Mutagenicity and genotoxicity evaluation of 15 nitrosamine drug substance-related impurities in human TK6 cells.

Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT).

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test.

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity.

Comparative Risk Assessment of Formulation Changes in Generic Drug Products: A Pharmacology/Toxicology Perspective.

This review highlights general toxicology concerns caused by formulation differences between generic and innovator drugs. It underscores the importance of a scientific, clinically oriented, evidence-based comparative safety evaluation procedure for generic drugs and discusses representative case studies from a pharmacology-toxicology perspective. For consideration by generic drug industry stakeholders, this article provides an overview of comparative risk assessments for generic drug products.

SDF-1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis.

Multiple human malignancies rely on C-X-C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF-1/CXCL12 (stroma cell-derived factor 1/C-X-C motif chemokine 12), to metastasize. CXCR4 inhibitors promote the mobilization of bone marrow stem cells, limiting their clinical application for metastasis prevention. We investigated the CXCR4-initiated signaling circuitry to identify new potential therapeutic targets.

A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors.

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-β.

Vandetanib for the treatment of symptomatic or progressive medullary thyroid cancer in patients with unresectable locally advanced or metastatic disease: U.S. Food and Drug Administration drug approval summary.

On April 6, 2011, the U.S. Food and Drug Administration approved vandetanib (Caprelsa tablets; AstraZeneca Pharmaceuticals LP) for the treatment of symptomatic or progressive medullary thyroid cancer in patients with unresectable, locally advanced, or metastatic disease.

Decreased lymphangiogenesis and lymph node metastasis by mTOR inhibition in head and neck cancer.

Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression.

G(12/13) signaling pathways substitute for integrin αIIbβ3-signaling for thromboxane generation in platelets.

Background: We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G(12/13) signaling pathways.

Hematopoietic lineage cell-specific protein-1 (HS1) regulates PAR-mediated ERK activation and thromboxane generation in platelets.

Thrombin-induced platelet activation leads to tyrosine phosphorylation of hematopoietic lineage cell-specific protein-1 (HS1), a 75 kDa adapter protein expressed exclusively in cells of hematopoietic lineage. We have shown HS1 to be a functionally important signaling molecule downstream of PAR-4 and GPVI collagen receptor. We have thus begun to elucidate PAR signaling pathway of HS1 phosphorylation, and its functional implications.

Role of P2Y receptor subtypes in platelet-derived microparticle generation.

Microparticles are shed from the platelet membrane upon platelet activation by strong agonists, and they aid in clot formation. As the P2Y1 and the P2Y12 receptors differentially contribute to different platelet functions, we studied the relative contribution of the P2Y1 and P2Y12 receptors to microparticle formation from platelets. The P2Y12 receptor antagonist AR-C 69931MX, but not the P2Y1 receptor antagonist MRS2179, caused a significant decrease in the number microparticles formed by convulxin and thrombin.

Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors.

G-protein-coupled receptors and cancer.

G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in signal transmission, have recently emerged as crucial players in tumour growth and metastasis. Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, invade their surrounding tissues and disseminate to other organs. This Review will address our current understanding of the many roles of GPCRs and their signalling circuitry in tumour progression and metastasis.

Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets.

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner.

Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets.

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets.

Differential requirements for calcium and Src family kinases in platelet GPIIb/IIIa activation and thromboxane generation downstream of different G-protein pathways.

G(12/13) or G(q) signaling pathways activate platelet GPIIb/IIIa when combined with G(i) signaling. We tested whether combined G(i) and G(z) pathways also cause GPIIb/IIIa activation and compared the signaling requirements of these events. Platelet aggregation occurred by combined stimulation of G(i) and G(z) pathways in human platelets and in P2Y1-deficient and G alpha(q)-deficient mouse platelets, confirming that the combination of G(i) and G(z) signaling causes platelet aggregation.

Central role of the P2Y12 receptor in platelet activation.

Platelet activation occurs in response to vessel injury and is important for the arrest of bleeding. Platelet activation during disease states leads to vascular occlusion and ischemic damage. The P2Y(12) receptor, activated by ADP, plays a central role in platelet activation and is the target of P2Y(12) receptor antagonists that have proven therapeutic value.

Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets.

Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets.

ADP receptors--targets for developing antithrombotic agents.

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target.

Platelet purinergic receptors.

Activation of P2Y(1) and P2Y(12) receptors, through secreted ADP that is stimulated by agonists such as thrombin, thromboxane and collagen, is a major mechanism of platelet activation. P2X(1) receptors also participate in platelet shape change and potentiation of calcium mobilization. The cloning of the P2Y(12) receptor and its subsequent knockout in mice promises further understanding of its downstream signaling events.

Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270.

Human platelets express 2 G protein-coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y(1)) via heterotrimeric guanosine 5-triphosphate (GTP)-binding protein G(q), and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y(12)) via heterotrimeric GTP-binding protein G(i). Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y(12) receptor and are predicted to interact with the extracellular cysteine residues on the P2Y(12) receptor.

Comparative analysis of various platelet glycoprotein IIb/IIIa antagonists on shear-induced platelet activation and adhesion.

Platelet accretion into arterial thrombus in stenotic arterial vessels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium concentration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system.

Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets.

Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G(q), G(i), and G(z). Although activated by several platelet agonists, including thrombin and thromboxane A(2), the contribution of the G(12/13) signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G(12/13) activation to platelet fibrinogen receptor activation.

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator.