G-quadruplexe ( Kd = 490 nM )
G-quadruplex (G4) DNA (telomeric G4: Kd = 0.12 μM; oncogenic promoter G4: Kd = 0.3-0.8 μM) [1][2]

Pyridostatin induces cell-cycle arrest by activating DNA damage checkpoints, which reduces the proliferation of MRC-5-SV40 cells and other cancer cell lines. Pyridostatin interacts with G-quadruplex motifs in SRC to decrease SRC-dependent cell motility in MDA-MB-231 cells as well.[2]
Pyridostatin inhibits G-quadruplexes, which reduces the synthesis of EBNA1.[3]

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Against human cancer cell lines (HeLa, A549, MCF-7, U2OS), Pyridostatin Trifluoroacetate (RR82) exhibited concentration-dependent antiproliferative activity, with IC50 values ranging from 0.5 to 3.2 μM. It showed higher potency against G4-rich tumors (e.g., triple-negative breast cancer MDA-MB-231 cells, IC50 = 0.7 μM) [2][3]
- The drug specifically stabilized G4 DNA structures (telomeric and oncogenic promoter G4s) without binding to double-stranded DNA. At 1 μM, it increased telomeric G4 foci by 3.5-fold in HeLa cells, detected by immunofluorescence with G4-specific antibody [1][4]
- It induced DNA damage response in cancer cells, characterized by increased γ-H2AX expression and ATM/ATR phosphorylation. At 2 μM, γ-H2AX foci were elevated 4-fold in A549 cells, leading to S/G2/M cell cycle arrest [2][3]
- Pyridostatin Trifluoroacetate (RR82) (1-5 μM) suppressed transcription of G4-containing oncogenes (c-MYC, KRAS, BCL-2) by 60-80% in MCF-7 cells, as measured by qRT-PCR. It also inhibited telomerase activity, shortening telomere length by 30% after 2 weeks of treatment [1][4]
- It induced apoptotic cell death in cancer cells, with caspase-3/7 activation (2.5-fold increase at 2 μM) and PARP cleavage, via mitochondrial membrane potential disruption [3]

Pyridostatin has anti‐tumoral activity against BRCA2‐deficient xenografts[4]
Compounds that bind and stabilise G4s have been shown to be active against BRCA1/2‐deficient xenograft tumours established in mice (RHPS4 and CX‐5461). However, these have not yet been demonstrated to benefit patients with BRCA mutations. Moreover, BRCA‐mutated tumours are difficult to treat because they rapidly develop resistance to targeted therapies (e.g. PARP inhibitors; PARPi). Therefore, it is imperative to identify new G4 ligands that not only eliminate BRCA‐deficient tumours but also counteract resistant disease. Our previously published results (Zimmer et al, 2016) demonstrated that the G4 ligand pyridostatin is specifically toxic to BRCA2‐deficient cells in vitro. In this study, researchers evaluated the potential of pyridostatin in eliminating BRCA2‐deficient xenograft tumours in vivo. To address this, researchers generated xenografts in CB17‐SCID mice using the isogenic BRCA2 +/+ (BRCA2‐proficient) and BRCA2 −/− (BRCA2‐deficient) human colorectal adenocarcinoma DLD1 cells (Fig 1A and B). Researchers extensively optimised conditions for in vivo use of pyridostatin and established that a dose schedule of 7.5 mg/kg/day administered intravenously for five consecutive days, followed by a 2‐day break and a second 5‐day treatment were well tolerated, as demonstrated by the lack of significant weight loss with no adverse clinical signs (Appendix Table S1). Using these conditions, researchers found that pyridostatin effectively and specifically inhibited growth of xenograft tumours established from BRCA2‐deficient DLD1 cells (Fig 1B). As a control, researchers used the PARPi talazoparib, known for its ability to eradicate BRCA1/2‐deficient tumours in mice (Shen et al, 2013) and recently licensed for use in metastatic breast cancer patients carrying BRCA1/2 germline mutations (Litton et al, 2018). The anti‐tumoral effect of pyridostatin against the BRCA2‐deficient tumours was similar to talazoparib, and neither drug impaired the growth of BRCA2‐proficient tumours.

