Combination of Enhanced Depth Imaging Optical Coherence Tomography and Fundus Images for Glaucoma Screening.

Glaucoma is an eye disease that damages the optic nerve and can lead to irreversible loss of peripheral vision gradually and even blindness without treatment. Thus, diagnosing glaucoma in the early stage is essential for treatment. In this paper, an automatic method for early glaucoma screening is proposed. The proposed method combines structural parameters and textural features extracted from enhanced depth imaging optical coherence tomography (EDI-OCT) images and fundus images. The method first segments anterior the lamina cribrosa surface (ALCS) based on region-aware strategy and residual U-Net and then extracts structural features of the lamina cribrosa, such as lamina cribrosa depth and deformation of lamina cribrosa. In fundus images, scanning lines based on disc center and brightness reduction are used for optic disc segmentation and brightness compensation is utilized for segmenting the optic cup. Afterward, the cup-to-disc ratio (CDR) and textural features are extracted from fundus images. Hybrid features are used for training and classification to screen glaucoma by gcForest in the early stage. The proposed method has given exceptional results with 96.88% accuracy and 91.67% sensitivity.

Phase I study of glasdegib (PF-04449913), an oral smoothened inhibitor, in Japanese patients with select hematologic malignancies.

The hedgehog signaling pathway regulates multiple morphogenetic processes during embryogenesis. Aberrant activation of the hedgehog pathway signal transduction in adult tissues is associated with the pathogenesis of hematologic malignancies and solid tumors. We report findings from an open-label, multicenter phase I trial of the selective, small-molecule hedgehog signaling inhibitor glasdegib (PF-04449913) in Japanese patients with select advanced hematologic malignancies. Glasdegib was administered as once-daily oral doses (25, 50 and 100 mg) in 28-day cycles after a lead-in dose on Day -5. The primary objectives were to determine first-cycle dose-limiting toxicities, safety, vital signs and laboratory test abnormalities. Secondary objectives included evaluation of pharmacokinetics, pharmacodynamics and preliminary evidence of clinical activity of glasdegib. No dose-limiting toxicities were noted in the 13 patients in the present study. All patients experienced at least one treatment-emergent, all-causality adverse event. The most frequent treatment-related adverse events (observed in ≥3 patients) were dysgeusia (n = 9), muscle spasms (n = 5), alopecia, decreased appetite (n = 4 each), and increased blood creatinine phosphokinase, constipation and diarrhea (n = 3 each). Two deaths occurred during the study and were deemed not to be treatment-related due to disease progression. Glasdegib demonstrated dose-proportional pharmacokinetics, marked downregulation of the glioma-associated transcriptional regulator GLI1 expression in normal skin, and evidence of preliminary clinical activity, although data are limited. Glasdegib was safe and well tolerated across the dose levels tested. It is confirmed that the 100-mg dose is safe and tolerable in Japanese patients, and this dose level will be examined in the future clinical trial.

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein.

PURPOSE: Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability. METHODS: The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram. RESULTS: The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA. CONCLUSIONS: These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

The cGAS-STING signaling in cardiovascular and metabolic diseases: Future novel target option for pharmacotherapy.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling exert essential regulatory function in microbial-and onco-immunology through the induction of cytokines, primarily type I interferons. Recently, the aberrant and deranged signaling of the cGAS-STING axis is closely implicated in multiple sterile inflammatory diseases, including heart failure, myocardial infarction, cardiac hypertrophy, nonalcoholic fatty liver diseases, aortic aneurysm and dissection, obesity, etc. This is because of the massive loads of damage-associated molecular patterns (mitochondrial DNA, DNA in extracellular vesicles) liberated from recurrent injury to metabolic cellular organelles and tissues, which are sensed by the pathway. Also, the cGAS-STING pathway crosstalk with essential intracellular homeostasis processes like apoptosis, autophagy, and regulate cellular metabolism. Targeting derailed STING signaling has become necessary for chronic inflammatory diseases. Meanwhile, excessive type I interferons signaling impact on cardiovascular and metabolic health remain entirely elusive. In this review, we summarize the intimate connection between the cGAS-STING pathway and cardiovascular and metabolic disorders. We also discuss some potential small molecule inhibitors for the pathway. This review provides insight to stimulate interest in and support future research into understanding this signaling axis in cardiovascular and metabolic tissues and diseases.

