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

Capivasertib (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]

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In order to understand the compoundʼs selectivity profile, 64/Capivasertib was assayed against a larger enzyme panel of 75 kinases, of which 35 were also AGC family kinases. Significant activity, defined herein as >75% inhibition at a fixed concentration of 1 μM, was seen for just 15 kinases, of which 14 were unsurprisingly from the AGC family. In addition to Akt1–3, these were ROCK2, MKK1, MSK1, MSK2, PKCγ, PKGα, PKGβ, PRKX, RSK2, RSK3, P70S6K, and PKA. Only the latter two kinases, P70S6K and PKA, were inhibited with enzyme IC50 values comparable to Akt1–3 inhibition, at 6 and 7 nM, respectively. However, in cellular end points of these two kinases, activity was relatively reduced compared to the primary Akt pharmacology. The cellular IC50 against P70S6K was approximately 5 μM, as measured by inhibition of S6 phosphorylation in TSC1 null RT4 bladder cancer cells, while activity against PKA was around 1 μM, as determined by inhibition of VASP phosphorylation in A431 cells. Activity against related ROCK1 isoform was much reduced relative to ROCK2, with an IC50 of 470 nM. Compound 64/Capivasertib was also very effective at inhibiting the phosphorylation of downstream Akt substrates in a variety of cell lines (Table 7). Potent inhibition was seen against pGSK3β and pPRAS40 as direct markers of Akt cell activity. The growth inhibitory effect of 64 was also examined across a much larger in-house cellular panel of 182 tumor cell lines in standard proliferation assay format. Sensitive cell lines were defined as those inhibited with an IC50 of 3 μM or less. A majority of breast cell lines proved to be sensitive (64%), with gastric, endometrial, prostate, and hematologic lines showing intermediate sensitivity (24–33% responsive). Lines that showed a poor response to 64 were derived from lung (12% sensitive), colorectal (7%), and bladder (0%) cells. The degree of sensitivity of a line could be correlated with a variety of oncogenic markers. Specifically, activating mutations in PIK3CA, loss or inactivation of tumor suppressor PTEN, or HER2 amplification all were significantly predictive of responsiveness to therapy. Additionally, correlation was also seen between the RAS mutation status of cell lines and resistance to 64. [1]

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Capivasertib (AZD5363) is a potent inhibitor of AKT in vitro [2]
In isolated enzyme assays, AZD5363 inhibited all 3 isoforms of AKT, with an IC50 < 10 nmol/L. P70S6K and PKA were inhibited with similar potency to the AKT isoforms, but a lower potency was shown against the Rho kinases ROCK1 and ROCK2 (Table 1). Further insight into selectivity was obtained by screening the compound at a concentration of 1 μmol/L in a panel of 75 kinases, which included 35 members of the AGC kinase family. AZD5363 had significant activity (>75% inhibition at 1 μmol/L) against 15 kinases, 14 of which were members of the AGC family. These enzymes were AKT1, AKT2, AKT3, P70S6K, PKA, ROCK2, MKK1, MSK1, MSK2, PKCγ, PKGα, PKGβ, PRKX, RSK2, and RSK3 (data not shown). The activity of Capivasertib (AZD5363) in cells was determined by its ability to inhibit phosphorylation of its substrates PRAS40 and GSK3β in BT474c (Her2+ PIK3CA mutant breast) and LNCaP (PTEN-null prostate) cancer cells using Western blotting, and in MDA-MB-468 (PTEN-null breast) cancer cells, by an immunofluorescence-based (Acumen) assay. AZD5363 inhibited phosphorylation of these substrates with an IC50 value of 0.06 to 0.76 μmol/L in the 3 cell lines (Table 1). The phosphorylation status of AKT, and several proteins downstream of AKT in the signaling network, were also monitored by Western blotting in BT474c and LNCaP cells. AZD5363 effectively inhibited phosphorylation of S6 and 4E-BP1 in these cell lines, whereas it increased phosphorylation of AKT at both ser473 and thr308 (Fig. 2B). The activity of AZD5363 was also measured by its ability to induce nuclear translocation of FOXO3a in BT474c cells. Inhibition of AKT prevents phosphorylation of FOXO3a; this results in translocation of FOXO3a to the nucleus, where it is able to switch on the expression of genes such as p27, FasL, and BIM, which collectively induce cell-cycle arrest and/or apoptosis. In BT474c cells, AZD5363 induced FOXO3a nuclear translocation with a half-maximal effective concentration (EC50) value of 0.69 μmol/L; a concentration of 3 μmol/L was sufficient to almost completely localize FOXO3a to the nucleus (Fig. 2C). To show P70S6K pharmacology of AZD5363 in cells, we used the RT4 bladder cancer cell line. These cells have a homozygous deletion in TSC1 and very low TSC2 expression; hence, AKT is largely uncoupled from P70S6K in these cells. In this cell line, AZD5363 inhibited S6 phosphorylation with an IC50 value of approximately 4.8 μmol/L, whereas the allosteric inhibitor MK-2206 was much less active (IC50 > 30 μmol/L; Supplementary Fig. S1).

