生物活性

Pifithrin-μ is a cell-permeable sulfonamide inhibitor of p53 binding and an anti-apoptotic. Inhibits p53 binding to mitochondria by reducing its affinity for antiapoptotic proteins Bcl-2 and Bcl-XL. Direct inhibition of p53 binding to mitochondria as well as to Bcl-xL and Bcl-2 proteins by Pifithrin-μ has been observed. Pifithrin-μ selectively inhibits p53 translocation to mitochondria without affecting the transactivation function of p53. Pifithrin-μ has been shown to reduce γ-radiation induced cell death in vitro. Unlike pifithrin-α (sc-45050), pifithrin-μ targets only the mitochondrial branch of the p53 pathway without affecting the important transcriptional functions of p53. Selectively inhibits heat shock protein 70 (HSP70) activity. Displays no effect on the transactivational or cell cycle checkpoint control function of p53. Potentially increases reprogramming efficiency of human somatic cells to induced pluripotent stem cells (iPSCs) by silencing p53. Reduces cell death induced by γ-radiation in vitro and protects mice from doses of radiation that cause lethal hematopoietic syndrome.

体外研究

体内研究

1% DMSO+30% polyethylene glycol+1% Tween 80

激酶实验

细胞实验

In Vitro Cell Culture[1]

Three prostate cancer cell lines; PC-3,LNCaP and DU145 and 3 colorectal cancer cell lines; HT29, LoVo, and HCT116 wereprepared.

LNCaP cells were cultured in RPMI 1640medium supplemented with 10% Heat-inactivated

Foetal Bovine Serum (HI-FBS), PC-3 cells inHam’s F12 Kaighn’s modified medium supplemented with 10% HI-FBS and DU145 cellsin Modified Eagle Medium (MEM) supplemented with 10% HI-FBS.

HT29 and LoVo cell lines were cultured inDMEM (Dulbecco’s Modified Eagle Medium) supplemented with 1%penicillin-streptomycin, 1% L-glutamine and 10% FBS. The HCT-116 cells werecultured in RPMI 1640 supplemented with 1% penicillin-streptomycin, 1%L-glutamine and 10% FBS.

In all cases cells were kept in anincubator set at 37^oC with 5% CO₂. Together the CO₂ environment and the hydrogen carbonate from the medium generate a physiology pHof 7.4.

InVitro Cytotoxicity Assays

One hundred microliters of stock solutionsof cisplatin and oxaliplatin were made freshly in medium and diluted out withmedium to required concentrations. A 20mM DMSO pifithrin-μ stocksolution was diluted out with medium to required concentrations with final DMSOconcentration of ≤0.2%. 0.2% DMSO inmedium was used as a control for experiments pertaining to pifithrin-μ. Cellgrowth was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt, (MTS tes), a colorimetric assay based on the ability of viablecells to reduce a soluble yellow tetrazolium salt to blue formazan. 110⁴ prostate cancer cells or 3 x10⁴ colorectal cancer cells were seededper well onto 96-well plates in 100μL of the appropriate culturemedium. Twenty-four hours after seeding, the medium was removed and the cellswere treated by adding 100μL of the test compound solutions at appropriate concentrations.

After 72 h of treatment, 20μLof the MTS reagent was added to each well and the plates incubated for 2hr at37^oC. The absorbance was measured at 490 nm using a Wallac 1420Victor 3 V plate reader. The percentage of surviving cells relative to untreatedcontrols was then determined. The IC50 value defined as the drug concentrationrequired inhibiting cell growth by 50% was estimated graphically fromdose-response plots using GraphPad Prisim, a scientific 2D graphing andstatistics software.

动物实验

Animals[2]

C57BL/6J male mice were used in this study.Mice were bred in house and were allowed water and food ad libitum. Housing waskept constant at 22 ± 2^oC, anda 12/12 hours reverse dark–light cycle (dark 1,000–2,200 hours) was employed.Video recording of animal behavior was performed under red light using a Sony HandycamDCR-SR100.

Injections

Cisplatin (2.3 mg/kg) or PBS was administeredintraperitoneally daily for 5 days, followed by a 5-day rest with no injectionand then another 5-day injection cycle. To investigate the effects of pifithrin(PFT)-μ oncognitive function, PFT-μ (8 mg/kg) or vehicle (5% dimethyl sulfoxide in saline) wasadministered intraperitoneally 1 hour before cisplatin injection.

Novelobject/place recognition and Y-maze tests

To assess cognitive function, we subjectedmice to novel object/place recognition (NOPR) and Y-maze tests 7 days after thefinal cisplatin injection. All behavioral assays were performed in a blindedsetup. NOPR was performed. In brief, mice were transferred to a testing arena(46.99 cm x 25.4 cm) containing two identical objects placed against one sideof the arena for 5 minutes (training phase) and then returned to their homecages for30 minutes. Mice were transferred back to the arena, now containingone familiar object placed at the same location as in training, and one novelobject placed on the opposite end of the arena (testing phase). Investigativebehavior toward either object during the 5-minute testing period was evaluatedusing EthoVision XT 10.1 video tracking software. Discrimination index wasdetermined by the equation

For the Y-maze test, spontaneousalternations were performed. In brief, mice were placed in a symmetricalthree-arm, gray plastic Y-maze (35 cm length x 5 cm width x 15.5 cm height perarm, with an arm angle of 120^o) with external spatial room cues.Mice were randomly placed in one of the arms. Movement was recorded for 5minutes, and mouse exploration was evaluated. Perfect alternations were definedas exploration of all three arms sequentially before reentering a previouslyvisited arm. All four paws must have been within the arm to be counted as an entrance.Results are represented as the ratio of the number of perfect alternations tothe total number of possible alternations.

Locomotion

Spontaneous locomotor activity was measured.In brief, mice were video recorded for 5 minutes in their home cage. Distancetravelled was quantified using EthoVision XT 10.1.

Bodyweight

Body weights were measured daily at thetime of injection. The percentage of change from baseline (day 0) wascalculated.

Tumorresponse to chemoradiation

A heterotopic syngeneic murine model ofhuman papillomavirus (HPV)–related head and neck cancer was used to assess whetherPFT-m might potentially interfere with the tumor's response to chemoradiation.Male C57BL/6J mice were injected in the hind leg with 1 x 10⁶ cellsderived from C57BL/6 oropharyngeal epithelial cells that were transfected withoncogenes E6/7 of HPV 16 and hRAS. Mice were injected with PFT-μ (8 mg/kg) or vehicle 24 hours before a curative regimen of cisplatin (5.28mg/kg intraperitoneally) and local irradiation (8 Gy provided by a small-animalcesium137 irradiator that collimates parallel opposed radiation beams to a 3-cmfield) starting 12 days after tumor implantation and repeated weekly for 3weeks. Tumor volume was determined using Vernier calipers from three mutuallyorthogonal tumor diameters, where volume = (π/6)(d1xd2xd3).

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] McKeon AM, Egan A, Chandanshive J, McMahon H, Griffith DM. Novel Improved Synthesis of HSP70 Inhibitor, Pifithrin-mu. In Vitro Synergy Quantification of Pifithrin-mu Combined with Pt Drugs in Prostate and Colorectal Cancer Cells. Molecules. 2016;21(7).
[2] Chiu GS, Maj MA, Rizvi S, et al. Pifithrin-mu Prevents Cisplatin-Induced Chemobrain by Preserving Neuronal Mitochondrial Function. Cancer Res. 2017;77(3):742-752.
[3] Hagn, Franz., et al., 2010. BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain. The Journal of biological chemistry. 285(5): 3439-50.

体内溶解度
10 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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