Pharmacokinetic-pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available heat shock protein 90 inhibitor in a human tumor xenograft mouse model.

PF04942847 [2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide] was identified as an orally available, ATP-competitive, small-molecule inhibitor of heat shock protein 90 (HSP90). The objectives of the present study were: 1) to characterize the pharmacokinetic-pharmacodynamic relationship of the plasma concentrations of PF04942847 to the inhibition of HSP90-dependent protein kinase, AKT, as a biomarker and 2) to characterize the relationship of AKT degradation to tumor growth inhibition as a pharmacological response (antitumor efficacy). Athymic mice implanted with MDA-MB-231 human breast cancer cells were treated with PF04942847 once daily at doses selected to encompass ED(50) values. Plasma concentrations of PF04942847 were adequately described by a two-compartment pharmacokinetic model. A time delay (hysteresis) was observed between the plasma concentrations of PF04942847 and AKT degradation; therefore, a link model was used to account for the hysteresis. The model reasonably fit the time courses of AKT degradation with the estimated EC(50) of 18 ng/ml. For tumor growth inhibition, the signal transduction model reasonably fit the inhibition of individual tumor growth curves with the estimated EC(50) of 7.3 ng/ml. Thus, the EC(50) for AKT degradation approximately corresponded to the EC(50) to EC(80) for tumor growth inhibition, suggesting that 50% AKT degradation was required for significant antitumor efficacy (50-80%). The consistent relationship between AKT degradation and antitumor efficacy was also demonstrated by applying an integrated signal transduction model for linking AKT degradation to tumor growth inhibition. The present results will be helpful in determining the appropriate dosing regimen and guiding dose escalation to achieve efficacious systemic exposure in the clinic.

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone.

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K(d)=1.3 nM, K(i)=15 nM, and cellular IC(50)=0.5 µM).

Solution-phase parallel synthesis of Hsp90 inhibitors.

As part of an oncology chemistry program directed toward discovery of orally bioavailable inhibitors of the 90 kDa heat shock protein (Hsp90), several solution-phase libraries were designed and prepared. A 2 x 89 library of racemic resorcinol amides was prepared affording 131 purified compounds. After evaluation in a binding assay, followed by an AKT-Luminex cellular assay, three potent analogs had functional activity between 0.1 and 0.3 microM. Resolution by preparative chiral SFC chromatography led to (+)-15, (+)-16, and (+)-17 having functional IC(50) = 27, 43, and 190 nM, respectively. (+)-15 exhibited high clearance in human hepatocytes driven primarily by glucuronidation as confirmed by metabolite identification. A second 8 x 14 exploratory library was designed to investigate heterocyclic replacements of the resorcinol ring. The second library highlights the use of the (-)-sparteine-mediated enantioselective Pd-catalyzed alpha-arylation of N-Boc-pyrrolidine to prepare chiral 2-arylpyrrolidines in parallel.

Synergy between celecoxib and radiotherapy results from inhibition of cyclooxygenase-2-derived prostaglandin E2, a survival factor for tumor and associated vasculature.

Previous work has demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors can act synergistically with radiotherapy to improve tumor debulking and control in preclinical models. The underlying mechanism of this remarkable activity has not yet been determined. Here, we report that radiation can elevate intratumoral levels of COX-2 protein and its products, particularly prostaglandin E(2) (PGE(2)). Furthermore, inhibition of COX-2 activity or neutralization of PGE(2) activity enhances radiotherapy even in tumors where COX-2 expression is restricted to the tumor neovasculature. Direct assessment of vascular function by direct contrast enhancement-magnetic resonance imaging showed that the combination of radiation and celecoxib lead to enhanced vascular permeability. These observations suggest that an important mechanism of celecoxib-induced radiosensitization involves inhibition of COX-2-derived PGE(2), thus removing a survival factor for the tumor and its vasculature.