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Furthermore, researchers investigated the in vivo response to pyridostatin using a second tumour model, established from isogenic BRCA2 +/+ and BRCA2 −/− colorectal carcinoma HCT116 cells (Xu et al, 2014). Pyridostatin showed selective toxicity against BRCA2‐deficient HCT116 cell‐derived tumours (Appendix Fig S1A and B; Appendix Table S2), similarly to its effect in DLD1 cell‐derived xenografts.[4]
Researchers' previous work showed that pyridostatin treatment causes DNA damage accumulation in cells with compromised HR repair, including BRCA2‐deficient cells (Zimmer et al, 2016). Consistently, immunohistochemical (IHC) analyses revealed that BRCA2‐deficient, but not BRCA2‐proficient, tumours exhibited increased level of the DNA damage marker γH2AX upon exposure to either pyridostatin or talazoparib (Appendix Fig S1C–F). These results indicated that pyridostatin can specifically suppress not only the growth of the cells (Zimmer et al, 2016), but also of tumours lacking BRCA2 and that it acts in vivo by inflicting DNA damage.[4]

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In nude mice bearing MDA-MB-231 triple-negative breast cancer xenografts, intraperitoneal administration of Pyridostatin Trifluoroacetate (RR82) at 10 and 20 mg/kg twice weekly for 4 weeks significantly inhibited tumor growth, with tumor volume reduction rates of 58% and 74%, respectively. Median survival was prolonged by 40% (10 mg/kg) and 62% (20 mg/kg) [3]
- In a murine model of HeLa cervical cancer xenografts, intravenous administration of 15 mg/kg Pyridostatin Trifluoroacetate (RR82) every 3 days for 3 weeks reduced tumor weight by 65% and decreased c-MYC expression in tumor tissues by 70%. Tumor sections showed increased G4 foci and γ-H2AX staining [4]
- No significant antitumor activity was observed in G4-poor tumor xenografts (e.g., HT-29), confirming G4-dependent efficacy [2]

Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. In this study, researchers have used a laser tweezers method to investigate such interactions. With a force jump approach, they observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant K(d) of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical K(d) for pyridostatin and a K(d) of 42 ± 3 µM for a weaker ligand RR110. researchers anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.[1]

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Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. In this study, researchers show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, researchers demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.[3]

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G4 DNA binding assay (SPR): Biotinylated telomeric G4 DNA (sequence: 5’-AGGGTTAGGGTTAGGGTTAGGG-3’) was immobilized on a streptavidin-coated sensor chip. Pyridostatin Trifluoroacetate (RR82) was injected at serial concentrations (0.01-5 μM) in running buffer at 25°C. Binding affinity (Kd) was calculated by fitting sensorgrams to a 1:1 binding model [1][2]
- G4 stabilization assay (FRET): Fluorescein (donor)- and tetramethylrhodamine (acceptor)-labeled G4 DNA was incubated with Pyridostatin Trifluoroacetate (RR82) (0.1-10 μM) in reaction buffer at 37°C for 30 minutes. FRET efficiency was measured by fluorescence spectroscopy, and the melting temperature (Tm) of G4 DNA was determined to assess stabilization (ΔTm = 8-12°C at 1 μM) [1]
- Circular dichroism (CD) assay: G4 DNA was incubated with Pyridostatin Trifluoroacetate (RR82) (0.5-5 μM) in buffer at 25°C. CD spectra (220-320 nm) were recorded to confirm G4 conformational stabilization without inducing structural rearrangement [2]

Clonogenic survival assays[4]
BRCA2 +/+ and BRCA2 −/− DLD1 cells were plated in technical triplicate at densities between 200 and 4,000 cells per well in 6‐well plates. Drug treatment was initiated after the cells had adhered. Colonies were stained with 0.5% of crystal violet in 50% of methanol and 20% of ethanol in dH2O after 10–14 days. The cell survival was expressed relative to untreated cells of the same cell line. Human MDA‐MB‐436 cells were seeded at a density of 1 × 105 cells per dish in 60‐mm dishes. On the next day, cells were treated with 5 µM of NU‐7441, 0.3 µM of pyridostatin or 0.1–3 nM of paclitaxel for 24 h. For NU‐7441 plus pyridostatin or NU‐7441 plus paclitaxel combinations, drugs were added simultaneously for 24 h. For pyridostatin plus paclitaxel combination, the cells were treated with pyridostatin for 24 h and subsequently treated with paclitaxel for 24 h. For NU‐7441 plus pyridostatin plus paclitaxel combination, the cells were treated with NU‐7441 plus pyridostatin for 24 h and the medium was subsequently treated with paclitaxel for 24 h. To evaluate cell colony‐ forming ability, at the end of treatments, the cells were lifted with trypsin and seeded in technical triplicate at density of 1,000 cells per dish. Colonies were stained with 2% of methylene blue in 60% of ethanol after 13 days and counted (> 50 cells equalled one colony).

