WEE1 (IC50 = 24 nM); Myt1 (IC50 = 72 nM); Chk1 (IC50 = 3.433 μM)

In seven out of seven cancer cell lines, PD0166285 (0.5 μM) significantly suppresses irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14[1]. In p53 mutant HT29 cells and the E6-transfected, p53-null ovarian cancer cell line PA-1, PD0166285 sensitizes radiation-induced cell killing; however, in p53 wild-type PA-1 cells, this effect is less pronounced. Radiation-induced G2 arrest is reversed by PD0166285 and mitotic cell populations are markedly increased[1]. PD0166285 has a sensitivity enhancement ratio of 1.23 and functions as a radiosensitizer to make cells more sensitive to radiation-induced cell death[1].

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The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repair prior to undergoing mitosis. We hypothesized that the G(2) checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G(2) arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 microM PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G(2) arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G(2) checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.[1]

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PD0166285 is transported by P-gp, but not BCRP, in vitro [2]
CETAs investigating translocation of PD0166285 revealed transport activity of P-gp, but not BCRP, in vitro. In none of the BCRP-expressing cell lines, translocation was observed (Fig. 3). In contrast to BCRP, both Abcb1a and ABCB1-expressing cell lines were found to transport PD0166285 while the parental porcine cell line was not. Again, loss of translocation in presence of the P-gp inhibitor zosuquidar is a further confirmation that P-gp was responsible for the observed PD0166285 transport.

P-gp, but not BCRP, limits the brain penetration of PD0166285 in vivo [2]
A similar pharmacokinetic experiment as described above for AZD1775 was conducted investigating the brain penetration of PD0166285. In this experiment, approximately 5-fold increased brain levels were observed in both Abcb1a/b−/− and Abcb1a/b;Abcg2−/− mice compared to wild type mice (Fig. 4b). Thus, this effect appeared to be solely caused by P-gp, since further elevated PD0166285 brain levels were not observed when Abcg2−/− was also absent. These differences in brain levels were also reflected in the brain-plasma ratios since plasma levels were similar in all genetic backgrounds. In summary, these results indicate that the brain penetration of PD0166285 in vivo is limited by P-gp, but not BCRP.

Concentration equilibrium transport assays [2]
Concentration equilibrium transport assays (CETAs) were carried out using 500 nM of the Wee1 inhibitors and 5 μM of zosuquidar or elacridar was used to block transport, as described previously. To prepare CETA samples for subsequent HPLC analysis, medium samples were mixed with two volumes of acetonitrile. After centrifugation, the supernatant was diluted 3-fold with water and the concentration of AZD1775 or PD0166285 was measured by High Performance Liquid Chromatography (HPLC) coupled to a UV detector using a GraceSmart RP18 5 μm column (150 × 2 mm) (Grace, Deerfield, IL). AZD1775 was detected at 340 nm using isocratic conditions with 45% acetonitrile in 0.1% (v/v) formic acid in water delivered at a flow rate of 0.2 mL/min. PD0166285 was detected at 360 nm using the same column eluted with a gradient of methanol and 0.1% (v/v) formic acid in water ranging from 30% to 70% delivered at a flow rate of 0.2 mL/min.

Western Blot Analysis[1]
Cell Types: Human and mouse cancer cell lines (HCT116, HT29, DLD-1, HCT8, H460, HeLa, C 26).
Tested Concentrations: 0.5 μM.
Incubation Duration: 4 h.
Experimental Results: Inhibited Cdc2Y15 and CdcT14 phosphorylation.

Animal/Disease Models: Wild-type, Abcg2-/-, Abcb1a/b-/- and Abcb1a/b;Abcg2-/- FVB mice[2].
Doses: 5 mg/kg.
Route of Administration: IV.
Experimental Results: Cmax is about 400 ng/mL. P-gp, but not BCRP, limited the brain penetration of PD0166285.

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Pharmacokinetic studies [2]
We used wildtype, Abcg2−/−, Abcb1a/b−/− and Abcg2;Abcb1a/b−/− FVB mice. PD0166285 (5 mg/kg) and AZD1775 (20 mg/kg) were administered i.v. in DMSO. Blood was collected by cardiac puncture 1 h after injection under isoflurane anesthesia, followed by brain tissue collection. Plasma was obtained by centrifugation (5 min, 5000 rpm, 4 °C). Brains were weighed and homogenized using a FastPrep®-24 in 1% (w/v) bovine serum albumin in water. All samples were stored at −20 °C until analysis. AZD1775 and PD0166285 were extracted using diethyl ether and AZD8055 was used as internal standard. Organic phases were separated and dried by vacuum. Samples were reconstituted in methanol:water (20:80 v/v) and measured in an LC-MS/MS setup consisting of an Ultimate 3000 LC System and an API 4000 mass spectrometer. Separation was performed on a ZORBAX Extend-C18 column. Mobile phase A (0.1% formic acid in water) and B (methanol) was used in a 5 min gradient from 30 to 95%B maintained for 3 min followed by re-equilibration at 30%B. Multiple reaction monitoring (MRM) ion traces were 501.5 / 442.4 (AZD1775) and 512.2 / 438.9 (PD0166285) and 466.2 / 450.1 (AZD8055).

