MDM2 (IC50 = 3.8 nM; Ki = 1 nM); Alrizomadlin targets the mouse double minute 2 (MDM2) protein, also known as HDM2 in humans. MDM2 is a negative regulator of the p53 tumor suppressor pathway and is often overexpressed in cancer cells. Alrizomadlin binds to MDM2 with high affinity, exhibiting an IC50 (half-maximal inhibitory concentration) of 3.8 ± 1.1 nM and a Ki (dissociation constant of binding affinity) of less than 1 nM. By binding to MDM2, alrizomadlin blocks the interaction between MDM2 and p53, thereby preventing MDM2-mediated ubiquitination and proteasomal degradation of p53.

Alrizomadlin demonstrates significant dose-dependent antiproliferative effects in TP53 wild-type (TP53wt) cancer cell lines. In TP53wt acute myeloid leukemia (AML) cell lines, alrizomadlin exhibits IC50 values of 26.8 nM for MOLM-13 cells, 165.9 nM for MV-4-11 cells, and 315.6 nM for OCI-AML-3 cells. In uveal melanoma cell lines, alrizomadlin exerts moderate growth inhibitory effects with IC50 values ranging from 0.151 to 1.857 µM. Mechanistically, alrizomadlin blocks the MDM2-p53 interaction, leading to increased p53 protein levels, upregulation of downstream p21 expression, and induction of G0/G1 cell cycle arrest and apoptosis in a p53-dependent manner. At 1 µM concentration, alrizomadlin induces apoptosis in up to 96.8% of MOLM-13 cells. Alrizomadlin also primes cancer cells to BCL-2 inhibition-induced apoptosis by downregulating anti-apoptotic proteins MCL-1 and BCL-xL and upregulating pro-apoptotic BAX. When combined with the BCL-2 inhibitor lisaftoclax (APG-2575), alrizomadlin exhibits synergistic antiproliferative and pro-apoptotic activities in TP53wt AML cell lines.

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With concentration-dependent effects, alrizomadlin (0.001-100 μM; 72 hours) suppresses cell proliferation in AGS and MKN45 cells, with IC50s of 18.9 ± 15.6 nM and 103.5 ± 18.3 nM, respectively [3]. To improve the effects of irradiation at various radiation doses, alrizomadlin (0.02 μM, 0.2 μM; Alrizomadlin (0.02 μM, 0.2 μM; 48) was induced by induction with wild-type p53 [3] at 48 h in G0/G1 phase in AGS and MKN45 cells. impact against ischemia[3]. In AGS and MKN45 cells, alrizomadlin (0.02 μM, 0.2 μM; 24 hours) activates p53 to improve radiation direction; stable p53 knockdown removes MDM2, p53, p21, PUMA, BAX, and Cleaved-caspase3 cells to influence progression. In p53 wide cell lines (TPC-1, KTC-1), alrizomadlin (0.3 μM, 1 μM, 3 μM, 10 μM; 24 hours) induces the production of the G2/M phase focused cell cycle marker γH2AX [3].

In uveal melanoma xenograft models (MP41), oral administration of alrizomadlin at doses of 25, 50, and 100 mg/kg induces dose-dependent antitumor activity, with tumor growth inhibition (TGI) rates of 24.8%, 38%, and 39.1%, respectively. Combination therapy of alrizomadlin with the MEK inhibitor selumetinib or the FAK inhibitor APG-2449 results in synergistic antitumor effects, with TGI rates of 65.4% and 47.5%, respectively. Mechanistically, alrizomadlin increases p53, MDM2, and p21 expression in tumor tissues while inhibiting ERK phosphorylation. In AML cell line-derived and patient-derived xenograft models, the combination of alrizomadlin with lisaftoclax (APG-2575) produces deep antitumor responses and prolonged survival, and this combination also resensitizes venetoclax-resistant tumors to apoptosis. In a Phase I clinical trial of patients with advanced solid tumors, alrizomadlin demonstrated promising antitumor activity, particularly in patients with MDM2 amplification and wild-type TP53, with an overall response rate of 25% (2/8) and a disease control rate of 100% (8/8).

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In vivo radioresistance of adenocarcinoma tumors is improved by alrizomadlin (oral formulation; 100 mg/kg; once daily; 10 days) [3].

The MDM2 binding affinity of alrizomadlin was determined using biochemical binding assays. The IC50 (half-maximal inhibitory concentration) value of alrizomadlin for MDM2 was measured as 3.8 ± 1.1 nM, and the Ki (dissociation constant of binding affinity between inhibitor and enzyme) was determined to be less than 1 nM. These values indicate high-affinity binding of alrizomadlin to the MDM2 protein, effectively blocking the MDM2-p53 interaction. The specific assay format (e.g., fluorescence polarization, time-resolved fluorescence resonance energy transfer, or surface plasmon resonance) is not detailed in the available literature.

