Phase I study of alpelisib (BYL719), an α-specific PI3K inhibitor, in Japanese patients with advanced solid tumors.

This phase I study aimed to determine tolerability and preliminary efficacy of single-agent alpelisib (BYL719) in Japanese patients with advanced solid malignancies. The primary objective of the study was to estimate the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) of oral alpelisib in patients with advanced solid tumors who had progressed despite standard therapy. The expansion part included patients with PIK3CA mutation/amplification; safety, preliminary efficacy, pharmacokinetic (PK)/pharmacodynamic profile, and food effect on the PK profile of alpelisib at the MTD/RP2D were determined. Oral alpelisib was given as a single agent on a continuous 28-day treatment cycle once daily. Overall, 33 patients received alpelisib. Dose-limiting toxicities were observed in 2 patients in the escalation part (at 400 mg/day) and 1 patient in the expansion part (at 350 mg/day). The RP2D of alpelisib was determined as 350 mg/day based on overall safety profile in the dose escalation part and previous data from a Western population; the MTD was not determined. The most common all-grade treatment-suspected adverse events were hyperglycemia and maculopapular rash (48.5% each) and diarrhea (45.5%). The PK of alpelisib in the Japanese population was similar to that reported in the Western population. The overall response rate, disease control rate, and median progression-free survival at 350 mg/day were 3%, 57.6%, and 3.4 months, respectively. Alpelisib as single agent showed a favorable safety profile and encouraging preliminary efficacy in Japanese patients with advanced solid tumors.

Phase I dose-escalation study of buparlisib (BKM120), an oral pan-class I PI3K inhibitor, in Japanese patients with advanced solid tumors.

Buparlisib (BKM120) is an oral pan-phosphatidylinositol 3-kinase inhibitor, targeting all four isoforms of class I PI3K (α, ß, γ and δ). This open-label Phase I dose-escalation study was conducted to determine the maximum tolerated dose of continuous daily buparlisib in Japanese patients with advanced solid tumors. Secondary objectives included safety and tolerability, pharmacokinetics, antitumor activity and pharmacodynamic marker changes. Fifteen patients were treated at 25 mg/day (n = 3), 50 mg/day (n = 3) and 100 mg/day (n = 9) dose levels. One dose-limiting toxicity of Grade 4 abnormal liver function occurred at 100 mg/day. Considering the safety profile and the maximum tolerated dose in the first-in-man study of buparlisib in non-Japanese patients, further dose escalation was stopped and 100 mg/day was declared the recommended dose. The most common treatment-related adverse events were rash, abnormal hepatic function (including increased transaminase levels), increased blood insulin levels and increased eosinophil count. Hyperglycemia was experienced by two patients, one Grade 1 and one Grade 4, and mood alterations were experienced by three patients, two Grade 1 and one Grade 2. Pharmacokinetic results showed that buparlisib was rapidly absorbed in a dose-proportional manner. Best overall response was stable disease for six patients, including one unconfirmed partial response. In these Japanese patients with advanced solid tumors, buparlisib had a manageable safety profile, with similar pharmacokinetics to non-Japanese patients. The recommended dose of 100 mg/day will be used in future studies of buparlisib in Japanese patients.

Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach.

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

Roles of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases 1 in acute encephalopathy following prolonged febrile seizures.

Prolonged febrile seizures may be followed by acute encephalopathy with neurological sequelae. To investigate the function of the blood-brain-barrier (BBB) in acute encephalopathy following prolonged febrile seizures with neurological sequelae (AEPFS), the concentrations of serum matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinases 1 (TIMP-1) were measured by ELISA in 10 children with AEPFS, 16 with prolonged febrile seizures without encephalopathy (PFS), 20 with simple febrile seizures (SFS), 23 with convulsive status epilepticus (CSE), and 18 with West syndrome. Serum MMP-9 levels in AEPFS and PFS patients were significantly higher than those in SPS and West syndrome patients and in controls, and those in CSE patients were significantly higher than in controls. Serum TIMP-1 levels in AEPFS patients were significantly lower than those in PFS, SFS, CSE and West syndrome patients and in controls. Serum MMP-9 levels and MMP-9/TIMP-1 ratios in AEPFS patients with motor paralysis were significantly higher than for those without motor paralysis. Our results suggest that prolonged seizures are related to high serum MMP-9 levels, and that an increased MMP-9/TIMP-1 ratio in AEPFS might induce dysfunction of the BBB. Furthermore, an imbalance of serum MMP-9 and TIMP-1 levels in patients with AEPFS may be associated with severe neurological sequelae.

