Wee1 (IC50 = 5.2 nM)

MK-1775 blocks Wee1 kinase in a way that is competitive with ATP. In contrast to Wee1, MK-1775 exhibits >100-fold selectivity over human Myt 1, another kinase that phosphorylates cyclin-dependent kinase 1 (CDC2) at a different site (Thr14), 2- to 3-fold less potency against Yes with an IC50 of 14 nM, and 10-fold less potency against seven other kinases with >80% inhibition at 1 μM. MK-1775 treatment inhibits the basal phosphorylation of CDC2 at Tyr15 (CDC2Y15) with an EC50 of 49 nM and suppresses gemcitabine-, carboplatin-, or cisplatin-induced phosphorylation of CDC2 and cell cycle arrest in a dose-dependent manner, with EC50 of 82 nM and 81 nM, 180 nM and 163 nM, as well as 159 nM and 160 nM, respectively. These effects are achieved by disrupting the DNA damage checkpoint in WiDr cells bearing mutated p53. While MK-1775 treatment at 300 nM, which is sufficient to inhibit Wee1 by >80%, exhibits moderate but significant antiproliferative effects by 34.1% in WiDr cells and 28.4% in H1299 cells, MK-1775 treatment at 30-100 nM has no significant antiproliferative effect in WiDr and H1299 cells.[1]

In rats with a T/C of 69% on day 3, MK-1775 treatment alone at ~20 mg/kg shows negligible antitumor effects against WiDr xenografts. In the TOV21G-shp53 and nude rat HeLa-luc xenograft models, MK-1775 alone has a moderate antitumor efficacy.

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Adavosertib (AZD-1775; MK-1775) enhances H1299 xenograft tumor response to fractionated radiotherapy [2]
Based on the substantial radiosensitization by Adavosertib (AZD-1775; MK-1775) in the p53-defective NSCLC cell lines (Table 1), we determined whether this effect extended to the in vivo situation. We performed a series of experiments to examine this question using xenograft tumors growing in nude mice made from one of the p53-defective NSCLC lines and treated with the combination of MK-1775 and external beam radiation where tumor growth delay was used as the endpoint for analysis. The Calu-6 cell line was chosen for this study based on its substantial radiosensitization by MK-1775 in the in vitro survival curve analysis (Fig. 1A and Table 1). Various treatment protocols were investigated including testing different sequences of drug and radiation, different doses of drug and different radiation fractionation schemes. Many of these protocols indicated that tumor growth delay was significantly enhanced by the drug/radiation combination compared to radiation alone. The greatest response was observed when tumors were irradiated twice a day with 1 Gy for 5 days and 60 mg/kg given twice a day on the same days as irradiation. The results of this experiment are presented in Figure 4. The enhancement factor for this treatment protocol was 3.2 (p<0.01). These results underscore the importance of sequencing the drug and radiation treatment close in time and demonstrate that the radiosensitizing effect of MK-1775 extends to the in vivo setting.

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Adavosertib (AZD-1775; MK-1775) treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. Conclusions: These results indicate that Adavosertib (AZD-1775; MK-1775) selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts [3].

Wee1 is a human recombinant protein. A kinase reaction is carried out using 10 μM ATP, 1.0 μCi of [γ-₃₃P]ATP, and 2.5 μg of poly(Lys, Tyr) as a substrate, all in the presence of increasing MK-1775 concentrations, at 30°C for 30 minutes. Radioactivity that has been mixed into the substrate is trapped on MultiScreen-PH plates and quantified using a liquid scintillation counter.

The entire protein is extracted from the cell pellet using a lysis solution that contains 0.4 mol/L NaCl, 1 mM EDTA, and 50 mM HEPES (pH 7.9). The protein is then fortified with protease inhibitor, 1% NP-40, and 10 µL/mL phosphatase inhibitor cocktail 1 and 2. The Bio-Rad protein assay yields the protein concentration of the lysates. On an Immobilon membrane, equal volumes of protein are separated using 12% SDS-PAGE. Buffered saline (150 mM, pH 7.4) with 0.1% Tween (TBS-T) and 5% nonfat dry milk block nonspecific binding sites on the membrane. Protein signals are identified by soaking the membrane in 5% nonfat dry milk with a primary antibody overnight at 4°C. This is followed by 45 minutes of incubation in the corresponding secondary antibody that has been peroxidase-conjugated. After that, the membrane is developed using a Typhoon 9400 scanner and enhanced chemiluminescence with ECL and Western Blotting Detection Reagents.

