Akt1 (IC50 = 3 nM); Akt2 (IC50 = 7 nM); Akt3 (IC50 = 7 nM); ROCK2 (IC50 = 60 nM); ROCK1 (IC50 = 470 nM); PKA (IC50 = 7 nM); P70S6K (IC50 = 6 nM); Autophagy

AZD5363 is a potent Akt inhibitor with IC50 of 3 nM, 8 nM and 8 nM for Akt1, Akt2 and Akt3, respectively.[1] With a potency of roughly 0.3 to 0.8 μM, AZD5363 prevents the phosphorylation of AKT substrates in cells. With a potency of less than < 3 μM, AZD5363 prevents the growth of 41 of 182 solid and hematologic tumor cell lines.[2] Significantly predicting factors for responsiveness to AZD5363 include activating mutations in PIK3CA, loss or inactivation of the tumor suppressor PTEN, and HER2 amplification. Furthermore, a connection is seen between a cell line's RAS mutation status and its resistance to AZD5363.[1]

Oral administration of AZD5363 (100, 300 mg/kg) to naked mice results in reversible increases in blood glucose levels, dose-dependent decreases in 2[18F]fluoro-2-deoxy-d-glucose (18F-FDG) uptake in U87-MG xenografts, and dose-dependent reductions in PRAS40, GSK3, and S6 phosphorylation in BT474c xenografts. Chronic oral administration of AZD5363 (130, 200, and 300 mg/kg) results in dose-dependent growth inhibition of xenografts derived from various tumor types, including trastuzumab-resistant HER2+ breast cancer models. Additionally, in breast cancer xenografts, AZD5363 significantly increases the antitumor activity of docetaxel, lapatinib, and trastuzumab.[2]

The ability of AZD5363 and other compounds to inhibit the activity of AKT1, AKT2, and AKT3 is evaluated by the Caliper Off-Chip Incubation Mobility Shift assay. Active recombinant AKT1, AKT2, or AKT3 are incubated with a 5-FAM-labeled custom-synthesized peptide substrate together with increasing concentrations of inhibitor. The final reactions contained 1 to 3 nM AKT1, AKT2, or AKT3 enzymes, 1.5 mM peptide substrate, ATP at Km for each AKT isoform, 10 mM MgCl2, 4 mM DTT, 100 mM HEPES, and 0.015% Brij-35. The reactions are allowed to proceed for an hour at room temperature before being stopped with the addition of a buffer containing 40 mM EDTA, 5% DMSO, 0.1% coating reagent, 0.1% Brij-35 solution, and 100 mM HEPES. After that, plates are examined on a Caliper LC3000, allowing for the electrophoretic separation of the peptide substrate and phosphorylated product and the subsequent detection and quantification of laser-induced fluorescence.

MTS and Sytox Green are the two methods used to measure cell proliferation. Briefly, cells are plated in 96-well dishes and incubated at 37 °C with 5% CO2 for an entire night. Following that, cells are subjected to AZD5363 concentrations ranging from 30 to 0.003 μM for 72 hours. Cell proliferation is assessed for the MTS endpoint using the CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent in accordance with the manufacturer's instructions. The Sytox Green endpoint, Sytox Green nucleic acid dye to cells at a final concentration of 0.13 μM and counting the number of dead cells using an Acumen Explorer. Following saponin permeabilization (0.03% final concentration, diluted in TBS-EDTA buffer), cells are incubated for an overnight period to determine the total cell count. Predose measurements are taken for Sytox Green and MTS endpoints, and the concentration needed to cut treated cell growth in half compared to untreated cell growth is calculated using live cell counts or MTS absorbance readings.

