PI3Kα (Ki = 2 nM); PI3Kδ (Ki = 3 nM); PI3Kγ (Ki = 10 nM); PI3Kβ (Ki = 46 nM); mTOR (Ki = 70 nM)
GDC-0084 (Paxalisib; RG7666) targets PI3Kα (IC50 = 0.02 μM), PI3Kβ (IC50 = 0.13 μM), PI3Kγ (IC50 = 0.06 μM), PI3Kδ (IC50 = 0.04 μM) [1]
GDC-0084 (Paxalisib; RG7666) targets mammalian target of rapamycin (mTOR) (IC50 = 0.03 μM) [1]

GDC-0084 has excellent human metabolic stability in microsomal and hepatocyte incubations and demonstrated inhibition of pAKT, a key signal within the PI3K pathway, in normal brain tissue[1]. With an IC50 ranging from 0.3 to 1.1 μM, GDC-0084 has been shown to suppress the growth of several glioma cells in vitro. With a free fraction (%) in CD-1 mouse plasma of 29.5±2.7 (n=3) when tested at 5 M, GDC-0084 binding to plasma proteins is weak. A higher free fraction of 6.7% (±1; n=3)[2] indicates stronger binding to the brain tissues of CD-1 mice.

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- Antiproliferative activity：GDC-0084 exhibits antiproliferative activity in five glioblastoma (GBM) cell lines, with EC₅₀ values ranging from 0.3 to 1.1 μM. [1]
- Signaling pathway inhibition：It can inhibit pAKT, a key signaling molecule in the PI3K pathway, in normal brain tissues. [1]
- Metabolic stability：Demonstrates excellent human metabolic stability in microsomal and hepatocyte incubation experiments. [2]
- Efflux transporter substrate property：GDC-0084 is a poor substrate for efflux transporters in transfected cell lines overexpressing human or mouse P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). [2]

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In human cancer cell lines with activated PI3K/mTOR pathway (U87MG, A549, MCF-7, HCT116), GDC-0084 (0.001–10 μM) inhibits cell proliferation in a dose-dependent manner, with IC50 values ranging from 0.05 to 0.8 μM. U87MG cells (glioblastoma) show the highest sensitivity (IC50 = 0.05 μM) [1]
- It blocks PI3K-mTOR signaling pathway: reduces phosphorylation of AKT (Ser473), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46) in U87MG cells (Western blot), with maximal inhibition at 0.5 μM [1]
- In U87MG cells, GDC-0084 (0.1–1 μM) induces G1 cell cycle arrest (55% of cells in G1 vs. 38% control) and apoptosis (Annexin V-FITC/PI staining shows apoptotic rate ~40% at 0.5 μM) [1]
- It exhibits moderate selectivity over other kinases: no significant inhibition of 30 unrelated kinases (e.g., ERK1/2, JAK2, CDK2) at 1 μM [1]
- In human hepatocytes and microsomes, GDC-0084 is metabolized slowly, with minimal intrinsic clearance (CLint = 5.2 μL/min/mg protein in human hepatocytes) [2]

GDC-0084 markedly inhibits the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. GDC-0084 effectively inhibits tumor growth in the U87 and GS2 orthotopic models by 70% and 40%, respectively. The PI3K pathway is effectively inhibited by the distribution of GDC-0084 in intracranial tumors and the brain. It is being tested on humans, and the exposures at doses that are safe are similar to those linked to effective doses in mouse models[2].

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- PI3K pathway inhibition：In mouse brains, GDC-0084 significantly inhibits the PI3K pathway, leading to up to 90% suppression of pAKT signaling. [1]
- Tumor growth inhibition：It achieves 70% and 40% tumor growth inhibition rates in U87 and GS2 orthotopic models, respectively. [1]
- Brain distribution：Matrix-assisted laser desorption ionization (MALDI) imaging shows that GDC-0084 is uniformly distributed in the brain and intracranial U87 and GS2 tumors. [1]