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Cell cycle analysis [2]
Cells were treated for 1 h with 200 nmol/L Adavosertib (AZD-1775; MK-1775), irradiated at 7.5 Gy, and then harvested at 0, 4, 8, 12, 16, and 24 h later. The cells were then washed with PBS and fixed in 70% ethanol in PBS overnight at 4°C. The fixed cells were washed in Buffer A (0.5% bovine serum albumin (BSA) and 2% FBS in PBS) and then incubated in lysis buffer (0.1% Triton X-100, 0.5% BSA, and 2% FBS in PBS) on ice for 5 min. The cells were pelleted by centrifugation and incubated in Buffer B (2% BSA and 10% FBS in PBS.) Again, cells were pelleted by centrifugation and then incubated with p-HH3 antibody at a dilution of 1:50 in Buffer A overnight at 4°C. The cells were then washed with Buffer A at room temperature and incubated for 1 h in anti-mouse FITC secondary antibody at a dilution of 1:100 in Buffer A. Cells were again washed with Buffer A, pelleted by centrifugation, and incubated in 2% BSA, 2% Tween-20, 5 µg/mL propidium iodide (PI), and 2 µg/mL RNAse A for 1 h in the dark, and flow cytometric analysis was performed immediately thereafter. Flow cytometry was performed using a Beckman Coulter EPICS-ALTRA with Hypersort system equipped with a water-cooled Argon laser emitting at 488 nm. Analysis was performed using EXPO32 software. p-HH3 was measured using a 525-nm band pass filter. A minimum of 10000 events were collected for analysis. Gates were set to exclude cellular debris, and the fluorescence intensity of events within the gated region was measured.

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Immunofluorescence [2]
A549 or H1299 cells were cultivated on coverslips placed in 35-mm dishes and treated with 0.2 µg/mL nocodazole, irradiated with 1 Gy, and treated with 200 nmol/L Adavosertib (AZD-1775; MK-1775) as indicated. The medium was then aspirated, and the cells were rinsed briefly in PBS and then fixed with 2% paraformaldehyde for 15 min. Permeabilization was achieved by a 10-min incubation with 100% methanol at −20°C. After three 5-min rinses in PBS, the cells were incubated in blocking buffer (1X PBS, 50 µL/mL normal goat serum, and 0.3% Triton X-100) for 1 h at room temperature. Next, the cells were incubated in γ-H2AX primary antibody in antibody dilution buffer (1X PBS, 10 mg/mL bovine serum albumin, 0.3% Triton X-100) overnight at 4°C with gentle shaking. After being washed with PBS, primary antibodies were visualized after a 2-h incubation with the appropriate Alexa Fluor-conjugated secondary antibody (goat anti-rabbit FITC or goat anti-mouse Alexa Fluor 594) at a 1:500 dilution. Nuclei were counterstained with 1:500 4’6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS, and the coverslips were mounted on slides with Vectashield. Slides were examined using a Leica fluorescence microscope equipped with a CCD camera and images were imported into Advanced Spot Image analysis software. To quantify γ-H2AX foci, 50 nuclei were evaluated. Micronucleated cells were identified by DAPI staining and quantified (200 cells/coverslip).

Inoculation of 1×10⁶ Calu-6 cells in 10 µL results in the production of tumor xenografts in the leg. Tumors with a diameter of 8 mm are treated with radiation and Adavosertib (AZD-1775; MK-1775) for 5 days. Unanesthetized mice are given gamma-rays locally at a dose rate of 5 Gy/min via a small-animal irradiator that consists of two parallel-opposed ¹³⁷Cs sources for their tumor-bearing legs. Tumors are exposed to radiation twice a day, six hours apart. Give adavosertib (MK-1775) by gavage in volumes of 0.1 mL one hour prior to and two hours following the initial daily radiation dosage.

