Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Abstract

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials.

Figure 1

Chemical structures of phosphatidylinositide 3-kinase inhibitors and their molecular and cellular effects on cancer cells in vitro. A, chemical structures of PI-103, PI-540, PI-620, and GDC-0941. B, in vitro IC₅₀ values for PI-103, PI-540, PI-620, and GDC-0941 against recombinant phosphatidylinositide 3-kinase enzymes. C, effects of agents on tumor cell and human umbilical vein endothelial cell (HUVEC) proliferation. Data shown as the mean of three independent determinations of GI₅₀ following 96-h continuous exposure to compound. The tumor cell lines used exhibit a variety of mechanisms of deregulation of the phosphatidylinositide 3-kinase pathway: PC3 and U87MG are PTEN null; IGROV-1 has a hetT319F deletion and frameshift in PTEN, a p85 binding domain hetR38C mutation of p110α, and an additional hetX1069W mutation that extends the C-terminus of p110α by four amino acids; and Detroit 562 and SKOV-3 have a hetH1047R mutation of the p110α kinase domain. D, schematic of some key proteins in the phosphatidylinositide 3-kinase pathway. E, effects of agents on molecular biomarkers. Electrochemiluminescent immunoassay (Meso Scale Discovery) analysis of the phosphorylation of AKT Thr³⁰⁸, AKT Ser⁴⁷³, GSK3β Ser⁹, p70S6K Thr⁴²¹/Ser⁴²⁴, and S6 ribosomal protein Ser235/Ser²³⁶. U87MG cells were treated for 2 or 8 h with 5 to 500 nmol/L phosphatidylinositide 3-kinase inhibitor or 10 to 2,500 nmol/L rapamycin. Values shown are the mean IC₅₀ ±SD.

Figure 2

Pharmacokinetics of PI-540 and PI-620 in mice. A, plasma and spleen concentration-time profile of PI-540 following 10 mg/kg administration i.v. and p.o. to female BALB/c mice. Pharmacokinetic parameters are derived from WinNonlin noncompartmental analysis model 201 and 200 for i.v. and p.o., respectively. Concentrations were measured by liquid chromatography tandem mass spectrometry in A to D. B, plasma and spleen concentration versus time profile of PI-620 following 10 mg/kg administration i.v. and p.o. to female BALB/c mice. Pharmacokinetic parameters are derived from WinNonlin noncompartmental analysis model 201 and 200 for i.v. and p.o., respectively. C, plasma concentration versus time profiles following i. p. administration of PI-540 at 25, 50, and 100 mg/kg to female BALB/c mice. Pharmacokinetic parameters are derived from WinNonlin noncompartmental analysis model 200. D, plasma concentration versus time profiles following i.p. administration of PI-620 at 12.5, 25, and 50 mg/kg to female BALB/c mice. Pharmacokinetic parameters are derived from WinNonlin noncompartmental analysis model 200. Values are mean of n = 3 animals per time point.

Figure 3

In vivo pharmacokinetic and pharmacodynamic properties of PI-540 and PI-620 in mice. A and B, concentrations of PI-540 (A) or PI-620 (B)in plasma and tumors of NCr athymic mice bearing well-established U87MG human glioblastoma xenografts. Animals were given a single i.p. administration of compound. Plasma and tumors were collected at 0.25, 2, and 4 h following compound administration. Concentrations were measured by liquid chromatography tandem mass spectrometry. C and D, U87MG human glioblastoma xenografts were grown s.c. bilaterally in female NCr athymic mice. Animals with well established tumors were given 4 d of treatment with PI-540 at 50 mg/kg b.i.d or 100 mg/kg u.i.d i.p. At 1, 4, 6, and 8 h after the last dose, tumors were excised, snap frozen, and assayed for levels of total and Thr³⁰⁸ phosphorylated AKT (C) and total and Ser⁴⁷³ phosphorylated AKT (D). Results were obtained by electrochemiluminescent immunoassay (Meso Scale Discovery). For pharmacokinetic data, results are mean of n = 3 animals per time point. For pharmacodynamic biomaker data, results are mean ± SE of n =6.