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Capivasertib (AZD5363) inhibits in vitro growth of a subset of tumor cell lines [2]
The activity of monotherapy AZD5363 was measured by its ability to inhibit growth of a panel of 182 cell lines derived from solid and hematologic tumors, by a standard proliferation assay. Tumor cell lines that were inhibited with a GI50 < 3 μmol/L were classified as sensitive whereas those with a GI50 > 3 μmol/L were classified as resistant. Forty-one cell lines (23%) were classified as sensitive; 25 of these lines (14%) were inhibited with a GI50 < 1 μmol/L and were classified as highly sensitive. The highest frequency of sensitivity was seen in cell lines derived from breast cancers (14 of 22; 64%); HER2+ and ER+ breast cancer cell lines were consistently sensitive (Fig. 3A). Cell lines derived from endometrial, gastric, hematologic, and prostate cancers all showed a frequency of response of 24% to 33%, although only 6 unique prostate cancer cell lines were screened. Cell lines derived from lung and colorectal tumors showed a lower frequency of response at 12% and 7% respectively, whereas all the cell lines derived from bladder cancers were classified as resistant. There appeared to be a correlation between sensitivity to AZD5363 and either the presence of activating PIK3CA mutations, PTEN loss or inactivating mutation, or HER2 amplification. Nineteen of 25 (76%) cell lines classified as highly sensitive and 30 of 41 (73%) the cell lines classified as sensitive carried at least one of these genetic defects (Fig. 3B). When data from the whole-cell panel were analyzed, regardless of other mutations, a significant relationship was found between the presence of PIK3CA mutations and sensitivity to AZD5363 (P = 0.0059; t test). When mutations in the helical and catalytic domains of PIK3CA were analyzed separately, a significant correlation was found between both types of mutation and sensitivity to AZD5363 (P = 0.024 and 0.0047 for helical and kinase domain mutations, respectively). A significant correlation was also found between PTEN mutation (loss or gene sequence mutation) and sensitivity to AZD5363 (P = 0.0099; t test; Supplementary Fig. S2). A significant correlation between the presence of a RAS mutation (collectively analyzing K-, N-, or H-RAS mutations) and resistance to AZD5363 was also found (P = 0.038; t test). When cell lines with coincident RAS mutations were excluded from the analysis, the relationship between PIK3CA mutation and AZD5363 sensitivity and PTEN mutation and AZD5363 sensitivity was very highly significant (P < 10−5 in both cases; Supplementary Fig. S3).

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Mechanism of action of Capivasertib (AZD5363) [2]
To determine whether AZD5363 has a predominantly antiproliferative or proapoptotic mechanism of action, a Sytox Green assay was carried out on a panel of breast and prostate cancer cell lines, which showed sensitivity to AZD5363 in the standard proliferation assay. The Sytox Green assay enables the generation of a dose–response curve based on cell number and determination of percentage of dead cells at a fixed concentration of 1 μmol/L. The GI50 values in the Sytox Green assay were generally similar to the proliferation assay values; the values in the 2 assays were all within 5-fold and 8 of 14 (57%) of the cell lines were within 2-fold. However, more than 10% cell death was only seen in 3 of 14 cell lines following incubation with 1 μmol/L AZD5363; these cell lines were BT474c breast and 2 prostate cancer cell lines (LNCaP and PC346C-Flut1; Supplementary Fig. S4A). The induction of cell death was confirmed in the BT474c cell line by exposing the cells to increasing concentrations of AZD5363 and monitoring cleaved caspase-3 and cleaved PARP. A dose proportional increase in both these apoptotic markers was observed in BT474c cells at 48 hours.