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Tumor xenografts were produced in the leg by im inoculation of 1 × 106 Calu-6 cells in 10 µL. Irradiation and Adavosertib (AZD-1775; MK-1775) treatment were started when tumors reached 8 mm diameter and continued for 5 days. Gamma-rays were delivered locally to the tumor-bearing legs of unanesthetized mice using a small-animal irradiator consisting of two parallel-opposed 137Cs sources, at a dose rate of 5 Gy/min. Tumors were irradiated twice daily separated by 6 h. Adavosertib (AZD-1775; MK-1775) was given by gavage in 0.1 mL volumes 1 h before and 2 h after the first daily radiation dose.[2]
Three mutually orthogonal tumor diameters were measured 2–3 times/week with a Vernier caliper and means calculated for plotting tumor growth delay. Mice were euthanized when tumors grew to 15 mm diameter. Tumor regrowth was expressed as the time in days for tumors in the treated groups to grow from 8 mm to 12 mm diameter minus the time for control tumors to reach the same size (absolute growth delay [AGD]). For treatment with both Adavosertib (AZD-1775; MK-1775) and radiation, normalized growth delay (NGD) was determined as time for tumors in the combined therapy group to grow to 12 mm minus time for tumors treated with drug alone to grow to 12 mm. Radiation enhancement factor (EF) was determined by dividing NGD for MK-1775 plus radiotherapy by the AGD for irradiation plus vehicle. p values for EFs were determined by Student’s t-test comparing NGD for Adavosertib (AZD-1775; MK-1775) plus irrradiation versus AGD for irradiation plus vehicle. [2]

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Nine pancreatic cancer xenografts (six with p53-deficient and 3 with p53-wild type status) were allowed to grow separately on both flanks of athymic mice. When tumors reached a volume of ~200 mm3, mice were individually identified and randomly assigned to treatment groups, with 5–6 mice (8–10 evaluable tumors) in each group: 1) control; 2) Adavosertib (AZD-1775; MK-1775) (30 mg/kg. p.o., once daily for 4 weeks; 3) GEM (100 mg/kg, i.p., twice weekly on days 1 and 4) for 4 weeks; 4) GEM followed 24 h later by Adavosertib (AZD-1775; MK-1775) in the above mentioned dose. Tumor growth was evaluated twice per week by measurement of two perpendicular diameters of tumors with a digital caliper. Individual tumor volumes were calculated as V = a × b2/2, a being the largest diameter, b the smallest. Relative tumor growth index (TGI) on day 28 was calculated using the formula: (mean tumor volume of drug-treated group/mean tumor volume of control group) × 100. Number of tumors that regressed more than 50% of its initial size in each xenograft was noted. Animals were sacrificed 1 h after the last dose of GEM or MK-1775 and tumors were harvested for analysis except three mice each from GEM and combination treatment group, which were kept longer to check tumor re-growth after the treatment. Mice kept for the re-growth study were sacrificed when the tumors reached the size of control tumors in that xenograft.[3]

The geometric mean plasma concentration profiles over 24 h of 250 mg and 200 mg adavosertib in Cycle 1, Day 1 and Day 5 are shown in Fig. 1. Adavosertib was steadily absorbed following the first and fifth QD doses over 5 days. The median tmax was 4.03 and 2.08 h after the first dose and 2.82 and 1.90 h after the fifth dose, in the 250 and 200 mg cohorts, respectively (Table 3). Adavosertib was slowly eliminated and generally similar between the two treatment cohorts; the mean t1/2λz was 7.36 and 7.30 h after the first dose and 10.55 and 8.88 h after the fifth dose, in the 250 and 200 mg cohorts, respectively. The accumulation of adavosertib in plasma following multiple QD doses for 5 days was generally minimal with mean accumulation ratios based on AUC0–24 of 1.63 and 1.73 in the 250 and 200 mg cohorts, respectively.[4]
Systemic exposure to adavosertib increased in a slightly more than dose-proportional manner. A 1.25-fold increase in dose (200 mg to 250 mg) resulted in 1.70- and 1.65-fold increases in the geometric mean of the Cmax and AUC0–24, respectively, after the first dose and 1.38- and 1.62-fold increases in the geometric mean of the Cmax and AUC0–24, respectively, after the fifth dose.[4]

