Appraisal of ICH E14/S7B Q&As adopted in February 2022 using thorough QT/QTc study data for α4-integrin antagonist carotegrast methyl in Japanese healthy subjects.

We investigated how a lack of placebo control affects the interpretation of results of thorough QT/QTc (TQT) study. Results of TQT study in 48 healthy Japanese subjects assessing the effects of 480 and 960 mg of carotegrast methyl (test drug) and 400 mg of moxifloxacin (positive control) on the time-matched changes in corrected QT from baseline (ΔQTcF) and the placebo-adjusted ΔQTcF (ΔΔQTcF) were analyzed with central-tendency and concentration-response analyses. In central-tendency analysis, moxifloxacin prolonged ΔQTcF and ΔΔQTcF with the largest mean values (90% confidence interval) of 12.1 ms (9.3, 14.8) and 15.4 ms (12.6, 18.1), respectively. Meanwhile, carotegrast methyl hardly altered ΔQTcF and ΔΔQTcF with the largest mean values of 0.8 ms (-2.3, 3.9) and 2.1 ms (-0.7, 4.8) for the low dose, and -0.2 ms (-3.4, 3.0) and 1.6 ms (-0.9, 4.2) for the high dose, respectively. In concentration-response analysis, moxifloxacin attained the estimated mean values for ΔQTcF and ΔΔQTcF of 11.4 ms (8.5, 14.4) and 16.7 ms (14.0, 19.4) at the mean Cmax, whereas carotegrast methyl provided those of -4.6 ms (-7.3, -1.9) and 0.7 ms (-1.4, 2.8), respectively. Thus, lack of placebo control did not influence the interpretation of TQT study with either of the analysis in line with updated E14/S7B Q&As.

Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging.

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

Natalizumab en el tratamiento de la esclerosis múltiple / Natalizumab in the treatment of multiple sclerosis

Revisar y actualizar la información sobre el mecanismo de acción del natalizumab y su eficacia en el tratamientode la esclerosis múltiple (EM). Desarrollo. El natalizumab, un anticuerpo humanizado monoclonal frente a la integrina alfa-4, se une a su receptor en la superficie de los linfocitos e impide la transmigración de éstos a las zonas inflamadasdel tejido cerebral. Además, parece que el natalizumab disminuye la activación de los linfocitos T que ocurre tras su infiltración en el parénquima cerebral y puede contribuir a la apoptosis de los linfocitos T en estos tejidos. Se han llevado a cabo dosgrandes ensayos clínicos multicéntricos de dos años de duración (AFFIRM y SENTINEL), que demuestran una eficacia superior a la conocida hasta ahora en la prevención de las recaídas y de la progresión de la EM. El natalizumab redujo la frecuencia anual de las recaídas en un 68 y 54% en estos ensayos respectivamente (p < 0,001). Además, también disminuyó significativamenteel riesgo de progresión de la discapacidad en un 42 y un 24%, respectivamente. Según los resultados del estudioAFFIRM, los acontecimientos adversos que fueron significativamente más frecuentes en el grupo tratado con natalizumab que en el grupo placebo fueron la fatiga (27 frente a 21%) y las reacciones alérgicas (9 frente a 4%). La incidencia de reaccionesde hipersensibilidad graves descritas como anafilácticas o anafilactoides fue baja (< 1%) y respondieron adecuadamente al tratamiento habitual. En el estudio SENTINEL se diagnosticaron dos casos de leucoencefalopatía multifocal progresiva(LMP), uno de ellos mortal, en el grupo tratado con natalizumab asociado a interferón beta-1a. Conclusiones. El natalizumab ha demostrado reducir el riesgo de progresión sostenida de discapacidad y la frecuencia de recaídas clínicamente detectadas en pacientes con EM remitente recurrente. A pesar de su bajo riesgo de producción de reacciones adversas, debe vigilarse alos pacientes tratados con natalizumab a intervalos regulares para detectar cualquier aparición o empeoramiento de signos o síntomas neurológicos que pudieran ser indicativos de LMP, y su empleo debe restringirse a las indicaciones aprobadas

