cIAP1 (Ki = 1.9 nM); cIAP2 (Ki = 5.1 nM); XIAP (Ki = 66.4 nM)

Cell growth of the breast cancer MDA-MB-231 and the ovarian cancer SK-OV-3 cell lines is effectively inhibited by xevinanapant (AT-406) hydrochloride, with IC50 values of 144 nM and 142 nM, respectively. Potently inducing cell death in a time- and dose-dependent manner is xevinanapant hydrochloride (0-3 μM; 0-48 hours) [1].

Xevinanapant (AT-406) hydrochloride is highly efficient in suppressing tumor growth in the MDA-MB-231 xenograft model with minimum toxicity in animals [1]. The pharmacokinetic (PK) parameters of Xevinanapant hydrochloride were investigated in mice, rats, non-human primates, and canines [1].

FL-AT-406 (the fluorescently tagged AT-406) is employed to develop a set of new FP assays for determination of the binding affinities of Smac mimetics to XIAP, cIAP-1, and cIAP-2 BIR3 proteins. Titration experiments using a fixed concentration of FL-AT-406 and varying concentrations of the protein up to full saturation are used to calculate the Kd value of FL-AT-406 to each IAP protein. A Microfluor 2 96-well, black, round-bottom plate is used to measure the fluorescence polarization values using an Infinite M-1000 plate reader. For experiments with XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3, FL-AT-406 (2, 1, and 1 nM for each well, respectively) and various protein concentrations are added to a final volume of 125 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 g/mL bovine -globulin, 0.02% sodium azide, with 4% DMSO). After being thoroughly combined, the plates are gently shaken for two to three hours at room temperature. At an excitation wavelength of 485 nm and an emission wavelength of 530 nm, the polarization values in millipolarization units (mP) are measured. Then, using Graphpad Prism 5.0 software, equilibrium dissociation constants (Kd) are calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations. In competitive binding tests for XIAP3 BIR3, AT-406 is incubated with 20 nM XIAP BIR3 protein and 2 nM FL-AT-406 in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine γ-globulin; 0.02% sodium azide). 3 nM protein and 1 nM FL-AT-406 are used in experiments to determine competitive binding for the cIAP1 BIR3 protein. 5 nM protein and 1 nM FL-AT-406 are used in competitive binding tests for cIAP2 BIR3. Using an Infinite M-1000 plate reader, polarization values are determined for each competitive binding experiment after two to three hours of incubation. Using nonlinear least-squares analysis, the IC50 value, or inhibitor concentration at which 50% of the bound tracer is displaced, is extracted from the plot. The PRISM program is used to fit curves.[1]

In this study, researchers evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. They demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies.[2]

Animal/Disease Models: SCID (severe combined immunodeficient) mouse bearing MDA-MB-231 xenograft tumors [1]
Doses: 30 and 100 mg/kg
Route of Administration: Oral; 5 days per week for 2 weeks
Experimental Results: 30 and 100 mg/kg Strong Inhibits tumor growth, completely inhibiting tumor growth during treatment at 100 mg/kg.

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In vivo efficacy studies of AT-406[2]
AT-406 was dissolved in DMSO as a stock solution at 200mg/ml. The stock solution was diluted in the vehicle solution, which consists of 10mg/ml hypromellose and 1μl of Tween 80 in PBS, to achieve the final concentration of 10 mg/ml. 5 × 106 OVCAR-3ip cells were injected intraperitoneally (i.p.) into each immunocompromised B6.129S7-Rag1tmMom mouse. Seven days after tumor implantation, the tumor bearing mice were randomly divided into the following treatment groups: control (n = 9), carboplatin (n = 7), AT-406 (n = 5), and combination of AT-406 and carboplatin (n = 7). These groups of mice were treated as follows: each control mouse received 0.2ml of vehicle solution alone by oral gavage every day for 10 d, followed by a 3 d break, and 6 subsequent oral gavage treatments for a total of 16 treatments. AT-406 treatment mice received AT-406 (100 mg/kg) by oral gavage as described for the control treatment mice and the carboplatin treatment mice received carboplatin (40 mg/kg) through intraperitoneal injection twice weekly for two cycles. The combination group received AT-406 and carboplatin treatments simultaneously adhering to the protocols described above for the individual treatment groups. Following an approved IACUC protocol, mouse survival analysis was performed and mice were sacrificed when they appeared moribund or displayed signs of distress, at which time, the mice were considered as dead. At the conclusion of the experiment, mouse tumors and vital organs were removed, fixed, and sectioned for further analyses.[2]

[1]. A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment. J Med Chem. 2011;54(8):2714-2726.
[2]. AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer. Cancer Biol Ther. 2012;13(9):804-811.