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Introduction: We aimed to assess the safety, pharmacokinetic profile, and antitumor activity of adavosertib monotherapy in Japanese patients with advanced solid tumors. Materials and methods: This was a single-center, open-label, phase I study with two consecutive cohorts (250 mg and 200 mg cohorts). Patients received adavosertib at 250 mg or 200 mg, orally once daily for 5 days on and 2 days off for Weeks 1 and 2 of a 21-day cycle. Results: Dose-limiting toxicities (Grade 3 febrile neutropenia) occurred in 2/6 patients in the 250 mg cohort. None of the three patients in the 200 mg cohort developed dose-limiting toxicities. The most frequent treatment-emergent adverse event was nausea (250 mg: 83.3 %; 200 mg: 100.0 %). Median time to peak drug concentration was 4.03 and 2.08 h after the first dose and 2.82 and 1.90 h after multiple dosing in the 250 and 200 mg cohorts, respectively; respective mean terminal elimination half-lives were 7.36 and 7.30 h (first dose) and 10.55 and 8.88 h (multiple dosing). Systemic exposure increased in a slightly more than dose-proportional manner. No RECIST v1.1 response was observed. Disease control rate was 0 % and 33.3 % in the 250 and 200 mg cohorts, respectively. One patient (33.3 %) in the 200 mg cohort showed a best overall response of stable disease at ≥ 8 weeks; the rest showed progressive disease. Conclusions: Adavosertib 200 mg once daily was well tolerated in this patient population and no safety concerns were raised. Exposure increased in a slightly more than dose-proportional manner and limited antitumor activity was shown.[4]

Safety and tolerability [4]
Overall, 8/9 patients (88.9 %) reported at least one AE, including 5/6 patients (83.3 %) in the 250 mg cohort and all three patients (100.0 %) in the 200 mg cohort. In the overall study population, the most commonly reported TEAEs were nausea (8/9; 88.9 %), followed by decreased appetite, constipation, diarrhea, vomiting, and platelet count decreased (4/9; 44.4 % each) (Table 2). In the 250 mg cohort (n = 6), the most commonly reported TEAEs were nausea (5/6 patients, 83.3 %), followed by vomiting and decreased appetite (4/6 patients, 66.7 % each). In the 200 mg cohort, the most commonly reported TEAEs were nausea (3/3 patients, 100.0 %), followed by diarrhea and hypoalbuminemia (2/3 patients, 66.7 % each). [4]
No AEs leading to discontinuation of treatment or death were reported in the study. Overall, 2/9 patients (22.2 %), both from the 250 mg cohort, reported serious AEs, including Grade 3 febrile neutropenia (2/9 patients, 22.2 %) and Grade 4 platelet count decreased (1/9 patients, 11.1 %). All three serious AEs were assessed by the investigator as possibly related to the study drug and all three were resolved. [4]
Four out of nine patients (44.4 %) reported CTCAE Grade ≥ 3 AEs that were assessed by the investigator as possibly related to the study drug. In the 250 mg cohort, 3/6 patients (50 %) reported CTCAE Grade ≥ 3 AEs that were assessed by the investigator as possibly related to the study drug, including Grade 4 events (neutropenia, white blood cell count decreased, platelet count decreased [serious AE], and neutrophil count decreased in 1/6 patients, 16.7 % each) and Grade 3 events (febrile neutropenia and anemia in 2/6 patients [33.3 %] each and white blood cell count decreased, lymphocyte count decreased, and decreased appetite in 1/6 patients, 16.7 % each). In the 200 mg cohort, 1/3 patients (33.3 %) reported one Grade 3 hypoalbuminemia event that was assessed by the investigator as possibly related to the study drug, while no Grade 4 events were reported. [4]
Grade 3 febrile neutropenia DLTs were reported in 2/6 patients in the 250 mg cohort. No DLTs were reported in the 200 mg cohort. [4]
No clinically meaningful changes in mean values over time were noted for any laboratory parameter, electrocardiogram parameters, or vital signs.

[1]. MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-FU. Cancer Biol Ther. 2010 Apr;9(7):514-22.

[2]. MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells. Clin Cancer Res. 2011 Sep 1;17(17):5638-48.

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[3]. MK-1775, a potent Wee1 inhibitor, synergizes with NSC 613327 to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.Clin Cancer Res. 2011 May 1;17(9):2799-806.

[4]. Safety, tolerability, pharmacokinetics, and antitumor activity of adavosertib in Japanese patients with advanced solid tumors: A phase I, open-label study. Cancer Treat Res Commun . 2024:39:100809.