Synthesis and structure-activity relationships of beta- and alpha-piperidine sulfone hydroxamic acid matrix metalloproteinase inhibitors with oral antitumor efficacy.

alpha-Piperidine-beta-sulfone hydroxamate derivatives were explored that are potent for matrix metalloproteinases (MMP)-2, -9, and -13 and are sparing of MMP-1. The investigation of the beta-sulfones subsequently led to the discovery of hitherto unknown alpha-sulfone hydroxamates that are superior to the corresponding beta-sulfones in potency for target MMPs, selectivity vs MMP-1, and exposure when dosed orally. alpha-Piperidine-alpha-sulfone hydroxamate 35f (SC-276) was advanced through antitumor and antiangiogenesis assays and was selected for development. Compound 35f demonstrates excellent antitumor activity vs MX-1 breast tumor in mice when dosed orally as monotherapy or in combination with paclitaxel.

Targeting small cell lung cancer harboring PIK3CA mutation with a selective oral PI3K inhibitor PF-4989216.

PURPOSE: Constitutive activation of phosphoinositide 3-kinase (PI3K) occurs frequently in many human tumors via either gene mutation in the p110α catalytic subunit of PI3K or functional loss of tumor suppressor PTEN. Patients with small-cell lung cancer (SCLC) have very poor prognosis and survival rates such that an effective targeted therapy is in strong demand for these patients. In this study, we characterized the highly selective oral PI3K inhibitor, PF-4989216, in preclinical SCLC models to investigate whether targeting the PI3K pathway is an effective targeted therapy option for SCLCs that harbor a PIK3CA mutation. EXPERIMENTAL DESIGN: A panel of SCLC cell lines with PIK3CA mutation or PTEN loss were treated with PF-4989216 in several in vitro assays, including PI3K pathway signaling, cell viability, apoptosis, cell-cycle progression, and cell transformation. SCLC cell lines that were sensitive in vitro to PF-4989216 were further evaluated by in vivo animal studies to determine the pharmacokinetic/pharmacodynamic relationship and tumor growth inhibition (TGI) by PF-4989216 treatment. RESULTS: PF-4989216 inhibited PI3K downstream signaling and subsequently led to apoptosis induction, and inhibition in cell viability, transformation, and xenograft tumor growth in SCLCs harboring PIK3CA mutation. In SCLCs with PTEN loss, PF-4989216 also inhibited PI3K signaling but did not induce BCL2-interacting mediator (BIM)-mediated apoptosis nor was there any effect on cell viability or transformation. These results implicate differential tumorigenesis and apoptosis mechanisms in SCLCs harboring PIK3CA mutation versus PTEN loss. CONCLUSIONS: Our results suggest that PF-4989216 is a potential cancer drug candidate for patients with SCLC with PIK3CA mutation but not PTEN loss.

miR-122 regulates tumorigenesis in hepatocellular carcinoma by targeting AKT3.

MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer.

miR-221 promotes tumorigenesis in human triple negative breast cancer cells.

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis. TNBCs are characterized by their resistance to apoptosis, aggressive cellular proliferation, migration and invasion, and currently lack molecular markers and effective targeted therapy. Recently, miR-221/miR-222 have been shown to regulate ERα expression and ERα-mediated signaling in luminal breast cancer cells, and also to promote EMT in TNBCs. In this study, we characterized the role of miR-221 in a panel of TNBCs as compared to other breast cancer types. miR-221 knockdown not only blocked cell cycle progression, induced cell apoptosis, and inhibited cell proliferation in-vitro but it also inhibited in-vivo tumor growth by targeting p27(kip1). Furthermore, miR-221 knockdown inhibited cell migration and invasion by altering E-cadherin expression, and its regulatory transcription factors Snail and Slug in human TNBC cell lines. Therefore, miR-221 functions as an oncogene and is essential in regulating tumorigenesis in TNBCs both in vitro as well as in vivo.

Effective targeting of triple-negative breast cancer cells by PF-4942847, a novel oral inhibitor of Hsp 90.

