Discovery of a potent dual inhibitor of wild-type and mutant respiratory syncytial virus fusion proteins through the modulation of atropisomer interconversion properties.

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.

Design and Synthesis of 2-(1-Alkylaminoalkyl)pyrazolo[1,5-a]pyrimidines as New Respiratory Syncytial Virus Fusion Protein Inhibitors.

Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infections and a significant pathogen for both adults and children. Although two drugs have been approved for the treatment of RSV infections, the low therapeutic index of these drugs have led pharmaceutical companies to develop safe and effective small-molecule anti-RSV drugs. The pyrazolo[1,5-a]pyrimidine series of compounds containing a piperidine ring at the 2-position of the pyrazolo[1,5-a]pyrimidine scaffold are known as candidate RSV fusion (F) protein inhibitor drugs, such as presatovir and P3. The piperidine ring has been revealed to facilitate the formation of an appropriate dihedral angle between the pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide bond for exertion of anti-RSV activity. A molecular-dynamic study on newly designed compounds with an acyclic chain instead of the piperidine ring proposed and demonstrated a new series of pyrazolo[1,5-a]pyrimidine derivatives, such as 9c with a 1-methyaminopropyl moiety, showing similar dihedral angle distributions to those in presatovir. Compound 9c exhibited potent anti-RSV activity with an EC50 value of below 1 nM, which was similar to that of presatovir. A subsequent optimization study on the benzene ring of 9c led to the potent RSV F protein inhibitor 14f with an EC50 value of 0.15 nM. The possibility of improving the biological properties of anti-RSV agents by modification at the 7-position of pyrazolo[1,5-a]pyrimidine is also discussed.

Quantitative diagnosis of diffuse alveolar damage using extravascular lung water.

OBJECTIVES: Acute respiratory distress syndrome is characterized by diffuse alveolar damage and increased extravascular lung water levels. However, there is no threshold extravascular lung water level that can indicate diffuse alveolar damage in lungs. We aimed to determine the threshold extravascular lung water level that discriminates between normal lungs and lungs affected with diffuse alveolar damage. DESIGN: A retrospective analysis of normal lungs and lungs affected with diffuse alveolar damage was performed. SETTING: Normal lung cases were taken from published data. Lung cases with diffuse alveolar damage were taken from a nationwide autopsy database. All cases of autopsy followed hospital deaths in Japan from more than 800 hospitals between 2004 and 2009; complete autopsies with histopathologic examinations were performed by board-certified pathologists authorized by the Japanese Society of Pathology. PATIENTS: Normal lungs: 534; lungs with diffuse alveolar damage: 1,688. INTERVENTIONS: We compared the postmortem weights of both lungs between the two groups. These lung weights were converted to extravascular lung water values using a validated equation. Finally, the extravascular lung water value that indicated diffuse alveolar damage was estimated using receiver operating characteristic analysis. MEASUREMENTS AND MAIN RESULTS: The extravascular lung water values of the lungs showing diffuse alveolar damage were approximately two-fold higher than those of normal lungs (normal group, 7.3±2.8 mL/kg vs diffuse alveolar damage group 13.7±4.5 mL/kg; p<0.001). An extravascular lung water level of 9.8 mL/kg allowed the diagnosis of diffuse alveolar damage to be established with a sensitivity of 81.3% and a specificity of 81.2% (area under the curve, 0.90; 95% CI, 0.88-0.91). An extravascular lung water level of 14.6 mL/kg represented a 99% positive predictive value. CONCLUSIONS: This study may provide the first validated quantitative bedside diagnostic tool for diffuse alveolar damage. Extravascular lung water may allow the detection of diffuse alveolar damage and may support the clinical diagnosis of acute respiratory distress syndrome. The best extravascular lung water cut-off value to discriminate between normal lungs and lungs with diffuse alveolar damage is around 10 mL/kg.

Disseminated aspergillosis associated with tsunami lung.