To review and update the mechanism of action of natalizumab and its efficacy in the treatment of multiplesclerosis (MS). Development. Natalizumab, an anti-alfa-4 integrin monoclonal humanized antibody, binds to lymphocyte surface receptors to prevent transmigration of lymphocytes to areas of inflammation into the brain tissue. Furthermore, natalizumab appears to reduce T-cell activation following their infiltration of the brain parenchyma and may contribute toT-cell apoptosis in these tissues. Two large two-year, multicenter phase III trials (AFFIRM and SENTINEL) have been completed and demonstrate previously unseen efficacy in preventing MS relapses and disease progression. Natalizumabreduced the rate of clinical relapse at one year by 68 and 54% respectively in these trials (p < 0.001). Moreover, natalizumab reduced significantly the risk of sustained progression of disability by 42 and 24% respectively. Based on results from theAFFIRM study, the adverse events that were significantly more frequent in the natalizumab group than in the placebo group were fatigue (27 vs. 21%) and allergic reaction (9 vs. 4%). There was a low incidence (< 1%) of serious systemic hypersensitivityreactions described as anaphylactoid or anaphylactic, and they appear to be effectively managed by post-treatment observation and by timely and appropriate medical treatment. In the SENTINEL study, two cases of progressive multifocal leukoencephalopathy (PML), one of which was fatal, were diagnosed in natalizumab plus interferon beta-1a treated patients.Conclusions. Natalizumab reduced the risk of the sustained progression of disability and the rate of clinical relapse in patients with relapsing MS. In spite of their low risk of adverse reactions, patients must be monitored at regular intervals for any newor worsening neurological symptoms or signs that may be suggestive of PML, and natalizumab use must be restricted to the indicated patients

Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Murine neuronal progenitor cells are preferentially recruited to tumor vasculature via alpha4-integrin and SDF-1alpha-dependent mechanisms.

Recent studies have described neuronal progenitor cell recruitment to tumors in vivo, however, the mechanisms mediating this recruitment are not yet understood. When C17.2 murine neuronal progenitors stably expressing luciferase (C17.2-luc) were adoptively transferred into mice carrying subcutaneous Lewis lung carcinomas they accumulated at 1% injected dose/g of tumor tissue. C17.2-luc demonstrated significantly greater accumulation and transmigration on tumor-derived endothelium (TEC) than on normal endothelium under physiologically relevant flow conditions. Function blocking of alpha4-integrin reduced recruitment of C17.2-luc cells to normal endothelium but not to TEC, however, function blocking of SDF-1alpha reduced overall accumulation of C17.2-luc on TEC and specifically reduced transendothelial migration. Together, these data suggest that recruitment of C17.2-luc cells to TEC is mediated via SDF-1alpha/CXCR4 activation that results in modification of alpha4-integrin and results in improved recruitment of C17.2-luc cells.

Role of alpha4 integrin and its ligand VCAM-1 in the specific extravasation of a tumor-specific TH2 clone into tumor tissue that initiates its rejection.

