Open-label phase II study of the efficacy of nivolumab for cancer of unknown primary.

BACKGROUND: Cancer of unknown primary (CUP) has a poor prognosis. Given the recent approval of immune checkpoint inhibitors for several cancer types, we carried out a multicenter phase II study to assess the efficacy of nivolumab for patients with CUP. PATIENTS AND METHODS: Patients with CUP who were previously treated with at least one line of systemic chemotherapy constituted the principal study population. Previously untreated patients with CUP were also enrolled for exploratory analysis. Nivolumab (240 mg/body) was administered every 2 weeks for up to 52 cycles. The primary endpoint was objective response rate in previously treated patients as determined by blinded independent central review according to RECIST version 1.1. RESULTS: Fifty-six patients with CUP were enrolled in the trial. For the 45 previously treated patients, objective response rate was 22.2% [95% confidence interval (CI), 11.2% to 37.1%], with a median progression-free survival and overall survival of 4.0 months (95% CI, 1.9-5.8 months) and 15.9 months (95% CI, 8.4-21.5 months), respectively. Similar clinical benefits were also observed in the 11 previously untreated patients. Better clinical efficacy of nivolumab was apparent for tumors with a higher programmed death-ligand 1 expression level, for those with a higher tumor mutation burden, and for microsatellite instability-high tumors. In contrast, no differences in efficacy were apparent between tumor subgroups based on estimated tissue of origin. Adverse events were consistent with the known safety profile of nivolumab. No treatment-related death was observed. CONCLUSIONS: Our results demonstrate a clinical benefit of nivolumab for patients with CUP, suggesting that nivolumab is a potential additional therapeutic option for CUP.

A randomized phase III trial of oral S-1 plus cisplatin versus docetaxel plus cisplatin in Japanese patients with advanced non-small-cell lung cancer: TCOG0701 CATS trial.

BACKGROUND: Platinum-based two-drug combination chemotherapy has been standard of care for patients with advanced nonsmall-cell lung cancer (NSCLC). The primary aim was to compare overall survival (OS) of patients with advanced NSCLC between the two chemotherapy regimens. Secondary end points included progression-free survival (PFS), response, safety, and quality of life (QoL). PATIENTS AND METHODS: Patients with previously untreated stage IIIB or IV NSCLC, an Eastern Cooperative Oncology Group performance status of 0-1 and adequate organ function were randomized to receive either oral S-1 80 mg/m(2)/day on days 1-21 plus cisplatin 60 mg/m(2) on day 8 every 4-5 weeks, or docetaxel 60 mg/m(2) on day 1 plus cisplatin 80 mg/m(2) on day 1 every 3-4 weeks, both up to six cycles. RESULTS: A total of 608 patients from 66 sites in Japan were randomized to S-1 plus cisplatin (n = 303) or docetaxel plus cisplatin (n = 305). OS for oral S-1 plus cisplatin was noninferior to docetaxel plus cisplatin [median survival, 16.1 versus 17.1 months, respectively; hazard ratio = 1.013; 96.4% confidence interval (CI) 0.837-1.227]. Significantly higher febrile neutropenia (7.4% versus 1.0%), grade 3/4 neutropenia (73.4% versus 22.9%), grade 3/4 infection (14.5% versus 5.3%), and grade 1/2 alopecia (59.3% versus 12.3%) were observed in the docetaxel plus cisplatin than in the S-1 plus cisplatin. There were no differences found in PFS or response between the two arms. QoL data investigated by EORTC QLQ-C30 and LC-13 favored the S-1 plus cisplatin. CONCLUSION: Oral S-1 plus cisplatin is not inferior to docetaxel plus cisplatin and is better tolerated in Japanese patients with advanced NSCLC. CLINICAL TRIAL NUMBER: UMIN000000608.

Association between skin toxicity and efficacy of necitumumab in squamous non-small-cell lung cancer: a pooled analysis of two randomized clinical trials-SQUIRE and JFCM.