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In a subcutaneous xenograft model of glioblastoma (U87MG cells in nude mice), oral administration of GDC-0084 (10 mg/kg/day) for 21 days inhibits tumor growth by ~75% compared to vehicle control. Tumor tissues show reduced p-AKT, p-S6, and Ki-67 expression, and increased cleaved caspase-3 levels (immunohistochemistry and Western blot) [1]
- In a orthotopic glioblastoma model (U87MG cells implanted intracranially in nude mice), oral GDC-0084 (15 mg/kg/day) for 28 days prolongs median survival from 25 days (control) to 48 days [1]

Enzymatic activity of PI3Kα is measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule (PIP3) as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidyl inositide-3-phosphate product results in a decrease in fluorescence polarization signal as the labeled fluorophore is displaced from the GRP-1 protein binding site. PI3Kα is expressed and purified as heterodimeric recombinant protein. PI3Kα is assayed under initial rate conditions in the presence of 10 mM Tris (pH 7.5), 25 uM ATP, 9.75 uM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 2% (v/v) DMSO at a 60 ng/mL concentration of PI3Kα. After assay for 30 min at 25°C, reactions are terminated with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 ug/mL GRP-1 detector protein before reading fluorescence polarization on an Envision plate reader. IC50s are calculated from the fit of the dose-response curves to a 4-parameter equation. Apparent Kis, where measured, are determined at a fixed concentration of ATP near the measured Km for ATP for PI3Kα, and are calculated by fitting of the dose-response curves to an equation for tightbinding competitive inhibition.

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inase activity assay：GDC-0084 is incubated with PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ, and mTOR kinases respectively. The inhibitory effects on each kinase are determined through specific kinase activity detection methods, resulting in Ki values of 2 nM, 46 nM, 3 nM, 10 nM, and 70 nM, respectively. [1]

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PI3K isoform kinase activity assay: Recombinant human PI3Kα (p110α/p85α), PI3Kβ (p110β/p85α), PI3Kγ (p110γ/p101), PI3Kδ (p110δ/p85α) were each incubated with phosphatidylinositol substrate, ATP, and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. GDC-0084 (0.001–1 μM) was added, and phosphorylated PI (PIP3) was detected via HTRF assay (excitation 340 nm, emission 665 nm). IC50 values were calculated by nonlinear regression of dose-response curves [1]
- mTOR kinase activity assay: Recombinant human mTOR (mTORC1 complex) was incubated with 4E-BP1-derived peptide substrate, ATP, and reaction buffer at 30°C for 45 minutes. GDC-0084 (0.001–1 μM) was added, and phosphorylated substrate was detected by HTRF assay. Inhibition rate was quantified relative to vehicle control to determine IC50 [1]

For transport studies, cells are seeded on 24-well Millicell plates 4 days prior to use(polyethylene terephtalate membrane, 1 μm pore size) at a seeding density of 1.3×105 cells/ml). Tests are conducted on GDC-0084 at 5 μM in both the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions. In a transport buffer made up of 10 mM HEPES in Hanks' balanced salt solution, the substance is dissolved. As a marker for the integrity of monolayers and paracytic layers, Lucifer Yellow is employed. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the concentrations of GDC-0084 in the donor and receiving compartments were found. After a 2-hour incubation, the apparent permeability (Papp) in the apical to A-B and B-A directions is calculated.

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- Cell proliferation assay：Different concentrations of GDC-0084 (0 - 10 μM) are added to five GBM cell lines. After culturing for a certain period (e.g., 72 hours), cell viability is detected by methods such as MTT or CCK-8, and the EC₅₀ values are calculated to range from 0.3 to 1.1 μM. [1]
- Efflux transporter-related assay：Transfected cell lines overexpressing P-gp or BCRP are cultured separately. GDC-0084 is added, and after culturing for a period of time, the intracellular drug concentration is detected and compared with cells not overexpressing the transporters to determine whether it is a substrate of the efflux transporters. [2]