Many survivors of the tsunami that occurred following the Great East Japan Earthquake on March 11, 2011, contracted a systemic disorder called "tsunami lung," a series of severe systemic infections following aspiration pneumonia caused by near drowning in the tsunami. Generally, the cause of aspiration pneumonia is polymicrobial, including fungi and aerobic and anaerobic bacteria, but Aspergillus infection is rarely reported. Here we report a case of tsunami lung complicated by disseminated aspergillosis, as diagnosed during autopsy.

[Intraarterial chemotherapy combined with the concept of pharmacoangiogram using noradrenalin].

Abrams predicted that the pharmacoangiogram theory may improve intraarterial chemotherapy. We provide the following evidence to support this hypothesis from an experimental study on Walker-256 xenograft tumors: (1) selective drug delivery to tumor vessels, (2) increased injection pressure, and (3) low tumor blood flow, which are induced by noradrenalin to enhance anti-tumor activity. Walker-256 tumors are divided into 2 groups, hypovascular and hypervascular tumors that arise 2 and 4 days after tumor inoculation, respectively. Noradrenalin mediates anti-tumor activity against hypervascular tumors. The concentrations of Evans blue in Walker-256 tumors are increased in the hypervascular phase and decreased in the hypovascular phase by 4 µg/mL noradrenalin. These data indicate that norepinephrine may decrease capillary circulation, which may increase the blood flow to hypervascular tumor tissues that lack sphincter muscles due to the contraction of precapillary vessels. Mitomycin C concentrations in hypervascular tumors are increased in tumor tissues and decreased in normal tissues. A higher injection pressure than interstitial pressure(20 mmHg) in hypervascular tumors may improve the delivery of antitumor agents to the tumor interstitium and enhance anti-tumor activity. Low blood flow and reperfusion of the tumor due to the contraction reflex of the tumor-feeding artery enhance anti-tumor activity against hypervascular tumors induced by continuous infusion of noradrenalin. Therefore, noradrenalin decreased the tumor tissues blood flow and induced the tumor necrosis.

Validation of extravascular lung water measurement by single transpulmonary thermodilution: human autopsy study.

INTRODUCTION: Gravimetric validation of single-indicator extravascular lung water (EVLW) and normal EVLW values has not been well studied in humans thus far. The aims of this study were (1) to validate the accuracy of EVLW measurement by single transpulmonary thermodilution with postmortem lung weight measurement in humans and (2) to define the statistically normal EVLW values. METHODS: We evaluated the correlation between pre-mortem EVLW value by single transpulmonary thermodilution and post-mortem lung weight from 30 consecutive autopsies completed within 48 hours following the final thermodilution measurement. A linear regression equation for the correlation was calculated. In order to clarify the normal lung weight value by statistical analysis, we conducted a literature search and obtained the normal reference ranges for post-mortem lung weight. These values were substituted into the equation for the correlation between EVLW and lung weight to estimate the normal EVLW values. RESULTS: EVLW determined using transpulmonary single thermodilution correlated closely with post-mortem lung weight (r = 0.904, P < 0.001). A linear regression equation was calculated: EVLW (mL) = 0.56 × lung weight (g) - 58.0. The normal EVLW values indexed by predicted body weight were approximately 7.4 ± 3.3 mL/kg (7.5 ± 3.3 mL/kg for males and 7.3 ± 3.3 mL/kg for females). CONCLUSIONS: A definite correlation exists between EVLW measured by the single-indicator transpulmonary thermodilution technique and post-mortem lung weight in humans. The normal EVLW value is approximately 7.4 ± 3.3 mL/kg. TRIAL REGISTRATION: UMIN000002780.

Discovery of a Potent Dual Inhibitor of Wild-Type and Mutant Respiratory Syncytial Virus Fusion Proteins.

A novel series of macrocyclic pyrazolo[1,5-a]pyrimidine derivatives as respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors were designed and synthesized based on docking studies of acyclic inhibitors. This effort resulted in the discovery of several macrocyclic compounds, such as 12b, 12f, and 12h, with low nanomolar to subnanomolar activities against the wild-type RSV F protein A2. In addition, 12h showed a single-digit nanomolar potency against the previously reported drug-resistant mutant D486N. Molecular modeling and computational analyses suggested that 12h binds to the D486N mutant while maintaining a rigid bioactive conformation via macrocyclization and that it interacts with a hydrophobic cavity of the mutant using a new interaction surface of 12h. This report describes the rational design of macrocyclic compounds with dual inhibitory activities against wild-type and mutant RSV F proteins.

