Discovery and pharmacological characterization of novel positive allosteric modulators acting on skeletal muscle-type nicotinic acetylcholine receptors.

Skeletal muscle-type nicotinic acetylcholine receptors (m-nAChRs) are ligand-gated ion channels that open after activation by ACh and whose signals cause muscle contraction. Defects in neurotransmission are reported in disorders such as myasthenia gravis (MG) and congenital myasthenia syndromes (CMS). Although treatments for these disorders exist, therapies which significantly increase muscle strength have yet to be reported. Positive allosteric modulators (PAMs), which promote ACh signaling through AChRs, are expected to be promising therapeutic agents. In this study, we identified an m-nAChR PAM called AS3513678 by high-throughput screening using human myotube cells and modified it to obtain novel compounds (AS3566987 and AS3580239) that showed even stronger PAM activity. AS3580239 caused a leftward shift in the ACh concentration-response curve and was 14.0-fold potent at 10 µM compared with vehicle. Next, we examined the effect of AS3580239 on electrically-induced isometric contraction of the extensor digitorum longus (EDL) muscle in wild-type (WT) and MG model rats. AS3580239 enhanced EDL muscle contraction in both WT and MG model rats at 30 µM. These data suggest that AS3580239 improved neurotransmission and enhanced muscle strength. Thus, m-nAChR PAMs may be a useful treatment for neuromuscular diseases.

Discovery of highly potent and selective biphenylacylsulfonamide-based beta3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the beta2-adrenergic receptor: part 6.

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human beta(3)-adrenergic receptor (AR) agonists. Result of structure-activity relationships (SAR) and cassette-dosing evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray structure of the beta(2)-adrenergic receptor.

Discovery of a novel series of biphenyl benzoic acid derivatives as potent and selective human beta3-adrenergic receptor agonists with good oral bioavailability. Part I.

A novel class of biphenyl analogues containing a benzoic acid moiety based on lead compound 8i have been identified as potent and selective human beta 3 adrenergic receptor (beta 3-AR) agonists with good oral bioavailability and long plasma half-life. After further substituent effects were investigated at the terminal phenyl ring of lead compound 8i, we have discovered that more lipophilic substitution at the R position improved potency and selectivity. As a result of these studies, 10a and 10e were identified as the leading candidates with the best balance of potency, selectivity, and pharmacokinetic profiles. In addition, compounds 10a and 10e were evaluated to be efficacious for a carbachol-induced increase of intravesical pressure, such as an overactive bladder model in anesthetized dogs. This represents the first demonstrated result dealing with beta 3-AR agonists.

Discovery of novel series of benzoic acid derivatives containing biphenyl ether moiety as potent and selective human beta(3)-adrenergic receptor agonists: Part IV.

Identification and SAR study of novel series of beta(3)-AR agonists with benzoic acid are described. Conversion of ether linkage position of phenoxybenzoic acid derivative 2b led to compound 7b with moderate beta(3)-AR activity. Further modification in right, center and left parts of compound 7b was investigated to improve the beta(3)-AR potency and selectivity. Compounds 7g and 7k, with the bulky aliphatic-substituted group at 2-position of benzoic acid moiety, were identified as potent and selective beta(3)-AR agonists. In addition, in vivo efficacy of compounds 7g and 7k was exhibited on dog OAB model.

The selective Na+-Ca2+ exchange inhibitor attenuates brain edema after radiofrequency lesion in rats.

2-[4-[(2,5-Difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a specific inhibitor of the Na+-Ca2+ exchanger, exerts cytoprotective action and reduces brain infarct volume after cerebral ischemia. We examined the effect of SEA0400 on vasogenic brain edema in rats. Histological observations showed that radiofrequency current caused brain infarct and extravasation of endogenous albumin in the brain. SEA0400 (3 and 10 mg/kg, i.v.) significantly suppressed the increase in brain water content with attenuation of Evans blue dye and sodium fluorescein extravasation after radiofrequency lesion. The findings suggest that the Na+-Ca2+ exchanger plays a role in vasogenic edema formation after radiofrequency lesion.

Mast cell infiltration around gastric cancer cells correlates with tumor angiogenesis and metastasis.