Mice: Specific, pathogen-free, female nude mice (nu/nu: Alpk) and male SCID mice (SCID/CB17; 786-0 xenograft studies) are used. The mice are randomly assigned to control and treatment groups once the mean tumor sizes reach about 0.2 cm3. The treatment groups received RP-56976, which was dissolved in 2.6% ethanol in injectable water, once on day 1, at 15 or 5 mg/kg once a week, and Capivasertib (AZD5363), which was dissolved in a 10% DMSO 25% w/v Kleptose HPB (Roquette) buffer by oral gavage. When used in conjunction with Capivasertib (AZD5363), RP-56976 is given an hour before the oral dose. The control group received the DMSO/Kleptose buffer alone, twice daily by oral gavage. For the duration of the study, tumor volumes (as determined by caliper), animal weight, and tumor condition are noted twice a week. By using CO2 euthanasia, mice are sacrificed. Using the formula: (length×width)×√(length×width)×(π/6), the tumor volume is calculated by considering length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter. By comparing the variations in tumor volume between the control and treated groups, growth inhibition from the onset of treatment is evaluated.

Absorption, Distribution and Excretion
The capivasertib steady-state AUC is 8,069 h·ng/mL (37%) and Cmax is 1,371 ng/mL (30%). Steady-state concentrations are predicted to be attained on the 3rd and 4th dosing day of each week, starting week 2. Capivasertib plasma concentrations are approximately 0.5% to 15% of the steady-state Cmax during the off-dosing days. Capivasertib AUC and Cmax are proportional with doses over a range of 80 to 800 mg (0.2 to 2 times the approved recommended dosage). Tmax is approximately 1-2 hours. The absolute bioavailability is 29%. No clinically meaningful differences in capivasertib pharmacokinetics were observed following the administration of capivasertib with a high-fat meal (approximately 1,000 kcal; fat 60%) or a low-fat meal (approximately 400 kcal; fat 26%).
Following a single radiolabeled oral dose of 400 mg, the mean total recovery was 45% from urine and 50% from feces.
The steady-state oral volume of distribution is 1,847 L (36%).
The steady-state oral clearance of capivasertib is 50 L/h (37% CV), and renal clearance was 21% of total clearance.

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Metabolism / Metabolites
Capivasertib is primarily metabolized by CYP3A4 and UGT2B7.

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Biological Half-Life
The half-life of capivasertib is 8.3 hours.

Protein Binding
Capivasertib plasma protein binding is 22% and the plasma-to-blood ratio is 0.71.

[1]. J Med Chem. 2013 Mar 14;56(5):2059-73.

[2]. Mol Cancer Ther. 2012 Apr;11(4):873-87.

Capivasertib is an aminopiperidine that is piperidine substituted by 7H-pyrrolo[2,3-d]pyrimidin-4-yl, amino, and [(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]aminocarbonyl groups at positions 1, 4, and 4, respectively. It is a pan-AKT kinase inhibitor used in combination with fulvestrant for the treatment of adult patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced or metastatic breast cancer with one or more PIK3CA/AKT1/PTEN-alterations. It has a role as an antineoplastic agent and an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It is a pyrrolopyrimidine, an aminopiperidine, a piperidinecarboxamide, a member of monochlorobenzenes, a primary alcohol and a secondary carboxamide.
Hormone receptor (HR) positive, especially estrogen receptor-positive, HER2-negative breast cancer is the most common subtype of metastatic breast cancer, resulting in more than 400,000 deaths annually. Although endocrine-based therapy is the first line of treatment, resistance eventually emerges, leaving chemotherapy the only but often ineffective treatment left. Therefore, significant research has been put into developing genetically targeted treatments. The PIK3/AKT pathway is one of the most commonly activated pathways in breast cancer, mainly through the constitutively active mutation in AKT1, loss of function mutation in PTEN, a negative regulator of the PIK3/AKT pathway, or PIK3CA mutations. Therefore, targeting the PIK3/AKT pathway presents a promising approach for the treatment of breast cancer, leading to the development of capivasertib, a pan-AKT kinase inhibitor. On November 17th, 2023, capivasertib, under the brand name TRUQAP, was approved by the FDA for the treatment of adult patients HR-positive, HER2-negative locally advanced or metastatic breast cancer with one or more alterations in PIK3CA/AKT1/PTEN gene(s) in combination with [fulvestrant]. This approval is based on favorable results obtained from the CAPItello-291 trial, where the combination of capivasertib and [fulvestrant] reduced the risk of disease progression or death by 50% versus [fulvestrant] alone.
Capivasertib is a novel pyrrolopyrimidine derivative, and an orally available inhibitor of the serine/threonine protein kinase AKT (protein kinase B) with potential antineoplastic activity. Capivasertib binds to and inhibits all AKT isoforms. Inhibition of AKT prevents the phosphorylation of AKT substrates that mediate cellular processes, such as cell division, apoptosis, and glucose and fatty acid metabolism. A wide range of solid and hematological malignancies show dysregulated PI3K/AKT/mTOR signaling due to mutations in multiple signaling components. By targeting AKT, the key node in the PIK3/AKT signaling network, this agent may be used as monotherapy or combination therapy for a variety of human cancers.

