rRNA synthesis, MIA PaCa-2 cells ( IC50 = 54 nM ); rRNA synthesis, A375 cells ( IC50 = 113 nM ); rRNA synthesis, HCT-116 cells ( IC50 = 142 nM )

CX-5461 exhibits in vivo antitumor activity against solid tumors of the human body and is orally bioavailable in murine xenograft models. On day 31, CX-5461 exhibits significant MIA PaCa-2 TGI, with a TGI of 69%, which is similar to gemcitabine's TGI of 63%. A positive control, gemcitabine, is given intraperitoneally once every three days at a dose of 120 mg/kg. Similarly, on day 32, CX-5461 exhibits significant A375 TGI, with TGI equal to 79%.[1]

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In vivo antitumor activity of CX-5461 [1]
The antitumor activity of CX-5461 was evaluated in two murine xenograft models of human cancers, pancreatic carcinoma (MIA PaCa-2) and melanoma (A375). In these xenograft models, CX-5461 was administered orally (50 mg/kg) either once daily or every 3 days. Untreated animals received vehicle orally on the equivalent schedule, whereas the positive control gemcitabine was administered intraperitoneally once every 3 days at 120 mg/kg. CX-5461 demonstrated significant MIA PaCa-2 TGI with TGI equal to 69% on day 31 (Fig. 7A), comparable to that of gemcitabine (63% TGI). Likewise, CX-5461 demonstrated significant A375 TGI (Fig. 7B) with TGI equal to 79% on day 32. Unpaired t test revealed statistically significant differences between the vehicle-treated and CX-5461–treated groups throughout both studies. CX-5461 was well tolerated at all tested schedules as judged by the absence of significant changes in animal body weights. These data demonstrate that a selective inhibitor of Pol I transcription can produce in vivo antitumor responses against solid tumors, with a favorable therapeutic window.

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Inhibition of Pol I Transcription by CX-5461 Selectively Induces p53-Mediated Cell Death of Lymphoma Cells In Vivo while Sparing Normal B Cells [2]
We next examined whether inhibition of Pol I transcription and thus activation of the Rp-MDM2-p53 nucleolar stress pathway could be used to selectively kill malignant B cells in vivo. C57BL/6 mice with established disease from transplanted Eμ-Myc lymphoma that is wild-type for p53 (Clone 4242) were treated with a single oral dose of CX-5461 (50 mg/kg) or vehicle. The Eμ-Myc tumor cells infiltrating the lymph nodes showed marked sensitivity to CX-5461, with cells exhibiting an 84% repression in Pol I transcription at 1 hr posttreatment (Figure 5A), also confirmed by RNA chromogenic in situ hybridization (CISH) for 47S pre-rRNA levels in the spleen (Figures S5A and S5B). Moreover, CX-5461 induced a rapid reduction in tumor burden in the lymph nodes (3.1% GFP-tagged malignant cells ± 0.20 for CX-5461-treated versus 34% GFP-tagged malignant cells ± 5.5 for vehicle-treated mice at 24 hr post therapy, p < 0.01) (Figures 5B and S5C) and a concomitant reduction of spleen size to within the normal range (0.14 g ± 0.01 for CX-5461-treated versus 0.47 g ± 0.01 for vehicle-treated mice at 24 hr posttherapy, p < 0.001) (Figure 5C). Consistent with the in vitro studies, reduction in malignant B cell numbers in vivo in response to CX-5461 therapy was preceded by the rapid activation of p53 and p21 and markers of apoptosis within 6 hr of drug administration (Figures 5D–5F and S5D).