BACKGROUND: Efficacy of necitumumab [recombinant human monoclonal antibody that blocks the ligand binding epidermal growth factor receptor (EGFR)] in patients with squamous (SQ) non-small-cell lung cancer (NSCLC) has been confirmed in two randomized clinical trials (SQUIRE and JFCM). This study evaluated the association between efficacy and initial skin toxicity with necitumumab treatment by analyzing pooled data from two clinical trials (SQUIRE and JFCM). MATERIALS AND METHODS: Data of 635 patients with SQ-NSCLC (intent-to-treat population) treated with necitumumab plus gemcitabine and cisplatin (N + GC) were pooled from two clinical trials (SQUIRE and JFCM). The relationship between skin toxicities developed by the end of the second cycle and efficacy was evaluated. Efficacy endpoints included overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Univariate and multivariate analyses were carried out for these endpoints. RESULTS: OS and ORR were associated with skin toxicity, whereas PFS was not. Patients with grade ≥2 or grade 1 skin toxicity had significantly longer OS compared to patients without skin toxicity (grade 0) in the N + GC group [median = 15.0 (grade ≥2); 12.7 (grade 1); 9.4 (grade 0) months; hazard ratio (HR) = 0.51 (grade ≥2 to grade 0); 95% confidence interval (CI) 0.40-0.64, P < 0.001 and HR = 0.64 (grade 1 to grade 0); 95% CI 0.52-0.80, P < 0.001]. In multivariate analysis, OS was significantly associated with skin toxicity. CONCLUSIONS: A significant association was found between necitumumab-induced skin toxicity and efficacy. These results are consistent with the previously reported association between other EGFR inhibitors-induced skin toxicity and efficacy.

MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are involved in essential biological activities, and have been reported to exhibit differential expression profiles in various cancers. Our previous study demonstrated that intercellular adhesion molecule-2 (ICAM2) inhibition induces radiosensitisation in oral squamous cell carcinoma (OSCC) cells. Thus, we hypothesised that certain miRNAs play crucial roles in radioresistance in OSCC by regulating ICAM2 expression. METHODS: Because predicted target gene analyses revealed that microRNA-125b (miR-125b) potentially regulates ICAM2 mRNA expression, we examined the association between miR-125b and radioresistance. The expression of miR-125b was investigated by real-time quantitative reverse transcriptase-PCR. For a functional analysis, miR-125b was transfected to OSCC-derived cells. RESULTS: A downregulated expression of miR-125b was found in OSCC-derived cell lines and OSCC samples. The miR-125b-transfected cells showed a decreased proliferation rate, enhanced radiosensitivity to X-ray irradiation and diminished ICAM2 mRNA expression. Moreover, miR-125b expression correlated with OSCC tumour staging and survival. CONCLUSION: These findings suggested that the downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signalling. Thus, controlling the expression or activity of miR-125b might contribute to suppressing proliferation and overcoming radioresistance in OSCC.

Feasibility of cytological diagnosis of sarcoidosis with endobronchial US-guided transbronchial aspiration.

BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a high diagnostic value in sarcoidosis if the obtained histological specimen is indicative of a non-caseating epithelioid-cell granuloma. However, EBUS-TBNA in sacoidosis sometimes affords solely cytological specimens. OBJECTIVE: To investigate the relevance of EBUS-TBNA cytology specimens in diagnosing sarcoidosis. DESIGN: The study population comprised 72 patients with sarcoidosis and 116 patients who had thoracic malignancies and intrathoracic lymphadenopathy but were eventually proven to be metastasis-free (controls). The EBUS-TBNA samples obtained for these subjects were blindly evaluated for the presence of epithelioid cell clusters by 2 independent cytoscreeners and a pathologist. RESULTS: Interobserver variability in the specimen grading was minimal. The sensitivity and specificity were 65.3% and 94.0%, respectively. The sensitivity was high, at 87.5%, for the combined cytological and histological examinations. Of 7 controls whose cytological specimens showed epithelioid cell clusters, 3 were also deemed positive for sarcoidosis on histological examination, which indicated that they had sarcoid reaction to cancer. CONCLUSIONS: Cytological evaluation of the EBUS-TBNA specimens had higher sensitivity than histological evaluation alone for intrathoracic lymphadenopathy due to sarcoidosis. It should be recognized, however, that up to 6% of patients with thoracic malignancy may have sarcoid reaction in non-metastatic lymph nodes.

