, etc.), because these values are based on continuous exposure of tumor cells to the drug in vitro, on the other hand, tumors grown in vivo are exposed to the drug on a certain schedule (e.g., q.d. × 4 for 16 days) at varying concentrations of compound based on the pharmacokinetics and degree of protein binding of the drug in plasma. Also, the drug concentration in plasma does not necessarily reflect the concentration distributed to the tumor. Indeed, the relatively high steady-state volume of distribution (9,230 mL/kg) suggests that CRA-024781 may readily distribute into tissues. Nevertheless, the doses required for maximal efficacy (≥80 mg/kg) are >10 mg/kg, perhaps because HDAC enzymes must be inhibited for a more prolonged period of time or to a greater extent following each dose in order to achieve antitumor efficacy.

In a previous experiment, an oral dose of 200 mg/kg did not cause toxicity as measured by body weight loss when dosed for 3 consecutive days. The dose of 200 mg/kg in mice was also chosen because it is similar to the plasma area under the concentration–time curve expected in an ongoing clinical trial after an oral dose of 2.0 mg/kg.[2]

[1]. CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo. Mol Cancer Ther, 2006, 5(5), 1309-1317.

[2]. HDAC inhibitor PCI-24781 decreases RAD51 expression and inhibits homologous recombination. Proc Natl Acad Sci U S A, 2007, 104(49), 19482-19487.

[3]. Combining PCI-24781, a novel histone deacetylase inhibitor, with chemotherapy for the treatment of soft tissue sarcoma. Clin Cancer Res, 2009, 15(10), 3472-3483.

[4]. Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target. Nat Chem Biol. 2022 Aug;18(8):812-820.

3-[(dimethylamino)methyl]-N-[2-[4-[(hydroxyamino)-oxomethyl]phenoxy]ethyl]-2-benzofurancarboxamide is a member of benzofurans.
Abexinostat has been used in trials studying the treatment of Sarcoma, Lymphoma, Leukemia, Lymphocytic, and Hodgkin Disease, among others. It is a novel, broad-spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) with potential antineoplastic activity.
Abexinostat is an orally bioavailable hydroxamate-based pan-inhibitor of histone deacetylase (HDAC), with potential antineoplastic and radiosensitizing activities. Upon administration, abexinostat inhibits HDAC, resulting in an accumulation of highly acetylated histones, followed by the induction of chromatin remodeling; the selective transcription of tumor suppressor genes; and the tumor suppressor protein-mediated inhibition of tumor cell division and induction of tumor cell apoptosis. In addition, abexinostat decreases the expression of the DNA-repair protein RAD51, thereby reducing the RAD51 protein, preventing repair of DNA double-strand breaks and increasing sensitivity of tumor cells to DNA damaging agents. HDAC, upregulated in many tumor types, is an enzyme that is responsible for the deacetylation of chromatin histone proteins.

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Mechanism of Action
Abexinostat is a novel histone deacetylase (HDAC) inhibitor. HDAC inhibitors target HDAC enzymes and inhibit the proliferation of cancer cells and induce cancer cell death, or apoptosis. Histone deacetylation is carried out by a family of related HDAC enzymes. Inhibition of these enzymes causes changes to chromatin structure and to gene expression patterns, which results in the inhibition of proliferation of cancer cells, and induction of apoptosis.

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CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.[1]

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Histone deacetylase (HDAC) inhibitors such as the phenyl hydroxamic acid PCI-24781 have emerged recently as a class of therapeutic agents for the treatment of cancer. Recent data showing synergy of HDAC inhibitors with ionizing radiation and other DNA-damaging agents have suggested that HDAC inhibitors may act, in part, by inhibiting DNA repair. Here we present evidence that HDAC enzymes are important for homologous recombinational repair of DNA double-strand breaks. Combination studies of PCI-24781 with the poly(ADP-ribose) polymerase inhibitor PJ34, an agent thought to produce lesions repaired by homologous recombination (HR), resulted in a synergistic effect on apoptosis. Immunofluorescence analysis demonstrated that HDAC inhibition caused a complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigations revealed that inhibition of HDAC enzymes by PCI-24781 led to a significant reduction in the transcription of genes specifically associated with HR, including RAD51. RAD51 protein levels were significantly decreased after 24 h of drug exposure both in vitro and in vivo. Consistent with inhibition of HR, treatment with PCI-24781 resulted in a decreased ability to perform homology directed repair of I-SceI-induced chromosome breaks in transfected CHO cells. In addition, an enhancement of cell killing was observed in Ku mutant cells lacking functional nonhomologous end joining compared with WT cells. Together these results demonstrate that HDAC enzymes are critically important to enable functional HR by controlling the expression of HR-related genes and promoting the proper assembly of HR-directed subnuclear foci. [2]

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Purpose: Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved. Experimental design: Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models. Results: PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy. [3]

C21H23N3O5

397.42

397.163

C, 63.46; H, 5.83; N, 10.57; O, 20.13

783355-60-2

11749858

White to off-white solid powder

1.3±0.1 g/cm3

1.624

1.68

3

6

8

29

550

0

O=C(NCCOC1=CC=C(C=C1)C(NO)=O)C2=C(C3=CC=CC=C3O2)CN(C)C

MAUCONCHVWBMHK-UHFFFAOYSA-N

InChI=1S/C21H23N3O5/c1-24(2)13-17-16-5-3-4-6-18(16)29-19(17)21(26)22-11-12-28-15-9-7-14(8-10-15)20(25)23-27/h3-10,27H,11-13H2,1-2H3,(H,22,26)(H,23,25)

3-[(dimethylamino)methyl]-N-[2-[4-(hydroxycarbamoyl)phenoxy]ethyl]-1-benzofuran-2-carboxamide

CRA024781; PCI24781; CRA 024781; ABEXINOSTAT; 783355-60-2; PCI-24781; PCI 24781; CRA-024781; CRA-02478; CRA 024781; Abexinostat [USAN]; PCI-24781; CRA024781; PCI 24781; CRA-024781

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 6 mg/mL (15.10 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)