Although CX-5461 activated p53 and induced apoptosis among malignant cells, it did not trigger these responses in the normal spleen cells of wild-type nontumor bearing mice (Figures 6A and 6B) and did not affect either spleen size or B cell numbers (Figures 6C and 6D). The lack of a cytotoxic effect in normal spleens cells was not due to lack of inhibition of Pol I transcription as CISH demonstrated robust reductions in 47S pre-rRNA levels in the spleens of wild-type nontumor bearing mice as observed in tumor bearing mice (Figures 6E and S6A) Further-more, the nucleolar integrity of normal bone marrow cells from mice treated with CX-5461 was maintained, as determined by FBL and B23 immunofluorescence (Figure 6F). By comparison, exposure of nontumor bearing mice to 5Gy of γ-irradiation, aclinically appropriate dose for hematologic malignancies, resulted in marked elevation of p53 levels, apoptosis, reduced spleen weight, and B cell numbers (Figures 6A–6D). We also examined the response of B220+ B cells from the spleens and bone marrow of 4- to 6-week-old premalignant Eμ-Myc mice. These cells also demonstrated profound sensitivity to CX-5461 as characterized by p53 activation and markers of apoptotic cell death (increased CASP3 cleavage and sub-G1 cells) whereas the normal B cells from age-matched littermate mice failed to show p53 activation or increased cell death (Figures S6B–S6E). Together these data demonstrate the therapeutic response to inhibition of Pol I transcription by CX-5461 in vivo is highly selective for premalignant and malignant cells, which differentiates CX-5461 from genotoxic therapies such as γ-irradiation.

To further demonstrate the therapeutic effect of CX-5461 in this model, mice with established transplanted p53 wild-type Eμ-Myc lymphoma (clone 107) were treated with three doses of CX-5461 at 50 mg/kg once every 3 days. This regimen significantly prolonged the survival of the tumor-bearing mice (Figure 7A, p < 0.0001) and restored the white blood cell count to within the normal range (Figure 7B). Indeed, the day following the last dose of CX-5461, there were few identifiable GFP-tagged tumor cells in the peripheral blood (Figures 7C and 7D). Notably, there was preservation and restoration of the recipient-derived nonmalignant normal B cell population, again indicating selective eradication of malignant disease (Figure 7C, bottom panel). Moreover, more prolonged dosing with CX-5461 also showed minimal effects on normal B cells (data not shown). CX-5461 had no discernible adverse effect on the health of the mice and the measured body weights showed minimal deviation below that at the commencement of therapy and in comparison to vehicle-treated mice (Figure 7E). To assess the ability of prolonged dosing of CX-5461 to extend survival, mice transplanted with p53 wild-type Eμ-Myc tumors (clone 4242) were maintained on continuous repeat dosing of CX-5461 at 40 mg/kg once every 3 days (Figure S7A). This treatment regimen reproducibly increased the average survival compared to the three repeat dose regimen with the mice going through a stage of apparent disease remission (no detectable tumor cells in peripheral blood at day 7/8) before eventually relapsing (Figures S7B– S7E). CX-5461 was also evaluated in a murine genetic model of acute myeloid leukemia (AML1/ETO9a+Nras) that reproduces many of the clinical aspects of leukemias in human patients that carry AML1/ETO fusion genes (Zuber et al., 2009). As with therapy in the Eμ-Myc model, transplanted AML1/ETO9a+Nras leukemic cells showed marked sensitivity to CX-5461 in vivo, undergoing p53-dependent apoptotic cell death within 6 hr of treatment of a single 40 mg/kg dose of CX-5461 (Figures S7F– S7I). This apoptotic response was comparable to 100 mg/kg cytarabine, a cytotoxic drug frequently used in treatment of patients with AML.

CX-5461-related effects on transcription are measured by qRT-PCR, which quantifies two short-lived RNA transcripts (half-lives ~20-30 minutes), one produced by Pol I and another by Pol II. As the Pol I transcript, the 45S pre-rRNA functioned, while the comparator Pol II transcript was the protooncogene c-myc mRNA. Cell stress in general is known to impact both Pol I and Pol II transcription. Cells are only exposed to test agents for a brief amount of time (2 hours) in order to reduce any possible effects of such stress. There is enough time for CX-5461 to impact the synthesis of these transcripts, resulting in a reduction of more than 90%.