PURPOSE: Triple-negative breast cancer (TNBC) patients have poor prognoses and survival outcomes such that the development of new targeted therapies is in strong demand. Mechanisms associated with high proliferation and aggressive tumor progression, such as PI3K/PTEN aberration, epidermal growth factor receptor (EGFR) overexpression, and cell-cycle upregulation, play important roles in TNBC. The molecular chaperone Hsp90 is required for the conformational maturation and stability of a variety of proteins in multiple pathways, such as EGFR, AKT, Raf, cdk4, etc. Therefore, an Hsp90 inhibitor may show therapeutic benefit in TNBC by targeting multiple pathways. EXPERIMENTAL DESIGN: The novel oral Hsp90 inhibitor PF-4942847 was characterized in multiple in vitro and in vivo assays to determine its antitumor activity in TNBC cell lines. In addition, the correlation of AKT degradation and Hsp70 induction in host peripheral blood lymphocytes (PBL) and xenograft tumors was determined. RESULTS: PF-4942847 induces degradation of multiple client proteins, cell-cycle block, apoptosis, and inhibits cell proliferation in TNBC lines, subsequently leading to tumor growth inhibition in mouse xenograft models. The correlation of AKT degradation and Hsp70 induction between PBLs and xenograft tumors reveals a differential modulation of Hsp90 activity between host and tumor tissues, and suggests that AKT degradation in PBLs may serve as a pharmacodynamic biomarker in future clinical development. CONCLUSIONS: The novel oral Hsp90 inhibitor, PF-4942847, is a candidate for clinical development in TNBC by collaboratively targeting multiple signaling pathways. In addition, AKT degradation in PBLs may serve as a biomarker in clinical development.

RETRACTED: A novel class of specific Hsp90 small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. Following an investigation by Pfizer, Figures 2, 5B and 5C appear to be duplications and hence the conclusions in the manuscript cannot be verified. The Authors apologize for this inconvenience.

PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity.

Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC(50) of 7.5-47 nmol/L and 3.8-20 nmol/L, respectively) and inhibited cell proliferation (IC(50) of 179-313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC(50) of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials.

Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

PF-03732010: a fully human monoclonal antibody against P-cadherin with antitumor and antimetastatic activity.

PURPOSE: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. EXPERIMENTAL DESIGN: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. RESULTS: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane ß-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. CONCLUSIONS: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials.

Orally active MMP-1 sparing α-tetrahydropyranyl and α-piperidinyl sulfone matrix metalloproteinase (MMP) inhibitors with efficacy in cancer, arthritis, and cardiovascular disease.

α-Sulfone-α-piperidine and α-tetrahydropyranyl hydroxamates were explored that are potent inhibitors of MMP's-2, -9, and -13 that spare MMP-1, with oral efficacy in inhibiting tumor growth in mice and left-ventricular hypertrophy in rats and in the bovine cartilage degradation ex vivo explant system. α-Piperidine 19v (SC-78080/SD-2590) was selected for development toward the initial indication of cancer, while α-piperidine and α-tetrahydropyranyl hydroxamates 19w (SC-77964) and 9i (SC-77774), respectively, were identified as backup compounds.

Discovery of novel benzothiazolesulfonamides as potent inhibitors of HIV-1 protease.

The human immunodeficiency virus (HIV) has been shown to be the causative agent for AIDS. The HIV virus encodes for a unique aspartyl protease that is essential for the production of enzymes and proteins in the final stages of maturation. Protease inhibitors have been useful in combating the disease. The inhibitors incorporate a variety of isosteres including the hydroxyethylurea at the protease cleavage site. We have shown that the replacement of t-butylurea moiety by benzothiazolesulfonamide provided inhibitors with improved potency and antiviral activities. Some of the compounds have shown good oral bioavailability and half-life in rats. The synthesis of benzothiazole derivatives led us to explore other heterocycles. During the course of our studies, we also developed an efficient synthesis of benzothiazole-6-sulfonic acid via a two-step procedure starting from sulfanilamide.