Design, synthesis, and biological evaluation of artificial macrosphelides in the search for new apoptosis-inducing agents.

Various artificial macrosphelides were designed and synthesized, including ring-enlarged analogues and epothilone-hybrid compounds. Syntheses were accomplished in an efficient manner by using a ring-closing metathesis (RCM) strategy in a key macrocyclization step. Biological evaluation of these new macrosphelide-based derivatives revealed that several epothilone hybrids, in which a thiazole-containing side chain was incorporated, exhibited potent apoptosis-inducing activity toward human lymphoma cells. These activities were considerably enhanced relative to those of natural macrosphelide compounds. Structure-activity relationship studies revealed that the "ene-dicarbonyl" substructure is apparently essential for bioactivity.

Nivolumab therapy for metastatic collecting duct carcinoma after nephrectomy: A case report.

RATIONALE: Collecting duct carcinoma (CDC) is a rare type of nonclear renal cell carcinoma, often presenting at an advanced stage of the disease, and standard treatment guidelines have not been established. PATIENT CONCERNS: A 73-year-old man was admitted to our hospital with complaints of fever and lower right back pain. DIAGNOSES: Computed tomography revealed a poorly defined tumor of the right kidney without metastasis. The patient underwent right radical nephrectomy and was diagnosed with clinical stage T1bN0M0 renal cancer; the pathological findings showed collecting duct carcinoma. INTERVENTIONS: After nephrectomy, multiple lung metastases were found in the following month, so first-line chemotherapy of gemcitabine (1000 mg/m on days 1 and 8, every 21 days) and cisplatin (70 mg/m on day 2, every 21 days) was administered. Due to disease progression, targeted therapy with axitinib (10 mg/body) and second-line chemotherapy of paclitaxel (200 mg/m on day 1, every 21 days) and carboplatin (area under the curve of 6 on day 1, every 21 days) were subsequently administered. However, the lung metastases progressed and new metastases spread to the right adrenal gland, liver, and lymph nodes. Based on the high expression of programmed death-ligand 1 in tumor cells, we treated the patient with the immune checkpoint inhibitor nivolumab. OUTCOMES: After 2 courses of treatment, he experienced a partial response and improved performance status, and thus was discharged from the hospital. To date, the patient is on his fifth course of treatment as an outpatient without disease progression. LESSONS: The findings of our study suggest that nivolumab may be effective even if the patient has highly progressive CDC with a low PS, if PD-L1 is highly expressed in the tumor cells.

Carbohydrate status detecting by PNA is changeable through cancer prognosis from primary to metastatic nodal site: A possible prognostic factor in patient with node-positive lung adenocarcinoma.

Carbohydrates antigens in cancer cells are considered to be important molecules, which may play a critical role for metastasis. To elucidate the prognostic relevance of the expression of peanut agglutinin (PNA) binding carbohydrates in patients with lung adenocarcinoma, we investigated the PNA binding carbohydrates immunohistochemically in both of primary tumors and involving nodal lesions. A total of 62 patients with node-positive primary lung adenocarcinoma, who had undergone complete resection and regional nodes dissection were subjected to this study. There were no significant correlations between PNA staining rates and clinicopathological variables. The survival rate of patients who had positive PNA staining in both of primary tumor and nodal lesion was significantly higher than those of patients in the other groups. Furthermore, the loss of the staining rate of PNA was an independent prognostic factor beside the lymphatic vessel invasion using multivariate analysis. The expression of PNA binding carbohydrates in tumor tissue and nodal lesion would be a novel significant prognostic factor for patients with node-positive lung adenocarcinomas.

Incidentally detected splenogonadal fusion in a laparoscopic transabdominal preperitoneal hernia repair operation: A case report.