A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice.

Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus.

Shenmai Injection Protects Against Doxorubicin-Induced Cardiotoxicity via Maintaining Mitochondrial Homeostasis.

Shenmai injection (SMI), as a patented traditional Chinese medicine, is extracted from Panax ginseng and Ophiopogon japonicus. It commonly used in the treatment of cardiovascular disease and in the control of cardiac toxicity induced by doxorubicin (DOX) treatment. However, its anti-cardiotoxicity mechanism remains unknown. The purpose of this study was to investigate the underlying mitochondrial protective mechanisms of SMI on DOX-induced myocardial injury. The cardioprotective effect of SMI against DOX-induced myocardial damage was evaluated in C57BL/6 mice and H9c2 cardiomyocytes. In vivo, myocardial injury, apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt)/glycogen synthase kinase 3 beta (GSK-3ß) signaling pathway related proteins were measured. In vitro, apoptosis, mitochondrial superoxide, mitochondrial membrane potential, mitochondrial morphology, levels of mitochondrial fission/fusion associated proteins, mitochondrial respiratory function, and AMP-activated protein kinase (AMPK) activity were assessed. To further elucidate the regulating effects of SMI on AMPK and PI3K/Akt/GSK-3ß signaling pathway, compound C and LY294002 were utilized. In vivo, SMI decreased mortality rate, levels of creatine kinase, and creatine kinase-MB. SMI significantly prevented DOX-induced cardiac dysfunction and apoptosis, decreased levels of Bax/Bcl-2 and cleaved-Caspase3, increased levels of PI3K, p-Akt, and p-GSK-3ß. In vitro, SMI rescued DOX-injured H9c2 cardiomyocytes from apoptosis, excessive mitochondrial reactive oxygen species production and descending mitochondrial membrane potential, which were markedly suppressed by LY294002. SMI increased ratio of L-OPA1 to S-OPA1, levels of AMPK phosphorylation, and DRP1 phosphorylation (Ser637) in order to prevent DOX-induced excessive mitochondrial fission and insufficient mitochondrial fusion. In conclusion, SMI prevents DOX-induced cardiotoxicity, inhibits mitochondrial oxidative stress and mitochondrial fragmentation through activation of AMPK and PI3K/Akt/GSK-3ß signaling pathway.

Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice.

Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

[Construction of minireplicon of human respiratory syncytial virus].

OBJECTIVE: The reverse genetics technology is an important way to develop genetically engineered attenuated living human respiratory syncytial virus (RSV) vaccine candidates. As the pilot experiment,it is necessary to prepare RSV minireplicon and investigate its biological activity. METHODS: After the gene start and gene end (GSGE) fragment containing the sequence of leader (Le), gene start (GS), multiple cloning sites (MCS), gene end (GE) and trailer (Tr) was synthesized and positioned under the control of T7 RNA promoter, we cloned this fragment further into px8deltaT vector. Then, the enhanced green fluorescent protein (EGFP) was cloned into the above recombinant px8deltaT vector, and RSV minireplicon plasmids px8deltaT/GSGE1/EGFP and px8deltaT/GSGE2/EGFP were finally obtained. Meanwhile, two ORFs of nucleocapsid proteins of large protein (L) and transcription elongation/antitermination factor (M2-1) were cloned to construct pcDNA3.1/L and pcDNA3.1/M2-1. At the end, we co-transfected a RSV minireplicon plasmid and four nucleocapsid protein plasmids into BSR T7/5 cell lines expressing T7 RNA polymerase by lipofectamine 2000, and analyed the expression of EGFP by inverted fluorescent microscopy and fluorescence activated cell sortor (FACS), respectively. RESULTS: The px8deltaT/GSGE1/EGFP, px8deltaT/GSGE2/EGFP, pcDNA3.1/L and pcDNA3.1/M2-1 were successfully constructed. After co-transfecting, EGFP can be observed in the transfected BSR T7/5 cells under fluorescent microscopy and by FACS. CONCLUSION: The constructed RSV minireplicon is able to replicate and transcript, which provides a solid foundation for further RSV vaccine development and drug selection by RSV reverse genetics.

