Effect of beta-1,6-glucan inhibitors on the invasion process of Candida albicans: potential mechanism of their in vivo efficacy.

Beta-1,6-glucan is a fungus-specific cell wall component that is essential for the retention of many cell wall proteins. We recently reported the discovery of a small molecule inhibitor of beta-1,6-glucan biosynthesis in yeasts. In the course of our study of its derivatives, we found a unique feature in their antifungal profile. D21-6076, one of these compounds, exhibited potent in vitro and in vivo antifungal activities against Candida glabrata. Interestingly, although it only weakly reduced the growth of Candida albicans in conventional media, it significantly prolonged the survival of mice infected by the pathogen. Biochemical evaluation of D21-6076 indicated that it inhibited beta-1,6-glucan synthesis of C. albicans, leading the cell wall proteins, which play a critical role in its virulence, to be released from the cell. Correspondingly, adhesion of C. albicans cells to mammalian cells and their hyphal elongation were strongly reduced by the drug treatment. The results of the experiment using an in vitro model of vaginal candidiasis showed that D21-6076 strongly inhibited the invasion process of C. albicans without a significant reduction in its growth in the medium. These evidences suggested that D21-6076 probably exhibited in vivo efficacy against C. albicans by inhibiting its invasion process.

[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.

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.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa.

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.

[Development of antituberculous drugs: current status and future prospects].