Serum and CSF levels of cytokines in acute encephalopathy following prolonged febrile seizures.

It is well known that an acute encephalopathy occasionally follows prolonged febrile seizures. We measured the concentrations of interferon-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, IL-10, and soluble TNF receptor 1 (sTNFR1) in serum and CSF during the acute stage in 13 children with acute encephalopathy following prolonged febrile seizures (AEPFS) and 23 with prolonged febrile seizures without encephalopathy (PFS) to investigate the pathogenesis of AEPFS. Serum IL-6, IL-10, sTNFR1, and CSF IL-6 levels were significantly higher in AEPFS and PFS compared with control subjects. CSF IL-6 levels in AEPFS were significantly higher than those in PFS, but not serum IL-6, IL-10, or sTNFR1. The CSF IL-6 levels were significantly higher than the serum levels in AEPFS, but not PFS. The serum levels of sTNFR1 and IL-10 were significantly higher than those in the CSF in AEPFS and PFS. The serum IL-10 and sTNFR1 levels in patients who did not experience a second seizure were significantly higher than those in patients who experienced a second seizure, which was characterized by clusters of complex partial seizures several days after the initial prolonged febrile seizure. Our results suggest that serum IL-6, IL-10, TNF-alpha, and CSF IL-6 are part of the regulatory system of cytokines in AEPFS.

West syndrome associated with mosaic Down syndrome.

We report a girl with West syndrome associated with mosaic Down syndrome. She had repetitive tonic spasms at 6 months and an electroencephalography (EEG) showed hypsarrhythmia. Her facial appearance was normal and she had no minor anomalies. Her karyotype was mosaic(46,XX/47,XX,+21). Adrenocorticotropin (ACTH) therapy was effective, and her developmental quotient was 76 at 17 months. This report re-emphasize that chromosomal analysis is recommended for epileptic patients with infantile onset when the cause is unclear.

[Analysis of 11 patients with profound multiple disabilities who underwent laryngotracheal separation].

We examined the clinical usefulness of laryngotracheal separation surgery for patients with profound multiple disabilities (PMD). The subjects were 11 severely retarded children who experienced repeated incidents of aspiration pneumonia or who were enough to have aspiration pneumonia easily. A retrospective investigation of their medical records was performed regarding pre- and post-operative data, including the number of times sputum suctionings were required, the number of times pneumonia developed, respiratory conditions, and nutrition methods. The guardians were interviewed regarding musle tone, spasm, sleep quality, internal medications, and changes in mood. After surgery, improvement was confirmed in the number of times sputum suctionings were required, the incidence of pneumonia and respiratory conditions, and oral intake of food in three children. The guardians were aware of improvements in their children's sleep quality and mood. Laryngotracheal separation surgery can reduce the burden of health care for patients with PMD by improving their respiratory conditions and methods of nutrition intake.

CD44 binding through the hemopexin-like domain is critical for its shedding by membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environment through its proteolytic activity and promotes migration, invasion, and proliferation of tumor cells. During cell migration, MT1-MMP binds to CD44H, a major hyaluronan receptor, through the hemopexin-like (HPX) domain and localizes at the migration front. MT1-MMP is also responsible for shedding CD44H, which supports CD44H-mediated cell migration. In this study, we asked whether the binding of MT1-MMP to CD44H is a prerequisite step for the successive shedding. Deletion of the HPX domain deprived MT1-MMP of its shedding activity. Furthermore, disruption of the CD44H/MT1-MMP complex by overexpressing the HPX fragments resulted in inhibition of the shedding. Thus, the CD44H in the complex appears to be the direct substrate of MT1-MMP for shedding. Interestingly, other members of the MT-MMP family showed varied extents of CD44H shedding. Domain swapping between MT1-MMP and other MT-MMPs revealed that the ability of the HPX domains to bind CD44H is conserved among them. However, the shedding activity was different depending on the catalytic domains. The conserved binding ability of the HPX domains suggests that CD44H may act as a core molecule assembling multiple MT-MMPs on the cell surface.