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Drug Indication
Capivasertib, in combination with fulvestrant, is indicated for the treatment of adult patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced or metastatic breast cancer with one or more PIK3CA/AKT1/PTEN-alteration as detected by an FDA-approved test following progression on at least one endocrine-based regimen in the metastatic setting or recurrence on or within 12 months of completing adjuvant therapy.
Treatment of breast cancer , Treatment of prostate cancer

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Mechanism of Action
Capivasertib is an inhibitor of all 3 isoforms of serine/threonine kinase AKT (AKT1, AKT2, and AKT3) and inhibits phosphorylation of downstream AKT substrates. AKT activation in tumors is a result of activation of upstream signaling pathways, mutations in AKT1, loss of phosphatase and tensin homolog (PTEN) function, and mutations in the catalytic subunit alpha of phosphatidylinositol 3-kinase (PIK3CA).

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Pharmacodynamics
In vitro, capivasertib reduced the growth of breast cancer cell lines including those with relevant PIK3CA or AKT1 mutations or PTEN alteration. In vivo, capivasertib alone and in combination with fulvestrant inhibited tumor growth of mouse xenograft models including estrogen receptor-positive breast cancer models with alterations in PIK3CA, AKT1, and PTEN. The exposure-response relationship and time course of pharmacodynamic response for the effectiveness of capivasertib has not been fully characterized. Exposure-response relationships were observed for diarrhea (CTCAE Grade 2 to 4), rash (CTCAE Grade 2 to 4), and hyperglycemia (CTCAE Grades 3 or 4) at doses of 80 to 800 mg (0.2 to 2 times the approved recommended dosage). At the recommended capivasertib dose, a mean increase in the QTc interval > 20 ms was not observed.

C21H25CLN6O2

428.9152

428.172

C, 58.81; H, 5.87; Cl, 8.27; N, 19.59; O, 7.46

1143532-39-1

(R)-Capivasertib;1143532-51-7

25227436

white solid powder

1.4±0.1 g/cm3

1.670

1.04

4

6

6

30

580

1

ClC1C([H])=C([H])C(=C([H])C=1[H])[C@]([H])(C([H])([H])C([H])([H])O[H])N([H])C(C1(C([H])([H])C([H])([H])N(C2C3C([H])=C([H])N([H])C=3N=C([H])N=2)C([H])([H])C1([H])[H])N([H])[H])=O

JDUBGYFRJFOXQC-KRWDZBQOSA-N

InChI=1S/C21H25ClN6O2/c22-15-3-1-14(2-4-15)17(6-12-29)27-20(30)21(23)7-10-28(11-8-21)19-16-5-9-24-18(16)25-13-26-19/h1-5,9,13,17,29H,6-8,10-12,23H2,(H,27,30)(H,24,25,26)/t17-/m0/s1

4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

Capivasertib; AZD-5363; AZD5363; AZD 5363; Truqap

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: 86~125 mg/mL (200.5~291.4 mM)
Ethanol: ~2 mg/mL (~4.0 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.85 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 20.8 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: ≥ 2.08 mg/mL (4.85 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 20.8 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: ≥ 2.08 mg/mL (4.85 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 20.8 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.)