INTRODUCTION: Splenogonadal fusion (SGF) is a rare congenital malformation in which the spleen is connected to the gonad. Few SGF cases have been reported in the English scientific literature, and we are unaware of any previous case reports of SGF with inguinal hernia by laparoscopic transabdominal preperitoneal hernia repair (TAPP). Here, we report a case of SGF that was incidentally detected during a TAPP procedure, with an uneventful postoperative course without complications. PRESENTATION OF CASE: A 76-year-old male presented with a 10-year history of left inguinal swelling. He was diagnosed with a left inguinal hernia, and we performed TAPP. Laparoscopy revealed the left inguinal hernia and two reddish-purple masses, one located close to the left inguinal ring. A cord of soft tissue extended cranially from the mass to the spleen, and passed through the left internal inguinal ring caudally. We cut the cord for mesh placement and to make an accurate diagnosis of the mass. Pathological and intraoperative findings indicated a diagnosis of continuous SGF. DISCUSSION: We observed two important clinical issues in this case. First, the potential for incidental diagnoses of SGF may be increasing. Second, to our knowledge, this is the first case report of a patient with SGF identified by TAPP. Such a therapeutic strategy for incidentally detected SGF has not been described; here we report a successful experience. CONCLUSION: To our knowledge, this is the first report of a patient with SGF diagnosed by a TAPP procedure. The postoperative course was uneventful using our method.

Prognostic impact of p53 protein overexpression in patients with node-negative lung adenocarcinoma.

Prognostic value of p53 protein expression in node-negative lung adenocarcinoma is still controversy. The expression of p53 protein was examined immunohistochemically in lung adenocarcinoma using monoclonal antibody BP53-12. A total 131 cases of primary lung adenocarcinoma were examined. Relationship between expression of p53 protein and clinicopathologic factors were studied. Overexpression of p53 protein was found in 19 patients (14.5%). Univariate and multivariate analysis showed that overexpression of p53 protein was an independent prognostic factor in node-negative lung adenocarcinoma. p53 alteration could be a valuable predictor for prognosis in node-negative lung adenocarcinoma.

Synthesis of macrosphelides with a thiazole side chain: new antitumor candidates having apoptosis-inducing property.

Hybrid compounds of macrosphelides and epothilones, both of which are natural macrolides having a 16-membered skeleton, were designed and synthesized using a ring-closing metathesis (RCM) strategy. Some of these hybrids were found to exhibit notable apoptosis-inducing activity against human lymphoma cells with higher potency than parent natural macrosphelides, and to be a promising lead compound for development of a new antitumor agent.

Organ Preference of Cancer Metastasis and Metastasis-Related Cell Adhesion Molecules Including Carbohydrates.

This review starts on one of our special interests, the organ preference of metastasis. We examined data on 1,117 autopsy cases and found that the organ distribution of metastasis of cancers of the lung, pancreas, stomach, colon, rectum, uterine cervix, liver, bile duct, and esophagus involved the lung, liver, adrenal gland, bone/bone marrow, lymph node, and pleura/peritoneum. Cancers of the kidney, thyroid, ovary, choriocarcinoma, and breast, however, manifested different metastatic patterns. The distribution of leukemia and lymphoma metastases was quite different from that of epithelial cancers. On the basis of experimental studies, we believe that the anatomical-mechanical hypothesis should be replaced by the microinjury hypothesis, which suggests that tissue microinjury induced by temporal tumor cell embolization is crucial for successful metastasis. This hypothesis may actually reflect the so-called inflammatory oncotaxis concept. To clarify the mechanisms underlying metastasis, we developed an experimental model system of a rat hepatoma AH7974 that embraced substrate adhesiveness. This model did not prove a relationship between substrate-adhesion potential and metastatic lung-colonizing potential of tumor cells, but metastatic potential was correlated with the expression of the laminin carbohydrate that was recognized by Griffonia (Bandeiraea) simplicifolia isolectin G4. Therefore, we investigated the relationship between carbohydrate expression profiles and metastasis and prognosis. We indeed found an intimate relationship between the carbohydrate expression of cancer cells and the progression of malignant tumors, organ preference of metastasis, metastatic potential of tumor cells, and prognosis of patients.

