Akt1 (IC50 = 5 nM); Akt3 (IC50 = 8 nM); Akt2 (IC50 = 18 nM); PKA (IC50 = 3100 nM)

Over 600-fold and over 100-fold, respectively, more selective for Akt1 at IC50 than for the closely related PKA and p70S6K, is exhibited by imatasertib diHClide. Ipatasertib diHClide inhibited only three protein conjugates (PRKG1α, PRKG1β, and p70S6K) by more than 70% at 1 μM in a panel of 230 protein conjugates, including 36 members of the human AGC family. ..For these three species, the IC50 values that were periodically measured were 98, 69, and 860 nM, in that order. Hence, in comparison to the next most potent non-Akt inhibitor, p70S6K Multiples panel above, ipatasertib dihydrochloride is 100 times more selective for Akt1 in the screen, with the exception of PKG1 (where ipatasertib dihydrochloride has >10-fold greater Akt1 selectivity). Three xenograft models demonstrating dose-dependent responses to drug treatment were used to study the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD) of ipatasertib dihydrochloride: MCF7-neo. The average cell viability IC50 of /HER2, TOV-21G.x1, and LNCaP Ipatasertib diHClide in these three cell lines were 2.56, 0.44, and 0.11 μM, respectively [2].

In transplantation models where Akt is triggered by genetic changes such as PTEN loss, PIK3CA mutations or mutations, or HER2 overexpression, imatinib diHClide is often successful. The tumors in these models developed slowly, were generated, or reverted at or below 100 mg/kg, the maximum well-tolerated dose observed in immunocompromised mice. When examined in vivo, the daily combination of RP-56976 with Ipatasertib diHClide produced analgesia and tumor regression in PC-3 and MCF7-neo/HER2 xenograft mice, but the toxin by itself was either ineffective or just caused the tumor to develop. gradually increasing dosages. Similarly, when Ipatasertib diHClide and NSC 241240 were coupled, a rise in TGI was seen in an OVCAR3 ovarian cancer xenograft model. Compared to treatment with extra chemotherapy, Ipatasertib diHClide in combination with RP-56976 or NSC 241240 alone resulted in less than 5% weight loss [2].

Enzymatic Assays[1]
The assay for the determination of Akt1/2/3 and PKA kinase activity employs the IMAP fluorescence polarization (FP) phosphorylation detection reagent to detect fluorescently labeled peptide substrates that have been phosphorylated by the respective kinases. The Akt enzymes employed in these studies consisted of recombinant baculovirus expressed, amino-terminal, polyhistidine-tagged, full-length, wild-type human forms and were obtained commercially. The PKA enzyme employed in these studies consisted of the recombinant untagged human isolated catalytic subunit of PKA expressed in Escherichia coli obtained commercially. Inhibitor, enzyme (9 nM Akt1 or 100 pM PKA), and substrate (100 nM Crosstide) were incubated with 5 μM ATP in assay buffer (10 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.1% BSA (w/v), final DMSO 2% (v/v)) for 60 min at ambient temperature in a 5 μL reaction volume. Reactions were initiated by addition of enzyme + peptide substrate to ATP solutions. IMAP binding reagent (15 μL) was added to terminate the reaction, and the stopped reactions were incubated for a minimum of 30 min at room temperature (rt).

Inhibition of cellular viability was measured in LNCaP cells plated in black, clear-bottomed 96-well plates at a density of 5000 cells/well and subsequently treated with 0–10 μM 28 (ipatasertib) for 72 h at 37 °C and 5% CO2. The extent of cell proliferation was determined by measuring the reduction of resazurin to resorufin as described in the manufacturer’s protocol using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Dose–response curves were generated using the four-parameter logistic model, and 50% inhibitory concentration (IC50) values were determined from these curve fits.[1]

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Briefly, after ipatasertib treatment, HCT116 WT or p53−/− were fixed with 1% formaldehyde and lysed in warm SDS lysis buffer. The genomic DNA was obtained and sheared to 200–1000 bp by sonication on ice. Samples were precleared with Protein A-Agarose/Salmon Sperm DNA (50% Slurry) for 1 h at 4 °C with agitation. Then anti-FoxO3a antibody or anti-p65 antibody was added and incubated overnight on a shaker at 4 °C. Normal rabbit IgG was used as a negative control. The protein agarose/salmon sperm DNA (50% slurry) bead was then added to precipitate the antibody/protein/DNA complexes. After washed with serial wash buffers, DNA–protein immunocomplexes were eluted from the beads by elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min. Finally, the protein–DNA cross-links were reversed to release DNA by incubation with 0.2 M NaCl at 65 °C for 4 h[3].

Female nude mice bearing LNCaP, PC3, KPL-4, or MCF7 tumor xenografts
~100 mg/kg/day
Orally

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For in vivo tumor xenograft studies, female nu/nu (nude) mice were inoculated subcutaneously in the right hind flank with PC3 cells suspended in Hank’s balanced salt solution (HBSS). When tumors reached a mean volume of 150 mm3, the animals were size matched and distributed into treatment groups consisting of 10 animals/group. Tumor volume was calculated as follows: tumor size (mm3) = (longer measurement × (shorter measurement)2) × 0.5. Following data analysis, p values were determined using Dunnett’s t test with JMP statistical software, version 7.0 (SAS Institute). Mouse body weights were recorded twice weekly using an Adventura Pro AV812 scale (Ohaus Corp.). Mice were promptly euthanized when the tumor volume exceeded 2000 mm3 or if body weight loss was ≥20% of the starting weight per IACUC protocol guidelines.[1]