BACKGROUND: Increased numbers of mast cells are found in various solid tumors. To investigate the role of mast cells in the vicinity of gastric cancer cells, we used special staining and an immunohistochemical technique.METHODS: Specimens were surgically obtained from 102 patients with gastric cancer. Mast cells around the tumor edge of gastric cancer nests were counted by staining with 0.05% toluidine blue solution. Blood vessels in these areas were also counted, by immunohistochemical staining of endothelial cells for factor VIII.RESULTS: The average number of mast cells and blood vessels in gastric cancer specimens was significantly higher than that in normal gastric tissue. Specimens from patients with advanced disease with metastases to lymph nodes had more mast cells than specimens from patients with early-stage disease. Mast cells in specimens from patients with metastatic lymph nodes were significantly increased in comparison with numbers in specimens from those without nodal metastases. Mast cell numbers in the specimens of patients with lymphatic or blood vessel invasion were significantly higher than numbers in specimens from patients without such invasion. Mast cells were localized near the new vessels around gastric cancer cells. Mast cell numbers increased as the number of blood vessels increased (correlation coefficient, 0.783). Postoperative survival curves revealed that patients with increased numbers of mast cells had a poor prognosis.CONCLUSIONS: All these results suggest that mast cell accumulation at the tumor site may lead to increased rates of tumor vascularization and, consequently, increased rates of tumor growth and metastasis.

Primary squamous cell carcinoma of the stomach.

Primary squamous cell carcinoma (SCC) of the stomach is extremely rare; only 32 cases are found in the Japanese literature. The pathogenesis of this neoplasm remains obscure and controversial. Furthermore, the optimal treatment, including adjuvant chemotherapy, remains unclear. We report herein a case of SCC of the stomach in a 70-year-old male with amazing effectiveness of neoadjuvant chemotherapy, low-dose FP chemotherapy. To our knowledge, no case of this disease has ever been reported that was given neoadjuvant chemotherapy and histologically showed its effectiveness. Our case demonstrated a striking effectiveness of chemotherapy in the neoplasm both radiologically and histologically.

Additional right parasternal incision without thoracotomy provides alternative access for hepatic resection.

BACKGROUND: Although hepatic resections are performed with various access approaches according to the location of the tumor, the extent of resection and patient's physical constitution, the exposure of the root of the hepatic veins is not optimal in some patients by any conventional incision. METHODS: An additional 5 cm of right parasternal skin incision followed by division of two costal cartilages was performed in 8 patients, which demonstrated poor exposure of the upper part of the liver by abdominal incision. RESULTS: In all cases, satisfactory exposure of the operative area was obtained without thoracotomy, and hepatic resection procedure was carried out without significant events. CONCLUSIONS: This simple technique without necessitating rotation of the operating table may be useful as an alternative method of access to the liver especially when fine isolation around the root of the hepatic veins is required.

Importance of the retro-mammary space as a route of breast cancer metastasis.

BACKGROUND: There are many cases of breast cancer with axillary lymph node metastases without lymphatic invasion. We hypothesized that in these cases cancer cells may pass through the retro-mammary space (RS) into lymph nodes and that axillary lymph node metastases may correlate with the tumor invasion of the RS. METHODS: A total of 127 patients who had undergone radical operation between April 1997 and April 2001 were studied. Whether or not the tumor had invaded the RS was histologically examined with hematoxylin and eosin staining of sections made at the point where the distance between the tumor and the fascia of the major pectoral muscle was the shortest. RESULTS: Eighty-five cases did not have lymphatic invasion. Twenty-nine of these 85 cases had RS invasion and 56 cases did not. Among the 29 cases with RS invasion, 14 cases had lymph node metastases. In contrast, of 56 cases without RS invasion only 3 cases had lymph node involvement. Of the 85 cases without lymphatic invasion, the relationship between RS invasion and lymph node invasion was statistically significant (RS(+) vs. RS(-), p<0.0001, chi-square test). For all 127 cases, if cases showing either lymphatic invasion or RS invasion were diagnosed with lymph node involvement, the sensitivity, specificity, accuracy, and negative predictive value were 93.5%, 65.4%, 75.6% and 94.6%, respectively. CONCLUSION: These data suggest that lymph node metastases may occur via the tumor cell migration through lymphatic vessels and the RS.

[A case of postoperative hepatic metastasis from gastric cancer responding to hepatic arterial infusion chemotherapy of paclitaxel].

A 70-year-old man underwent total gastrectomy for advanced gastric cancer in January 2000. He was treated with UFT of 300 mg/day after surgery. In December 2000, liver metastasis was detected. In January 2001, treatment was switched to oral administration of TS-1 at 100 mg/day. In April 2001, there were no extrahepatic lesions. However, the metastatic liver focus showed progression of the disease (PD). TS-1 was discontinued. In May 2001, a hepatic arterial injection reservoir was inserted. Thereafter, arterial injection chemotherapy with 5-FU was administered. However, in November, liver dysfunction was exacerbated, and computed tomography (CT) revealed PD. Therefore, in December, the above chemotherapy was switched to arterial injection of paclitaxel at 100 mg, which was administered once a week for 3 weeks and discontinued for the subsequent week. In March 2002, hepatopathy had subsided at the end of the 4th course, the performance status (PS) improved and CT revealed a minor response. In June 2002, the condition deteriorated to PD. However, there were no side effects other than alopecia. During the administration period, treatment at the outpatient clinic could be safely performed. These findings suggest that hepatic arterial injection therapy with paclitaxel for liver metastasis after surgery for gastric cancer is a useful second line for 5-FU-resistant patients.

