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

In mice, rats, non-human primates, and dogs, AT-406 has good oral bioavailability and pharmacokinetic (PK) characteristics. AT-406 effectively induces cIAP1 degradation, procaspase-8 processing, and PARP cleavage in tumor tissues at 100 mg/kg with good tolerance even at 200 mg/kg in the MDA-MB-231 xenograft. At 100 mg/kg, AT-406 significantly inhibits tumor growth, with a p value of 0.0012. [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.

At a density of (3-4) × 103 cells/well, cells are seeded in 96-well flat-bottom cell culture plates with AT-406 and incubated for 4 days. Three to four 103 cells/well of AT-406-seeded cells are placed in 96-well flat-bottom cell culture plates, and the cells are then incubated for four days. The rate of cell growth inhibition after treatment with different concentrations of AT-406 is determined by assaying with (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8). WST-8 is added to each well to a final concentration of 10%, and then the plates are incubated at 37 °C for 2−3 hours. Using a TECAN ULTRA reader, the samples' absorbance is calculated at 450 nm. By comparing absorbance in cells that were not treated and cells that were treated with AT-406, the concentration of AT-406 that inhibited cell growth by 50% (IC50) can be determined. A TECAN ULTRA reader is used to measure the samples' absorbance at 450 nm. By comparing the absorbance of treated and untreated cells, the concentration of AT-406 that 50% inhibited cell growth (IC50) can be determined.

MDA-MB-231 xenograft tumors in severe combined immune deficiency (SCID) mice
10 mg/kg (i.v.), 10 mg/kg (p.o.), 30 mg/kg (p.o.) and 100 mg/kg (p.o.)
Administered via intravenously (i.v.) or oral gavage (p.o.)

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In Vivo pharmacodynamic (PD) studies[1]
For in vivo PD studies, the MDA-MB-231 xenograft tumor model was employed. To develop xenograft tumors, 5 × 106 MDA-MB-231 cancer cells with matrigel were injected subcutaneously on the dorsal side of the severe combined immunodeficient mice (SCID mice from Charles River), one tumor per mouse. Mice bearing MDA-MB-231 xenograft tumors were administered with a single dose of AT406 (Xevinapant, SM406, ARRY334543) in its HCl salt form at 100 mg/kg via oral gavage, Taxotere at 7.5 mg/kg intravenously or vehicle control. Tumor tissues were harvested at indicated time points. Tumor tissues were analyzed using Western blotting to examine levels of cIAP1 and XIAP, caspase-8 processing and PARP cleavage in tumor tissues.

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In Vivo Pharmacokinetic studies in plasma and MDA-MB-231 tumor tissues in SCID mice[1]
To develop xenograft tumors, 5 × 106 MDA-MB-231 cancer cells with matrigel were injected subcutaneously on the dorsal side of the severe combined immunodeficient mice (SCID mice from Charles River), two tumors (left and right sides) per mouse. Mice bearing MDA-MB-231 xenograft tumors were administered with a single dose of compound 2 [AT406 (Xevinapant, SM406, ARRY334543)] in its HCl salt form at 100 mg/kg via oral gavage. Blood and tumor samples were collected from each mouse by terminal cardiac puncture at 0.25, 0.5, 1, 2, 4, 6, 8, 24 h post-dose. Samples were taken from three mice at each time point. Blood samples were collected into potassium heparin treated tubes and centrifuged at 2000g and 4°C for 10 min. Plasma was collected and stored at −80°C prior to analysis. Isolated tumor tissues were immediately frozen and ground with a mortar and pestle in liquid nitrogen, then stored at −80°C until analysis.

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In Vivo antitumor efficacy study[1]
SCID mice (8–10 per group) bearing MDA-MB-231 xenograft tumors were treated with different doses of AT406 (Xevinapant, SM406, ARRY334543), or 7.5 mg/kg of Taxotere or vehicle control daily, 5 days a week for 2 weeks. Tumor sizes and animal weights were measured 3 times a week during the treatment and twice a week after the treatment. Data are presented as mean tumor volumes ± SEM. Statistical analyses were performed by two-way ANOVA and unpaired two-tailed t test, using Prism (version 4.0, GraphPad, La Jolla, CA). P < 0.05 was considered statistically significant. The efficacy experiment was performed under the guidelines of the University of Michigan Committee for Use and Care of Animals.

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Pharmacokinetics of AT406 (Xevinapant, SM406, ARRY334543) in rats, dogs and non-human primates[1]
Pharmacokinetic (PK) studies in male Sprague Dawley rats, beagle dogs and cynomolgus monkeys (non-human primates) were performed a CRO company.
AT406 (Xevinapant, SM406, ARRY334543) in its hydrochloride salt form was used in pharmacokinetic (PK) evaluations and was dissolved in saline to yield final concentration at 25 mg/mL (pH≈7). The solution was administered to animals on preparation. The concentration of AT406 (Xevinapant, SM406, ARRY334543) in dosing solution was confirmed by HPLC.
For PK studies in rats, dogs and monkeys, animals were randomly assigned to the treatment groups and were carotid cannulated before the PK studies. The LC system comprised an Agilent liquid chromatograph equipped with an isocratic pump (1100 series), an autosampler (1100 series) and a degasser (1100 series). Mass spectrometric analysis was performed using an API3000 (triple-quadruple) instrument from AB Inc with an ESI interface. The data acquisition and control system were created using Analyst 1.4 software from ABI Inc. The concentrations in plasma below the limit of quantitation (LOQ = 5 ng/mL) were designated as zero. The pharmacokinetic data analysis was performed using noncompartmental analysis. Oral bioavailability was calculated as F(%)=(Dose(oral)×AUC(0-∞)(oral))/(Dose (iv)×AUC(0-∞) (iv))*100%.

[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.

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.
See also: Xevinapant Hydrochloride (is active moiety of).

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Drug Indication
Treatment of head and neck epithelial malignant neoplasms

C32H43N5O4

561.71

561.331

C, 68.42; H, 7.72; N, 12.47; O, 11.39

1071992-99-8

Xevinapant hydrochloride;1071992-57-8

25022340

White to off-white solid powder

1.2±0.1 g/cm3

840.5±65.0 °C at 760 mmHg

462.1±34.3 °C

0.0±3.1 mmHg at 25°C

1.603

2.09

3

5

9

41

896

4

O=C1[C@]([H])(C([H])([H])N(C(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])([H])[C@@]2([H])C([H])([H])C([H])([H])[C@@]([H])(C(N([H])C([H])(C3C([H])=C([H])C([H])=C([H])C=3[H])C3C([H])=C([H])C([H])=C([H])C=3[H])=O)N21)N([H])C([C@]([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O

LSXUTRRVVSPWDZ-MKKUMYSQSA-N

InChI=1S/C32H43N5O4/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)/t22-,25+,26-,27-/m0/s1

(5S,8S,10aR)-N-benzhydryl-5-[[(2S)-2-(methylamino)propanoyl]amino]-3-(3-methylbutanoyl)-6-oxo-1,2,4,5,8,9,10,10a-octahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide

DeBio-1143; DeBio1143; DeBio 1143; Xevinapant; AT 406; AT-406; AT406; SM406; SM 406; N65WC8PXDD; SM-406; UNII-N65WC8PXDD; Xevinapant; ARRY-334543; ARRY 334543; ARRY334543

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 (4.45 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 (4.45 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 (4.45 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: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL

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