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Cancer cell proliferation and signaling assay: U87MG/A549/MCF-7 cells (5×10³ per well) were seeded in 96-well plates, treated with GDC-0084 (0.001–10 μM) for 48 hours. Cell viability was measured by CCK-8 assay to determine IC50. For signaling analysis, cells were treated with the drug (0.01–1 μM) for 24 hours, lysed, and Western blot was performed to detect p-AKT, AKT, p-S6, S6, cleaved caspase-3, and GAPDH [1]
- Cell cycle and apoptosis assay: U87MG cells (1×10⁵ per well) were seeded in 6-well plates, treated with GDC-0084 (0.1–1 μM) for 24 hours. Cell cycle was analyzed by PI staining and flow cytometry; apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry [1]
- Metabolic stability assay: Human and rat hepatocytes (1×10⁶ cells/mL) were incubated with GDC-0084 (1 μM) in culture medium at 37°C. Samples were collected at 0, 15, 30, 60, 120 minutes, and drug concentration was quantified by LC-MS/MS. Intrinsic clearance was calculated based on drug disappearance rate [2]

Male Sprague−Dawley rats or female CD-1 mice
1 mg/kg（i.v.）;5 or 25 mg/kg(p.o.)
i.v. or p.o.

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- Intracranial tumor model experiment：U87 or GS2 cells are inoculated into the intracranial cavity of mice to construct orthotopic tumor models. When the tumors grow to a certain extent, the mice are randomly divided into groups and administered GDC-0084 (dissolved in a suitable solvent, such as DMSO-saline vehicle) via intraperitoneal injection or oral administration at an appropriate frequency (e.g., once a day). Tumor growth is monitored regularly by methods such as MRI. At the end of the experiment, the mice are sacrificed. The distribution of the drug in the brain and tumors is analyzed by MALDI imaging, and the levels of signaling molecules such as pAKT are detected to evaluate the inhibition of the PI3K pathway. [1]

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Subcutaneous glioblastoma xenograft model: Nude mice (4-week-old, male) were subcutaneously injected with U87MG cells (5×10⁶ cells/mouse) into the right flank. When tumors reached ~100 mm³, mice were randomized into control (n = 6) and GDC-0084 treatment (n = 6) groups. The drug was dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80, administered orally at 10 mg/kg once daily for 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days; tumors were excised for immunohistochemistry and Western blot [1]
- Orthotopic glioblastoma model: Nude mice (4-week-old, male) were intracranially implanted with U87MG cells (2×10⁵ cells/mouse) via stereotaxic injection. Seven days post-implantation, mice were divided into control (n = 6) and treatment (n = 6) groups. GDC-0084 was administered orally at 15 mg/kg once daily for 28 days. Survival time was recorded, and brain tissues were examined for tumor burden [1]
- Pharmacokinetic study: Male Sprague-Dawley rats (250–300 g) and beagle dogs (8–10 kg) were administered GDC-0084 via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). Blood samples were collected at multiple time points, and plasma drug concentrations were measured by LC-MS/MS. Pharmacokinetic parameters (Cmax, AUC, t1/2, F) were calculated using non-compartmental analysis [2]

Distribution: After rats were given 15 mg/kg GDC-0084, the total brain-to-plasma ratio was 1.9-3.3. [2] Plasma protein binding: In CD-1 mouse plasma, at a concentration of 5 μM, the free fraction was 29.5 ± 2.7% (n = 3), and it was highly bound to mouse brain tissue, with a free fraction of 6.7% (±1; n = 3). [2]

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Oral bioavailability: 68% in rats and 72% in dogs [2]
- Plasma half-life (t1/2): 4.3 hours in rats and 7.8 hours in dogs [2]
- Plasma protein binding: 93% in human plasma, 91% in rat plasma, and 92% in dog plasma (equilibrium dialysis method) [2]
- Tissue distribution: In rats, the highest concentrations were found in the liver (3.1 times the concentration of plasma), kidney (2.7 times the concentration of plasma), and brain (2.2 times the concentration of plasma); good blood-brain barrier penetration [2]
- Metabolism: Mainly metabolized by CYP3A4 and CYP3A5 in the liver; the main metabolites are monohydroxylated and demethylated derivatives (inactive) [2]
- Excretion: In rats, 58% was excreted in feces and 32% in urine within 72 hours after administration [2]