Xevinapant is an orally available mimetic of the natural second mitochondrial-derived activator of caspases (Smac) and inhibitor of Inhibitor of Apoptosis Proteins (IAPs), with potential immunomodulating, apoptotic-inducing, chemo-radio-sensitizing and antineoplastic activities. Upon oral administration,xevinapant targets and binds to the Smac binding groove on IAPs, including the direct caspase inhibitor X chromosome-linked IAP (XIAP), and the cellular IAPs 1 (c-IAP1) and 2 (c-IAP2). This inhibits the activities of these IAPs and promotes the induction of apoptosis. Additionally, as xevinapant inhibits the activity of IAPs, it may work synergistically with cytotoxic drugs and/or radiation to overcome tumor cell resistance to apoptosis. As IAPs regulate nuclear factor-kappa B (NFkB) signaling pathways, which drives the expression of genes involved in immune and inflammatory responses, xevinapant may enhance anti-tumor immune responses when administered with certain immunomodulating agents, such as immune checkpoint inhibitors. IAPs are overexpressed by many cancer cell types and suppress both intrinsic and extrinsic apoptosis by binding to and inhibiting active caspases via their baculoviral lAP repeat (BIR) domains. They contribute to chemo-radio-resistance of cancer cells to certain cytotoxic agents and radiation, promote tumor cell survival and are associated with poor prognosis in certain types of cancer. SMAC, a pro-apoptotic mitochondrial protein, is an endogenous inhibitor of the IAPs family of cellular proteins.

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The inhibitors of apoptosis proteins (IAPs) are a class of key apoptosis regulators overexpressed or dysregulated in cancer. SM-406/AT-406 is a potent and selective small molecule mimetic of Smac that antagonizes the inhibitor of apoptosis proteins (IAPs). A physiologically based pharmacokinetic and pharmacodynamic (PBPK-PD) model was developed to predict the tissue concentration-time profiles of SM-406, the related onco-protein levels in tumor, and the tumor growth inhibition in a mouse model bearing human breast cancer xenograft. In the whole body physiologically based pharmacokinetic (PBPK) model for pharmacokinetics characterization, a well stirred (perfusion rate-limited) model was used to describe SM-406 pharmacokinetics in the lung, heart, kidney, intestine, liver and spleen, and a diffusion rate-limited (permeability limited) model was used for tumor. Pharmacodynamic (PD) models were developed to correlate the SM-406 concentration in tumor to the cIAP1 degradation, pro-caspase 8 decrease, CL-PARP accumulation and tumor growth inhibition. The PBPK-PD model well described the experimental pharmacokinetic data, the pharmacodynamic biomarker responses and tumor growth. This model may be helpful to predict tumor and plasma SM-406 concentrations in the clinic. Biopharm Drug Dispos . 2013 Sep;34(6):348-59. https://pubmed.ncbi.nlm.nih.gov/23813446/

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Ovarian carcinoma is the most deadly gynecological malignancy. Current chemotherapeutic drugs are only transiently effective and patients with advance disease often develop resistance despite significant initial responses. Mounting evidence suggests that anti-apoptotic proteins, including those of the inhibitor of apoptosis protein (IAP) family, play important roles in the chemoresistance. There has been a recent emergence of compounds that block the IAP functions. Here, we evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. We demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death and increases survival of the experimental mice, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies. AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer. Cancer Biol Ther . 2012 Jul;13(9):804-11.

C32H44CLN5O4

597.31Elemental Analysis

597.308

C, 64.25; H, 7.41; Cl, 5.93; N, 11.71; O, 10.70

1071992-57-8

Xevinapant;1071992-99-8

25022339

Solid powder

4.473

C[C@@H](C(=O)N[C@H]1CN(CC[C@H]2CC[C@H](N2C1=O)C(=O)NC(C3=CC=CC=C3)C4=CC=CC=C4)C(=O)CC(C)C)NC

DBXTZCYPHKJCHF-ZZPLZQMBSA-N

InChI=1S/C32H43N5O4.ClH/c1-21(2)19-28(38)36-18-17-25-15-16-27(37(25)32(41)26(20-36)34-30(39)22(3)33-4)31(40)35-29(23-11-7-5-8-12-23)24-13-9-6-10-14-24/h5-14,21-22,25-27,29,33H,15-20H2,1-4H3,(H,34,39)(H,35,40)1H/t22-,25+,26-,27-/m0./s1

(5S,8S,10aR)-N-benzhydryl-5-((S)-2-(methylamino)propanamido)-3-(3-methylbutanoyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide hydrochloride

AT-406; AT 406; AT406; D 1143; Debio 1143; 73T1W2MF9C; UNII-73T1W2MF9C; Xevinapant Hydrochloride; AT-406 HCl; Xevinapant (hydrochloride); (5S,8S,10aR)-N-benzhydryl-5-((S)-2-(methylamino)propanamido)-3-(3-methylbutanoyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide hydrochloride; 1071992-57-8 (HCl);N65WC8PXDD; SM 406.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~175 mg/mL (~292.55 mM)

Solubility in Formulation 1: ≥ 8.75 mg/mL (14.63 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 87.5 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: ≥ 8.75 mg/mL (14.63 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 87.5 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: ≥ 8.75 mg/mL (14.63 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 87.5 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.)