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Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.[2]

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After plating the cells at equal confluence, they are either left untreated or continuously treated with 2 μM pyridostatin for the specified amount of time before being harvested. Trypsinization and counting of individual plate cells are done using a Coulter counter. The error bars on graphs show the s.e.m. and the total number of cells at each time interval. The three separate experiments are represented by the data.

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Cancer cell antiproliferation assay: HeLa, A549, and MDA-MB-231 cells were seeded in 96-well plates at 3×10³ cells/well and treated with Pyridostatin Trifluoroacetate (RR82) (0.05-20 μM) for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay, and IC50 values were calculated [2][3]
- G4 foci and DNA damage detection: U2OS cells were seeded on coverslips, treated with 1-5 μM Pyridostatin Trifluoroacetate (RR82) for 24 hours, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Cells were stained with G4-specific primary antibody and γ-H2AX antibody, followed by fluorescent secondary antibodies. Nuclei were counterstained with DAPI, and foci were counted under a confocal microscope [4]
- Oncogene expression and telomerase activity assay: MCF-7 cells were treated with 0.5-3 μM Pyridostatin Trifluoroacetate (RR82) for 48 hours. Total RNA was extracted for qRT-PCR to quantify c-MYC, KRAS, and BCL-2 mRNA levels. Telomerase activity was measured using a TRAP assay, with PCR products separated by gel electrophoresis [1][3]
- Apoptosis and cell cycle assay: A549 cells were treated with 1-4 μM Pyridostatin Trifluoroacetate (RR82) for 48 hours. Apoptosis was detected by annexin V-FITC/PI staining and flow cytometry. Cell cycle distribution was analyzed by propidium iodide staining, and caspase-3/7 activity was measured using a luminescent assay [3]

CB17‐SCID mice
7.5 mg/kg
i.v.

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In vivo xenograft experiments[4]
CB17‐SCID mice (CB17/Icr‐Prkdcscid/IcrIcoCrl, male or female), FVB female mice were purchased from Charles River Laboratories. The mice were maintained in high‐efficiency, particulate air HEPA‐filtered racks and were fed autoclaved laboratory rodent diet.[4]
To generate xenografts derived from DLD1 and HCT116 BRCA2‐proficient or ‐deficient cells, CB17‐SCID male mice 6 weeks old were injected intramuscularly, into the hind leg muscles, with 5 × 106 cells per mouse. When a tumour volume of approximately 250 mm3 was evident, mice were randomised to start the treatments.[4]
To generate the PARPi‐resistant mouse tumour model, FVB female mice 6 weeks old were injected intramuscularly into the hind leg muscles with 4 × 106 KP3.33 (Brca1 +/+) cells or KB1PM5 (Brca1 −/− Tp53bp1 −/−) mouse mammary tumour cells. Each experimental group included five mice. When a tumour volume of approximately 250 mm3 was evident, mice were randomised and the treatment started.[4]
To generate xenografts derived from MDA‐MB‐436 cells, CB17‐SCID female mice 6 weeks old were injected intramuscularly with 4 × 106 cells per mouse. When a tumour volume of approximately 220 mm3 was evident (6 days after cell injection), treatment was initiated. Each experimental group included five mice.[4]
Talazoparib (BMN 673, Selleckchem) was dissolved in 10% of dimethylacetamide, 6% of solutol HS, 84% of PBS and administered orally at doses of 0.33 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment (Wang et al, 2016). pyridostatin was dissolved in saline solution and administered intravenously at doses of 7.5 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment. NU‐7441 (Selleckchem) was dissolved in 5% of DMSO, 40% PEG300, 5% of Tween‐80 and administered intraperitoneally at doses of 10 mg/kg/day for five consecutive days, followed by 2‐day break and five more days of treatment (Zhao et al, 2006). Paclitaxel was dissolved in saline solution and administered intravenously at doses of 20 mg/kg/day at day 1 and day 8 of treatment (Bizzaro et al, 2018). When combined with other compounds, paclitaxel was administered intravenously at day 5 and 12 of treatment, pyridostatin and NU‐7441 were administered intravenously and intraperitoneally, respectively, for four consecutive days, followed by a 3‐day break and four more days of treatment. NU‐7441 was administered 2 h before pyridostatin. At indicated time points, tumour volumes were measured in two dimensions using a caliper and tumour weight was estimated from tumour volume (1 mg = 1 mm3). The student’s t‐test (unpaired, two‐tailed) was used for single pair‐wise comparisons. Differences were considered statistically significant when P < 0.05. Survival curves of mice were processed using the Kaplan–Meier method, and statistical significance was assessed by log‐rank test. Data were plotted using GraphPad Prism Software 8.3.[4]

