[Six-year survival with recurrent intrahepatic cholangiocarcinoma achieved by simple S-1 administration].

We report here a rare case of a patient with recurrent intrahepatic cholangiocarcinoma that was treated with simple S-1 chemotherapy, who is still alive 6 years later. A liver tumor was identified in segments 5 to 6 in a 60-year-old male asymptomatic hepatitis B carrier. The tumor was diagnosed as hepatocellular carcinoma by MRI and CT. However, following its resection by extended posterior segmentectomy of the liver, pathological findings identified it as an intrahepatic cholangiocarcinoma. The surgical margin was cancer-negative. No additional adjuvant chemotherapy was administered because of the patient 's impaired renal function. When tumor recurrence was found by MRI 30 months later, an additional liver resection was planned but a laparotomy was eventually performed. This was because intraoperative findings revealed Glissonian sheath invasion with involvement of the umbilical portion. S-1 treatment(100mg/body/day)was started. Although the dose had to be reduced(mostly 75mg/body/day)due to hyperbilirubinemia and there were some interruptions in the regimen, in total of 42 g of S-1 was administered. The patient is currently still alive, 6 years after the detection of the tumor recurrence. This represents a rare case in patients with intrahepatic cholangiocarcinoma.

Inhibition of ergosterol synthesis by novel antifungal compounds targeting C-14 reductase.

The limited number of clinically available antifungal drugs for life-threatening fungal infections has produced an increased demand for new agents. In the course of our screening for novel antifungals, we identified aminopiperidine derivatives which exhibit antifungal activities against the major pathogenic yeasts. Thin layer chromatography (TLC) analysis of the extracted non-saponifiable lipids from Candida albicans showed that these compounds inhibited the ergosterol production in the late step of the synthesis pathway. The results of an LC/Q-Tof MS analysis showed that abnormal sterols including predicted ignosterol, which is known to be accumulated in C. albicans ERG24 deleted mutant, were accumulated in C. albicans treated with one of these derivatives (Compound 1b). Furthermore, the partial disruption of the cell membrane of C. albicans treated with compound 1b was observed by electron microscopy analysis, suggesting its inhibition of ergosterol synthesis. Additionally, a genetic approach demonstrated that ERG24 gene would be responsible for the resistance of Saccharomyces cerevisiae against Compound 1b, strongly indicating that the enzyme targeted by Compound 1b is Erg24p. From all these data, we concluded that these aminopiperidine derivatives are novel antifungal compounds inhibiting C-14 reduction in the ergosterol synthesis pathway.

In vitro and in vivo antifungal activities of aminopiperidine derivatives, novel ergosterol synthesis inhibitors.

Aminopiperidine derivatives, Compound 1a and 1b, are novel small molecules that inhibit C-14 reduction catalyzed by Erg24p in ergosterol synthesis of Candida albicans. We evaluated the properties of the in vitro and in vivo activities of these compounds against pathogenic fungi and compared their activities with those of fluconazole. Compound 1a and 1b exhibited potent in vitro activities against clinically important fungi such as Candida species, including both of fluconazole-resistant strains of C. albicans and non-albicans Candida, Aspergillus fumigatus, and Cryptococcus neoformans. Against C. albicans, its mode of action was fungistatic. Furthermore, orally administered Compound 1b clearly prolonged the survival of infected mice in systemic lethal infection caused by C. albicans. These results suggest that aminopiperidine derivative is a promising lead compound for an orally available novel antifungal drug with a broad spectrum.

Quinolones with enhanced bactericidal activity induce autolysis in Streptococcus pneumoniae.

OBJECTIVES: DC-159a and sitafloxacin show greater bactericidal activity against Streptococcus pneumoniae than garenoxacin and other quinolones. We investigated whether the autolysis induced by these quinolones contributes to their rapid bactericidal activity. METHODS: Time-kill studies were conducted against a S. pneumoniae clinical isolate in broth with choline chloride, which is known to inhibit autolytic amidases, and lytA mutants. Western blot analysis was performed to examine LytA production. Scanning electron microscopy (SEM) was used to investigate morphological differences after exposure to quinolone. RESULTS: Bactericidal activity of DC-159a and sitafloxacin against S. pneumoniae at 2 h of exposure to twice the minimum inhibitory concentration (MIC) was found to decrease by approximately 1 log CFU/ml when autolytic amidases were blocked. Time-kill studies using lytA mutants showed that DC-159a exhibited slower killing than that against the lytA-positive strains. On exposure to the MIC and twice the MIC of DC-159a and sitafloxacin, R6 and a clinical isolate overexpressed LytA, while garenoxacin caused a less significant increase in LytA than DC-159a and sitafloxacin. Scanning electron microscopy images revealed that R6 treated with DC-159a underwent distinct morphological changes, while the lytA mutant did not. CONCLUSIONS: The present study demonstrated that quinolone-induced autolysis may provide quinolones more powerful bactericidal activity against S. pneumoniae.

[Novel model of replicative Legionella pneumophila lung infection in DBA/2 mice].