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For PK/PD studies, blood and tumor samples were collected at 1, 3, 8, and 24 h after a single dose of ipatasertib from PC3 tumor bearing mice. Blood samples (approximately 800 μL) were collected from each animal at the scheduled sample collection time by terminal cardiac puncture into tubes containing K2EDTA as an anticoagulant and centrifuged at 1500–2000g to isolate plasma. The concentration of ipatasertib in each plasma sample was determined by a nonvalidated LC/MS/MS assay in the DMPK Bioanalytical Department at Genentech. The assay lower limit of quantitation (LLOQ) was 0.005 μM. Tumor samples were dissociated in Tris lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100. Protein concentrations were determined using the BCA Protein Assay Kit. The human enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of total PRAS40 and PRAS40 phosphorylated at Thr246 (p-PRAS40). The assay quantifies protein levels on the basis of measurements of absorbance. The colored product is directly proportional to the concentration of p-PRAS40 and tPRAS40 present in the specimen. The Meso Scale Discovery Multi-Spot Biomarker Detection System was used to determine the levels of total S6RP and S6RP phosphorylated at Ser235/236 (pS6RP). These assays quantify protein levels on the basis of measurements of electrochemiluminescence intensity. Levels of phosphorylated protein were normalized to total protein levels in ipatasertib-treated tumors and compared to the vehicle control.[1]

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In vivo efficacy was evaluated in multiple tumor cell line- and patient-derived xenograft models. Cells or tumor fragments were implanted subcutaneously into the flank of immunocompromised mice. Female or male nude (nu/nu) or severe combined immunodeficient mice (SCID)/beige mice were used. For the MCF7-neo/HER2 model, 17β-estradiol pellets (0.36 mg/pellet, 60-day release, no. SE-121) were implanted into the dorsal shoulder before cell inoculation. Male mice were castrated before implantation of tumor fragments. After implantation of tumor cells or fragments into mice, tumors were monitored until they reached mean tumor volumes of 180 to 350 mm3 and distributed into groups of 8 to 10 animals/group. ipatasertib/GDC-0068 was formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT) and administered daily (QD), via oral (per os; PO) gavage.[2]

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HCT116 WT and PUMA−/− were harvested, and 1 × 10~6 cells in 0.2 ml of medium were implanted subcutaneously into the back of athymic nude female mice. Female 5-week-old nude mice were housed in a sterile environment with microisolator cages and allowed access to water and chow ad libitum. Mice were treated daily with ipatasertib/GDC-0068 at 40 mg/kg by oral gavage for 21 days treatment after 7 days. Calipers were used to monitor the tumor growth, volume was calculated by the formula: 0.5 × length × width2. Mice were euthanized when tumors reached ~1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin.[3]

[1]. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27.

[2]. Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models. Clin Cancer Res. 2013 Apr 1;19(7):1760-72

[3]. Ipatasertib, a novel Akt inhibitor, induces transcription factor FoxO3a and NF-κB directly regulates PUMA-dependent apoptosis. Cell Death Dis. 2018 Sep 5;9(9):911.

Colon cancer is one of the three common malignant tumors, with a lower survival rate. Ipatasertib, a novel highly selective ATP-competitive pan-Akt inhibitor, shows a strong antitumor effect in a variety of carcinoma, including colon cancer. However, there is a lack of knowledge about the precise underlying mechanism of clinical therapy for colon cancer. We conducted this study to determine that ipatasertib prevented colon cancer growth through PUMA-dependent apoptosis. Ipatasertib led to p53-independent PUMA activation by inhibiting Akt, thereby activating both FoxO3a and NF-κB synchronously that will directly bind to PUMA promoter, up-regulating PUMA transcription and Bax-mediated intrinsic mitochondrial apoptosis. Remarkably, Akt/FoxO3a/PUMA is the major pathway while Akt/NF-κB/PUMA is the secondary pathway of PUMA activation induced by ipatasertib in colon cancer. Knocking out PUMA eliminated ipatasertib-induced apoptosis both in vitro and in vivo (xenografts). Furthermore, PUMA is also indispensable in combinational therapies of ipatasertib with some conventional or novel drugs. Collectively, our study demonstrated that PUMA induction by FoxO3a and NF-κB is a critical step to suppress the growth of colon cancer under the therapy with ipatasertib, which provides some theoretical basis for clinical assessment.[3]

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Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling.[2]

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The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.[1]

C24H33CL2N5O2

494.45712351799

493.201

1489263-16-2

1001264-89-6;1489263-16-2 (HCl);1396257-94-5 (2HCl);1491138-23-8 (besylate); 1491138-24-9;

72188570

Typically exists as solid at room temperature

3

6

6

33

622

3

C[C@@H]1C[C@H](C2=C1C(=NC=N2)N3CCN(CC3)C(=O)[C@H](CNC(C)C)C4=CC=C(C=C4)Cl)O.Cl

DGGYVQQEWGRNDH-GJYOXNSLSA-N

InChI=1S/C24H32ClN5O2.ClH/c1-15(2)26-13-19(17-4-6-18(25)7-5-17)24(32)30-10-8-29(9-11-30)23-21-16(3)12-20(31)22(21)27-14-28-23/h4-7,14-16,19-20,26,31H,8-13H2,1-3H31H/t16-,19-,20-/m1./s1

(S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one hydrochloride.

GDC0068; GDC-0068; GDC 0068; RG7440; Ipatasertib HCl; 1489263-16-2; Ipatasertib hydrochloride; Ipatasertib monohydrochloride; M94BW9PF2L; Ipatasertib hydrochloride [JAN]; (2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-one;hydrochloride; Ipatasertib hydrochloride (JAN); RG-7440; RG 7440

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)