Cancer metastasis: characterization and identification of the behavior of metastatic tumor cells and the cell adhesion molecules, including carbohydrates.

This review focuses on the behavior of metastatic tumor cells and their specific adhesion molecules. Much of this review is based on the results from our researches over many years. Electron microscopic investigations of metastatic processes have demonstrated that desmosomes, tight junctions, or cell fusion-like structures are formed between tumor cells and other cells such as endothelial, mesothelial, hepatic, and nerve cells. These findings suggest that metastatic tumor cells acquire specific cell adhesion or recognition systems. To investigate these adhesion mechanisms, we established floating sublines from rat ascites hepatoma AH7974 in vitro and compared their adhesion molecules with those of their adherent counterparts. The anchorage independence of these tumor cells can be explained by reduced production of extracellular matrix proteins, decreased expression of cell surface integrin(s), or lack of heparan sulfate proteoglycans on the cell surfaces. Although the metastatic potential of these sublines for lung could not be explained by these properties, it may be explained by expression of 56 and 62 kDa laminin-like substances containing Griffonia Bandeirea simplicifolia isolectin B4-binding carbohydrate(s). We examined the relationship between carbohydrate expression and metastasis of human breast, pulmonary, colorectal, gastric, and other cancers by using a panel of lectins and monoclonal antibodies (MAbs). These studies revealed that several lectins and MAbs such as Vicia villosa agglutinin (VVA), Helix pomatia agglutinin, anti-sialyl Lewis(x-i) MAb, and others were useful not only for predicting metastasis and the prognosis of patients but also for understanding the routes of metastatic spread. VVA-binding carbohydrates, i.e. N-acetyl-D-galactosamine residues, especially those carried by atypical MUC1 protein, in aggressive cancer cells may serve as an important drug target.

Carbohydrate expression profile of colorectal cancer cells is relevant to metastatic pattern and prognosis.

Carbohydrate expression of cancer cells is closely related to the metastatic nature of colorectal cancer. In the present study we investigated the relevance of carbohydrate expression profiles of colorectal cancer cells in the primary lesion to metastatic distribution patterns as well as prognosis in 134 cases. Carbohydrate expression was estimated by histochemistry with 17 kinds of lectins and 3 kinds of Lewis-related monoclonal antibodies (MAbs), and correlations between the staining and clinicopathological parameters were examined. The results showed that lymphatic invasion, lymph node metastasis, and peritoneal metastasis correlated with staining with lectins that bind galactose/N-acetylgalactosamine residues (Gal/GalNAc) such as Maclura pomifera (MPA), Arachis hypogaea (PNA), Helix pomatia (HPA), and Vicia villosa (VVA). In contrast, hepatic metastasis correlated with staining with Anguilla anguilla lectin (AAA), anti-LewisX (LEX-2), anti-sialyl Lewisa (NS 19-9), and anti-sialyl-dimeric LewisX (FH-6) MAbs, all of which bind preferentially to fucosylated carbohydrate chains. The five-year survival rate of patients was related to the staining of cancers with MPA, HPA, FH-6 or NS19-9, and MPA- and FH-6 staining were independent prognostic factors. We conclude that carbohydrate expression profiles of cancer cells are relevant to the route of tumor cell dissemination, metastatic pattern as well as prognosis of colorectal cancer.

Expression of peanut agglutinin-binding carbohydrates correlates with nodal involvement in human lung adenocarcinoma.

The expression of 15 kinds of lectin-binding carbohydrates was examined histochemically in lung adenocarcinoma. The relation between expression of lectin binding carbohydrates and clinicopathologic factors was studied. The expression of peanut agglutinin (PNA) binding carbohydrates showed a high lymph node metastatic rate. Relation between PNA-binding carbohydrate expression and lymphatic vessel invasion was also recognized. Multivariate analysis showed that PNA-binding carbohydrate expression was an independent predictive factor of lymph node metastasis (odds ratio: 2.8) and of lymphatic vessel invasion (odds ratio: 5.2). The expression of PNA-binding carbohydrates would be a significant predictive factor for lymph node metastasis of lung adenocarcinomas.