We present here a new model of Legionella pneumophila lung infection in DBA/2 mice. By intranasal inoculation with 106 colony-forming units of L. pneumophila strain suzuki serogoup 1, persistent non-lethal lung infection was established as reflected by the detection of more than 10(4) CFU/lung of the organism 14 days after infection. Treatment of mice with cyclophosphamide before infection enhanced bacterial replication in the lungs and all cyclophosphamide-treated mice experienced lethal infection. Histopathologically, the course of non-lethal lung infection was characterized by early response of neutrophiles, then monocyte/macrophages response in the alveoli with disease progression, and diffuse alveolar wall thickening with lymphocyte migration at later phase of infection. Transmission electron microscopic evaluation of the lungs confirmed that L. pneumophila located intracellularly within neutrophiles and infrequently intracellular bacteria were observed undergoing binary fission. Therefore, the mouse model of replicative L. pneumophila lung infection provides method for evaluating pathogenesis of L. pneumophila lung infection and antibacterial therapy.

[Therapeutic efficacy of levofloxacin against a model of replicable Legionella pneumophila lung infection in DBA/2 mice].

The in vitro and in vivo antibacterial activities of levofloxacin (LVFX), a quinolone antibacterial, against clinically isolated Legionella pneumophila were investigated in comparison with those of existing antimicrobial agents approved for legionnaires disease. The minimum inhibitory concentrations (MICs) of the agents against 42 strains of L. pneumophila isolated in Japan were determined using agar dilution methods with buffered starch yeast extract agar. MIC90 of LVFX was 0.03 microg/ml and this activity was similar to ciprofloxacin and pazufloxacin, and higher than telithromycin and minocycline. Therapeutic efficacy of LVFX was studied against a pneumonia model induced by intranasal of L. pneumophila strain suzuki serogoup 1 in DBA/2 mice. Therapeutic doses in mice were selected that would closely match human exposure profile, area under the concentration-time curve (AUC) for a human oral dose of LVFX at 500 mg once a day. LVFX decreased significantly the bacterial burden in the lungs from the next day of commencing treatment. These results, including in vitro antibacterial activity against clinical isolates and therapeutic efficacy of a humanized dosing regimen, provide good evidence to support the use of LVFX at 500 mg once a day for treating patient with legionnaires disease.

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae.

OBJECTIVES: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. METHODS: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. RESULTS: DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. CONCLUSIONS: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.

Role of 18F-fluorodeoxyglucose positron emission tomography imaging in surgery for pancreatic cancer.

AIM: To evaluate the role of positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) in the surgical management of patients with pancreatic cancer, including the diagnosis, staging, and selection of patients for the subsequent surgical treatment. METHODS: This study involved 53 patients with proven primary pancreatic cancer. The sensitivity of diagnosing the primary cancer was examined for FDG-PET, CT, cytological examination of the bile or pancreatic juice, and the serum levels of carcinoembrionic antigens (CEA) and carbohydrate antigen 19-9 (CA19-9). Next, the accuracy of staging was compared between FDG-PET and CT. Finally, FDG-PET was analyzed semiquantitatively using the standard uptake value (SUV). The impact of the SUV on patient management was evaluated by examining the correlations between the SUV and the histological findings of cancer. RESULTS: The sensitivity of FDG-PET, CT, cytological examination of the bile or pancreatic juice, and the serum levels of CEA and CA19-9 were 92.5%, 88.7%, 46.4%, 37.7% and 69.8%, respectively. In staging, FDG-PET was superior to CT only in diagnosing distant disease (bone metastasis). For local staging, the sensitivity of CT was better than that of FDG-PET. The SUV did not correlate with the pTNM stage, grades, invasions to the vessels and nerve, or with the size of the tumor. However, there was a statistically significant difference (4.6 +/- 2.9 vs 7.8 +/- 4.5, P = 0.024) in the SUV between patients with respectable and unresectable disease. CONCLUSION: FDG-PET is thus considered to be useful in the diagnosis of pancreatic cancer. However, regarding the staging of the disease, FDG-PET is not considered to be a sufficiently accurate diagnostic modality. Although the SUV does not correlate with the patho-histological prognostic factors, it may be useful in selecting patients who should undergo subsequent surgical treatment.

A case report of extrahepatic portal vein aneurysm with thrombosis.

Extrahepatic portal vein aneurysm (PVA) is very rare with only 17 previously reported cases. Methods of treatment include resection, thrombectomy, and portal venous decompression. We report herein the first case of large PVA with thrombosis which has been managed without surgical treatment over a long period. A PVA was detected in a 78-year-old woman by abdominal ultrasonography. Computed tomography revealed an aneurysm of 6 cm in a diameter in the porta hepatis. Portal venography showed obstruction of the portal vein and developed collateral vessels around the aneurysm. Since the patient had no symptoms of portal hypertension, we decided to carefully manage her clinical course without surgical treatment. At present, this patient is healthy and has developed no complications over the 5 years since leaving our hospital. This case suggests that surgical treatment is not required for PVA without portal hypertension.