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]

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The effect of 64/Capivasertib in vivo was characterized first by measuring pharmacodynamic activity in a BT474c breast adenocarcinoma xenograft model. Following single oral doses of 100 and 300 mg/kg, 64 potently inhibited the phosphorylation of Akt downstream substrates pGSK3β and pPRAS40 as well as pS6 in a manner that was directly linked to plasma exposure (Figure 4). Potent inhibition of pPRAS40 and pGSK3β was seen out to 4 h, which started to recover at 8 h, and was back to basal levels by 24 h as compound was eliminated. The more distal cellular marker pS6 showed a similar exposure response despite overall less marked inhibition. The impact on tumor growth of continuous oral dosing of 64 was also assessed in the same model over 14 days. When dosed at 200 mg/kg once per day, 64 was less effective than dosing at 100 mg/kg twice per day (39% inhibition versus 80%). Greatest inhibition of growth was observed with a dose of 200 mg/kg twice per day, which led to 104% inhibition, and this proved to be the maximum well-tolerated continuous twice-daily dose (Figure 5). [1]

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Capivasertib (AZD5363) inhibits the growth of human tumor xenografts in vivo [2]
The effect of monotherapy AZD5363 on growth of xenografts was determined by continuous oral dosing to nude mice. Dose-dependent inhibition was observed in all models tested. In HER2+ amplified, PIK3CA mutant BT474c xenografts, oral administration at 100 mg/kg twice daily resulted in 80% inhibition (P < 0.0001); this schedule was more effective than 200 mg/kg every day (39% inhibition; P = 0.02) but less effective than the maximum well-tolerated dose of 200 mg/kg twice daily (104% inhibition; P < 0.0001; Fig. 4A). In the HER2+ amplified, PIK3CA mutant HCC-1954 breast cancer xenograft, AZD5363 at 150 mg/kg twice daily caused pronounced tumor regression (129% inhibition; P < 0.0001) whereas 75 mg/kg twice daily resulted in 111% inhibition (P < 0.001; Fig. 4B). In contrast, 30 mg/kg twice weekly trastuzumab was inactive in this HER2+ model. In 786-0 PTEN-null renal cancer xenografts, AZD5363 at 150 mg/kg twice daily resulted in partial regression (125% inhibition; P < 0.0001) whereas 75 mg/kg twice daily caused partial growth inhibition (56%; P = 0.001; Fig. 4C). AZD5363 also inhibited growth of PIK3CA mutant/PTEN-null HGC-27 gastric cancer xenografts at doses more than 50 mg/kg twice daily; in this model slight tumor regressions were observed at doses more than 100 mg/kg twice daily, and a dose-dependent time to progression was observed after cessation of dosing (108%, 106%, and 72% inhibition of growth; P < 0.0001, 0.001 and 0.003 by doses of 150, 100, and 50 mg/kg twice daily respectively; Fig. 4D).

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Capivasertib (AZD5363) has pharmacodynamic activity in vivo [2]
The pharmacodynamic activity of AZD5363 was determined in BT474c xenografts in nude mice, following acute doses of 300 and 100 mg/kg and related to plasma pharmacokinetics (Fig. 5A). Following a 300 mg/kg dose of AZD5363, phosphorylation of PRAS40, GSK3β, and S6 was significantly inhibited for at least 24 hours. pPRAS40 was most strongly inhibited, with approximately 90% inhibition at 1 and 2 hours, and this recovered to approximately 70% inhibition at 24 hours. Inhibition of GSK3β and S6 phosphorylation varied from approximately 80% at 1 hour to approximately 50% at 8 hours and approximately 40% at 24 hours. Total plasma exposure of AZD5363 (not corrected for protein binding) exceeded 10 μmol/L at 1 hour and remained more than 1 μmol/L for approximately 8 hours following a 300 mg/kg dose. Phosphorylation of all 3 biomarkers was significantly inhibited at for at least 8 hours following a 100 mg/kg dose of AZD5363, but the magnitude of inhibition was less than that observed following a 300 mg/kg dose (Fig. 5A). Plasma exposure of AZD5363 was approximately 1 μmol/L for at least 4 hours following a 100 mg/kg dose. Plotting the pharmacodynamic–pharmacokinetic relationship between PRAS40 phosphorylation of individual animals showed that 50% inhibition of pPRAS40 occurred at a total plasma exposure of approximately 0.1 μmol/L AZD5363 (Fig. 5B). A dose- and time-dependent relationship between dose of AZD5363 and blood glucose concentration was also seen in the nonfasted animals used for this study; the glucose concentration increased to approximately 20 mmol/L at 2 hours after a 300 mg/kg dose, and fell back to control levels by 16 hours whereas the glucose concentration increased by less than 2-fold following a 100 mg/kg dose, and fell to control levels by 8 hours (Fig. 5C).