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The World Health Organization estimates that about eight to ten million new TB cases occur annually worldwide and the incidence of TB is currently increasing. In this context, TB is in the top three, with malaria and HIV being the leading causes of death from a single infectious agent, and approximately two million deaths are attributable to TB annually. In particular, pulmonary TB, the most common form of TB, is a highly contagious and life-threatening infection. Moreover, enhanced susceptibility to TB in HIV-infected populations is another serious health problem throughout the world. In addition, multidrug-resistant TB (MDR-TB) has been increasing in incidence in many areas, not only in developing countries but industrialized countries as well, during the past decade. These situations, particularly the global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs and new protocols for efficacious clinical control of TB patients using ordinary antimycobacterial drugs. Concerning the development of new antituberculous drugs, the following points are of particular importance. (1) Development of drugs which display lasting antimycobacterial activity in vivo is desirable, since they can be administered with long intervals and consequently facilitate directly observed therapy and enhance patient compliance. (2) Development of novel antituberculosis compounds to combat MDR-TB is urgently needed. (3) The eradication of slowly metabolizing and, if possible, dormant populations of MTB organisms that cause relapse, using new classes of anti-TB drugs is very promising for prevention of TB incidence, because it will markedly reduce the incidence of active TB from persons who are latently infected with MTB. Unfortunately, no new drugs except rifabutin and rifapentine has been marketed for TB in the US and other countries during the 40 years after release of rifampicin. There are a number of constraints that have deterred companies from investing in new anti-TB drugs. The research is expensive, slow and difficult, and requires specialized facilities for handling MTB. There are few animal models that closely mimic the human TB disease. Development time of any anti-TB drug will be long. In fact, clinical trials will require the minimum six-month therapy, with a follow-up period of one year or more. In addition, it is hard to demonstrate obvious benefit of a new anti-TB agents over pre-existing drugs, since clinical trials involve multidrug combination therapy using highly effective ordinary anti-TB drugs. Finaly, there is the perceived lack of commercial return to companies engaged in the development of new anti-TB drugs, because over 95% of TB cases worldwide are in developing countries. In this symposium, we reviewed the following areas. 1. Critical new information on the entire genome of MTB recently obtained and increasing knowledge of various mycobacterial virulence genes are greatly promoting the identification of genes that code for new drug targets. In this context, Dr. Namba reviewed the status of new types of compounds which are being developed as anti-TB drug. He also discussed the development of new antimycobacterial drugs according to new and potential pharmacological targets and the best clinical development plans for new-TB drugs in relation to corporate strategy. 2. Using such findings for mycobacterial genomes, bioinformatics/genomics/proteomics-based drug design and drug development using quantitative structure-activity relationships may be possible in the near future. In this context, Dr. Suwa and Dr. Suzuki reviewed the usefulness of chemical genomics in searching novel drug targets for development of new antituberculous drugs. The authors reviewed (1) the history and present status of chemical genomics that is defined as the systemic search for a selective small molecular modulator for each function of all gene products, (2) recent studies of the authors on profiles of the interactions between various kinds of human proteins and small molecule modulators using the new technology devised by Reverse Proteomics Research Institute, and (3) future prospects of the development of new antituberculous drugs based on chemical genomics. 3. It appears also promising to develop new types of drug administration systems using drug vehicles, which enable efficacious drug delivery to their target in vivo. Dr. Izumikawa, Dr. Ohno and Dr. Kohno reviewed the usefulness of liposome- and polymer-based technologies, which enable efficacious delivery of encapsulated drugs at required doses for prolonged periods of time with only a single shot without toxicity, and also enable highly targeted delivery of drugs to their target in vivo. They indicated that the applications of drug delivery system using conventional anti-mycobacterial agents are challenging to improve the compliance of treatment and better clinical outcome. 4. Immunoadjunctive therapy appears to be promising in improving outcome of clinical control of refractory mycobacterial infections, including MDR-TB and M. avium complex infection. Dr. Shimizu, Dr. Sato and Dr. Tomioka reviewed the present status of immunotherapy of mycobacterial infections in combination with antimycobacterial drugs. They indicated that the development of new classes of immunomodulators other than cytokines (IL-2, IFN-gamma, GM-CSF, IL-12, etc.) particularly those with no severe side-effects, are urgently needed. Their review dealed with some promising immunoadjunctive agents, especially ATP and its analogues, which potentiate macrophage antimycobacterial activity via purinergic P2 receptors. The aim of this symposium is to address the future prospects of the development of new drugs and drug regimens for anti-TB chemotherapy. There are a number of difficulties in drug-design for the development of new drug formulations with increased potential for antimycobacterial effects, excellent pharmacokinetics, and tolerability. It should be emphasized that the most urgent goal of chemotherapy of TB and MAC infections, especially that associated with HIV infection, is to develop highly active, low-cost drugs which can be used not only in industrialized countries but also in developing countries, since the incidences of AIDS-associated intractable TB and MAC infections are rapidly increasing in the latter. We strongly wish a great advance of fundametal and practical studies in developing such kinds of new anti-TB drugs in the near future. 1. Prospects for non-clinical or clinical development of new antituberculous drugs in relation to corporate strategy: Kenji NAMBA (New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd.) Tuberculosis (TB) remains one of the deadliest threats to public health. No new anti-TB drugs have been brought into the clinic in the past 40 years. Current non-clinical works with progressed technology and Global Alliance for TB Drug Development, a non-profit organization established in 2000, accelerate research and development of faster-acting anti-TB compounds. We reviewed the status of new types of compounds which are being developed as anti-TB drug, such as diarylquinoline (TMC 207), nitroimidazole (PA-824 and OPC-67683), and moxifloxacin (MFLX). We also discussed the best clinical development plans for new-TB drugs in relation to corporate strategy. 2. Exploring novel drug targets through the chemical genomics approach and its possible application to the development of anti-tuberculosis drugs: Yorimasa SUWA (Reverse Proteomics Research Institute Co., Ltd.), Yohji SUZUKI (Teijin Ltd.) Recently, chemical genomics approach has been focused as an emerging technology for the drug discovery. In advance to a very large scale national project in US started last year, Reverse Proteomics Research Institute Co., Ltd. (REPRORI) has developed the core technologies for chemical genomics. Here we describe the outline of chemical genomics study, especially that of REPRORI, and discuss about its possible application to the development of anti-tuberculosis drugs. 3. Anti-mycobacterial agents and drug delivery: Koichi IZUMIKAWA, Hideaki OHNO, Shigeru KOHNO (Second Department of Internal Medicine, Nagasaki University School of Medicine) Mycobacterium infection is a major clinical concern in whole world. Since the newly developed anti-mycobacterial agents are few and still unavailable in clinical settings, the applications of drug delivery system using conventional anti-mycobacterial agents are challenging to improve the compliance of treatment and better efficacy. The efficacy of anti-mycobacterial agents modified by liposome or polymer based technology have been investigated and reported using various animal models. Drug delivery system increased and prolonged the drug concentrations at the blood and targeted organs and the duration of sustained drug release, respectively. These effects lead to decrease in the frequency of drug administrations dramatically and better efficacy rates. The studies, however, were performed only in animal models, the further investigations and evaluations in human are required for practical use. 4. Adjunctive immunotherapy of mycobacterial infections: Toshiaki SHIMIZU, Katsumasa SATO, Haruaki TOMIOKA (Department of Microbiology and Immunology, Shimane University School of Medicine) There is an urgent need to develop new antimicrobials and protocols for the administration of drugs that are potently efficacious against intractable mycobacterial infections. Unfortunately, development of the new drugs for solving this problem is not progressing. (ABSTRACT TRUNCATED)

In-vitro activity of sitafloxacin (DU-6859a), either singly or in combination with rifampin analogs, against Mycobacterium leprae.