Serum levels of matrix metalloproteinase-9 and its tissue inhibitor (TIMP-1) in acute disseminated encephalomyelitis.

In multiple sclerosis, there have been many reports on matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). However, MMPs and TIMPs have not been reported in acute disseminated encephalomyelitis (ADEM). We determined the relationship between the serum concentrations of MMP-9 and TIMP-1 and activity of lesions on MRI in 14 patients with ADEM to investigate the roles of MMP-9 and TIMP-1 in the pathogenesis of ADEM. Serum MMP-9 and TIMP-1 levels, measured by ELISA and gadolinium-enhanced (Gd+) brain MRI, were analyzed. Serum MMP-9 and TIMP-1 levels at the acute stage were higher than controls, and the serum MMP-9 levels at the acute stage were higher than those at the convalescent stage in ADEM. In seven patients with Gd+ lesions on brain MRI, serum MMP-9 levels and the MMP-9/TIMP-1 ratio at the acute stage were higher than those at the convalescent stage, and serum TIMP1 levels at the acute stage were lower than those at the convalescent stage. In seven patients without Gd+ lesions on brain MRI, serum TIMP-1 levels at the acute stage were higher than those at the convalescent stage. We speculated that MMP-9 is related to lesion formation at the early stage in ADEM and that TIMP-1 is induced to modulate MMP-9 activity. These findings suggest that MMP-9 and TIMP-1 secondarily play some roles in the inflammatory cascade of ADEM.

IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness.

IRSp53 has been characterized as an adaptor protein that links Rho-family small GTPases, such as Rac, to reorganization of the actin cytoskeleton. Here, we search for other binding partners for the IRSp53 SH3 domain and identify Eps8 as the major binding protein in fibroblasts and various cancer cell lines. Eps8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with Abi-1 and Sos-1, which seems essential for ruffling formation induced by oncogenic Ras. We confirm the IRSp53/Eps8 complex formation in vivo and the direct association between Eps8 NH(2)-terminal proline-rich sequence and IRSp53 SH3 domain. This complex synergistically activates Rac by reinforcing the formation of the Eps8/Abi-1/Sos-1 Rac-guanine nucleotide exchange factor complex, which mediates positive regulation of Rac activity. In addition, IRSp53/Eps8 complex formation as determined by fluorescent resonance energy transfer analysis, occurs at the leading edge of motile cells, and the motility and invasiveness of HT1080 fibrosarcoma cells are suppressed by inhibiting complex formation. These findings implicate the importance of the IRSp53/Eps8 complex in Rac activation and metastatic behavior of the malignant tumor cells.

Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase.

CD44 is a receptor for hyaluronan and mediates signaling that regulates complex cell behavior including cancer cell migration and invasion. Shedding of the extracellular portion of CD44 is the last step in the regulation of the molecule-releasing interaction between the ligand and cell. However, highly glycosylated forms of CD44 have hampered the identification of the exact cleavage sites for shedding and the responsible proteases. In this study, we found that expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) increased shedding of the 65-70 kDa CD44H (standard form) fragments and generated two additional smaller fragments. We purified the shed fragments and identified the cleaved sites by mass spectrometry. Specific antibodies that recognize the newly exposed COOH terminus by cleavage were prepared and used to analyze shedding at each site. Shedding of the 65-70 kDa fragments was inhibited by tissue inhibitor of metalloproteinase 3 (TIMP-3) but not by TIMP-1 and TIMP-2, suggesting involvement of a disintegrin and metalloproteinase (ADAM)-like proteases, although shedding is affected by MT1-MMP. Conversely, shedding of the two smaller fragments was inhibited by TIMP-2 and TIMP-3 but not TIMP-1, suggesting involvement of MT1-MMP itself. Shed fragments cleaved at these sites were also detected in human tumor tissues. Increased shedding at one of the MT1-MMP-sensitive sites was observed in the tumor compared with the surrounding normal tissue. However, no significant difference was observed with shedding by ADAM-like proteases. Thus, the cleavage sites for the shedding of CD44H were identified for the first time, and the results provide a basis for exploring the unknown biologic roles of shedding at different sites.