Pharmacokinetic experiments using wildtype and ABC transport knockout mice sampled for brain and plasma at 1 h after drug administration clearly show that these same transporters are responsible for the very low brain penetration of the Wee1 inhibitors in vivo (Fig. 4). Notably, in the absence of these transporters the brain-plasma ratio of both agents was remarkably high (approximately 25 for AZD1775 and 6 for PD0166285), whereas in wild type mice AZD1775 and PD0166285 could only achieve a brain-plasma ratio of 1.0 and 1.2, respectively.

[1]. Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator. Cancer Res. 2001 Nov 15;61(22):8211-7.

[2]. ATP-binding cassette transporters limit the brain penetration of Wee1 inhibitors. Invest New Drugs. 2018 Jun;36(3):380-387.

PD-0166285 is a small molecule drug with a maximum clinical trial phase of I.

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Introduction Wee1 is an important kinase involved in the G2 cell cycle checkpoint and frequently upregulated in intracranial neoplasms such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). Two small molecules are available that target Wee1, AZD1775 and PD0166285, and clinical trials with AZD1775 have already been started. Since GBM and DIPG are highly invasive brain tumors, they are at least to some extent protected by the blood-brain barrier (BBB) and its ATP-binding cassette (ABC) efflux transporters. Methods We have here conducted a comprehensive set of in vitro and in vivo experiments to determine to what extent two dominant efflux transporters in the BBB, P-gp (ABCB1) and BCRP (ABCG2), exhibit affinity towards AZD1775 and PD0166285 and restrict their brain penetration. Results Using these studies, we demonstrate that AZD1775 is efficiently transported by both P-gp and BCRP, whereas PD0166285 is only a substrate of P-gp. Nonetheless, the brain penetration of both compounds was severely restricted in vivo, as indicated by a 5-fold (PD0166285) and 25-fold (AZD1775) lower brain-plasma ratio in wild type mice compared to Abcb1a/b;Abcg2-/- mice. Conclusion The brain penetration of these Wee1 inhibitors is severely limited by ABC transporters, which may compromise their clinical efficacy against intracranial neoplasms such as DIPG and GBM. [2]
In summary, targeting Wee1 to treat intracranial neoplasms holds promise since Wee1 is overexpressed in various glioma types and several clinical trials have been started. However, since gliomas are highly invasive and thus to a considerable extent protected by the BBB, using a Wee1 inhibitor with sufficient brain penetration capacity is pivotal to the success of this treatment strategy. We demonstrate that both available Wee1 inhibitors, AZD1775 and PD0166285, are efficient substrates of ABC transporters in the BBB and it is therefore not very likely that they will be able to exhibit efficacy in patients.[2]

C26H27CL2N5O2

512.4309

511.154

C, 60.94; H, 5.31; Cl, 13.84; N, 13.67; O, 6.24

185039-89-8

PD0166285 dihydrochloride;212391-63-4; PD0166285;185039-89-8; 1933496-20-8 (HCl hydrate)

5311382

Light yellow to yellow solid powder

1.3±0.1 g/cm3

665.3±65.0 °C at 760 mmHg

356.2±34.3 °C

0.0±2.0 mmHg at 25°C

1.637

5.09

1

6

9

35

719

0

ClC1C=CC=C(C=1C1=CC2=CN=C(NC3C=CC(=CC=3)OCCN(CC)CC)N=C2N(C)C1=O)Cl

IFPPYSWJNWHOLQ-UHFFFAOYSA-N

InChI=1S/C26H27Cl2N5O2/c1-4-33(5-2)13-14-35-19-11-9-18(10-12-19)30-26-29-16-17-15-20(25(34)32(3)24(17)31-26)23-21(27)7-6-8-22(23)28/h6-12,15-16H,4-5,13-14H2,1-3H3,(H,29,30,31)

6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy)phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-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: ≥ 2.5 mg/mL (4.88 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 25.0 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: 2.17 mg/mL (4.23 mM) 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 21.7 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.)