AKT plays a key role in glucose metabolism; its substrates GSK3β and AS160 can modulate glycogen synthesis and glucose transporter function respectively, and signaling through the pathway can regulate glycolytic enzymes including hexokinase and phosphofructokinase. Indeed, AKT activation may, at least in part, explain the Warburg effect—the metabolic shift from oxidative phosphorylation to elevated glycolysis in tumors. Therefore, 18F-FDG-PET imaging has potential as a biomarker of pathway output following AKT inhibition. The effect of Capivasertib (AZD5363) on 18F-FDG uptake was tested in U87-MG xenografts in fasted nude mice, 4 hours after acute dosing. Doses of 130, 200, and 300 mg/kg AZD5363 all caused a significant decrease in tumor uptake of 18F-FDG compared with vehicle controls, but the blood glucose concentration was only significantly elevated at this time point after 200 and 300 mg/kg doses (Fig. 6A).

Given that it seems to be possible to induce cell death in the BT474c cell line in vitro, we compared the effects of a continuous and intermittent dosing schedule of Capivasertib (AZD5363) that delivered similar areas under curve (AUC) but different Cmax values on BT474c xenograft growth in vivo. Continuous dosing of 100 mg/kg twice daily, which achieved a steady-state exposure of approximately 1 μmol/L AZD5363, resulted in more than 90% inhibition of tumor growth, as shown previously, but no tumor regression (Supplementary Fig. S4B). However, a schedule of 300 mg/kg every 4 days on, 3 days off, which achieves 4-peak concentrations exceeding 10 μmol/L AZD5363, resulted in waves of tumor regression during the dosing period, followed by a recovery of tumor growth in the drug holiday period. When a pharmacodynamic analysis was carried out on these xenografts after short-term chronic dosing (3 days' dosing), there was a significant induction of cleaved caspase-3 at 2 hours following a 300 mg/kg dose of AZD5363, which was not observed after dosing at 100 mg/kg twice daily. In contrast, Ki-67 staining decreased significantly at 8 hours after dosing of both schedules of drug (Supplementary Fig. S4B). These experiments show that a high dose, intermittent schedule of AZD5363 has the potential to be more efficacious than a continuous one in tumors that are susceptible to apoptosis as a consequence of AKT inhibition.

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Capivasertib (AZD5363) enhances the activity of HER2 inhibitors and docetaxel in vivo [2]
The potential of AZD5363 to combine with therapeutic antibodies and small-molecule inhibitors of HER2 signaling was tested in the HER2+, PIK3CA mutant KPL4 breast cancer xenograft. This model shows suboptimal responses to lapatinib and trastuzumab. Monotherapy lapatinib at 100 mg/kg every day, trastuzumab at 15 mg/kg twice weekly, and AZD5363 150 mg/kg twice daily all inhibited tumor growth (37%, not significant; 69%, P = 0.002; and 65%, P = 0.004, respectively), but none achieved stasis. In contrast, combinations of AZD5363 and trastuzumab or AZD5363 and lapatinib were well tolerated and, respectively, resulted in tumor regressions of 107% and 109% (P < 0.0001). Moreover, the combinations both showed enhanced growth delays after cessation of dosing compared with the monotherapy groups (Fig. 7A). The combination of AZD5363 with docetaxel was tested in 2 different breast cancer xenografts: BT474c and HCC-1187. In BT474c xenografts, a single dose of 15 mg/kg docetaxel resulted in slight tumor regression (129% inhibition; P < 0.0001). When combined with 150 mg/kg twice daily of AZD5363, the tumors showed dramatic and greatly enhanced regressions (159% inhibition; P < 0.0001), with 6 of 9 tumors showing complete regressions where the tumors were nonmeasurable at the end of the experiment (Fig. 7B). The effect of combining weekly dosing cycles of 5 mg/kg docetaxel with AZD5363 was assessed in the HCC-1187 xenograft model. In the first experiment (Fig. 7C), the combination of 150 mg/kg twice daily of Capivasertib (AZD5363) and docetaxel was investigated. The combination was considerably more efficacious than either of the respective monotherapy groups (100% inhibition; P = 0.0003 for the combination compared with 71% P = 0.002 for monotherapy docetaxel and 79% P = 0.005 for monotherapy AZD5363) and showed evidence of increased apoptosis by cleaved caspase-3 staining (Supplementary Fig. S6). In the second experiment (Fig. 7D), the combination of 5 mg/kg once weekly docetaxel with 2 schedules of AZD5363 that deliver equivalent AUCs was investigated. Both schedules enhanced the efficacy of docetaxel monotherapy, and to a similar extent; at the end of the first dosing period docetaxel monotherapy resulted in 76% inhibition of tumor growth (P = 0.0003) whereas the combinations of docetaxel and the continuous and intermittent dosing schedules of AZD5363, respectively, resulted in 103% and 101% inhibition of tumor growth. All treatment groups showed regrowth when dosing was stopped. Following rechallenge with the same treatments, the docetaxel monotherapy–treated tumors slowly increased in size, whereas the combination groups showed progressive regression. The combination of docetaxel with an intermittent schedule of 300 mg/kg AZD5363 (4 days on, 3 days off) initially appeared to be slightly superior to the combination of docetaxel with a continuous dosing schedule of 100 mg/kg twice daily AZD5363, but the group sizes did not differ significantly from one another at the end of the experiment (Fig. 7D).