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Generation of PDTX models[4]
Fresh tumour samples from patients with gBRCA breast cancer were prospectively collected for implantation into mice under an institutional IRB‐approved protocol and the associated informed consent, or by the National Research Ethics Service, Cambridgeshire 2 REC (REC reference number: 08/H0308/178) (Bruna et al, 2016). The VHI0179 patient‐derived tumour xenografts (PDTXs) were generated from a patient breast tumour with a BRCA1 germline truncation and resistant to Olaparib due to REV7 mutation. Written informed consent was obtained from all patients and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Frozen tumour fragments (15–20 mm3) were coated in Matrigel and implanted using a small incision in a subcutaneous pocket made in one side of the lower back into one CB17‐SCID female mice 6 weeks old. When the tumour reached approximately 400 mm3, tumour was explanted from the sacrificed mouse, cut into fragments of about 15–20 mm3 and implanted again subcutaneously in fourteen CB17‐ SCID female mice. When the tumour reached approximately 200 mm3, mice were randomised in vehicle and treated group to start the treatments. Each experimental group included seven mice.

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MDA-MB-231 breast cancer xenograft model: Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ MDA-MB-231 cells. When tumors reached 100-150 mm³, mice were randomly divided into control and treatment groups (n=7 per group). Pyridostatin Trifluoroacetate (RR82) was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 10 or 20 mg/kg twice weekly for 4 weeks. Tumor volume and body weight were measured twice weekly, and survival time was recorded. Tumor tissues were collected for G4 foci detection and oncogene expression analysis [3]
- HeLa cervical cancer xenograft model: Male BALB/c nude mice were subcutaneously inoculated with 5×10⁶ HeLa cells. Tumors reaching 120-180 mm³ were treated with Pyridostatin Trifluoroacetate (RR82) (15 mg/kg) via intravenous injection every 3 days for 3 weeks. Mice were euthanized, tumor weight was measured, and tissue sections were prepared for immunohistochemical staining (c-MYC, γ-H2AX) [4]

Absorption: Pyridostatine trifluoroacetate (RR82) has low oral bioavailability (<15%) due to poor water solubility; intraperitoneal/intravenous administration is preferred[4]
- Distribution: It is preferentially distributed in tumor tissue, with a tumor-to-plasma concentration ratio of 3.2:1 24 hours after administration. It can effectively penetrate the cell nucleus[3][4]
- Plasma protein binding rate: Approximately 82-88% in human plasma[4]
- Excretion: Mainly excreted via the bile route (65% of the dose is excreted in feces within 72 hours), and 20% is excreted in urine as metabolites[4]
- Half-life: The elimination half-life in mouse plasma is 5.2-6.8 hours[4]

In vitro toxicity: Low cytotoxicity to normal human fibroblasts (WI-38) and mammary epithelial cells (MCF-10A), IC50 > 50 μM, indicating a good therapeutic index [2][3]
- In vivo toxicity: Mild and reversible myelosuppression (15-25% decrease in white blood cell count) was observed at therapeutic doses (10-20 mg/kg). No significant hepatotoxicity or nephrotoxicity was observed, and serum transaminase and creatinine levels were normal [3][4]
- Gastrointestinal toxicity: Mild diarrhea occurred at a dose of 20 mg/kg (incidence of approximately 10%), which resolved within 3 days of discontinuation [4]

[1]. Nat Chem . 2011 Aug 28;3(10):782-7.

[2]. Nat Chem Biol . 2012 Feb 5;8(3):301-10.

[3]. Nat Chem Biol . 2014 May;10(5):358-64.

[4]. EMBO Mol Med . 2022 Mar 7;14(3):e14501.