Sequence-specific silencing of MT1-MMP expression suppresses tumor cell migration and invasion: importance of MT1-MMP as a therapeutic target for invasive tumors.

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) has been believed a key enzyme in tumor invasion, because it is expressed in a variety of malignant human tumors, and overexpression of the enzyme enhances the ability of cellular invasiveness. However, it has not necessarily been clarified whether the endogenously expressed MT1-MMP in human tumors plays a critical role in their invasiveness. We used RNA silencing technology to downregulate the endogenous MT1-MMP expression in human tumor cells (fibrosarcoma HT1080 and gastric carcinoma MKN-28 cell lines), and evaluated the effect on the invasion of a reconstituted basement membrane (Matrigel). Transfection of a double-stranded RNA targeted to the MT1-MMP gene decreased the level of the enzyme to less than 10-20% without affecting production of other MMPs. According to the degree of silencing, activation of proMMP-2 was inhibited. CD44 shedding was also inhibited, but only in part. Decreased MT1-MMP levels were also reflected in reduced cell motility on hyaluronan (HA) and invasion in Matrigel. Thus, specific downregulation of MT1-MMP expression was sufficient to cause significant inhibition of the migration and invasion of tumor cells, even though other MMPs continued to be expressed.

Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy.

CD40 ligand (CD40L) is mainly expressed on activated platelets and CD4+T cells, and it can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L). Most sCD40L is derived from activated platelets. A previous paper revealed that the platelet number of patients with influenza virus-associated encephalopathy (IE) was correlated with the outcome. We determined the utility of sCD40L as a predictor for the prognosis of IE. We measured the serum concentration of sCD40L and the platelet number on the day of hospitalization in 34 patients with IE, 16 with influenza virus-associated febrile seizures (IFS), 19 with influenza virus infection without complications (Flu), and 7 with Epstein-Barr virus (EBV) infection. The serum sCD40L concentrations in IE and IFS were significantly lower than those in controls, Flu, and EBV infections. Serum sCD40L concentrations in the IE group were 0.70+/-0.43 ng/ml for deceased patients, 1.73+/-1.36 ng/ml for those with sequelae, and 3.85+/-2.91 ng/ml for those without sequelae. There was no significant difference in platelet number between IE patients with and without sequelae, while the platelet number of deceased patients with IE was significantly lower than in controls, Flu, and IFS. Serum sCD40L concentration on the day of hospitalization was more correlated with the outcome of IE than platelet number. Our findings suggest that the serum sCD40L concentration during acute IE is important for predicting the prognosis at an early stage.

[Low-dose therapy with carbamazepine for convulsions associated with mild gastroenteritis].

We investigated the effect of carbamazepine on convulsions associated with mild gastroenteritis. Sixteen infants and young children (aged 9 months to 3 years) who experienced repetitive convulsions associated with mild gastroenteritis were admitted to our hospital. We treated the sixteen affected patients with 5 mg/kg of carbamazepine once per day until the diarrhea had stopped. Thirteen of the sixteen patients were subjected to intravenous and/or suppository administration of diazepam (0.3-0.5 mg/kg/time), and one patient suppository administration of 0.5 mg/kg diazepam and 5.7 mg/kg phenobarbital before the administration of carbamazepine. In all patients who were given diazepam and/or phenobarbital, the convulsions recurred after the administration of these medicines. The convulsions occurred 2 to 8 times (mean, 4.1 times) before the administration of carbamazepine. Fifteen of the sixteen patients had no seizures after the administration of carbamazepine. One patient had one convulsion 15 min after the administration of carbamazepine. All patients were treated with 5 mg/kg of carbamazepine once per day until the diarrhea had stopped, i.e. for 2 to 9 days (mean, 6.4 days). Low dose therapy with carbamazepine once per day is thus effective for convulsions associated with mild gastroenteritis.

