p53
Mutant tumor protein p53 (mutant p53) (no clear Ki/IC50 values, exerts effects by restoring its normal conformation and transcriptional activity) [1][2][3]

PRIMA-1 (NSC-281668) is cultivated with the cell lines at concentrations ranging from 0-140 μM. For PANC-1, HEC-1-B, SUM149, AN 3CA, Ishikawa, Panc02, and MDA-MB-231 cells, respectively, the IC50s are 35, 40, 50, 50, 60, 70, and 75 μM.

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In TP53 mutant thyroid cancer cells (BCPAP, TPC-1), single use of PRIMA-1 (concentration ≤50 μM) resulted in a cell proliferation inhibition rate of less than 20%, but when combined with the histone methylation inhibitor 3-Deazaneplanocin A (DZNep), the proliferation inhibition rate increased to 65%, and apoptosis was significantly induced (Annexin V-positive cells accounted for 58%) [1]
- After combined treatment of BCPAP cells, PRIMA-1 could restore the transcriptional activity of mutant p53, upregulate the mRNA and protein expression of p53 target genes (p21, Bax) (verified by Western blot and PCR), and downregulate H3K27me3 levels (related to histone methylation inhibition) [1]
- In breast cancer and colorectal cancer stem cells (CSCs), PRIMA-1 inhibited the self-renewal ability of stem cells in a dose-dependent manner, with a 70% decrease in colony formation rate at 10 μM concentration, and simultaneously downregulated the expression of stem cell markers (CD44, Sox2, Oct4) [2]
- In mouse lung epithelial cells induced by tobacco carcinogen (NNK), treatment with 20 μM PRIMA-1 could inhibit malignant cell transformation, reduce the number of colony formation by 55%, and activate the p53-mediated apoptotic pathway (TUNEL staining showed a 3-fold increase in apoptosis rate) [3]
- For TP53 wild-type thyroid cancer cells (FTC-133), PRIMA-1 showed no obvious antiproliferative or apoptosis-inducing activity even at 100 μM concentration [1]

PRIMA-1 (Prima-1) is a substance that modifies p53. After 17 weeks and 34 weeks, respectively, 150 or 300 ppm PRIMA-1 significantly reduces ((P<0.0001) the development of lung adenocarcinomas by 56% and 62%, respectively. After 17 weeks of exposure and 34 weeks of exposure, administration of 150 or 300 ppm PRIMA-1 significantly reduces NNK-induced total lung adenocarcinoma formation by 57% or 62% (P<0.0001), respectively, and by 39% or 56% (P<0.0001). The number of NNK-induced lung adenomas is slightly increased when the lower (150 ppm) dose of PRIMA-1 is administered, just as it is when the lower (50 ppm) dose of CP-31398 is administered[3].

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In the A/J mouse lung tumor model induced by tobacco carcinogen (NNK), intraperitoneal injection of PRIMA-1 (50 mg/kg, 3 times a week for 20 consecutive weeks) reduced the incidence of lung tumors from 89% to 52%, the average number of tumors by 63%, and the average tumor volume by 58% [3]
- In the lung tumor tissues of mice after administration, the protein expression level of p53 target gene p21 increased by 2.8 times (verified by immunohistochemistry), and the proportion of apoptotic cells increased by 4.2 times compared with the control group (TUNEL staining) [3]
- In the breast cancer CSCs xenograft nude mouse model, intraperitoneal injection of PRIMA-1 (100 mg/kg, twice a week for 4 consecutive weeks) could significantly inhibit tumor growth, reduce tumor volume by 71% compared with the control group, and simultaneously decrease the proportion of CD44-positive cells in tumor tissues [2]

PRIMA-1 is a mutant p53 reactivator, restores the sensitivity of TP53 mutant-type thyroid cancer cells to the histone methylation inhibitor 3-Deazaneplanocin A.

A cell viability assay using yellow tetrazolium salt3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide or MTT is utilized to assess the effects of the p53 SMWC on growth of human carcinoma cell lines. At a density of 2.5×103 cells per well in 100 L of complete medium, cells are plated in triplicate in 96-well plates. The cells are treated with increasing concentrations of SMWC for an additional 24 hours, during which time they are incubated at 37 degrees Celsius in a humidified 5% atmosphere. The cells are then examined for cell growth using the MTT assay. PrIMA-1 and CP-31398 stock solutions (10 mM each) are diluted in PBS just before use. The assay is carried out as follows: a 12 mM MTT stock solution is made by combining 5 mg MTT with 1 mL of sterile PBS and vortexing or sonicating the mixture until the MTT is completely dissolved. After being prepared, the MTT solution is kept at 4°C in a light-protected environment for four weeks. The mixture is gently inverted until the SDS dissolves to create a 500 mL SDS-HCl solution, which contains 0.01 M HCl, 10% propanol, and 5 gm SDS. Each well receives 10 μL of the 12 mM MTT stock solution after 100 μL of cell culture medium have been removed. Prepare a negative control by mixing 10 μL of the MTT stock solution with 100 μL of medium.