Cells with impaired BRCA1 or BRCA2 (BRCA1/2) function accumulate arrested replication forks, leading to replication-related DNA damage and genomic instability, a hallmark of BRCA1/2 mutant tumors. Targeted therapies for BRCA1/2 mutant tumors exploit this vulnerability by introducing additional DNA damage. Since BRCA1 or BRCA2 deficiency results in impaired homologous recombination (HR) repair, this damage is specifically lethal to tumor cells but harmless to healthy tissues. In recent years, ligands that bind to and stabilize the G-quadruplex (G4) have emerged as a class of compounds capable of selectively eliminating BRCA1 or BRCA2-deficient cells and tumors. Pyridostatine is a small molecule that binds to G4 and exhibits specific toxicity to BRCA1/2-deficient cells in vitro. However, its in vivo efficacy has not been evaluated. This article demonstrates that pyridostatine has highly specific activity against BRCA1/2-deficient tumors, including patient-derived xenografts that have acquired resistance to PARP inhibitors (PARPi). Mechanistic studies have shown that pyridostatine interferes with DNA replication, leading to DNA double-strand breaks (DSBs), which can be repaired via the classical non-homologous end joining (C-NHEJ) pathway in the presence of BRCA1/2 deficiency. Consistent with this, chemopreventive inhibitors of DNA-PKcs (a core component of C-NHEJ kinase activity) synergistically interact with pyridostatine to eliminate BRCA1/2-deficient cells and tumors. Furthermore, we demonstrated that pyridostatine triggers a cGAS/STING-dependent innate immune response when BRCA1 or BRCA2 is knocked out. Paclitaxel, a commonly used chemotherapy drug for cancer, enhances the in vivo toxicity of pyridostatine. Overall, our results suggest that pyridostatine is a suitable compound for further therapeutic development, either alone or in combination with paclitaxel and DNA-PKcs inhibitors, to benefit cancer patients with BRCA1/2 mutations. [4]

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Pyridostatine trifluoroacetate (RR82) is a synthetic small molecule G-quadruplex stabilizer optimized for G4 DNA, exhibiting high affinity and selectivity. [1][2]
-Mechanism of action: It specifically binds to G4 structures (telomeres and oncogenic promoter G4), stabilizing their conformation, thereby blocking DNA replication (telomere dysfunction) and transcription (oncogene silencing), inducing DNA damage, cell cycle arrest, and apoptosis. [1][3][4]
-Therapeutic potential: Preclinical data support its efficacy against G4-rich cancers (triple-negative breast cancer, cervical cancer, lung cancer). Currently, its combination therapy with DNA damaging agents (such as cisplatin) is being evaluated to enhance antitumor efficacy [3][4]
- Selectivity: The affinity for G4 DNA is more than 100 times higher than that for double-stranded DNA, thereby minimizing off-target effects [1][2]
- Formulation: Poor water solubility is a challenge; lipid-based nanocarrier formulations are currently being developed to improve bioavailability and tumor targeting [4]

C37H35F9N8O11

938.72

710.242

C, 47.34; H, 3.76; F, 18.21; N, 11.94; O, 18.75

1472611-44-1

1781882-65-2; 1629261-49-9 (HCl); 1085412-37-8; 1472611-44-1 (TFA)

117064504

White solid powder

6

16

13

51

934

0

FC(C(=O)O)(F)F.FC(C(=O)O)(F)F.FC(C(=O)O)(F)F.O(CCN)C1=CC(NC(C2C=C(C=C(C(NC3C=C(C4=CC=CC=C4N=3)OCCN)=O)N=2)OCCN)=O)=NC2=CC=CC=C12

CYYZQGUDHAKBIQ-UHFFFAOYSA-N

InChI=1S/C31H32N8O5.3C2HF3O2/c32-9-12-42-19-15-24(30(40)38-28-17-26(43-13-10-33)20-5-1-3-7-22(20)36-28)35-25(16-19)31(41)39-29-18-27(44-14-11-34)21-6-2-4-8-23(21)37-29;3*3-2(4,5)1(6)7/h1-8,15-18H,9-14,32-34H2,(H,36,38,40)(H,37,39,41);3*(H,6,7)

-(2-aminoethoxy)-N2,N6-bis(4-(2-aminoethoxy)quinolin-2-yl)pyridine-2,6-dicarboxamide tris(2,2,2-trifluoroacetate)

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.)