Phase I Study of Ceritinib (LDK378) in Japanese Patients with Advanced, Anaplastic Lymphoma Kinase-Rearranged Non-Small-Cell Lung Cancer or Other Tumors.

INTRODUCTION: Anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) is sensitive to ALK inhibitors, but resistance develops. This study assessed the maximum-tolerated dose, safety, pharmacokinetics (PK), and antitumor activity of ceritinib, a novel ALK inhibitor (ALKi), in Japanese patients with ALK-rearranged malignancies. METHODS: This phase I, multicenter, open-label study (NCT01634763) enrolled adult patients with ALK-rearranged (by fluorescence in situ hybridization and/or immunohistochemistry) locally advanced/metastatic malignancy that had progressed despite standard therapy. The study comprised two parts: dose escalation and dose expansion. Ceritinib (single-dose) was administered orally in the 3-day PK run-in period, then once daily, in 21-day cycles. Adaptive dose escalations were guided by a Bayesian model. RESULTS: Twenty patients (80% with ALKi treatment history [ALKi-pretreated]; 19 NSCLC; one inflammatory myofibroblastic tumor) received ceritinib 300 to 750 mg (19 during dose escalation, one in dose expansion). Two dose-limiting toxicities occurred: grade 3 lipase increase (600 mg); grade 3 drug-induced liver injury (750 mg). The most common adverse events were gastrointestinal (nausea: 95%; diarrhea, vomiting: 75%). Ceritinib PK profile was dose proportional across 300 to 750 mg dosages; steady state was reached by day 15. Overall response rate was 55% (11 of 20 patients). Among patients with NSCLC, partial response was observed in two of four ALKi-naive patients, five of nine crizotinib-pretreated patients, two of four alectinib-pretreated patients, and one of two crizotinib and alectinib/ASP3026 pretreated patients. The ASP3026-pretreated inflammatory myofibroblastic tumor patient achieved partial response. CONCLUSIONS: Ceritinib maximum-tolerated dose was 750 mg once daily in Japanese patients. Antitumor activity was observed irrespective of prior ALKi treatment history. Dose expansion, examining the activity of ceritinib in alectinib-resistant patients, is ongoing.

Analysis of cytokine levels in cerebrospinal fluid in mumps meningitis: comparison with echovirus type 30 meningitis.

BACKGROUND: It is unclear whether or not the CSF cytokine profiles in viral meningitis differ with the kind of causative virus. METHODS: We measured the concentrations of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-10 in CSF during the acute stage in 15 children with mumps meningitis (MM), and 34 with echovirus type 30 meningitis (EM). RESULTS: The CSF IFN-gamma, IL-2, IL-6, and IL-10 levels were elevated in MM, and the CSF IFN-gamma, IL-2, and IL-6 levels were elevated in EM. The CSF IFN-gamma, IL-2, and IL-10 levels in MM were significantly higher than those in EM (p<0.0001, p<0.0001, and p<0.0001, respectively). The CSF IL-6 levels in EM were significantly higher than those in MM (p=0.0255). The CSF TNF-alpha and IL-4 levels were not elevated in MM or EM. In MM, the IL-6 level was correlated with the IL-2 and IL-10 levels in CSF (p=0.0347 and p=0.0120, respectively). In EM, the IFN-gamma level was correlated with the IL-10 level in CSF (p=0.0002). CONCLUSION: CSF cytokine profiles in MM were different from those in EM. Therefore, it is likely that the pathogenesis of MM is different from that of EM.