The ability of Capivasertib (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.

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Enzyme assays [2]
The ability of Capivasertib (AZD5363) to inhibit the activity of AKT1, AKT2, and AKT3 was evaluated by the Caliper Off-Chip Incubation Mobility Shift assay. Active recombinant AKT1, AKT2, or AKT3 were incubated with a 5-FAM–labeled custom-synthesized peptide substrate (Cambridge Research Biochemicals) together with increasing concentrations of inhibitor. Final reactions contained 1 to 3 nmol/L AKT1, AKT2, or AKT3 enzymes; 1.5 μmol/L peptide substrate; ATP at Km for each AKT isoform; 10 mmol/L MgCl2, 4 mmol/L dithiothreitol (DTT), 100 mmol/L HEPES, and 0.015% Brij-35. The reactions were incubated at room temperature for 1 hour and stopped by the addition of buffer containing 100 mmol/L HEPES, 0.015% Brij-35 solution, 0.1% coating reagent, 40 mmol/L EDTA, and 5% DMSO. Plates were then analyzed using a Caliper LC3000, allowing for separation of peptide substrate and phosphorylated product by electrophoresis with subsequent detection and quantification of laser-induced fluorescence. To determine the kinase selectivity profile, Capivasertib (AZD5363) was also tested against PKA, ROCK1, ROCK2, and P70S6K. PKA, ROCK1, and ROCK2 activity were determined by Caliper Off-Chip Incubation Mobility Shift Assay, as described earlier. Final reaction conditions for measuring ROCKI activity were 5 nmol/L active recombinant ROCK1, 1.5 μmol/L fluorescein isothiocyanate (FITC)-labeled custom peptide substrate, 7 μmol/L ATP, 1 mmol/L DTT, 5 mmol/L MgCl2, 100 mmol/L HEPES, 0.015% Brij-35, and 5 mmol/L β-glycerophosphate; final reaction for measuring ROCK2 activity contained 7.5 nmol/L active recombinant ROCK2, 1.5 μmol/L FAM-labeled custom peptide substrate, 7.5 μmol/L ATP, 1 mmol/L DTT, 10 mmol/L MgCl2, 100 mmol/L HEPES, 0.015% Brij-35, and 5 mmol/L β-glycerophosphate; and protein kinase A (PKA) activity was measured in a final reaction containing 0.0625 nmol/L PKA, 3 μmol/L FITC-labeled custom peptide substrate, 4.6 μmol/L ATP, 1 mmol/L DTT, 10 mmol/L MgCl2, 110 mmol/L HEPES, and 0.015% Brij-35. P70S6K activity was measured by a radioactive (33P-ATP) filter-binding assay. Recombinant S6K1 (T412E) was assayed against a substrate peptide (KKRNRTLTV) in a final volume of 25.5 μL containing 8 mmol/L MOPS, 200 μmol/L EDTA, 100 μmol/L substrate peptide, 10 mmol/L magnesium acetate, 20 μmol/L γ-33P-ATP (50–1,000 cpm/pmol), and increasing concentrations of Capivasertib (AZD5363). The reactions were incubated for 30 minutes at room temperature and terminated by the addition of 0.5 mol (3%) orthophosphoric acid. Reactions were then harvested onto a P81 UniFilter and product formation quantified. IC50 values for all enzyme assays were obtained by fitting data in Origin 7.0. To evaluate a broader selectivity profile, Capivasertib (AZD5363) was also tested across the Dundee Kinase Panel.

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.

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Cellular inhibition of AKT [2]
A high-throughput screening cell-based assay was developed to measure cellular AKT activity using the MDA-MB-468 breast cancer cell line. Cells were exposed to Capivasertib (AZD5363) at concentrations ranging from 3 to 0.003 μmol/L. After a 2-hour treatment, cells were fixed with formaldehyde, washed, permeabilized with 0.5% polysorbate 20 and then probed with a phospho-specific antibody against GSK3βser9. Levels of phosphorylated GSK3βser9 were measured with an Acumen Explorer laser scanning cytometer (TTP LabTech) and IC50 values estimates by fitting data in Origin 7.0.

