MDM2 (Kd = 11 nM)

RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. In vitro, MDM2's interactions with p53 are blocked by RG7112's highly specific binding of MDM2 (KD of 10.7 nM). The RG7112-MDM2 complex has been crystallized, and it shows that the small molecule mimics the interactions of crucial p53 amino acid residues by binding to MDM2's p53 pocket. By activating the p53 pathway, RG7112 causes cell-cycle arrest and apoptosis in cancer cells that express wild-type p53. A panel of solid tumor cell lines is sensitive to the antitumor effects of RG7112. However, the apoptotic activity of this drug varies greatly, with osteosarcoma cells that have MDM2 gene amplification showing the best response. [1]

---

RG7112 is a potent inhibitor of p53-MDM2 binding[2].
RG7112 stabilizes wild-type p53 and induces p53 signaling in cancer cells[2].
RG7112 effectively activates p53 functions in cancer cells[2].
Caspase inhibition does not affect the onset of RG7112-induced cell death[2].

In vivo, RG7112 causes tumor cells to undergo apoptosis and activates the p53 pathway. At nontoxic doses, oral administration of RG7112 to mice bearing human xenografts resulted in dose-dependent alterations in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, androgen deprivation and RG7112 have powerful synergistic effects in LNCaP xenograft tumors. [1]

---

Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.[2]

---

PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival. Conclusions: These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2-amplified models suggests that additional markers of response to MDM2 inhibitors must be identified.[3]

Homogeneous time-resolved fluorescence (HTRF) assay measures the signal generated by 2 components when they are in close proximity. The p53–MDM2 binding assay uses a biotinylated peptide derived from the MDM2-binding domain of p53 and a truncated N-terminal portion of recombinant human GST-tagged MDM2 protein containing the p53-binding domain. Proteins for crystal structure studies were expressed in E. coli strain BL21 using the helper plasmid pUBS 520 coding for the lacIq repressor and the rare tRNAArg [AGA/AGG]. For crystallization, the frozen protein was thawed and concentrated to 9.8 mg/mL using a Centricon concentrator (3,000 MW cutoff). The complex was then formed by combining the protein with a slight molar excess of the inhibitor (stock solution is 100 mmol/L in DMSO) and this solution was allowed to sit for 4 hours at 4°C. Cryopreserved crystals were used to collect diffraction data on beamline X8C at the National Synchrotron Light Source at Brookhaven National Laboratory[2].

Cell proliferation/viability was evaluated by the tetrazolium dye (MTT) assay. Cell growth kinetics were measured using the IncuCyte live cell imaging system. For cell-cycle analysis, cells were cultured in T75 flask with appropriate growth medium (106 cells/condition in 10 mL) and incubated overnight at 37°C. They were incubated with test compound RG7112 and processed as previously described. Apoptosis was determined using the Annexin V assay using the GuavaNexin apoptosis detection kit and percent apoptosis determined by using a Guava Personal Cell Analyzer following the manufacturer's protocol[2].

---

Proliferation assay[3]
For drug sensitivity assays of the cohort #1 cell lines, 96-well plates were coated with 10 μg/mL laminin at 37°C for 1 hour. Three thousand cells/well were then plated. RG7112 was resuspended as a 10 mM stock solution in DMSO and was added 24 h after plating. Seventy-two hours after drug addition, WST-1 reagent was added according to the manufacturer’s instructions. WST-1 salt is cleaved to a soluble formazan dye by a NAD(P)H-dependent reaction in viable cells. Plates were incubated for 3 h and read by spectrophotometry at 450 nm wavelength. For cohort #2 cell lines, cells were plated in 384-well format and a pin transfer robot was used to transfer the compound solution into each well, with 3 replicates per condition. Cell viability was measured after 72 hours of continuous drug exposure by CellTiter Glo luminescence assay. IC75, IC99, and IC100 (concentrations that induce a 75, 99, and 100% decrease in cell viability, respectively) were determined by least squares curve fitting using GraphPad® Prism 6.[3]

For SJSA-1, SJSA-1luc2, and MHM xenograft studies, female Balb/c nude mice were implanted subcutaneously in the right flank with 5 × 106 cells suspended in a 0.2 mL volume of a 1:1 mixture of Matrigel:PBS. For studies with hormone-dependent LNCaP xenografts, castrated male Balb/c nude were implanted with 12.5 mg sustained-release testosterone pellets 5 days before subcutaneous inoculation with 1 × 107 cells suspended in 0.2 mL of Matrigel:PBS. Mice were randomized into treatment groups (n = 10 per group) when mean tumor volume reached approximately 150 to 400 mm3. In all studies, mice received either vehicle (1% Klucel LF/0.1% Tween 80) or RG7112, administered as an oral suspension at the dose indicated (25–200 mg/kg). For assessment of androgen ablation treatment in combination with RG7112 in LNCaP xenograft-bearing mice, testosterone pellets were removed under ketamine/xylazine anesthesia. Tumor volume was monitored by caliper measurement and body weights were recorded 2 to 3 times weekly. Tumor volume (in mm3) was calculated as described previously [2].
For Western blot analysis, mice bearing established SJSA-1 subcutaneous xenografts received a single oral dose of vehicle or 50, 100, or 200 mg/kg RG7112, and tumors were harvested at 4 and 8 hours after dosing. Protein was extracted from tumor tissue with 1× radioimmunoprecipitation assay buffer containing protease inhibitors by homogenization. Equal amounts of total protein were resolved on 4% to 12% NuPAGE gradient gel and blotted with antibody dilutions as recommended by manufacturer. The chemiluminescent signal was generated with enhanced chemiluminescence Plus and detected with Fujifilm LAS-3000 imager. The densitometric quantitation of specific bands was determined using Multi Gauge Software. The complete methods can be found in the online Supplementary Information.[2]