Resurrection of a bull by cloning from organs frozen without cryoprotectant in a -80 degrees c freezer for a decade.

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

In vitro and in vivo antibacterial activities of DC-159a, a new fluoroquinolone.

DC-159a is a new 8-methoxy fluoroquinolone that possesses a broad spectrum of antibacterial activity, with extended activity against gram-positive pathogens, especially streptococci and staphylococci from patients with community-acquired infections. DC-159a showed activity against Streptococcus spp. (MIC(90), 0.12 microg/ml) and inhibited the growth of 90% of levofloxacin-intermediate and -resistant strains at 1 microg/ml. The MIC 90s of DC-159a against Staphylococcus spp. were 0.5 microg/ml or less. Against quinolone- and methicillin-resistant Staphylococcus aureus strains, however, the MIC 90 of DC-159a was 8 microg/ml. DC-159a was the most active against Enterococcus spp. (MIC 90, 4 to 8 microg/ml) and was more active than the marketed fluoroquinolones, such as levofloxacin, ciprofloxacin, and moxifloxacin. The MIC 90s of DC-159a against Haemophilus influenzae, Moraxella catarrhalis, and Klebsiella pneumoniae were 0.015, 0.06, and 0.25 microg/ml, respectively. The activity of DC-159a against Mycoplasma pneumoniae was eightfold more potent than that of levofloxacin. The MICs of DC-159a against Chlamydophila pneumoniae were comparable to those of moxifloxacin, and DC-159a was more potent than levofloxacin. The MIC 90s of DC-159a against Peptostreptococcus spp., Clostridium difficile, and Bacteroides fragilis were 0.5, 4, and 2 microg/ml, respectively; and among the quinolones tested it showed the highest level of activity against anaerobic organisms. DC-159a demonstrated rapid bactericidal activity against quinolone-resistant Streptococcus pneumoniae strains both in vitro and in vivo. In vitro, DC-159a showed faster killing than moxifloxacin and garenoxacin. The bactericidal activity of DC-159a in a murine muscle infection model was revealed to be superior to that of moxifloxacin. These activities carried over to the in vivo efficacy in the murine pneumonia model, in which treatment with DC-159a led to bactericidal activity superior to those of the other agents tested.

In vitro and in vivo antibacterial activities of DK-507k, a novel fluoroquinolone.

The antibacterial activities of DK-507k, a novel quinolone, were compared with those of other quinolones: ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, sitafloxacin, and garenoxacin (BMS284756). DK-507k was as active as sitafloxacin and was as active as or up to eightfold more active than gatifloxacin, moxifloxacin, and garenoxacin against Streptococcus pneumoniae, methicillin-susceptible and methicillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci. DK-507k was as active as or 4-fold more active than garenoxacin and 2- to 16-fold more active than gatifloxacin and moxifloxacin against ciprofloxacin-resistant strains of S. pneumoniae, including clinical isolates and in vitro-selected mutants with known mutations. DK-507k inhibited all ciprofloxacin-resistant strains of S. pneumoniae at 1 microg/ml. A time-kill assay with S. pneumoniae showed that DK-507k was more bactericidal than gatifloxacin and moxifloxacin. The activities of DK-507k against most members of the family Enterobacteriaceae were comparable to those of ciprofloxacin and equal to or up to 32-fold higher than those of gatifloxacin, levofloxacin, moxifloxacin, and garenoxacin. DK-507k was fourfold less active than sitafloxacin and ciprofloxacin against Pseudomonas aeruginosa, while it was two to four times more potent than levofloxacin, gatifloxacin, moxifloxacin, and garenoxacin against P. aeruginosa. In vivo, intravenous treatment with DK-507k was more effective than that with gatifloxacin and moxifloxacin against systemic infections caused by S. aureus, S. pneumoniae, and P. aeruginosa in mice. In a mouse model of pneumonia due to penicillin-resistant S. pneumoniae, DK-507k administered subcutaneously showed dose-dependent efficacy and eliminated the bacteria from the lungs, whereas gatifloxacin and moxifloxacin had no significant efficacy. Oral treatment with DK-507k was slightly more effective than that with ciprofloxacin in a rat model of foreign body-associated urinary tract infection caused by a P. aeruginosa isolate for which the MIC of DK-507k was fourfold higher than that of ciprofloxacin. Oral administration of DK-507k to rats achieved higher peak concentrations in serum and higher concentrations in cumulative urine than those achieved with ciprofloxacin. These data indicate the potential advantages of DK-507k over other quinolones for the treatment of a wide range of community-acquired infections.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 2: achieving activity in vivo through the use of alternative scaffolds.

Problems of low solubility, high serum protein binding, and lack of efficacy in vivo in first generation MexAB-OprM specific efflux pump inhibitors were addressed. Through the use of pharmacophore modelling, the key structural elements for pump inhibition were defined. Use of alternative scaffolds upon which the key elements were arrayed gave second generation leads with greatly improved physical properties and activity in the potentiation of antibacterial quinolones (levofloxacin and sitafloxacin) versus Pseudomonas aeruginosa in vivo.