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Western blot analysis [2]
LNCaP prostate cancer cells and BT474c breast adenocarcinoma cells were exposed to Capivasertib (AZD5363) at concentrations ranging from 10 to 0.03 μmol/L for 2 or 24 hours. Cells were then lysed on ice with a buffer containing 25 mmol/L Tris-HCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 50 mmol/L NaF, 2 mmol/L sodium orthovanadate, 0.27 mol/L sucrose, 10 mmol/L β-glycerophosphate, 5 mmol/L sodium pyrophosphate, and 0.5% Triton X-100 and protease and phosphatase inhibitors. Lysates were then diluted with sample loading buffer, separated on 4% to 12% Bis-Tris Novex gels, transferred onto nitrocellulose membranes, and probed with antibodies for phospho-PRAS40, phospho-GSK3β, phospho-S6, phospho-AKT, phospho-4E-BP1, total 4E-BP1, PARP, and caspase-3. After an overnight incubation with the primary antibody, membranes were washed and incubated with horseradish peroxidase-tagged secondary antibodies, followed by visualization of the immunoblotted proteins on a Syngene ChemiGenius with SuperSignal West Dura Chemiluminescence Substrate. Quantification was carried out using Syngene GeneTools, and IC50 values were estimated.

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FOXO3a translocation assay [2]
BT474c cells were seeded into a clear bottom, black wall 96-well plate, and incubated overnight at 37°C, 5% CO2 before being exposed to Capivasertib (AZD5363) at concentrations ranging from 3 to 0.003 μmol/L. After 2 hours of treatment, cells were fixed with formaldehyde, permeabilized with 0.5% polysorbate 20, and then probed with a primary antibody against FOXO3a overnight at 4°C. Following a wash step, cells were incubated with a secondary antibody conjugated to an Alexa Fluor 488 dye and imaged using a Cellomics ArrayScan. An algorithm measuring the ratio of nuclear to cytoplasmic fluorescence intensity was developed, and IC50 values were estimated.

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Proliferation assays [2]
Cell proliferation assay was determined by 2 methods, MTS and Sytox Green. Briefly, cells were seeded in 96-well plates (at a density to allow for logarithmic growth during the 72-hour assay) and incubated overnight at 37°C, 5% CO2. Cells were then exposed to concentrations of Capivasertib (AZD5363) ranging from 30 to 0.003 μmol/L for 72 hours. For the MTS endpoint, cell proliferation was measured by the CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent in accordance with the manufacturer's protocol. Absorbance was measured with a Tecan Ultra instrument. For the Sytox Green endpoint, Sytox Green nucleic acid dye diluted in TBS-EDTA buffer was added to cells (final concentration of 0.13 μmol/L) and the number of dead cells detected using an Acumen Explorer. Cells were then permeabilized by the addition of saponin (0.03% final concentration, diluted in TBS-EDTA buffer), incubated overnight and a total cell count measured. Predose measurements were made for both MTS and Sytox Green endpoints, and concentration needed to reduce the growth of treated cells to half that of untreated cells (GI50) values were determined using absorbance readings (MTS) or live cell counts.

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.

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Implantation of cells into mice [2]
For in vivo implants, cells were harvested from T225 tissue culture flasks by a 2- to 5-minute treatment with 0.05% trypsin (Invitrogen) in EDTA solution followed by suspension in basic medium and 3 washes in PBS. Only single-cell suspensions of greater than 90% viability, as determined by trypan blue exclusion, were used for injection. Tumor cells were injected subcutaneously in the left flank of the animal in a volume of 0.1 mL. For BT474c studies the animals were supplemented with 0.36 mg/60-d 17β estradiol pellets 1 day before cell implantation. For KPL-4 and HGC-27 antitumor studies, tumors were passaged as approximately 10 mm3 fragments into the flank before carrying out efficacy studies, to achieve more consistent take rates.

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Efficacy studies [2]
When mean tumor sizes reached approximately 0.2 cm3, the mice were randomized into control and treatment groups. The treatment groups received varying dose schedules of Capivasertib (AZD5363) solubilized in a 10% DMSO 25% w/v Kleptose HPB buffer by oral gavage, docetaxel solubilized in 2.6% ethanol in injectable water by intravenous injection once on day 1 at 15 or 5 mg/kg once weekly. When administered in combination, docetaxel was administered 1 hour before the oral dose of Capivasertib (AZD5363). The control group received the DMSO/Kleptose buffer alone, twice daily by oral gavage. Tumor volumes (measured by caliper), animal body weight, and tumor condition were recorded twice weekly for the duration of the study. Mice were sacrificed by CO2 euthanasia. The tumor volume was calculated (taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter) using the formula: (length × width) × √(length × width) × (π/6). Growth inhibition from the start of treatment was assessed by comparison of the differences in tumor volume between control and treated groups. Because the variance in mean tumor volume data increases proportionally with volume (and is therefore disproportionate between groups), data were log transformed to remove any size dependency before statistical evaluation. Statistical significance was evaluated using a one-tailed, 2-sample t test.