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Cell-free Pol I transcription assay [1]
A reaction mixture consisting of 30 ng/μL DNA template corresponding to (−160/+379) region on rDNA and 3 mg/mL nuclear extract isolated from HeLa S3 cells in a buffer containing 10 mmol/L Tris-HCl, pH 8.0, 80 mmol/L KCl, 0.8% polyvinyl alcohol, 10 mg/mL α-amanitin was combined with different amounts of test compounds and incubated at ambient for 20 minutes. Transcription was initiated by addition of rNTP mix to a final concentration of 1 mmol/L and was incubated for 1 hour at 30°C. Afterward, DNase I was added and the reaction was further incubated for 2 hours at 37°C. DNase digestion was terminated by the addition of EDTA to final concentration of 10 mmol/L, followed immediately by 10-minute incubation at 75°C, and then samples were transferred to 4°C. The levels of resultant transcript were analyzed by qRT-PCR on 7900HT Real Time PCR System.

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Chromatin immunoprecipitation [1]
Cells were treated with 2 μmol/L of CX-5461 for 1 hour and chromatin immunoprecipitation (ChIP) assay was performed as previously described.

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Electrophoretic mobility shift assay [1]
32P-labeled DNA probe corresponding to the rDNA promoter was produced by PCR using prHu3 plasmid as a template. The SL1 complex was isolated from HeLa S3 cells nuclear extract as described previously. For the competition studies, a mixture of 5 nmol/L of DNA probe and 0.6 μg SL1 complex was incubated in binding buffer for 15 minutes at ambient temperature with 0.4 to 12.5 μmol/L CX-5461 or CX-5447. The resulting complexes were resolved using Novex TBE DNA retardation PAGE. The gels were dried and exposed to X-ray film [1].

Cell viability assay [1]
Cells were plated on 96-well plates and treated the next day with dose response of drugs for 96 hours. Cell viability was determined using Alamar Blue and CyQUANT assays.

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Detection and quantification of acidic vesicular organelles with acridine orange staining [1]
Cells were plated on 10-cm dishes and treated the next day with indicated doses of CX-5461 for 24 or 48 hours. At the end of treatment, cells were stained for 15 minutes with 1 μg/mL of acridine orange, trypsinized, washed twice with ice-cold PBS, and analyzed on a BD LSR II flow cytometer. The analysis was performed as previously described.

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The following day, cells are treated with CX-5461 dose response for 96 hours after being plated on 96-well plates. Alamar Blue and CyQUANT assays are used to measure cell viability.

Mice: Female Balb/c athymic mice (NCr nu/nu fisol) aged 5 to 6 weeks are used in animal experiments. Hypnic (NCr nu/nu fisol) mice are injected subcutaneously in the right flank of the mice with 100 μL of cell suspension. Calculating the tumor volume involves applying the formula (l×w²)/2, where w represents the tumor's width and l its length in millimeters. Tumor measurements are carried out through caliper analysis. randomized to treatment groups with either vehicle (50 mM NaH₂PO₄, pH 4.5), NSC 613327, or CX-5461 for established tumors (approx 110-120 mm³). On the last day of the study, tumor growth inhibition (TGI) is calculated using the following formula: TGI (%)=[100 − (Vf^D− Vi^D)/ (Vf^V − Vi^V) × 100], where Vi^V represents the initial mean tumor volume in the vehicle-treated group, Vf^V represents the final mean tumor volume in the vehicle-treated group, Vi^D represents the initial mean tumor volume in the drug-treated group, and Vf^D represents the final mean tumor volume in the drug-treated group on the study day.

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In vivo efficacy in murine xenograft model [1]
Mice were inoculated with 5 × 106 in 100 μL of cell suspension subcutaneously in the right flank. Tumor measurements were performed by caliper analysis, and tumor volume was calculated using the formula (l × w2)/2, where w = width and l = length in mm of the tumor. Established tumors (∼110–120 mm3) were randomized into vehicle (50 mmol/L NaH2PO4, pH 4.5), gemcitabine, or CX-5461 treatment groups. Tumor growth inhibition (TGI) was determined on the last day of study according to the formula: TGI (%) = [100 − (VfD − ViD)/ (VfV − ViV) × 100], where ViV is the initial mean tumor volume in vehicle-treated group, VfV is the final mean tumor volume in vehicle-treated group, ViD is the initial mean tumor volume in drug-treated group, and VfD is the final mean tumor volume in drug-treated group.