Differential expression profile analysis of cisplatin­regulated miRNAs in a human gastric cancer cell line.

Cisplatin, one of the most commonly used drugs in combination chemotherapy, is an effective anti­tumor agent widely used for diverse tumor types. MicroRNAs (miRNAs/miRs) are involved in the occurrence, development, diagnosis and treatment of cancer. Therefore, the aim of the current study was to explore whether cisplatin exerts anticancer effects by causing differential expression of miRNAs in human gastric cancer cells. The human gastric cancer cell line NCI­N87 was cultured with a certain dose of cisplatin and high­throughput sequencing combined with reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to detect cisplatin­regulated miRNAs. miRNAs upregulated and downregulated following cisplatin exposure were analyzed. High­throughput sequencing revealed 33 upregulated and 16 downregulated miRNAs. A total of five significantly upregulated and five significantly downregulated miRNAs were identified by RT­qPCR. The expression levels of hsa­miR­1246 and hsa­miR­892b were consistent with the results obtained from high­throughput sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway clustering of cisplatin­regulated miRNAs revealed that the miRNAs regulated genes involved in several biological processes and signaling pathways. The results obtained in the current study suggested that cisplatin may exert an important anticancer effect in gastric cancer via complex biological processes and signaling pathways.

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

Oral respiratory syncytial virus (RSV) DNA vaccine expressing RSV F protein delivered by attenuated Salmonella typhimurium.

Human respiratory syncytial virus (RSV) is a major viral pathogen of the lower respiratory tract of infants and young children worldwide. No effective prevention measure is available. Attenuated Salmonella strains expressing heterologous antigens can be delivered by the oral route, triggering efficient antigen-specific humoral, cellular, and mucosal immunity. In this study, we orally administered attenuated Salmonella strain SL7207, carrying the plasmid pcDNA3.1/F expressing the RSV F gene, to BALB/c mice and showed significant elevations of serum anti-RSV IgG and bronchoalveolar lavage secretory IgA as compared with the control group carrying empty plasmid (p<0.001). The ratio of IgG1 and IgG2a was 0.96. The experimental group also showed a stronger cytotoxic T cell response (p<0.01 at effector:target ratios of 100:1 and 50:1) and a higher stimulation index value of T cell proliferation (p<0.05) than the respective control group. RSV titers in the lung homogenates of the experimental group on day 3 and day 5 postchallenge were lower than in the control group (p<0.05). Histopathological analysis showed obvious differences in infiltration of inflammatory cells and pulmonary alveolar wall thickness (p<0.01) between the two groups. In summary, our results demonstrate the potential of orally administered SL7207-based DNA vaccines against RSV infection.

Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034.