Final overall survival analysis from the phase III J-ALEX study of alectinib versus crizotinib in ALK inhibitor-naïve Japanese patients with ALK-positive non-small-cell lung cancer.

BACKGROUND: Mature progression-free survival (PFS) data from the phase III J-ALEX study showed superiority for alectinib versus crizotinib [hazard ratio (HR) 0.37, 95% confidence interval (CI) 0.26-0.52; median PFS 34.1 versus 10.2 months, respectively] in advanced ALK (anaplastic lymphoma kinase)-positive non-small-cell lung cancer (NSCLC). Overall survival (OS) data were immature (HR 0.80, 99.8799% CI 0.35-1.82) at the time of data cut-off (30 June 2018). We report final OS data after ≥5 years of follow-up. PATIENTS AND METHODS: ALK inhibitor naive Japanese patients who were chemotherapy naive or had received one prior chemotherapy regimen were enrolled. Patients were randomized to receive alectinib 300 mg (n = 103) or crizotinib 250 mg (n = 104) twice daily until progressive disease, unacceptable toxicity, death, or withdrawal. The primary endpoint was independent review facility-assessed PFS, with OS (not fully powered) as a secondary endpoint. RESULTS: Median duration of OS follow-up was 68.6 months with alectinib and 68.0 months with crizotinib. Treatment with alectinib did not prolong OS relative to crizotinib (HR 1.03, 95.0405% CI 0.67-1.58; P = 0.9105). Five-year OS rates were 60.9% (95% CI 51.4-70.3) with alectinib and 64.1% (95% CI 54.9-73.4) with crizotinib. In total, 91.3% (n = 95/104) of crizotinib-treated patients and 46.6% (n = 48/103) of alectinib-treated patients received at least one subsequent anticancer therapy. After study drug discontinuation, 78.8% of patients in the crizotinib arm switched to alectinib, while 10.7% of patients in the alectinib arm switched to crizotinib as a first subsequent anticancer therapy. Patients randomized to crizotinib tended to switch treatment earlier than those randomized to alectinib. CONCLUSION: Final OS analysis from J-ALEX did not show superiority of alectinib to crizotinib; this result was most likely confounded by treatment crossover. Alectinib remains a standard of care for the treatment of patients with advanced ALK-positive NSCLC.

The effects of FasL on inflammation and tumor survival are dependent on its expression levels.

The apoptosis-inducing Fas ligand (FasL) is expressed in a variety of human cancers and has been implicated in tumor immune evasion. Paradoxically, ectopic expression of FasL in experimental tumors triggers a neutrophil-mediated inflammatory response and tumor rejection. To resolve these conflicting findings, we have established B16 melanoma and P29 Lewis lung carcinoma lines expressing different levels of FasL and examined their tumorigenicity in vivo. While tumors with a high level of FasL were rapidly rejected as previously reported, those expressing a low level of FasL were not rejected but grew faster than did FasL-negative parental cells. The growth enhancement of FasL(low) tumors was not observed in T-cell-deficient nude mice, suggesting that FasL expressed in tumors at low levels counteracted against T-cell-dependent antitumor responses. In support of this notion, FasL(low) tumors were found to grow faster than parental cells in mice that had acquired tumor-specific immunity. Furthermore, histological examinations revealed apoptosis of lymphocytes in tissue sections of FasL(low) tumors. These results collectively suggest that FasL on tumors is a double-edged sword: at high levels it triggers tumor rejection whereas at low levels it facilitates tumor growth possibly by suppressing antitumor immune responses.

Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u.

Four kinds of thermostable chitinase were isolated from the cell-free culture broth of Bacillus licheniformis X-7u by successive column chromatographies on Butyl-Toyopearl, Q-Sepharose, and Sephacryl S-200. We named the enzymes chitinases I(89 kDa), II(76 kDa), III(66 kDa) and IV(59 kDa). Chitinases II, III and IV possessed extremely high optimum temperatures (70-80 degrees C), showing remarkable heat stability. Chitinases II, III and IV produced (GlcNAc)2 and GlcNAc from colloidal chitin and chitinase I predominantly produced (GlcNAc)2. The action pattern of chitinase I on PN-(GlcNAc)4 also showed a stronger propensity to cleave off the (GlcNAc)2 unit from the non-reducing end than the other three chitinases. Chitinases II, III and IV catalyzed a transglycosylation reaction that converted (GlcNAc)4 into (GlcNAc)6.

Visualization of the GDP-dependent switching in the growth polarity of microtubules.

Microtubules are filamentous polar polymers with plus and minus ends. This polarity plays a crucial role in a variety of cellular functions such as chromosome movement and organelle transport. To examine the relationship between the growth polarity of microtubules and guanine nucleotide dependence, we polymerized microtubules from axonemes of sea urchin sperm flagella either with GTP or with GTP and GDP, and observed individual microtubules by dark-field microscopy. Tubulin concentrations were adjusted in each case to grow microtubules from only one end of each axoneme. The growth polarity of microtubules was determined using N-ethylmaleimide-modified tubulin (NEM-tubulin). In the presence of GTP only and at low tubulin concentrations, microtubules grew from the plus ends of axonemes. Surprisingly, in the presence of GTP and GDP, microtubules grew from the minus ends, even at high tubulin concentrations. To confirm these results, we used a perfusion chamber to monitor the growth polarity of microtubules from the same axoneme under different conditions. Exchanging a solution containing only GTP for one containing GTP and GDP elicited a switch in the growth polarity of microtubules from the plus ends to the minus ends. These results suggest that GDP directly affects microtubule polymerization and inverts microtubule growth polarity, probably by inhibiting microtubule growth at the plus ends.

Adipogenesis inhibitory factor. A novel inhibitory regulator of adipose conversion in bone marrow.

Recombinant adipogenesis inhibitory factor (AGIF) was purified to homogeneity from the conditioned medium of COS-1 cells transfected with human AGIF cDNA. The amino-terminal sequence analysis of the mature AGIF revealed that AGIF was produced as a precursor consisting of 199 amino acids and processed into a mature form of 178 amino acids by a cleavage between Ala(-1) and Pro(+1). The purified AGIF inhibited the process of adipogenesis in mouse 3T3-L1 preadipocytes, indicating that AGIF directly acts on the cells. AGIF acted as an adipogenic antagonist not only on the extramedullary cell line 3T3-L1 but also on the mouse bone marrow stroma-derived cell line H-1/A, suggesting that this cytokine may regulate adipogenesis in bone marrow.

Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with interleukin-11.

A cDNA encoding a novel adipogenesis inhibitory factor (AGIF) that inhibits the process of adipogenesis in mouse 3T3-L1 preadipocytes was cloned from a cDNA library of the human bone marrow-derived stromal cell line KM-102. The cloned cDNA contains an open reading frame coding for an AGIF precursor of 199 amino acids. Analysis of the sequence of this cDNA revealed identity of this factor with a recently reported novel cytokine, designated interleukin-11 (IL-11). AGIF/IL-11 may play an important role in stromal cell-associated hematopoeisis through its regulatory action on adipocyte differentiation in the bone marrow microenvironment.

Evaluation of metastatic ability at specific times during primary tumor growth: a novel spontaneous metastasis assay.