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Fluorescence Prolarization (FP)-Based Protein Binding Assay [5]
The binding affinity of MDM2 inhibitors was determined by an optimized, sensitive, and quantitative FP-based binding assay, using a recombinant human His-tagged MDM2 protein (residues 1–118) and a FAM tagged p53-based peptide as the fluorescent probe. The design of the fluorescent probe was based upon a previously reported high affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2).31 This tagged peptide was named as PMDM6-F. The equilibrium dissociation constant (Kd) of PMDM6-F to the MDM2 protein was determined to be 1.4 ± 0.3 nM by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the MDM2 protein with increasing concentrations up to full saturation. Fluorescence polarization values were measured using the Infinite M-1000 plate reader in Microfluor 1 96-well, black, round-bottom plates. In the saturation experiments, 1 nM of PMDM6-F and increasing concentrations of proteins were added to each well to a final volume of 125 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide (Invitrogen), with 0.01% Triton X-100 and 4% DMSO). Plates were mixed and incubated at room temperature for 30 min with gentle shaking to ensure equilibrium. The polarization values in millipolarization units (mP) were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The Kd value was then calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software. IC50 and Ki values of tested compounds were determined in a dose-dependent competitive binding experiment. Mixtures of 5 μL of the tested compound with different concentrations in DMSO and 120 μL of pre-incubated protein/fluorescent probe complex with fixed concentrations in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide, with 0.01% Triton X-100) were added into assay plates and incubated at room temperature for 30 min with gentle shaking. Final concentrations of the protein and fluorescent probe in the competitive assays were 10 and 1 nM, respectively, and final DMSO concentration was 4%. Negative controls containing protein/fluorescent probe complex only (equivalent to 0% inhibition), and positive controls containing free fluorescent probe only (equivalent to 100% inhibition), were included in each assay plate. FP values were measured as described above. IC50 values were determined by nonlinear regression fitting of the sigmoidal dose-dependent FP decreases as a function of total compound concentrations using Graphpad Prism 6.0 software. Ki values of competitive inhibitors were obtained directly by nonlinear regression fitting as well, based upon the Kd values of the probe to different proteins and concentrations of the proteins and probes in the competitive assays.

In vitro cell-based assays were performed using TP53 wild-type (TP53wt) and TP53 mutant/deficient cancer cell lines. Cells were seeded in appropriate culture media and treated with various concentrations of alrizomadlin (ranging from 0.001 to 100 µM) for 72 hours. Cell viability was assessed using standard cell viability assays (e.g., ATP quantification or MTT assays), and IC50 values were calculated from concentration-response curves. Cell cycle distribution was analyzed by flow cytometry following propidium iodide staining, and apoptosis was evaluated by Annexin-V staining or measurement of Caspase-3/7 activity. For combination studies, alrizomadlin was tested together with other agents such as the BCL-2 inhibitor lisaftoclax (APG-2575) or MEK inhibitors in TP53wt AML and uveal melanoma cell lines. In T cell activation studies, CD4+ T cells isolated from mouse spleens were exposed to 250 nM alrizomadlin for 3, 6, and 24 hours, and cell viability and activation markers (CD25highCD62Llow) were assessed by flow cytometry.

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Cell proliferation assay [3]
Cell Types: AGS and MKN45 cells
Tested Concentrations: 0.0001μM, 0.001μM, 0.01μM, 0.1μM, 1μM, 10μM, 100μM
Incubation Duration: 72 hrs (hours)
Experimental Results: and S phase reduction [4]. Inhibits cell proliferation in a concentration-dependent manner.

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RT-PCR[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.02 μM, 0.2 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: MDM2, p21, PUMA and BAX mRNA expression increased.

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Cell cycle analysis[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.02 μM, 0.2 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Cell arrest in G0/G1 phase.

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Apoptosis analysis [4]
Cell Types: DePTC p53 wide
Cell Types: TPC-1 cells, KTC-1 cells
Tested Concentrations: 0.3μM, 1μM, 3μM, 10μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished number of cells in S phase, while cells accumulate in the G2/M phase.

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Western Blot Analysis[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.2 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: MDM2 and p53 expression were enhanced, and stable knockdown of p53 eliminated them.

Animal/Disease Models: 4weeks old male BALB/c athymic nude mice, MKN45 cells [3]
Doses: 100 mg/kg
Route of Administration: po (po (oral gavage)) one time/day; 10-day
Experimental Results: Xenograft tumor growth diminished.

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In vivo efficacy studies were conducted using xenograft mouse models. For the uveal melanoma model (MP41), tumor-bearing mice were orally administered alrizomadlin at doses of 25, 50, and 100 mg/kg. Tumor volumes were measured regularly using digital calipers, and tumor growth inhibition (TGI) rates were calculated. For combination studies, alrizomadlin was administered together with selumetinib or APG-2449. In AML xenograft models, both cell line-derived and patient-derived xenograft models were used to evaluate the combination of alrizomadlin with lisaftoclax (APG-2575). Tumor growth, survival (Kaplan-Meier analysis), and biomarker expression in harvested tumor tissues (e.g., p53, MDM2, p21, phosphorylated ERK) were assessed. Alrizomadlin was administered orally, and the vehicle formulation details are not specified in the available literature.