Figure 4

In vivo pharmacodynamic effects of PI-540 and PI-620 in mice. U87MG human glioblastoma xenografts were grown s.c. bilaterally in female NCr athymic mice. Animals with well-established tumors were given 4 d of treatment with PI-540 at 50 mg/kg b.i.d i.p. or 100 mg/kg u.i.d i.p. (A and B) and PI-620 at 25 mg/kg b.i.d i.p. or 50 mg/kg u.i.d i.p. (C and D). At 1, 4, 6, and 8 h after the last dose, tumors were excised, snap frozen, and assayed for levels of total and Ser⁹ phosphorylated GSK3β (A), total and Thr⁴²¹/Ser⁴²⁴ phosphorylated P70S6K(B), total and Thr³⁰⁸ phosphorylated AKT (C), and total and Ser⁴⁷³ phosphorylated AKT (D). Results were obtained by electrochemiluminescent immunoassay (Meso Scale Discovery). Results are mean ± SE of n =6.

Figure 5

In vivo pharmacodynamic properties of PI-620 and antitumor activity of PI-540 and PI-620 in mice. A and B, U87MG human glioblastoma xenografts were grown s.c. bilaterally in female NCr athymic mice. Animals with well established tumors were given 4 d of treatment with PI-620 at 25 mg/kg b.i.d i.p. or 50 mg/kg u.i.d i.p. At 1, 4, 6, and 8 h after the last dose, tumors were excised, snap frozen, and assayed for levels of total and Ser⁹ phosphorylated GSK3β (A) or total and Thr⁴²¹/Ser⁴²⁴ phosphorylated P70S6K(B). Results were obtained by electrochemiluminescent immunoassay (Meso Scale Discovery). C, relative mean tumor volume (percentage of initial volume before therapy) following daily administration of PI-540 (50 mg/kg b.i.d i.p.) and PI-620 (50 mg/kg u.i.d i.p and 25 mg/kg b.i.d i.p.) for 14 d. Established tumors were grown s.c. bilaterally in female NCr athymic mice, and controls received equivalent volumes of vehicle. D, mean final tumor weights following administration of PI-540 (50 mg/kg b.i.d i.p.) and PI-620 (50 mg/kg u.i.d i.p. and 25 mg/kg b.i.d i.p.) for 14 d. For pharmacodynamic biomarker data, results are mean ± SE of n = 6. For therapy data, the results are mean ± SE of n = 16.

Figure 6

In vivo pharmacokinetic properties and pharmacodynamic effects of GDC-0941 in mice. A, mean plasma and tumor concentration versus time profile of GDC-0941 following a single oral administration of 50 mg/kg to female NCr athymic mice bearing s.c. U87MG human glioblastoma xenografts. B-E, U87MG human glioblastoma xenografts were grown s.c. bilaterally in female NCr athymic mice. Animals with well established tumors were given 4 d of oral treatment with GDC-0941 at 50 mg/kg or 150 mg/kg u.i.d. p.o. At 1, 4, 6, and 8 h after the last dose, tumors were excised, snap frozen, and assayed for total and Thr³⁰⁸ phosphorylated AKT (B), total and Ser⁴⁷³ phosphorylated AKT (C), total and Ser⁹ phosphorylated GSK3β (D), and total and Thr⁴²¹/Ser⁴²⁴ phosphorylated P70S6K(E). Results were obtained by electrochemiluminescent immunoassay (Meso Scale Discovery). For pharmacokinetic data, results are mean ± SD of n = 3. For pharmacodynamic biomarker data, results are mean ± SE of n =6.

Figure 7

In vivo oral antitumor activity and associated pharmacokinetic and pharmacodynamic properties of GDC-0941 in U87MG human glioblastoma xenografts. A, relative mean tumor volume (percentage of initial volume before therapy) following 25, 50, 100, and 150 mg/kg daily doses of GDC-0941 p.o. for 19 d in the established human U87MG human glioblastoma xenograft model. Tumors were grown s.c. bilaterally in female NCr athymic mice, and controls received equivalent volumes of vehicle. B, mean final tumor weights for the U87MG human glioblastoma xenograft model following 25, 50, 100, and 150 mg/kg daily GDC-0941 p.o. for 19 d. C, pharmacodynamic effects in the U87MG human glioblastoma xenograft model. Tumors were from the same efficacy study as shown in A and B and were taken 4 and 8 h following the final dose. AKT phosphorylation Ser⁴⁷³ and total AKT were measured by electrochemiluminescent immunoassay (Meso Scale Discovery). D, tumor concentrations of GDC-0941 in the U87MG human glioblastoma xenograft model measured by liquid chromatography tandem mass spectrometry following 19 d daily oral dosing with 25, 50, 100, and 150 mg/kg in the efficacy study. Tumors were collected 4 and 8 h after the final dose. For therapy data, results are mean ± SE of n = 16. For pharmacodynamic biomarker data, results are mean ± SE of n = 6. For pharmacokinetic data, results are mean ± SD of n = 3.