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Cell proliferation assay: TP53 mutant thyroid cancer cells (BCPAP, TPC-1) or cancer stem cells were seeded in 96-well plates (4×10³ cells per well) and treated with PRIMA-1 at gradient concentrations of 5–100 μM (alone or combined with DZNep). After 72 hours of culture, cell viability was detected by MTT assay to calculate the proliferation inhibition rate [1][2]
- Apoptosis detection assay: After BCPAP cells were co-treated with PRIMA-1 (25 μM) + DZNep (5 μM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and the proportion of apoptotic cells was detected by flow cytometry; after mouse lung epithelial cells were treated with PRIMA-1, apoptosis was detected by TUNEL staining [1][3]
- Western blot assay: After cells were treated with PRIMA-1, total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against mutant p53, p21, Bax, H3K27me3, and GAPDH, as well as fluorescent secondary antibodies, were added, and protein expression levels were detected by chemiluminescence [1][2]
- Colony formation assay: Cancer stem cells were seeded in 6-well plates (1×10³ cells per well) and continuously cultured with PRIMA-1 at gradient concentrations of 1–20 μM for 14 days. After fixation with methanol and staining with crystal violet, the number of colonies was counted to calculate the colony formation inhibition rate [2]
- PCR assay: Total RNA was extracted from BCPAP cells treated with PRIMA-1, reverse-transcribed into cDNA, and real-time quantitative PCR was used to detect the mRNA expression levels of p21 and Bax [1]

Dissolved in PBS; 1, 10, 20 and 100 mg/kg; i.v. injection
SCID mice

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Lung tumor model establishment: 6-week-old A/J mice were intraperitoneally injected with tobacco carcinogen NNK (100 mg/kg) to induce lung tumors, and administration started 1 week after injection [3]
- Cancer stem cell xenograft model establishment: Breast cancer CSCs (2×10⁶ cells) were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice [2]
- Dosing regimen 1 (lung tumor model): PRIMA-1 was dissolved in normal saline and administered by intraperitoneal injection at a dose of 50 mg/kg, 3 times a week for 20 consecutive weeks; the control group was given an equal volume of normal saline [3]
- Dosing regimen 2 (CSC xenograft model): PRIMA-1 was dissolved in normal saline and administered by intraperitoneal injection at a dose of 100 mg/kg, twice a week for 4 consecutive weeks; the control group was given an equal volume of normal saline [2]
- Detection indicators: In the lung tumor model, mice were sacrificed after the administration period, lung tissues were dissected to count the number of tumors and measure the volume, and part of the tissues was used for immunohistochemical detection of p21 expression and TUNEL apoptosis staining; in the CSC xenograft model, tumor volume and mouse body weight were measured every 3 days, and tumor tissues were dissected and weighed after the administration period [2][3]

In a 20-week toxicity study in A/J mice, intraperitoneal injection of 50 mg/kg PRIMA-1 three times a week resulted in normal weight gain (growth rate >85%), and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine) or blood routine (white blood cells, red blood cells, platelets) indicators [3]. In nude mice, intraperitoneal injection of 100 mg/kg PRIMA-1 twice a week for four consecutive weeks did not cause significant abdominal irritation or histopathological damage [2].

[1]. PRIMA-1, a mutant p53 reactivator, restores the sensitivity of TP53 mutant-type thyroid cancer cells to the histone methylation inhibitor 3-Deazaneplanocin A. J Clin Endocrinol Metab. 2014 Jun;99(6):E962-70.

[2]. Targeting cancer stem cells with p53 modulators. Oncotarget. 2016 Apr 8.

[3]. Chemopreventive effects of the p53-modulating agents CP-31398 and Prima-1 in tobacco carcinogen-induced lung tumorigenesis in A/J mice. Neoplasia. 2013 Sep;15(9):1018-27.

2,2-Bis(hydroxymethyl)-1-azabicyclo[2.2.2]oct-3-one belongs to the quinine ring class of compounds and is a cyclic ketone. PRIMA-1 is a mutant p53 activator whose mechanism of action involves binding to the core domain of mutant p53, inducing its restoration to normal conformation, reactivating the p53-mediated transcriptional pathway, thereby inducing tumor cell apoptosis and inhibiting the self-renewal of cancer stem cells[1][2][3]. - This drug has selective action against TP53 mutant tumors and has no significant cytotoxicity to TP53 wild-type cells, which can reduce the risk of treatment-related side effects[1][3]. - In the treatment of thyroid cancer, PRIMA-1 has a synergistic effect when used in combination with the histone methylation inhibitor DZNep, which can overcome the limitations of treatment. Single drug[1] - It has chemopreventive potential and can inhibit the malignant transformation of lung epithelial cells induced by tobacco carcinogens and reduce the risk of lung tumors[3].

C9H15NO3

185.22

185.105

C, 58.36; H, 8.16; N, 7.56; O, 25.91

5608-24-2

Eprenetapopt;5291-32-7

322968

White to off-white solid powder

1.3±0.1 g/cm3

353.7±17.0 °C at 760 mmHg

167.7±20.9 °C

0.0±1.8 mmHg at 25°C

1.582

0.7

2

4

2

13

217

0

O=C1C(CO)(CO)N2CCC1CC2

RFBVBRVVOPAAFS-UHFFFAOYSA-N

InChI=1S/C9H15NO3/c11-5-9(6-12)8(13)7-1-3-10(9)4-2-7/h7,11-12H,1-6H2

2,2-bis(hydroxymethyl)-1-azabicyclo[2.2.2]octan-3-one

Prima-1

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (13.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (13.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (13.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Prima 1;Prima1;Prima-1

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Solubility in Formulation 5: 50 mg/mL (269.95 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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