The effectiveness of anticancer immunotherapeutic strategies involving the transfer of tumor-specific T cells depends on appropriate lymphocyte-endothelial cell interactions that facilitate the migration of lymphocytes into tumor. Here, we investigated the molecular mechanisms underlying the migration of the antigen-specific Th2 CD4(+) T-cell clone YS1093 into S1509a tumor tissue. YS1093 is specific for the S1509a tumor but does not recognize the S713a tumor. Transfer of YS1093 cells into mice bearing both S1509a and S713a tumors caused only the S1509a tumor to regress. This regression was markedly inhibited by pretreating YS1093 cells with an anti-alpha4 integrin MAb and administering an anti-VCAM-1 MAb at T-cell transfer. Since vascular endothelial cells in S1509a tumor tissues express VCAM-1 and the MHC class II (I-E(k)) molecule restricting YS1093 activity, labeled YS1093 cells migrated specifically into the S1509a tumor, and this migration was also blocked by the anti-TCRbeta F(ab')(2) and anti-I-E(k) MAbs. Furthermore, in vitro assays revealed that anti-CD3 MAb-mediated TCR cross-linkage initiated the binding of alpha4 integrin on YS1093 cells to VCAM-1. This adhesive activity was completely blocked by the anti-alpha4 integrin MAb. These results strongly suggest that i.v.-transferred YS1093 cells act in tumor regression by specifically recognizing their tumor antigen peptide in the context of I-E(k) on vascular endothelial cells in the S1509a tumor, which activates the binding of alpha4 integrin to VCAM-1 on the endothelial cells, facilitating YS1093 extravasation into the tumor. It is likely that this initial migration of specific CD4(+) T cells into tumor tissues promotes the subsequent infiltration into the tumor of other immunocytes that effect tumor destruction.

CD44, alpha(4) integrin, and fucoidin receptor-mediated phagocytosis of apoptotic leukocytes.

Various types of phagocytes mediate the clearance of apoptotic cells. We previously reported that human and murine high endothelial venule (HEV) cells ingest apoptotic cells. In this report, we examined endothelial cell fucoidin receptor-mediated phagocytosis using a murine endothelial cell model mHEV. mHEV cell recognition of apoptotic leukocytes was blocked by fucoidin but not by other phagocytic receptor inhibitors such as mannose, fucose, N-acetylglucosamine, phosphatidylserine (PS), or blocking anti-PS receptor antibodies. Thus, the mHEV cells used fucoidin receptors for recognition and phagocytosis of apoptotic leukocytes. The fucoidin receptor-mediated endothelial cell phagocytosis was specific for apoptotic leukocytes, as necrotic cells were not ingested. This is in contrast to macrophages, which ingest apoptotic and necrotic cells. Endothelial cell phagocytosis of apoptotic cells did not alter viable lymphocyte migration across these endothelial cells. Antibody blocking of CD44 and alpha4 integrin on the apoptotic leukocyte inhibited this endothelial cell phagocytosis, suggesting a novel function for these adhesion molecules in the removal of apoptotic targets. The removal of apoptotic leukocytes by endothelial cells may protect the microvasculature, thus ensuring that viable lymphocytes can successfully migrate into tissues.

Alpha 4 integrin increases anoikis of human osteosarcoma cells.

Cell motility, growth, and proliferation are regulated by adhesion to the extracellular matrix. Detachment of adherent cells from extracellular matrix results in induction of apoptosis ("anoikis"). Transformed cells often show an anchorage-independent growth that enables them to acquire a motile, invasive phenotype. This phenotype has been associated with the altered expression and function of the integrin family of transmembrane proteins that mediate cell adhesion to the extracellular matrix. Although alpha4 integrin is normally expressed on leukocyte subpopulations, a number of metastatic melanomas and sarcomas express it as well. In this study, we demonstrated the expression of alpha4 integrins on the human osteosarcoma cell line SAOS and on metastatic osteosarcoma lesions from the lung and pericardium. We further demonstrated that alpha4 integrin is coupled to the beta1 subunit by biochemical analysis and by using a mAb directed against a combinatorial epitope unique to the alpha4beta1 molecule. SAOS cells undergo anoikis when adherence is denied. Anoikis involved the activation of caspase 3 and the release of cytochrome c from mitochondria. Treatment of non-adherent SAOS with an anti-alpha4 mAb increased anoikis while anti-beta1 integrin mAbs did not alter anoikis, thus indicating a novel function for the alpha4 subunit in the control of cell death. Since integrins can control cell migration, proliferation, and apoptosis these results demonstrate a potential role for alpha4 integrin during multiple aspects of osteosarcoma metastasis.

VLA-4-dependent and -independent pathways in cell contact-induced proinflammatory cytokine production by synovial nurse-like cells from rheumatoid arthritis patients.

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.