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]

Figure 1 shows the geometric mean plasma concentrations of 250 mg and 200 mg adavoritetinib over 24 hours on days 1 and 5 of cycle 1. Adavoritetinib was stably absorbed within 5 days following the first and fifth once-daily doses. The median time to peak concentration (tmax) was 4.03 hours and 2.08 hours after the first dose in the 250 mg and 200 mg groups, respectively, and 2.82 hours and 1.90 hours after the fifth dose (Table 3). Elimination of adavoritetinib was slow but generally similar in both treatment groups; the mean half-life (t1/2λz) was 7.36 hours and 7.30 hours after the first dose in the 250 mg and 200 mg groups, respectively, and 10.55 hours and 8.88 hours after the fifth dose. After five consecutive days of once-daily dosing, plasma accumulation of adavosetin was generally very low, with mean accumulation ratios of 1.63 and 1.73 based on AUC0-24 in the 250 mg and 200 mg groups, respectively. [4]
Systemic exposure to adavosetin was slightly higher than that of dose-proportional increases. A 1.25-fold increase in dose (from 200 mg to 250 mg) resulted in a 1.70-fold and 1.65-fold increase in the geometric mean of Cmax and AUC0-24 after the first dose, respectively, and a 1.38-fold and 1.62-fold increase in the geometric mean of Cmax and AUC0-24 after the fifth dose, respectively. [4]

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Introduction: We aimed to evaluate the safety, pharmacokinetic characteristics, and antitumor activity of adavosetin monotherapy in Japanese patients with advanced solid tumors. Materials and Methods: This was a single-center, open-label phase I clinical study comprising two consecutive cohorts (250 mg and 200 mg groups). Patients received adavoriteinib 250 mg or 200 mg orally once daily for 5 consecutive days, followed by a 2-day break, during weeks 1 and 2 of a 21-day treatment cycle. Results: Two of the six patients in the 250 mg group experienced dose-limiting toxicity (grade 3 febrile neutropenia). No dose-limiting toxicity was observed in the three patients in the 200 mg group. The most common adverse event during treatment was nausea (250 mg group: 83.3%; 200 mg group: 100.0%). The median time to peak concentration after the first dose was 4.03 hours and 2.08 hours in the 250 mg and 200 mg groups, respectively, and 2.82 hours and 1.90 hours after multiple doses, respectively; the mean terminal elimination half-life was 7.36 hours and 7.30 hours (first dose) and 10.55 hours and 8.88 hours (multiple doses), respectively. Systemic exposure was slightly higher than the dose-proportional increase. No RECIST v1.1 efficacy evaluation was observed. Disease control rates were 0% and 33.3% in the 250 mg and 200 mg groups, respectively. One patient (33.3%) in the 200 mg group achieved best overall efficacy (disease stabilization) at ≥ 8 weeks; the remaining patients experienced disease progression. Conclusion: Once-daily 200 mg Adavosertib was well tolerated in this study population, and no safety issues were found. Exposure increased slightly above dose proportion and showed limited antitumor activity. [4]

Safety and Tolerability [4] Overall, at least one adverse event (AE) was reported in 8 out of 9 patients (88.9%), including 5 out of 6 patients (83.3%) in the 250 mg group and all 3 patients (100.0%) in the 200 mg group. In the overall study population, the most common treatment-related adverse events (TEAEs) during treatment were nausea (8/9; 88.9%), followed by decreased appetite, constipation, diarrhea, vomiting and decreased platelet count (4/9; 44.4% each) (Table 2). In the 250 mg group (n = 6), the most common treatment-related adverse events (TEAEs) during treatment were nausea (5/6 patients, 83.3%), followed by vomiting and decreased appetite (4/6 patients, 66.7% each). In the 200 mg dose group, the most frequently reported treatment-interventional adverse events (TEAEs) were nausea (3/3 patients, 100.0%), followed by diarrhea and hypoalbuminemia (both 2/3 patients, 66.7%). [4] No adverse events leading to treatment interruption or death were reported in the study. Overall, 2 out of 9 patients (22.2%) reported serious adverse events, all from the 250 mg dose group, including grade 3 febrile neutropenia (2/9 patients, 22.2%) and grade 4 thrombocytopenia (1/9 patients, 11.1%). The investigators assessed that all 3 serious adverse events were likely related to the study drug and all resolved. [4] 4 out of 9 patients (44.4%) reported CTCAEs ≥ grade 3, which the investigators assessed were likely related to the study drug. In the 250 mg dose group, 3 out of 6 patients (50%) reported grade ≥ 3 CTCAE adverse events, which investigators assessed as potentially related to the study drug. These included grade 4 events (neutropenia, decreased white blood cell count, decreased platelet count [serious adverse events], and decreased neutrophil count, each in 16.7% of the 6 patients) and grade 3 events (febrile neutropenia and anemia in 2 out of 6 patients [each in 33.3%], and decreased white blood cell count, decreased lymphocyte count, and decreased appetite in 1 out of 16.7% of the 6 patients). In the 200 mg dose group, 1 out of 3 patients (33.3%) reported a grade 3 hypoalbuminemia event, which investigators assessed as potentially related to the study drug. No grade 4 events were reported. [4]
Two out of six patients in the 250 mg dose group reported grade 3 febrile neutropenia dose-limiting toxicity (DLT). No DLT was reported in the 200 mg dose group. [4]
No clinically significant changes were observed in the mean values of any laboratory parameters, 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-hydroxypropyl-2-yl)-2-pyridyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2-propyl-2-enyl-3-pyrazolo[3,4-d]pyrimidinone belongs to the piperazine class of compounds. MK-1775 has been used in clinical trials for the treatment of various cancers, including lymphoma, tumors, ovarian cancer, tongue cancer, and adult glioblastoma. Adavosertib is a small molecule tyrosine kinase inhibitor of WEE1 with potential antitumor 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 CDC2 phosphorylation and damages the G2 phase DNA damage checkpoint. This may lead to apoptosis in cells after treatment with DNA-damaging chemotherapy drugs. Unlike normal cells, most p53-deficient or mutated human cancers lack the G1 phase checkpoint because p53 is a key regulator of the G1 phase checkpoint, and these cells rely on the G2 phase checkpoint for DNA repair. Therefore, the absence of the G2 checkpoint may make p53-deficient tumor cells more vulnerable to anti-tumor drugs and enhance their cytotoxicity.

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Drug Indications
Treatment of malignant endometrial tumors, treatment of pancreatic cancer

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Adavosertib belongs to the pyrazolopyrimidine class of compounds. Its structure is 1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one, substituted at positions 1, 2, and 6 by 6-(2-hydroxypropyl-2-yl)pyridin-2-yl, prop-1-en-3-yl, and [4-(4-methylpiperazin-1-yl)phenyl]amino, respectively. It is a potent and selective oral WEE1 kinase inhibitor. It is an antitumor drug and an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It is a pyrazolopyrimidine compound, belonging to the pyridine class, and is also a tertiary alcohol, secondary amino compound, N-arylpiperazine, and N-methylpiperazine. MK-1775 has been used in clinical trials for the treatment of various cancers, including lymphoma, tumors, ovarian cancer, tongue cancer, and adult glioblastoma. Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antitumor 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 CDC2 phosphorylation and damages the G2 phase DNA damage checkpoint. This may lead to apoptosis in cells after treatment with DNA-damaging chemotherapy drugs. Unlike normal cells, most p53-deficient or mutated human cancers lack the G1 phase checkpoint because p53 is a key regulator of the G1 phase checkpoint, and these cells rely on the G2 phase checkpoint for DNA repair. Therefore, failure of the G2 phase checkpoint may make p53-deficient tumor cells more susceptible to anti-cancer drugs and enhance their cytotoxic effects. ADAVOSERTIB is a small molecule drug with up to Phase II clinical trials (covering all indications) and 29 investigational indications. MK-1775 is a potent and selective small molecule Wee1 inhibitor. We have previously demonstrated that MK-1775 can block gemcitabine, carboplatin, and cisplatin-induced DNA damage checkpoints and selectively enhance the anti-cancer efficacy of these drugs in p53-deficient tumor cells. Currently, MK-1775 is undergoing a Phase I clinical trial in combination with these anticancer drugs. This study aimed to investigate the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging drugs with different mechanisms of action. Results showed that MK-1775 enhanced the cytotoxicity of 5-FU in p53-deficient human colon cancer cells. Furthermore, MK-1775 inhibited intracellular CDC2 Y15 phosphorylation, blocked 5-FU-induced DNA damage checkpoints, and promoted premature mitosis by inducing histone H3 phosphorylation. The enhancing effect of MK-1775 was specific to p53-deficient cells because the compound did not sensitize p53 wild-type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 enhanced the antitumor efficacy of 5-FU or its prodrug capecitabine at tolerable doses. These enhancements were closely related to the inhibition of CDC2 phosphorylation and the induction of histone H3 phosphorylation in tumors. In addition, MK-1775 also enhanced the cytotoxicity of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA damage agents in cancer patients. [1]