1-[6-(2-hydroxypropan-2-yl)-2-pyridinyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2-prop-2-enyl-3-pyrazolo[3,4-d]pyrimidinone is a member of piperazines.
MK-1775 has been used in trials studying the treatment of LYMPHOMA, Neoplasms, Ovarian Cancer, Tongue Carcinoma, and Adult Glioblastoma, among others.
Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antineoplastic sensitizing activity. Adavosertib selectively targets and inhibits WEE1, a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1, CDC2) to inactivate the CDC2/cyclin B complex. Inhibition of WEE1 activity prevents the phosphorylation of CDC2 and impairs the G2 DNA damage checkpoint. This may lead to apoptosis upon treatment with DNA damaging chemotherapeutic agents. Unlike normal cells, most p53 deficient or mutated human cancers lack the G1 checkpoint as p53 is the key regulator of the G1 checkpoint and these cells rely on the G2 checkpoint for DNA repair to damaged cells. Annulment of the G2 checkpoint may therefore make p53 deficient tumor cells more vulnerable to antineoplastic agents and enhance their cytotoxic effect.

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Drug Indication
Treatment of malignant endometrial neoplasms, Treatment of pancreatic cancer

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Adavosertib is a member of the class of pyrazolopyrimidines that is 1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one substituted by 6-(2-hydroxypropan-2-yl)pyridin-2-yl, prop-1-en-3-yl, [4-(4-methylpiperazin-1-yl)phenyl]amino groups at positions 1, 2, and 6, respectively. It is a potent and selective oral inhibitor of WEE1 kinase. 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 pyrazolopyrimidine, a member of pyridines, a tertiary alcohol, a secondary amino compound, a N-arylpiperazine and a N-methylpiperazine.
MK-1775 has been used in trials studying the treatment of LYMPHOMA, Neoplasms, Ovarian Cancer, Tongue Carcinoma, and Adult Glioblastoma, among others.

Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antineoplastic sensitizing activity. Adavosertib selectively targets and inhibits WEE1, a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1, CDC2) to inactivate the CDC2/cyclin B complex. Inhibition of WEE1 activity prevents the phosphorylation of CDC2 and impairs the G2 DNA damage checkpoint. This may lead to apoptosis upon treatment with DNA damaging chemotherapeutic agents. Unlike normal cells, most p53 deficient or mutated human cancers lack the G1 checkpoint as p53 is the key regulator of the G1 checkpoint and these cells rely on the G2 checkpoint for DNA repair to damaged cells. Annulment of the G2 checkpoint may therefore make p53 deficient tumor cells more vulnerable to antineoplastic agents and enhance their cytotoxic effect.
ADAVOSERTIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 29 investigational indications.

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MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.[1]

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Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Experimental design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G₂ block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G₂ block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.[2]

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Purpose: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. Experimental design: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. Results: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. Conclusions: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. [3]

C27H32N8O2

500.6

500.264

C, 64.78; H, 6.44; N, 22.38; O, 6.39

955365-80-7

24856436

Yellow solid powder

1.3±0.1 g/cm3

723.8±70.0 °C at 760 mmHg

391.5±35.7 °C

0.0±2.5 mmHg at 25°C

1.655

0.5

2

9

7

37

795

0

O=C1N(CC=C)N(C2C=CC=C(C(C)(C)O)N=2)C2C1=CN=C(NC1C=CC(N3CCN(C)CC3)=CC=1)N=2

BKWJAKQVGHWELA-UHFFFAOYSA-N

InChI=1S/C27H32N8O2/c1-5-13-34-25(36)21-18-28-26(29-19-9-11-20(12-10-19)33-16-14-32(4)15-17-33)31-24(21)35(34)23-8-6-7-22(30-23)27(2,3)37/h5-12,18,37H,1,13-17H2,2-4H3,(H,28,29,31)

1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.87 mg/mL (5.73 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.16 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 3: ≥ 2.08 mg/mL (4.16 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 4: ≥ 2.08 mg/mL (4.16 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 corn oil and mix evenly.

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Solubility in Formulation 5: 2% DMSO +30% PEG 300 +5% Tween+ddH2O: 5 mg/mL

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Solubility in Formulation 6: 5 mg/mL (9.99 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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