UDP-GlcNAc2-epimerase regulates cell surface sialylation and cell adhesion to extracellular matrix in Burkitt's lymphoma.

Alteration of cell surface glycoconjugates plays a crucial role in many biological phenomenon, including invasion and metastasis. In the present study, we investigated the relationship between cell surface sialylation and the adhesive properties of two clones (3G3 and 3D2) of a human Burkitt's lymphoma cell line, HBL-8 to extracellular matrix. By flow cytometric analysis we found that the cell surface of 3G3 clone was highly sialylated and that of the 3D2 clone were hyposialylated. Moreover, cell surface sialic acid content was significantly greater in the 3G3 clone than in the 3D2 clone. In vitro adhesion assays showed that cell surface sialylation in the 3G3 clone cells might reduce their attachment to collagen type IV and fibronectin, compared to 3D2 clone. Sialic acid metabolic complementation assays using several precursors of sialic acid suggested that hyposialylation in the 3D2 clone resulted from low activities of uridine diphosphate-N-acetylglucosamine-2-epimerase (UDP-GlcNAc2-epimerase) which is a key enzyme in sialic acid biosynthesis. From RT-PCR analysis the expression of UDP-GlcNAc2-epimerase mRNA was found to be correlated with sialic acid content in the two clones of HBL-8. Therefore, expression of UDP-GlcNAc2-epimerase mRNA may regulate the expression of sialoglycoconjugates which affect the adhesion of lymphoma cells to collagen type IV and fibronectin.

New strategy for the total synthesis of macrosphelides a and B based on ring-closing metathesis.

[reaction: see text] A new total synthesis of macrosphelides A and B using ring-closing metathesis (RCM) as a macrocyclization step is described. The substrate of the RCM could be synthesized from readily available chiral materials, methyl (S)-(+)-3-hydroxybutyrate and methyl (S)-(-)-lactate, with a high efficiency. The RCM proceeded in the presence of Grubbs' Ru-complex, providing a new effective synthetic route to these natural products.

Alpha-2,6-sialylation of L-PHA reactive oligosaccharides and expression of N-acetylglucosaminyltransferase V in human diffuse large B cell lymphoma.

Cell surface sialylation and beta1-6 branching of L-PHA reactive oligosaccharides play an important role in metastatic capacities of various tumor cell lines. We analyzed the expression and sialylation of L-PHA reactive oligosaccharides in human diffuse large B cell lymphoma (DLBCL). DLBCL was grouped into three types; i). Group A, non-reactive type with no expression of L-PHA reactive oligosaccharides, ii). Group B, sialylated type with expression of sialylated L-PHA reactive oligosaccharides and iii). Group C, non-sialylated type with expression of non-sialylated L-PHA reactive oligosaccharides. To clarify the linkage of sialic acid residues in L-PHA reactive oligosaccharides of Group B cases, L-PHA lectin histochemistry after treatment with two different neuraminidases was performed. In all Group B cases, L-PHA binding reactivity was found after treatment with Vibrio cholerae neuraminidase. But not after treatment with Newcastle disease virus neuraminidase. These data indicate that alpha2,6-linked sialic acid residues were predominantly involved in sialylation of L-PHA reactive oligosaccharides of Group B. To clarify the relationship between expression of N-acetylglucosaminyltransferase V (GnT-V), which catalyzes beta1-6 branching of L-PHA reactive oligosaccharides, and L-PHA reactivities in DLBCL, we investigated the expression of GnT-V using immunohistochemical methods. Most of the Group B and C cases expressed GnT-V while 33% of Group A cases showed no expression of GnT-V. These data suggest that expression of GnT-V is not always correlated with the expression of L-PHA reactive glycoconjugates. Furthermore, survival of patients in Group A which showed no expression of GnT-V was significantly shorter than that of patients in Group C which expressed GnT-V. Therefore, loss of non-sialylated L-PHA reactive oligosaccharides due to lack of expression of GnT-V in lymphoma cells may be associated with aggressiveness of DLBCL.