A transformed NIH 3T3 fibroblast cell line, Cl-e, normally does not produce spontaneous metastasis from subcutaneous or footpad tumors in nude mice. However, pulmonary tumor nodules are formed when more than 1 x 10(3) cells are injected intravenously into nude mice. Co-injection of 1 x 10(6) heavily irradiated and inactivated cells increases the clonogenic ability of the viable cells in that tumor colonies then occur with as few as 1 x 10(2) viable cells. Utilizing the action of these inactivated cells to enhance the lung colonizing ability of a relatively small number of viable tumor cells, we have developed a novel experimental model of spontaneous metastasis. In this model, a footpad tumor of the nude mouse metastasizes to the lungs following intravenous injection of 1 x 10(6) inactivated cells at a specific time of tumor growth and following tumor foot amputation, whereas no spontaneous metastasis develops without injection of inactivated cells. This model enables us to detect metastatic ability which would otherwise be too low to detect using other assays. In addition, it allows us to evaluate metastatic ability at a specific time point during primary tumor growth, since no metastases can develop during the periods before inactivated cell injection and after tumor amputation. Using this model, we have determined that the metastatic ability of Cl-e tumors in the footpad is constant throughout the exponential and stationary growth phases, even though cells isolated from exponentially growing tumors possess a 3.3-fold greater lung colonizing ability following intravenous injection than those from stationary tumors. This new experimental model may be applicable to other tumor cell lines and to other analyses where metastatic ability during a defined interval of tumor growth is of importance.

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts.

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2-4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.

Lattices of type I collagen select invasive variants of K-ras oncogene-transfected NIH3T3 with less cellular fibronectin.

A clone of NIH3T3 transformant (H3) can yield subcutaneous tumors and experimental pulmonary metastasis in nude mice. Compared to H3 in culture, the cells after in vivo tumor growth (H3-N) acquired enhanced tumorigenicity and metastatic ability. Also, indirect immunofluorescence revealed that cellular fibronectin (c-FN) of H3-N was decreased remarkably. We have studied the interactions between H3 and extracellular matrices to elucidate these phenomena. In the present study, we observed the effect of NIH3T3, H3, and H3-N cultured in type I collagen gel. Morphologically in the collagen gel, NIH3T3 assumed an extensive elongated fiber-like shape, H3 assumed a moderately elongated shape, and H3-N assumed a round or spindle shape with short pseudopodia. Compared to conventional cultures on dishes, cell proliferation of all three types was suppressed in collagen gel, but the degree of the suppression was least in H3-N. As a result, H3-N grew fastest in collagen gel. The variants which acquired growth advantage in the subcutaneum of mice also kept it in collagen gel. H3 cells were cultured in type I collagen gel for 4 weeks, a period comparable to that of tumor formation in nude mice. The cells after this long-term culture (H3-C) acquired enhanced tumorigenicity and metastatic ability nearly equal to that of H3-N. FACS analysis revealed that the c-FN of H3-C had decreased to a value comparable to that of H3-N. This means that type I collagen gel as well as subcutaneous tissues could select variants of H3 with less c-FN through proliferation. Moreover, it is suspected that lattices of type I collagen regulate cell proliferation of fibroblast via c-FN.

Aortic cholesterol esterase and other lysosomal enzyme activities in DOCA-salt, renal and spontaneous hypertension in the rat.

In spontaneously hypertensive rats, prolonged hypertension caused a decrease in aortic cholesterol esterase activity with N-acetyl-beta-D-glucosaminidase activity increased and acid phosphatase activity unchanged [3]. The present study was undertaken to compare these changes with those caused by other experimentally induced types of hypertension. Treatment with DOCA-salt for one month significantly elevated both aortic cholesterol esterase and acid phosphatase activities. In contrast, to spontaneous hypertension, venous changes were also observed. An intake of 1% NaCl ad libitum produced results similar to those with the DOCA-salt treatment, despite the fact that blood pressure did not increase. This suggested that humoral factors were the main cause of the elevated enzyme activities in DOCA-salt hypertension. In rats made hypertensive by unilateral renal arterial constriction with contralateral nephrectomy (one clip--one kidney hypertension) or without contralateral nephrectomy (one clip--two kidney hypertension), aortic cholesterol esterase activities were unchanged, while aortic N-acetyl-beta-D-glucosaminidase, and aortic and venous acid phosphatase activities were increased. These results show distinct differences in the response of lysosomal enzymes during the three hypertensive states.