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MV 4;11Xenograft Model [1]
Female immunocompromised mice CrTac:Ncr-Foxn1nu (6–8 weeks old) were maintained under clean room conditions in sterile filter top cages. Xenografts were initiated by subcutaneous injection of 5 × 106 MV4;11 cells into the right hind flank region of each mouse. When tumors reached a designated volume of ∼150 mm3, animals were randomized and divided into Vehicle (50 mM NaH2PO4, pH 4.5) or CX-5461 treatment groups of 10 mice per group. CX-5461 was administered intraperitoneally once a week at 125 mg/kg for the length of 25 days. Tumor volumes and body weights were measured twice weekly. The length and width of the tumor were measured with calipers and the volume calculated using the following formula:tumor volume = (length × width2)/2. Tumor growth inhibition (TGI) was determined on the last day of study according to the formula: TGI (%) = [100 – (VfD – ViD)/(VfV – ViV) × 100], where ViV is the initial mean tumor volume in vehicle-treated group, VfV is the final mean tumor volume in vehicle-treated group, ViD is the initial mean tumor volume in drug-treated group, and VfD is the final mean tumor volume in drug-treated group.

[1]. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth. Cancer Res. 2011 Feb 15;71(4):1418-30.

[2]. Inhibition of RNA Polymerase I as a Therapeutic Strategy to Promote Cancer-Specific Activation of p53. Cancer Cell. 2012 Jul 10;22(1):51-65.

CX-5461 is an organic heterotetracyclic compound that is 5-oxo-5H-[1,3]benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxylic acid substituted by a 4-methyl-1,4-diazepan-1-yl group at position 2 and in which the carboxy group at position 6 is substituted by a [(5-methylpyrazin-2-yl)methyl]nitrilo group. An inhibitor of ribosomal RNA (rRNA) synthesis which specifically inhibits RNA polymerase I-driven transcription of rRNA in several cancer cell lines. It is currently in phase I/II clinical trials for advanced hematologic malignancies and triple-negative breast cancer with BRCA1/2 mutation. It has a role as an antineoplastic agent, an apoptosis inducer and an EC 2.7.7.6 (RNA polymerase) inhibitor. It is a diazepine, an organic heterotetracyclic compound, a member of pyrazines, a secondary carboxamide and a naphthyridine derivative.
Pidnarulex is an orally bioavailable inhibitor of RNA polymerase I (Pol I), with potential antineoplastic activity. Upon oral administration, pidnarulex selectively binds to and inhibits Pol I, prevents Pol I-mediated ribosomal RNA (rRNA) synthesis, induces apoptosis, and inhibits tumor cell growth. Pol I, the multiprotein complex that synthesizes rRNA, is upregulated in cancer cells and plays a key role in cell proliferation and survival. Hyperactivated rRNA transcription is associated with uncontrolled cancer cell proliferation.

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Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I-driven transcription relative to Pol II-driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment.[1]

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Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.[2]

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RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy. Oncotarget. 2016 Aug 2;7(31):49800-49818.

C27H27N7O2S

513.61

513.194

C, 63.14; H, 5.30; N, 19.09; O, 6.23; S, 6.24

1138549-36-6

1138549-36-6; 2101314-20-7 (HCl)

25257557

White solid powder

1.5±0.1 g/cm3

739.9±60.0 °C at 760 mmHg

401.3±32.9 °C

0.0±2.4 mmHg at 25°C

1.740

-0.81

1

9

4

37

915

0

S1C2=C([H])C([H])=C([H])C([H])=C2N2C1=C(C(N([H])C([H])([H])C1C([H])=NC(C([H])([H])[H])=C([H])N=1)=O)C(C1C([H])=C([H])C(=NC2=1)N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])C1([H])[H])=O

XGPBJCHFROADCK-UHFFFAOYSA-N

InChI=1S/C27H27N7O2S/c1-17-14-29-18(15-28-17)16-30-26(36)23-24(35)19-8-9-22(33-11-5-10-32(2)12-13-33)31-25(19)34-20-6-3-4-7-21(20)37-27(23)34/h3-4,6-9,14-15H,5,10-13,16H2,1-2H3,(H,30,36)

2-(4-methyl-1,4-diazepan-1-yl)-N-[(5-methylpyrazin-2-yl)methyl]-5-oxo-[1,3]benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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