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Pharmacodynamic studies [2]
When mean tumor size reached 0.5 cm3, the mice were randomized into control (n = 8 animals) and treatment groups (n = 5 animals per group). The treatment groups received 300 or 100 mg/kg acute dose of Capivasertib (AZD5363)solubilized in a DMSO/Kleptose buffer, by oral gavage . The control group received the DMSO/Kleptose buffer alone, once by oral gavage. At 2, 4, 8, 16, or 24 hours after dosing, the animals were humanely killed and the tumor was snap frozen in liquid nitrogen and stored at −80°C. Total blood was collected by intracardiac puncture and plasma prepared and immediately frozen at −20°C for pharmacokinetic analysis. Frozen tumors were homogenized using Fastprep methodology lysis matrix A and lysates generated using adjusted lysis buffer (1% Triton X-100). Equivalent amounts of protein (12 μg per lane) were resolved by 4% to 15% gradient SDS-PAGE premade gels and transferred to nitrocellulose membranes.

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Radiotracer preparation and positron emission tomography imaging [2]
2[18F]Fluoro-2-deoxy-d-glucose (18F-FDG) was supplied by PETNET solutions. Specific activity was approximately 185 GBq/mmol. Radiochemical purity (determined by thin-layer chromatography) was greater than 95%. Imaging was carried out using the Inveon positron emission tomography (PET) and computed tomography-docked system from Siemens Medical Solutions. Data were acquired with IAW software Version 1.4.3 and analyzed with IRW software version 3.0. The performance of the scanner has been documented previously.
Images were reconstructed using the MAP/3D algorithm (quality control measurement was carried out for the PET scanner before commencement of imaging). Following imaging, tumors were snap frozen in liquid nitrogen and stored at −80°C. Mice received on average 7.96 MBq of radioactivity as a bolus intravenous injection via the tail vein under isoflurane anesthesia. Food was withdrawn on the day of imaging in half-hour intervals so each mouse was fasted for 4 hours before injection of 18F-FDG. Mice were dosed with either vehicle or Capivasertib (AZD5363) (130, 200, or 300 mg/kg) and were imaged 4 hours after drug dosing. Mice were anesthetized in preparation for 18F-FDG injection 3 hours and 15 minutes after dosing, and anesthesia was then maintained for a 45-minute uptake period followed by a 20-minute PET scan under isoflurane anesthesia. Anesthesia was induced using isoflurane delivered in 100% oxygen (∼1.5% isoflurane, 3 L oxygen). Respiration and temperature were monitored throughout, with body temperature being maintained at 36°C–37°C. Following imaging, tumors were snap frozen in liquid nitrogen and stored at −80°C.
Images were reconstructed using the MAP/3D algorithm (quality control measurement was carried out for the PET scanner before commencement of imaging). Regions of interest (ROI) were manually drawn on the 3-dimensional visualization package of Inveon Research Workplace software, to determine radioactivity uptake in the whole tumor. Data were expressed as the maximum standardized uptake value (SUV). Max SUV was calculated using the following formula described by Gambhir, where ID is the injected dose.
Blood glucose concentration was measured before vehicle or Capivasertib (AZD5363) dosing and after PET scanning. Blood glucose concentrations were measured with an Accu Chek meter. Data are reported as mean ± SEM unless otherwise stated. Statistical analyses were conducted using GraphPad prism (v. 4.02). An ANOVA allowing for treatment group was carried out as well as group means, which were compared using a 2-sided t test.

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.

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Pharmacokinetic Profiling [1]
The DMPK profile of 64/Capivasertib is highlighted in Table 6. Protein binding remained low across all species, consistent with initial lead 3. Compound 64/Capivasertib is extensively distributed outside of blood, with volumes of distribution ranging from 2 to 4 L/kg in preclinical species. Oral bioavailability in mouse remains high despite higher clearance, which may indicate a saturation of first-pass metabolism with the oral dose or extrahepatic metabolism. The profile in rat is somewhat worse, however: whole blood clearance is relatively high, and consequently bioavailability remains a modest 13%. Optimization of the critical parameters of cell potency, ROCK selectivity, and absolute hERG margin of 3 has been achieved, but here at the expense of some of the favorable pharmacokinetic properties the early lead demonstrated. The profile in dog appears more balanced, with moderate clearance and moderate bioavailability. As with the initial lead, in vitro intrinsic hepatic clearance (Clint) measured in hepatocytes is generally low, with turnover in human cells only measurable by an assay with a 2 h incubation.