Hemodynamic effects on aortic enzyme activities in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHRSP and SHR) and normotensive WKR were treated with hypotensive drugs, and arterial and venous enzyme activities were compared between treated and nontreated hypertensive groups. With the 4 month experiment, cholesterol esterase activity in the aorta from hypertensive SHRSP and SHR was significantly lower than that in the respective treated groups, whereas venous activity did not differ. By contrast, aortic NAGA activity was significantly higher in the hypertensive groups without any changes in venous activity. Acid phosphatase activity was unaltered. No effects of treatment were observed in the normotensive WKR. Accompanying a decrease in aortic cholesterol esterase, there was a marked increase in aortic cholesteryl esters accompanying hypertension. Aortic phosphodiesterase activity was significantly elevated in the hypertensive SHRSP and SHR compared with the respective treated groups. These results suggest that hypertension of long duration specifically decreased aortic cholesterol esterase activity with a consequent accumulation of cholesteryl esters in the aorta, and that this hemodynamic effect seemed to be partly mediated by cyclic AMP with an effect on the lysosomal membrane. These results could provide the biochemical bases for the relationship between hypertension and atherosclerosis.

A chemiluminescent detection of superoxide radical produced by adherent leucocytes to the subendothelium following thrombolysis: studies with a photochemically induced thrombosis model in the guinea pig femoral artery.

Reocclusion following thrombolysis is a major limitation of thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) because denuded vessel wall exposed to blood following thrombolysis is a favourable surface for platelet and leucocyte deposition. We have applied a chemiluminescence technique to detect superoxide radical (0(-2)) produced by leucocytes adherent to the femoral artery 24 h after photochemically induced thrombogenesis in the guinea pig in vivo and subsequent thrombolysis by rt-PA. Intravenous administration of MCLA, a specific chemiluminescence reagent for detecting O(-2), markedly increased photon emission. the photon emission was markedly potentiated by phorbol myristate acetate and was suppressed by superoxide dismutase. Reocclusion 24 h after rt-PA induced thrombolysis was observed in 10 of 16 animals. Histological observations revealed extensive polymorphonuclear leucocytes adherent to the vessel wall at the site of thrombogenesis and thrombolysis. A higher level of 0(-2) could be detected from the arteries in which thrombolysis was induced compared with those without thrombolysis. Further, the level 0(-2) detected was greater in reoccluded arteries compared with those in which reflow was established. These observations suggest that 0(-2) is produced by adherent leucocytes at the site of thrombolysis and that leucocytes are involved in reocclusion after thrombolysis.

GPIIb/IIIa integrin antagonists with the new conformational restriction unit, trisubstituted beta-amino acid derivatives, and a substituted benzamidine structure.

Ethyl N-[3-(2-fluoro-4-(thiazolidin-3-yl(imino)methyl)benzoyl)amino-2, 2-dimethylpentanoyl]piperidine-4-acetate 40 (NSL-96184) is a highly potent and orally active fibrinogen receptor antagonist, which is characterized by the presence of the trisubstituted beta-amino acid residue, 3-ethyl-2,2-dimethyl-beta-alanine. This compound was developed on the basis of the SAR study of N-[3-(N-4-amidinobenzoyl)amino-2, 2-dimethyl-3-phenylpropionyl]piperidine-4-acetic acid 1(NSL-95301) with the derivatization focused on the central trisubstituted beta-amino acid unit as well as the basic amidinobenzoyl unit, and the esterification of the carboxyl group for prodrug composition. Compound 1, which was reported in our previous study, was discovered by the application of combinatorial chemistry. The molecular modeling study suggests that the trisubstituted beta-amino acid unit is responsible for fixing the molecule to its active conformation. Compound 40 showed an excellent profile in the in vitro and in vivo studies for its human platelet aggregation inhibitory activity and oral availability in guinea pigs. This oral availability largely depends on the modification of the amidino group with a cyclic secondary amine, i.e., thiazolidine in 40. In in vivo studies, the onset of the antiplatelet action of 40 is very fast after oral administration, whereas its duration of action is relatively short. These results suggest that 40 has an excellent therapeutic potential, especially for antithrombotic treatment in the acute phase. 3-Substituted-2,2-dimethyl-beta-amino acid residues would serve as new and useful linear templates to restrict the conformational flexibility of peptidomimetics.