In a first-in-human Phase I study of patients with advanced solid tumors, alrizomadlin demonstrated approximately linear pharmacokinetics over the dose range of 100 to 200 mg. The maximum tolerated dose (MTD) was established at 150 mg, and the recommended Phase II dose (RP2D) was 100 mg administered once daily every other day for 21 days of a 28-day cycle (i.e., 21 days on and 7 days off). Plasma samples were collected at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours post-dose for pharmacokinetic analysis, and parameters were calculated using noncompartmental analysis. Alrizomadlin administration was associated with increased plasma levels of macrophage inhibitory cytokine-1 (MIC-1), a biomarker indicative of p53 pathway activation.

In a Phase I clinical trial of patients with advanced solid tumors, alrizomadlin demonstrated an acceptable safety profile. The most common grade 3/4 treatment-related adverse events were thrombocytopenia (33.3%), lymphocytopenia (33.3%), neutropenia (23.8%), and anemia (23.8%). One patient in the 200-mg cohort experienced dose-limiting toxicities of thrombocytopenia and febrile neutropenia. In a Phase II study of alrizomadlin with or without toripalimab in patients with advanced solid tumors, common treatment-related adverse events included nausea, decreased appetite, thrombocytopenia, neutropenia, and anemia. Grade ≥3 thrombocytopenia and neutropenia were observed, and one patient discontinued treatment due to grade 4 thrombocytopenia; no treatment-related deaths were reported. No dose-limiting toxicity was observed in the combination arm at the 150 mg dose level.

[1]. 4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-fluorophenyl)-1′-ethyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development. J Med Chem. 2017 Apr 13; 60(7): 2819–2839.
[2]. A phase Ib/II study of APG-115 in combination with MK-3475 in patients with unresectable or metastatic melanomas or advanced solid tumors, Ann Oncol. 2019 Feb 1; 30(Supplement_1). pii: mdz027.
[3]. A novel small molecule inhibitor of MDM2-p53 (APG-115) enhances radiosensitivity of gastric adenocarcinoma, J Exp Clin Cancer Res. 2018 May 2;37(1):97.
[4]. Restoration of p53 using the novel MDM2-p53 antagonist APG115 suppresses dedifferentiated papillary thyroid cancer cells. Oncotarget. 2017 Jun 27;8(26):43008-43022.
[5]. Design of Chemically Stable, Potent, and Efficacious MDM2 Inhibitors That Exploit the Retro-Mannich Ring-Opening-Cyclization Reaction Mechanism in Spiro-oxindoles. J Med Chem. 2014 Dec 26; 57(24): 10486–10498.

Alrizomadlin is a novel MDM2 inhibitor that blocks the interaction between MDM2 and p53. It is currently being researched for cancer treatment. Alrizomadlin is an orally administered inhibitor of human dual microsome 2 homologs (HDM2; mouse dual microsome 2 homologs; MDM2) with potential antitumor activity. After oral administration, alrizomadlin binds to HDM2, preventing the HDM2 protein from binding to the transcriptional activation domain of the tumor suppressor protein p53. By blocking the HDM2-p53 interaction, it inhibits proteasome-mediated p53 enzymatic degradation and restores p53 transcriptional activity. This may lead to the restoration of the p53 signaling pathway and ultimately p53-mediated tumor cell apoptosis. HDM2 is a zinc finger protein and a negative regulator of the p53 pathway, frequently overexpressed in cancer cells. It is closely related to cancer cell proliferation and survival.

C34H38CL2FN3O4

642.5934

641.222

C, 63.55; H, 5.96; Cl, 11.03; F, 2.96; N, 6.54; O, 9.96

1818393-16-6

1818393-16-6

91972012

White to off-white solid

1.4±0.1 g/cm3

805.8±65.0 °C at 760 mmHg

441.1±34.3 °C

0.0±3.0 mmHg at 25°C

1.658

7.63

3

6

5

44

1160

3

ClC1C=CC2=C(C=1)NC([C@@]12[C@@H](C2C=CC=C(C=2F)Cl)[C@H](C(NC23CCC(C(=O)O)(CC2)CC3)=O)N(CC)C21CCCCC2)=O

YJCZPJQGFSSFOL-MNZPCBJKSA-N

InChI=1S/C34H38Cl2FN3O4/c1-2-40-27(28(41)39-32-16-13-31(14-17-32,15-18-32)30(43)44)25(21-7-6-8-23(36)26(21)37)34(33(40)11-4-3-5-12-33)22-10-9-20(35)19-24(22)38-29(34)42/h6-10,19,25,27H,2-5,11-18H2,1H3,(H,38,42)(H,39,41)(H,43,44)/t25-,27+,31?,32?,34+/m0/s1

4-((3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2''-oxodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indoline]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic acid

AA-115; APG115; AA 115; APG115; AA115; APG-115

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)

DMSO : ~100 mg/mL (~155.62 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (7.78 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 50.0 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: ≥ 5 mg/mL (7.78 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 50.0 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 (3.89 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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 (Please use freshly prepared in vivo formulations for optimal results.)