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Objective: Radiotherapy is a common treatment for a variety of solid tumors. However, the treatment rate for multiple disease sites needs to be improved, which prompts us to evaluate the potential of molecular targeted therapy as a radiosensitizer. This study aimed to evaluate the radiosensitizing effect of the wee1 kinase inhibitor MK-1775 on human tumor cells. Experimental design: The radiosensitizing effect of MK-1775 on human tumor cells derived from lung cancer, breast cancer and prostate cancer was detected using a clonogenic survival assay. The experiment included p53 wild-type and p53-deficient cell lines. Flow cytometry and detection of γ-H2AX foci were used to determine whether MK-1775 could relieve radiation-induced G2 phase arrest, thereby allowing cells carrying DNA damage to enter the mitotic phase earlier. In this study, the in vivo efficacy of MK-1775 combined with radiotherapy was evaluated using a nude mouse xenograft tumor model and a tumor growth delay assay of human lung cancer cell lines. Results: Colony formation survival analysis showed that nanomolar concentrations of MK-1775 enhanced the radiosensitivity of p53-deficient human lung, breast, and prostate cancer cells, but had no effect on similar cell lines with wild-type p53. Consistent with its radiosensitivity-enhancing effect, MK-1775 eliminated radiation-induced G₂ phase arrest in p53-deficient cells, but had no effect on p53 wild-type cell lines. Tumor growth delay assays showed that MK-1775 also significantly enhanced the in vivo antitumor efficacy of radiotherapy, and this effect also applied to p53-deficient tumors. Conclusion: These results indicate that the potent and selective wee1 kinase inhibitor MK-1775 significantly enhances the radiosensitivity of p53-deficient human tumor cells in both in vitro and in vivo experiments. In summary, our findings strongly support the clinical evaluation of MK-1775 in combination with radiotherapy. [2]

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Objective: To investigate the efficacy and pharmacodynamics of monotherapy with the potent Wee1 inhibitor MK-1775 and its combination therapy with gemcitabine (GEM) using a group of p53-deficient and p53-wild-type human pancreatic cancer xenograft models. Experimental design: Nine independent patient-derived pancreatic cancer xenograft models (six p53-deficient and three p53-wild-type) were selected from the PancXenoBank database of Johns Hopkins University. Patients were treated with MK-1775, GEM, or GEM for 24 hours followed by MK-1775 treatment for 4 weeks. Tumor growth/regression rates were calculated on day 28. Target regulation was assessed using Western blotting and immunohistochemistry. Results: MK-1775 treatment inhibited Wee1 kinase and reduced the inhibitory phosphorylation level of its substrate Cdc2. Compared with the control group and the gemcitabine (GEM) treatment group, the combination of MK-1775 and GEM relieved cell cycle checkpoint arrest, promoted cell entry into mitosis, and promoted tumor cell death. MK-1775 monotherapy did not induce tumor regression. However, in p53-deficient tumors, the combination of MK-1775 and GEM produced significant antitumor activity and significantly enhanced the tumor regression response (4.01-fold), which was superior to GEM monotherapy. The tumor regeneration curves plotted after the drug treatment showed that the effect of the combination therapy was more durable than that of GEM monotherapy. None of the drugs caused regression of p53 wild-type xenografts. Conclusion: These results indicate that MK-1775 and gemcitabine (GEM) have a selective synergistic effect and can selectively cause regression of 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.)