Comparison of the inhibitory effects of the TXA2 receptor antagonist, vapiprost, and other antiplatelet drugs on arterial thrombosis in rats: possible role of TXA2.

The antithrombotic effect of the thromboxane A2 receptor antagonist, vapiprost, was compared with those of other antiplatelet drugs using an arterial thrombosis model which utilized photochemical reaction in the rat femoral artery. Vapiprost prolonged the time required to occlude the artery with thrombus and inhibited collagen-induced rat platelet aggregation in whole blood ex vivo, in a dose-dependent manner. The potency ranking of antithrombotic effect was vapiprost > ketanserin (serotonin 5-HT2 receptor antagonist) >> ticlopidine (inhibitor of ADP-induced platelet aggregation) = dipyridamole (adenosine uptake inhibitor) > aspirin (cyclooxygenase inhibitor). On the other hand, the ranking of antiplatelet effect was ticlopidine > or = vapiprost > or = aspirin. Ketanserin and dipyridamole were ineffective. Relative to their antiplatelet effect, vapiprost and ketanserin had powerful antithrombotic effects. It is possible that the potent antithrombotic effects of vapiprost and ketanserin in vivo reflect the ability of these drugs to inhibit mediator-induced vascular contractions in addition to platelet aggregation. The results of the present study also suggest that TXA2 may play an important role in thrombogenesis in rats.

Comparison of antithrombotic effects of GPIIb-IIIa receptor antagonist and TXA2 receptor antagonist in the guinea-pig thrombosis model: possible role of TXA2 in reocclusion after thrombolysis.

The effects of Ro 44-9883, a new specific antagonist of platelet glycoprotein IIb-IIIa receptor, on thrombus formation and reocclusion after thrombolysis induced by tissue-type plasminogen activator (t-PA) were compared with those of vapiprost, a thromboxane (TX) A2 receptor antagonist, using a photochemically-induced thrombosis model in the guinea-pig femoral artery. Pretreatment with Ro 44-9883 (5, 10 and 20 micrograms/kg/min, i.v.) prolonged the time required to occlude the artery in a dose-dependent manner. Ro 44-9883 at 10 and 20 micrograms/kg/min significantly inhibited ex vivo platelet aggregation in whole blood induced by collagen, ADP or U46619. Vapiprost 0.3 mg/kg inhibited thrombus formation and platelet aggregation induced by collagen or U46619, to the same extent as Ro 44-9883 at the higher doses. In the thrombolysis study, Ro 44-9883 at the higher doses given as comedication with t-PA reduced the time to achieve reperfusion and increased the vascular patency after successful reperfusion. Vapiprost also significantly reduced the time to reperfusion and prevented reocclusion. However, the vascular patency after thrombolysis by t-PA with vapiprost was significantly increased compared with Ro 44-9883. Ro 44-9883 inhibited platelet aggregation, but did not prevent TXA2 formation in platelets. Thus, vascular contraction mediated by platelet-derived TXA2 may be responsible for lower efficacy of Ro 44-9883 against reocclusion compared with vapiprost. These results indicate that not only platelet aggregation but also vasoconstriction may contribute to reocclusion after t-PA-induced thrombolysis in the guinea-pig.