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Plasma exposure of Capivasertib (AZD5363) was approximately 1 μmol/L for at least 4 hours following a 100 mg/kg dose. Plotting the pharmacodynamic–pharmacokinetic relationship between PRAS40 phosphorylation of individual animals showed that 50% inhibition of pPRAS40 occurred at a total plasma exposure of approximately 0.1 μmol/L AZD5363 (Fig. 5B). A dose- and time-dependent relationship between dose of AZD5363 and blood glucose concentration was also seen in the nonfasted animals used for this study; the glucose concentration increased to approximately 20 mmol/L at 2 hours after a 300 mg/kg dose, and fell back to control levels by 16 hours whereas the glucose concentration increased by less than 2-fold following a 100 mg/kg dose, and fell to control levels by 8 hours (Fig. 5C).[2]

Protein Binding
Capivasertib plasma protein binding is 22% and the plasma-to-blood ratio is 0.71. Hepatotoxicity
In the prelicensure trials of capivasertib in combination with fulvestrant as therapy of advanced or metastatic breast cancer, liver test abnormalities were frequent, with elevations in ALT levels in 23% compared to 13% of those on placebo and fulvestrant. However, the enzyme elevations were usually transient, mild-to-moderate in severity and not associated with symptoms or jaundice. ALT elevations above 5 times the upper limit of normal (ULN) arose in 3% of patients on capivasertib vs 0% with placebo. Because capivasertib was always given in combination with a hormonal agent, it was not always possible to attribute the liver test abnormalities to capivasertib alone. There were no discontinuations of capivasertib because of liver test abnormalities and no episodes of clinically apparent liver injury or deaths from liver failure. Since approval and clinical availability of capivasertib, there have been no published case reports of clinically apparent liver injury with jaundice, but clinical experience with its use has been limited.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of capivasertib during breastfeeding. Because of its potential toxicity in the breastfed infant, the manufacturer recommends that breastfeeding be discontinued during capivasertib therapy. With a half-life of 8.3 hours the drug should be cleared from the maternal bloodstream in 2 days. However, capivasertib is used in combination with fulvestrant, which may increase the risk to the infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[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.

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Wide-ranging exploration of analogues of an ATP-competitive pyrrolopyrimidine inhibitor of Akt led to the discovery of clinical candidate AZD5363, which showed increased potency, reduced hERG affinity, and higher selectivity against the closely related AGC kinase ROCK. This compound demonstrated good preclinical drug metabolism and pharmacokinetics (DMPK) properties and, after oral dosing, showed pharmacodynamic knockdown of phosphorylation of Akt and downstream biomarkers in vivo, and inhibition of tumor growth in a breast cancer xenograft model. Compound 3 served as a lead Akt inhibitor with an acceptable DMPK profile in preclinical species and in vivo antitumor efficacy with modulation of biomarkers following oral dosing. Nevertheless, it had an unfavorably low ROCK selectivity, only modest cell activity, and unwanted activity at the hERG ion channel. A crystal structure of this compound bound to Akt1 suggested a possible vector for further substitution, and this position was ultimately explored with a range of diverse substituents and chain lengths, leading ultimately to compound 64, AZD5363. This agent inhibits all Akt isoforms with a potency of <10 nM in vitro and is a potent inhibitor of phosphorylation of the Akt substrates GSK3β, PRAS40, and S6 in a range of cell lines. It has good selectivity over both the hERG ion channel and closely related AGC kinase ROCK, and it shows pharmacodynamic and xenograft activity in vivo. It has potential in cancer therapy and is currently in phase 1 clinical trials. [1]

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AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2(+) breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials. [2]

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In conclusion, AZD5363 is a potent inhibitor of AKT with a pharmacologic profile consistent with its mechanism of action in vitro and in vivo. Tumor types with PIK3CA mutation, PTEN mutation, or HER2 amplification, without coincident RAS mutation, show the highest frequency of response to AZD5363 in vitro; in such tumor types, stasis or regression is achievable by monotherapy dosing in vivo. AZD5363 also has potential to overcome resistance or increase sensitivity to HER2 inhibitors in breast cancer, and greatly sensitizes to docetaxel chemotherapy, resulting in tumor regression in vivo. AZD5363 is currently in phase I clinical trials. [2]

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; apivasertib; 1143532-39-1; Truqap; 4-Amino-N-[(1s)-1-(4-Chlorophenyl)-3-Hydroxypropyl]-1-(7h-Pyrrolo[2,3-D]pyrimidin-4-Yl)piperidine-4-Carboxamide; 4-Piperidinecarboxamide, 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-; 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.)