CI-1034, an endothelin-A receptor antagonist was being developed for pulmonary hypertension. Drug-drug interaction studies using human hepatic microsomes were conducted to assess CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 inhibition potential; CYP3A4 induction potential was evaluated using primary human hepatocytes. CI-1034 moderately inhibited CYP2C9 (IC(50) 39.6 microM) and CYP3A4 activity (IC(50) 21.6 microM); CYP3A4 inhibition was metabolism-dependent. In human hepatocytes, no increase in CYP3A4 activity was observed in vitro, while mRNA was induced 15-fold, similar to rifampin, indicating that CI-1034 is both an inhibitor and inducer of CYP3A4. A 2-week clinical study was conducted to assess pharmacokinetics, pharmacodynamics and safety. No significant changes were observed in [formula: see text] between days 1 and 14. However, reversible elevations of serum liver enzymes were observed with a 50mg BID dose and the program was terminated. To further understand the interactions of CI-1034 in the liver and possible mechanisms of the observed hepatotoxicity, we evaluated the effect of CI-1034 on bile acid transport and previously reported that CI-1034 inhibited biliary efflux of taurocholate by 60%, in vitro. This indicated that inhibition of major hepatic transporters could be involved in the observed hepatotoxicity. We next evaluated the in vitro inhibition potential of CI-1034 with the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2 and OCT. CI-1034 inhibited OATP1B1 (K(i) 2 microM), OATP1B3 (K(i) 1.8 microM) and OATP2B1 activity (K(i) 3.3 microM) but not OCT, MDR1 or MRP2 mediated transport. Our data indicates that CI-1034 is an inhibitor of major hepatic transporters and inhibition of bile efflux may have contributed to the observed clinical hepatotoxicity. We recommend that in vitro drug-drug interaction panels include inhibition and induction studies with transporters and drug metabolizing enzymes, to more completely assess potential in vivo interactions or toxicity.

Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts.

PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 micromol/L) and Cdk6 (IC50, 0.016 micromol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

Preferential interactions between protein and arginine: effects of arginine on tertiary conformational and colloidal stability of protein solution.

The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals' forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process.

PEST domain mutations in Notch receptors comprise an oncogenic driver segment in triple-negative breast cancer sensitive to a γ-secretase inhibitor.

PURPOSE: To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014. EXPERIMENTAL DESIGN: We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014. RESULTS: We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014. CONCLUSIONS: This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs.

A phase I study of PF-04449913, an oral hedgehog inhibitor, in patients with advanced solid tumors.

PURPOSE: To estimate the maximum tolerated dose (MTD) of single-agent PF-04449913, and to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity in patients with advanced tumors. EXPERIMENTAL DESIGN: A 3+3 design was used in this open-label, multicenter, phase I study and dose escalation/de-escalation applied until identification of the MTD. PF-04449913 was orally administered once daily in continuous 28-day treatment cycles. The starting dose was 80 mg. RESULTS: A total of 23 patients were enrolled; 19 were evaluable for first-cycle dose-limiting toxicity (DLT). The first-cycle DLT rate at the 640 mg dose level was 33.3%, and the MTD was estimated to be 320 mg once daily. The recommended phase II dose was not determined. PF-04449913 was generally well tolerated at doses of 80 to 320 mg once daily. The most common treatment-related adverse events (AE) were grade 1-2 dysgeusia, fatigue, decreased appetite, nausea, dizziness, dehydration, and diarrhea. Treatment-related grade 3 AEs only occurred in patients receiving PF-04449913 640 mg once daily. No treatment-related grade 4-5 AEs were reported. Pharmacokinetic analysis indicated a generally dose-proportional kinetics with biphasic elimination, supporting once-daily dosing. PF-04449913 modulated hedgehog signaling at the dose levels tested, as demonstrated by >80% downregulation of GLI1 expression in the skin of treated patients. Eight patients (34.8%) achieved stable disease; none had complete or partial response. Three patients with disease progression at enrollment had prolonged disease stabilization (≥6 months). CONCLUSIONS: The results obtained in this study support further evaluation of PF-04449913 in patients with advanced solid tumors.

Treatment with PF-04449913, an oral smoothened antagonist, in patients with myeloid malignancies: a phase 1 safety and pharmacokinetics study.

