Molecule

Description

Sarcoma ,Lymphoma

Description

PCI-24781 (CRA-024781) is a novel broad spectrum HDAC inhibitor targeting HDAC1, HDAC2, HDAC3, HDAC6, HDAC8 and HDAC10 with K_i of 7 nM, 19 nM, 8.2 nM, 17 nM, 280 nM, 24 nM, respectively.

Targets

IC₅₀

In Vitro

PCI-24781 exhibits potent antitumor activity against a variety of tumor cell lines with GI50 ranging from 0.15 μM to 3.09 μM. PCI-24781 also has an antiproliferative effect on HUVEC endothelial cells with GI50 of 0.43 μM. PCI-24781 treatment causes dose-dependent accumulation of both acetylated histones and acetylated tubulin in HCT116 or DLD-1 cells, induces expression of p21, and leads to PARP cleavage and accumulation of the γH2AX. ^[1] Inhibition of HDAC enzymes by PCI-24781 leads to a significant reduction in the transcription of genes specifically associated with HR, including RAD51. Consistent with inhibition of HR, PCI-24781 treatment results in a decreased ability to perform homology directed repair of I-SceI-induced chromosome breaks in transfected CHO cells. ^[2] PCI-24781 induces S phase depletion, G2 cell cycle arrest, and apoptosis in soft tissue sarcoma (STS) cells. PCI-24781 induces Rad51 transcriptional repression in STS cells potentially mediated via enhanced E2F1 binding to the Rad51 proximal promoter. ^[3] PCI-24781 induces caspase and reactive oxygen species-dependent apoptosis through NF-κB mechanisms in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. ^[4]

In Vivo

Administration of PCI-24781 at 200 mg/kg once daily every other day (q.o.d.) significantly inhibits the growth of HCT116 and DLD-1 xenografts in mice by 69% and 59%, respectively. Administration of PCI-24781 at 20 mg/kg, 40 mg/kg, 80 mg/kg, or 160 mg/kg once daily for 4 consecutive days followed by 3 days without treatment each week (q.d. × 4 per week) in the HCT116 model causes inhibition of tumor growth by 48%, 57%, 82.2%, or 80.0%, respectively. ^[1]

Clinical Trials

A Phase I study of PCI-24781 in subjects with lymphoma is currently ongoing.

Features

Description

The sequential combination of PCI-24781 (0.1 μM) and Doxorubicin (0.5 μg/mL) causes growth reductions of 77%, 81%, and 65% in HT1080, RD, and SKLMS1 cells, respectively. PCI-24781 in combination with Cisplatinum also shows a statistically significant superior effect. Combining PCI-24781 (50 mg/kg/day) with low-dose Doxorubicin (1.2 mg/kg/biweekly) results in significant reduction of human leiomyosarcoma (SKLMS1) growth in SCID mice. Combining PCI-24781 (50 mg/kg/day) with low-dose Cisplatinum (2 mg/kg/biweekly) or Doxorubicin (1.2 mg/kg/biweekly) results in significant reduction of human fibrosarcoma (HT1080) local and metastatic growth in nude mice. ^[3] Combined PCI-24781 and Bortezomib results in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and is additive in Hodgkin lymphoma and primary CLL/SLL cells. ^[4]

HDAC Activity

HDAC activity is measured using a continuous trypsin-coupled assay. For inhibitor characterization, measurements are done in a reaction volume of 100 μL using 96-well assay plates. For each isozyme, the HDAC protein in reaction buffer [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)] is mixed with PCI-24781 at various concentrations and allowed to incubate for 15 minutes. Trypsin is added to a final concentration of 50 nM, and acetyl-Gly-Ala-(N-acetyl-Lys)-AMC is added to a final concentration of 25 μM (HDAC1, 3, and 6), 50 μM (HDAC2 and 10), or 100 μM (HDAC8) to initiate the reaction. Negative control reactions are done in the absence of PCI-24781 in replicates of eight. Reactions are monitored in a fluorescence plate reader. After a 30-minute lag time, the fluorescence is 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 is used as the measure of the reaction rate. Inhibition constants K_i(app) are obtained using the program BatchKi.

Cell Lines

HCT116, HCT-15, BT-549, NCI-H226, CWR-22RV1, MCF-7, NCI-PC3, DLD-1, SKOV-3, and OVCAR-3

Concentrations

Dissolved in DMSO, final concentrations ~10 μM

Incubation Time

48, 72, 96, or 120 hours

Methods

Cells are cultured for at least two doubling times, and growth is monitored at the end of PCI-24781 exposure using an Alamar blue fluorometric cell proliferation assay. PCI-24781 is assayed in triplicate wells in 96-well plates at nine concentrations using half-log intervals ranging from 0.0015 μM to 10 μM. The final DMSO concentration in each well was 0.15%. The concentration required to inhibit cell growth by 50% (GI50) and 95% confidence intervals are estimated from nonlinear regression using a four-parameter logistic equation.

Animal Models

Female BALB/c nu/nu mice implanted s.c. with HCT116 and DLD-1 tumor cells

Formulation

Formulated in 30% HP-cyclodextrin in water

Doses

~200 mg/kg

Administration

Administered via i.v.