The in-vitro antibacterial activity of sitafloxacin (DU-6859a) against Mycobacterium leprae was evaluated and compared with those of ofloxacin, levofloxacin, and ciprofloxacin. Two biochemical indicators (intracellular ATP and uptake of [(3)H]-thymidine) were used to measure the in-vitro growth of M. leprae in Dhople-Hanks (DH) medium. Sitafloxacin was found to be more potent than the other three commonly used fluoroquinolones, with the minimum inhibitory concentration (MIC) against M. leprae being 0.1875 microg/ml and the action being bactericidal. The MICs of ofloxacin, levofloxacin, and ciprofloxacin were 1.5, 0.75, and 3.0 microg/ml, respectively. Similar to ofloxacin and levofloxacin, sitafloxacin also exhibited synergistic activity when combined with either rifabutin or KRM-1648, but not with rifampin. Thus, further studies on the incorporation of sitafloxacin in multidrug therapy regimens in treating leprosy patients are suggested.

In vivo susceptibility of Mycobacterium leprae to sitafloxacin (DU-6859a), either singly or in combination with rifampicin analogues.

The antimicrobial effects of sitafloxacin (DU-6859a) against Mycobacterium leprae, either singly or in combination with either rifampicin, rifabutin or KRM-1648, were studied using a mouse footpad assay technique and the results were compared with those obtained with ofloxacin. When used singly, the minimum concentrations of sitafloxacin and ofloxacin needed to inhibit completely the growth of M. leprae were 25 and 100 mg per kg body weight per day, respectively, and the effects were bactericidal. Both sitafloxacin and ofloxacin exhibited excellent synergistic effects when combined with either rifabutin or KRM-1648, but not with rifampicin. Thus, incorporation of sitafloxacin and rifabutin (or KRM-1648) in the multidrug regimen for treating leprosy patients is suggested.

Quantitative evaluation of the neuroprotective effects of hypothermia ranging from 34 degrees C to 31 degrees C on brain ischemia in gerbils and determination of the mechanism of neuroprotection.

OBJECTIVE: The present study was designed to determine whether the predominant factor responsible for neuroprotection of hypothermia ranging from 31 to 34 degrees C is prolongation of onset of ischemic depolarization or suppression of neuronal injury during ischemic depolarization and to quantitatively determine the neuroprotective effects of hypothermia of 34 degrees C and 31 degrees C. DESIGN: Prospective animal study. SETTING: A university research laboratory. SUBJECTS: Eighty-nine gerbils. INTERVENTIONS: Bilateral common carotid arteries were occluded for 3-20 mins. The brain temperature was set at 37 degrees C, 34 degrees C, or 31 degrees C before and during ischemic depolarization. MEASUREMENTS AND MAIN RESULTS: DC potentials were measured in the CA1 region, where histologic evaluation was performed 7 days later. Onset times of ischemic depolarization were 1.3 +/- 0.2, 1.6 +/- 0.4, and 2.4 +/- 0.7 mins at 37 degrees C, 34 degrees C, and 31 degrees C, respectively. The logistic regression curve demonstrated a close relationship between duration of ischemic depolarization and neuronal damage and showed a rightward shift by lowering the brain temperature. In the 37 degrees C, 34 degrees C, and 31 degrees C groups, the durations of ischemic depolarization causing 50% neuronal damage were estimated to be 8.0, 14.2, and 26.0 mins, respectively, and the ischemia times causing 50% neuronal damage were estimated to be 4.9, 8.1, and 14.2 mins, respectively. CONCLUSIONS: The onset of ischemic depolarization was prolonged in the 34 degrees C and 31 degrees C groups by only 0.3 and 1.1 mins, respectively, compared with that in the 37 degrees C group. Most of the neuroprotection by hypothermia was attributed to the suppression of neuronal injury during ischemic depolarization, suggesting that hypothermia has neuroprotective effects if it is initiated during the ischemic depolarization period. The results also indicate that the neuroprotective effect at 31 degrees C is about three times greater than that at 34 degrees C and that neuronal cells can withstand 2.9 times longer duration of ischemia at 31 degrees C than at 37 degrees C.

In vitro activity of sitafloxacin (DU-6859a) alone, or in combination with rifampicin, against Mycobacterium ulcerans.

The antimicrobial effect of sitafloxacin (DU-6859a), used either singly or in combination with rifampicin, was evaluated in vitro against Mycobacterium ulcerans. Growth of M. ulcerans was measured by plate counts and the BACTEC radiometric method. The MICs and MBCs of sitafloxacin for M. ulcerans were in the range 0.125-0.5 mg/L. The values for other fluoroquinolones were two- to four-fold higher than for sitafloxacin. Combination of sitafloxacin and rifampicin exhibited synergy with five of the eight strains, whereas the combination of ofloxacin and rifampicin resulted in additive effects only. These results suggest that the combination of sitafloxacin and rifampicin has potential in the treatment of M. ulcerans infection.