BACKGROUND: Activation of the Hedgehog signalling pathway contributes to cancer progression and the development of myeloid leukaemia stem cell therapeutic resistance. We aimed to identify the maximum tolerated dose (MTD) and the recommended phase 2 dose of the selective Hedgehog antagonist PF-04449913 in myeloid malignancies. METHODS: We undertook an open-label, dose-finding, standard 3+3 design phase 1 study of PF-04449913 in adult patients with acute myeloid leukaemia, chronic myeloid leukaemia, chronic myelomonocytic leukaemia, myelodysplastic syndrome, or myelofibrosis who were refractory, resistant, or intolerant to previous treatments, at three centres in the USA and one in Italy. Patients who had newly diagnosed, untreated disease were included if they were not eligible for standard treatment options or if standard treatments were not deemed appropriate. Patients received PF-04449913 once daily continuously until disease progression, unacceptable toxic effects, or patient withdrawal for up to 12 28-day cycles. Additional cycles were given if patients showed evidence of clinical benefit. The starting dose was 5 mg and was increased by 100% until the first dose-limiting toxic effect (DLT) and by 50% thereafter, in keeping with a 3+3 clinical trial statistical design. The primary endpoint was first-cycle DLTs. Secondary endpoints were safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity. This trial is registered with ClinicalTrials.gov, number NCT00953758. FINDINGS: Between March 24, 2010, and Sept 7, 2012, 47 patients were enrolled and included in the study: 28 with acute myeloid leukaemia, six with myelodysplastic syndrome, five with chronic myeloid leukaemia (two with chronic-phase and three with blast-phase disease), one with chronic myelomonocytic leukaemia, and seven with myelofibrosis. Patients received PF-04449913 once daily at 5 mg (n=3), 10 mg (n=3), 20 mg (n=4), 40 mg (n=4), 80 mg (n=8), 120 mg (n=3), 180 mg (n=3), 270 mg (n=5), 400 mg (n=9), and 600 mg (n=5). Two patients experienced DLTs (one each in the 80 mg and 600 mg dose groups). The MTD for PF-04449913 was established to be 400 mg once daily. Of the 47 patients enrolled, 28 (60%) experienced treatment-related adverse events, three of which were grade 4 in severity. The most common treatment-related adverse events included dysgeusia (13 [28%] patients), decreased appetite (nine [19%]), and alopecia (seven [15%]). None of the 15 deaths reported were treatment related. Pharmacokinetics seemed to be dose proportional. The mean half-life was 23·9 h (SD 14·0) in the MTD group. Some suggestion of clinical activity was noted in 23 (49%) of 47 patients with haematological malignancies. Based on these results, the recommended phase 2 dose was 200 mg or lower once daily. INTERPRETATION: Based on these findings, PF-04449913 is being tested in phase 2 studies in patients with myelodysplastic syndrome, acute myeloid leukaemia, and myelofibrosis. FUNDING: Pfizer.

A Phase I, dose-finding study in patients with advanced solid malignancies of the oral γ-secretase inhibitor PF-03084014.

PURPOSE: To estimate the maximum tolerated dose (MTD) for continuous oral administration of the γ-secretase inhibitor PF-03084014, determine the recommended phase II dose (RP2D), and evaluate safety and preliminary activity in patients with advanced solid tumors. EXPERIMENTAL DESIGN: This open-label, phase I study consisted of a dose-finding portion based on a 3+3 design, followed by an expansion cohort. PF-03084014 was administered orally, twice daily (BID) for 21 continuous days. Tested doses ranged from 20 to 330 mg BID. In the expansion cohort, patients were to receive the estimated MTD or a lower dose of PF-03084014. RESULTS: A total of 64 patients received treatment. The MTD was estimated to be 220 mg BID. The RP2D was determined to be 150 mg BID, based on the better safety profile versus the 220-mg BID dose, given comparable NOTCH-related target inhibition. The most common treatment-related adverse events were diarrhea, nausea, fatigue, hypophosphatemia, vomiting, rash, and decreased appetite, which were generally mild to moderate in severity. One patient with advanced thyroid cancer had a complete response, and five of seven response-evaluable patients with desmoid tumor achieved a partial response (71.4% objective response rate). Tumor responses were mostly durable, ranging from 1.74+ to 24+ months. PF-03084014 demonstrated a generally dose-dependent pharmacokinetic profile at doses ranging from 20 to 330 mg BID. Consistent downmodulation of NOTCH-related HES4 gene expression was observed in peripheral blood from all evaluable patients. CONCLUSION: Further development of PF-03084014 for the treatment of patients with advanced solid tumors is warranted and currently under evaluation.