PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia

Abstract

Imatinib mesylate is highly effective in newly diagnosed chronic myeloid leukemia (CML), but BCR/ABL (breakpoint cluster region/abelson murine leukemia)-positive progenitors persist in most patients with CML treated with imatinib mesylate, indicating the need for novel therapeutic approaches. In this study, we have used the murine CML-like myeloproliferative disorder as a platform to characterize the pharmacokinetic, signal transduction, and antileukemic properties of PD166326, one of the most potent members of the pyridopyrimidine class of protein tyrosine kinase inhibitors. In mice with the CML-like disease, PD166326 rapidly inhibited Bcr/Abl kinase activity after a single oral dose and demonstrated marked antileukemic activity in vivo. Seventy percent of PD166326-treated mice achieved a white blood cell (WBC) count less than 20.0 x 10(9)/L (20,000/microL) at necropsy, compared with only 8% of imatinib mesylate-treated animals. Further, two thirds of PD166326-treated animals had complete resolution of splenomegaly, compared with none of the imatinib mesylate-treated animals. Consistent with its more potent antileukemic effect in vivo, PD166326 was also superior to imatinib mesylate in inhibiting the constitutive tyrosine phosphorylation of numerous leukemia-cell proteins, including the src family member Lyn. PD166326 also prolonged the survival of mice with imatinib mesylate-resistant CML induced by the Bcr/Abl mutants P210/H396P and P210/M351T. Altogether, these findings demonstrate the potential of more potent Bcr/Abl inhibitors to provide more effective antileukemic activity. Clinical development of PD166326 or a related analog may lead to more effective drugs for the treatment of de novo and imatinib mesylate-resistant CML.

Figure 1.

Chemical structures for the pyrido[2,3-d]pyrimidine PD166326 and the 2-phenylaminopyrimidine imatinib mesylate.

Figure 2.

PD166326 is a more potent inhibitor of Bcr/Abl- and stem cell factor–dependent proliferation than imatinib mesylate. (A) The P210-expressing MO7e cell line R10(-) growing in the absence of exogenous cytokine, and (B) parental MO7e cells growing in the presence of stem cell factor (MO7e/KL) were treated with the indicated concentrations of PD166326 (•) and imatinib mesylate (▴) for 48 hours and then analyzed by [³H]thymidine incorporation as described in “Materials and methods.” Results are expressed as the counts per minute (CPM) relative to cells treated with diluent alone (DMSO) versus the concentration (log₁₀ scale) of PD166326 or imatinib mesylate. The calculated IC₅₀ for each drug is shown. The results depicted are representative of multiple independent experiments.

Figure 3.

Orally administered PD166326 rapidly reaches concentrations sufficient to inhibit Bcr/Abl tyrosine phosphorylation in vivo. (A) PD166326 was administered to Balb/c mice at the indicated doses (▪, 25 mg/kg; ▵, 35 mg/kg; •, 50 mg/kg; □, 60 mg/kg) twice a day by oral gavage, and plasma PD166326 concentrations were determined at 2, 8, and 15 hours after the last dose at steady state. The error bars indicate the standard error from 3 mice at each dose level. (B) Peripheral blood leukemia cells from mice with the CML-like disease were analyzed by anti-phosphotyrosine (ptyr) or anti-Abl Western immunoblot after a single dose of PD166326 (50 mg/kg), imatinib mesylate (100 mg/kg), or placebo. The ratio of tyrosine-phosphorylated P210 (pB-a) to P210 (B-a) signal was determined by densitometry and is shown above each lane. Ba/F3 cells expressing P210 (+) or not (-) are shown as controls at far left. Anti-actin immunoblot serves as a loading control. Note that the level of P210 expression is not necessarily identical in all animals.

Figure 4.

PD166326 prolongs the survival of mice with the CML-like disease. (A) Mice reconstituted with P210-transduced BM cells received PD166326 (PD16), imatinib mesylate, or placebo according to the indicated treatment schema bid indicates twice a day. (B) Survival of the indicated treatment cohort (N) is depicted as the number of days after bone marrow (BM) reconstitution (day 0). The start of drug treatment (Rx) is indicated at the arrow. PD166326-(▴) and imatinib mesylate (•)–treated animals were electively killed on day 33 to 37 for analysis. ▪ indicates mice receiving placebo. The results depicted are from 2 independent experiments.

Figure 5.

Superior control of leukemic burden in PD166326-treated CML mice. (A) Spleen weight and (B) peripheral blood white blood cell (WBC) count of PD166326 (PD16; •)–, imatinib mesylate (▴)–, or placebo (▪)–treated CML mice at necropsy are shown by scatter plot. The median for each treatment group is depicted by the horizontal line. The P values for the differences in median spleen weight are PD166326 versus placebo (P < .001), PD166326 versus imatinib mesylate (P < .05), and imatinib mesylate versus placebo (P < .05). In the WBC count analysis only the PD166326 versus placebo comparison reached statistical significance (P < .01).

Figure 6.

Oligoclonal CML in mice treated with PD166326 or imatinib mesylate. Genomic DNA was prepared from the spleens of mice with the CML-like illness after 3 to 4 weeks of treatment with the indicated drug or placebo and analyzed for the number of unique BCR/ABL proviral integration sites by Southern blot as described in “Materials and methods.” The number of leukemic clones for each sample is indicated at the top. Genomic DNA from a clonal P210 and parental Ba/F3 cell line are shown at left as controls. DNA size markers are indicated at left.

Figure 7.

The effect of PD166326 on leukemic cell signaling in vivo. (A) Bone marrow protein lysates from mice with the CML-like disease on chronic PD166326 (PD16), imatinib mesylate, or placebo therapy were analyzed by anti-phosphotyrosine immunoblot. A protein size marker (M) is shown at far left. (B) Spleen protein lysates from PD16-, imatinib mesylate–, or placebo-treated mice were subjected to anti–Crk-L immunoblot (IB) as a surrogate marker of Bcr/Abl kinase activity in vivo. The positions of the upper (phosphorylated; p-Crk-L) and lower (Crk-L; nonphosphorylated) forms are shown at right. The ratio of p-Crk-L/Crk-L was quantitated by densitometry and is shown at the top of each lane. Parental or P210-Ba/F3 cells are shown in the bottom panel as a control. (C) The same samples in panel B were immunoprecipitated with anti-Lyn antibody and probed with an antibody that recognizes activated (pLyn) or total Lyn protein. (D) The same samples in panel B were probed with an antibody that recognizes activated (pERK) or total ERK1/2. The average pERK/ERK ratio by densitometry was 0.8 for PD16, 0.5 for imatinib mesylate, and 0.6 for placebo samples. Lysates from parental (p) and Bcr/Abl-expressing (B) Ba/F3 cells, or NIH 3T3 cells (-) are shown at far left in each panel as controls. The positions of P210^BCR/ABL, Lyn, and ERK1/2 are indicated at the arrows.

Figure 8.

PD166326 prolongs survival in a murine model of imatinib mesylate–resistant CML. Mice with the CML-like disease induced by the imatinib mesylate–resistant Bcr/Abl mutants H396P (A), M351T (B), or T315I (C) were randomly assigned to treatment with PD166326 (25 mg/kg orally twice a day; ▴), imatinib mesylate (100 mg/kg orally twice a day; •), or placebo (▪) and analyzed for survival according to the method of Kaplan and Meier. The number of animals (N) in each cohort is shown at right. Imatinib mesylate did not prolong the survival of any of the groups. PD166326 prolonged the survival of P210/H396P (P < .001) and P210/M351T (P = .03) animals, but not T315I/P210.