[Two cases of recurrent lung cancer successfully treated with concurrent radiochemotherapy with vinorelbine plus cisplatin].

Case 1:A 62-year-old man, who had undergone right upper lobectomy for lung squamous cell carcinoma (pT2pN2M0, stage IIA) 4 months earlier, was diagnosed as mediastinal lymph node recurrence by chest CT. Case 2: A 63-year-old woman, who had undergone right middle-lower lobectomy for lung squamous cell carcinoma (pT2pN1M0, stage IIB) 16 months earlier, was diagnosed as hilar lymph node recurrence by chest CT. Both patients underwent radiochemotherapy with 2 cycles of cisplatin (CDDP) 80 mg/m2 on day 1 and vinorelbine (VNR) 15 mg/m2 on day 1 and another day (day 8-16), and concurrent radiation (60 Gy/30 fr) for mediastinum. After this treatment, a partial response (PR) in case 1 and a complete response (CR) in case 2 were achieved, and neither patient showed any relapse after 3 years of the treatment. Adverse reactions were grade 3 neutropenia in both patients and transient hemiparalysis in case 2. Concurrent radiochemotherapy with cisplatin and vinorelbine is considered effective without serious side effects for postoperative recurrence in localized mediastinal lymph nodes of non-small-cell lung cancer.

[Reasonable approach for prophylactic hepatic arterial infusion chemotherapy after potentially curative surgery for colorectal liver metastases].

The authors discuss the reasonable management of implantable ports and catheters after cessation of adjuvant hepatic arterial infusion (HAI) chemotherapy following curative resection of colorectal liver metastases. Local recurrence in the residual liver was observed in only 4 patients of thirty-two patients (13%). Although heparin administration into the port was regularly performed in 17 patients to prevent its occlusion, the ports were successfully maintained in only 9 patients at a median time of 11.8 months postoperatively, and only one patient received further regional chemotherapy for recurrent disease. These findings indicate that heparin administration to maintain the port brings little benefit. In the most recent 3 cases, we used a new Piolax W Spiral catheter and removed the catheter and port after cessation of adjuvant chemotherapy. No complication related to the procedure occurred, and patients' quality of life was well preserved, suggesting that this approach for HAI is reasonable and beneficial.

[Hepatectomy with an additional parasternal incision without thoracotomy].

A 74-year-old woman was diagnosed with hepatocellular carcinoma in S8 and underwent partial hepatectomy. A 5-cm right parasternal skin incision was added to the median incision, followed by division of the two costal cartilages, which resulted in excellent exposure of the subphrenic portion of the liver including the root of hepatic veins. The authors used this access technique in 7 patients over the past 12 months, and a satisfactory operative field was achieved in all cases. This simple technique without thoracotomy may be useful as an alternative access to the liver, especially when fine isolation around the root of the hepatic veins is required.

Discovery of highly potent and selective biphenylacylsulfonamide-based beta3-adrenergic receptor agonists and evaluation of physical properties as potential overactive bladder therapies: part 5.

As an extension of research conducted on beta(3)-adrenergic receptor agonists as potential drugs for treating overactive bladder (OAB), novel series containing an acylsulfonamide moiety instead of the carboxylic acid moiety were evaluated. These compounds have been identified as potent and selective human beta(3)-AR agonists with improved oral bioavailability compared to the previous series. Results of structure-activity relationship (SAR) studies and cassette dosing evaluation in dogs showed several analogues (namely, 6h, 6j, 6o, 7e, and 9e) to have an excellent balance of in vitro potency and selectivity, pharmacokinetic (PK) profile, and an in vivo OAB model. Here we examined the relaxation response in dog detrusor muscle strips to a KCl induced tonic concentration. Results showed that the potency of in vitro relaxation response was not mirrored in the potency of the cAMP accumulation in CHO cell lines. Surprisingly, the EC(50) values of 6e and 7e found to induce relaxation of isolated bladder strips were over 50-fold higher than the cAMP accumulation in cell line. In general, increased lipophilicity led to decreased potency for the bladder relaxation compared with cAMP accumulation in CHO cell lines, indicating that lipophilicity is crucial for OAB drug candidates to improve beta(3) activity.