---

For the heterotopic (subcutaneous) model, 2x106 cells were resuspended in Hank’s Buffered Salt Solution, mixed with an equal volume of Matrigel and injected into both flanks of eight-week-old NU/NU mice. Animals were randomly assigned to treatment or vehicle arm when tumors measured a volume of 200 mm3. For both orthotopic and heterotopic models, animals were treated by gavage with 100 mg/kg of RG7112formulation (100 mg/mL RG7112, 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water) or vehicle once per day, 5 days/week for 3 weeks. For the evaluation of GBM blood-brain barrier (BBB) integrity only, 1.2 mg of Hoechst 33342 diluted in PBS was injected intravenously (iv) prior to termination. Mice were terminated by asphyxiation when they showed signs of tumor-associated illness or before reaching maximum subcutaneous tumor burden.[3]
Pharmacokinetics studies[3]
GBM cells were inoculated in the brain of Athymic Nude mice as described below and animals were assigned to different pharmacokinetics time points when bioluminescence signal reached 1.108 photon/second. This threshold was selected to ensure that tumor volumes were as significant as possible without causing symptoms of pain or illness. The dose treatment solution of RG7112 (100 mg/mL RG7112) was prepared in a vehicle composed of 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water . Mice were sacrificed at 0, 1h, 2h, 4h, 8h, 24h and 48h post-gavage (3 mice per time point). Blood was collected via live cardiac puncture in polyethylene tubes using a heparinized syringe. Samples were immediately centrifuged at 5000 rpm for 15 min and plasma was removed and stored at −80°C until analysis. Whole brains were collected, rinsed with 0.9% sodium chloride. The right and left brain hemispheres were harvested separately and labeled as tumor hemisphere and counter hemisphere, respectively, and were frozen at −80°C. RG7112 levels in mice plasma, and brains were measured using validated liquid chromatography coupled with mass tandem spectrometry methods. [3]

---

1% Klucel LF/0.1% Tween 80; 200 mg/kg; oral taken

SJSA-1, SJSA-1luc2, and MHM xenografted Balb/c nude mice

[1]. Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development. ACS Med Chem Lett. 2013 Apr 2;4(5):466-9.

[2]. MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models. Cancer Res. 2013 Apr 15;73(8):2587-97.

[3]. Preclinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 1;22(5):1185-96.

RO-5045337 is under investigation in clinical trial NCT01164033 (A Study of RO5045337 in Patients With Solid Tumors).
MDM2 Antagonist RO5045337 is an MDM2 (human homolog of double minutes-2; HDM2) antagonist with potential antineoplastic activity. RO5045337 binds to MDM2, thereby preventing the binding of the MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By preventing this MDM2-p53 interaction, the proteasome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored, which may result in the restoration of p53 signaling and thus the p53-mediated induction of tumor cell apoptosis. MDM2, a zinc finger protein, is a negative regulator of the p53 pathway; often overexpressed in cancer cells, it has been implicated in cancer cell proliferation and survival.

C38H48CL2N4O4S

727.78

726.277

C, 62.71; H, 6.65; Cl, 9.74; N, 7.70; O, 8.79; S, 4.41

939981-39-2

57406853

White to off-white solid powder

1.2±0.1 g/cm3

790.4±70.0 °C at 760 mmHg

431.8±35.7 °C

0.0±2.8 mmHg at 25°C

1.598

6.67

0

6

10

49

1260

2

C[C@]1([C@@](C2C=CC(Cl)=CC=2)(C)N=C(C2C=CC(C(C)(C)C)=CC=2OCC)N1C(N1CCN(CCCS(=O)(=O)C)CC1)=O)C1C=CC(Cl)=CC=1

QBGKPEROWUKSBK-QPPIDDCLSA-N

InChI=1S/C38H48Cl2N4O4S/c1-8-48-33-26-29(36(2,3)4)14-19-32(33)34-41-37(5,27-10-15-30(39)16-11-27)38(6,28-12-17-31(40)18-13-28)44(34)35(45)43-23-21-42(22-24-43)20-9-25-49(7,46)47/h10-19,26H,8-9,20-25H2,1-7H3/t37-,38+/m0/s1

[(4S,5R)-2-(4-tert-butyl-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazol-1-yl]-[4-(3-methylsulfonylpropyl)piperazin-1-yl]methanone

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: ≥ 10 mg/mL (13.74 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 100.0 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.

---

Solubility in Formulation 2: ≥ 10 mg/mL (13.74 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 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

View More

Solubility in Formulation 3: ≥ 5 mg/mL (6.87 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: ≥ 2.5 mg/mL (3.44 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

---

Solubility in Formulation 5: 1% CMC Na : 14mg/mL

---

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