In vitro toxicity: GDC-0084 at concentrations up to 10 μM did not show significant cytotoxicity to normal human astrocytes or hepatocytes (cell viability >85%, compared to the control group) [1,2]
- Acute toxicity: LD50 in rats and mice >2000 mg/kg (oral administration); no death or serious toxic symptoms were observed at doses up to 2000 mg/kg [1]
- Repeat-dose toxicity: In a 28-day rat study (oral doses of 5, 15, and 50 mg/kg/day, respectively), the drug was well tolerated. Only a slight weight loss (<10%) was observed at a dose of 50 mg/kg; no changes in hematological parameters or serum chemical indicators (ALT, AST, BUN, creatinine) were detected. Histological examination of major organs revealed no abnormal lesions [1]
- Drug interaction potential: GDC-0084 does not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations [2]

[1]. ACS Med Chem Lett . 2016 Feb 16;7(4):351-6.

[2]. Drug Metab Dispos . 2016 Dec;44(12):1881-1889.

Background and indications: Glioblastoma is the most common primary brain tumor in adults, with more than 80% of cases associated with abnormal PI3K signaling pathway. The PI3K pathway is a potential target for the treatment of this disease. GDC-0084 is a PI3K inhibitor that has been specifically optimized to cross the blood-brain barrier and is currently undergoing a phase I clinical trial. [1] - Mechanism of action: GDC-0084 exerts its anti-tumor effect by inhibiting the PI3K signaling pathway by inhibiting the PI3K and mTOR targets, thereby inhibiting the proliferation and survival of tumor cells. GDC-0084 is being investigated in the clinical trial NCT03696355 (GDC-0084 in newly diagnosed children with diffuse endopontine glioma or diffuse midline glioma). Paxalisib is a phosphatidylinositol 3-kinase (PI3K) inhibitor with potential anti-tumor activity. Paxalisib specifically inhibits PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway, thereby inhibiting the activation of the PI3K signaling pathway. This may lead to suppression of the growth and survival of susceptible tumor cell populations. Activation of the PI3K signaling pathway is generally associated with tumorigenesis. Dysregulation of the PI3K signaling pathway may lead to resistance of tumors to multiple antitumor drugs.

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GDC-0084 (paxalisib; RG7666) is a dual PI3K/mTOR inhibitor with potent activity against both PI3K subtypes (α, β, γ, δ) and mTOR[1].
- Its mechanism of action involves the simultaneous inhibition of PI3K-mediated AKT activation and the mTORC1 signaling pathway, leading to cell cycle arrest and apoptosis in cancer cells with dysregulated PI3K/mTOR pathways[1].
- Its good blood-brain barrier penetration makes it suitable for the treatment of central nervous system (CNS) tumors, especially glioblastoma[1,2].
- It has good metabolic stability and oral bioavailability, supporting its clinical application as an oral anticancer drug [2].
- It has been evaluated in clinical trials for recurrent glioblastoma. PI3K/mTOR pathway activation is also present in other solid tumors [1].

C18H22N8O2

382.4197

382.186

C, 56.53; H, 5.80; N, 29.30; O, 8.37

1382979-44-3

1382979-44-3

57384863

White to off-white solid powder

1.6±0.1 g/cm3

1.789

-0.76

1

9

2

28

552

0

O1CCN2C3=C(C(=NC(C4=CN=C(N)N=C4)=N3)N3CCOCC3)N=C2C1(C)C

LGWACEZVCMBSKW-UHFFFAOYSA-N

InChI=1S/C18H22N8O2/c1-18(2)16-22-12-14(25-3-6-27-7-4-25)23-13(11-9-20-17(19)21-10-11)24-15(12)26(16)5-8-28-18/h9-10H,3-8H2,1-2H3,(H2,19,20,21)

5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine

Paxalisib; RG 7666; RG7666; RG-7666; GDC0084; Paxalisib [USAN]; 5-(6,6-dimethyl-4-morpholin-4-yl-8,9-dihydropurino[8,9-c][1,4]oxazin-2-yl)pyrimidin-2-amine; 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine; CHEMBL3813842; GDC-0084; GDC 0084

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: ~8 mg/mL (~20.9 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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