Discovery of a novel series of benzoic acid derivatives as potent and selective human beta3 adrenergic receptor agonists with good oral bioavailability. 3. Phenylethanolaminotetraline (PEAT) skeleton containing biphenyl or biphenyl ether moiety.

We designed a series of benzoic acid derivatives containing the biphenyl ether or biphenyl template on the RHS and a phenylethanolaminotetraline (PEAT) skeleton, which was prepared by highly stereoselective synthesis, to generate two structurally different lead compounds ( 10c, 10m) with a good balance of potency, selectivity, and pharmacokinetic profile. Further optimization of the two lead compounds to improve potency led to several potential candidates (i.e., 11f, 11l, 11o, 12b). In particular, biphenyl analogue 12b exhibited an excellent balance of high potency (EC50 = 0.38 nM) for beta3, high selectivity over beta1 and beta2, and good pharmacokinetic properties in rats, dogs, and monkeys.

Discovery of a novel series of biphenyl benzoic acid derivatives as highly potent and selective human beta3 adrenergic receptor agonists with good oral bioavailability. Part II.

The left-hand side (LHS) and central part of our first generation biphenyl (FGB) series was modified to improve in vitro and in vivo beta3-AR potency without loss of oral bioavailability. First, in this study, we focused our efforts on replacement of the 3-chlorophenyl moiety in the LHS of FGB analogues with 3-pyridyl ring analogues to adjust the lipophilicity. Second, we investigated the replacement of the central part of this series and discovered that introduction of a methyl group into the alpha-position of the phenethylamine moiety greatly enhanced potency keeping good oral availability. Finally, the replacement of the two carbon linker of the central part with an ethoxy-based linker provided improved potency and PK profiles. As a result of these studies, several analogues (i.e., 9h, 9k, 9l, 10g, 10m, 10p, 10r, 11b, and 11l) were identified that displayed an excellent balance of very higher human beta3-AR potency compared to the FGB compounds, high selectivity, and good pharmacokinetic profiles. Furthermore, these several compounds showed high in vivo efficacy in an overactive bladder (OAB) model. These findings suggest that our selected second generation biphenyl (SGB) series compounds may be attractive new successful therapeutic candidates for the treatment of OAB.

Inhibitory effect of zacopride on Cisplatin-induced delayed emesis in ferrets.

We evaluated the antiemetic effect of zacopride, a potent 5-HT3-receptor antagonist with 5-HT4-receptor agonist properties, on delayed emesis caused by cisplatin (5 mg/kg, i.p.) in ferrets, compared with granisetron, a selective 5-HT3-receptor antagonist. Multiple intravenous injections of zacopride at 1 mg/kg, a dose that completely inhibited acute emesis caused bycisplatin (10 mg/kg, i.v.), significantly reduced delayed emesis. Granisetron (3.2 mg/kg) also reduced delayed emesis but this failed to reach statistical significance. The present study suggests that a combined 5-HT3-receptor antagonist/5-HT4-receptor agonist, like zacopride, may be useful against both acute and delayed emesis induced by cancer chemotherapy.

Efficacy of absorbable clips compared with metal clips for cystic duct ligation in laparoscopic cholecystectomy.

PURPOSE: This study was conducted to examine the usefulness and safety of absorbable clips in laparoscopic cholecystectomy (LC). METHODS: We retrospectively compared the clinical data of 328 patients who underwent LC using absorbable clips for cystic duct ligation and 444 patients who underwent LC using metal clips for cystic duct ligation. RESULTS: The mean operative time in the absorbable clip group was significantly shorter than that in the metal clip group. The mean intraoperative blood loss and hospital stay were not significantly different between the two groups. The rate of conversion to open laparotomy in the absorbable clip group was significantly higher than that in the metal clip group, at 6.7% vs 2.3%, respectively. The most common reason for conversion was difficult adhesions and inflammation around the gallbladder. The morbidity of the metal clip group was significantly higher than that of the absorbable clip group, at 8.4% vs 4.0%, respectively. The incidence of major postoperative complications requiring laparotomy did not differ between the two groups. There were no deaths in the absorbable clip group, but 1 of the 444 patients (0.23%) in the metal clip group died from disseminated intravascular coagulation on postoperative day 3. CONCLUSION: The results of this study suggest that absorbable clips are as safe and effective as standard metal clips for vessel and duct ligation in LC.

Laparoscopic wedge resection for gastric perforation after endoscopic mucosal resection: report of a case.

We performed laparoscopic wedge resection to treat gastric perforation that occurred as a complication of endoscopic mucosal resection (EMR) in a 53-year-old man. We believe this is an appropriate procedure to treat gastric perforation after EMR for early gastric cancer because it is safe and minimally invasive, and faster recovery can be expected than after conservative therapy.