Haruaki Tomioka

Current status of some antituberculosis drugs and the development of new antituberculous agents with special reference to their in vitro and in vivo antimicrobial activities.

Tuberculosis (TB) is a growing international health concern, since it is the leading infectious cause of death in the world today. In particular, the increasing prevalence of multidrug-resistant (MDR)-TB has greatly contributed to the increased difficulties in the control of TB. Because of the global health problems of TB, the increasing rate of MDR-TB and the high rate of a co-infection with HIV, the development of potent new anti-TB drugs without cross-resistance with known antimycobacterial agents is urgently needed. This article deals with the following areas. First, it briefly reviews some recent findings on the pharmacological status of fluoroquinolones and rifamycin derivatives. Second, it describes other types of new agents, such as oxazolidinones (linezolid, PNU-100480), nitroimidazoles (nitroimidazopyran PA-824, metronidazole), 2-pyridone, riminophenazines and diarylquinolines, which are being developed as anti-TB drugs. In addition, the future development of new antitubercular drugs is briefly discussed according to the potential pharmacological targets. New critical information on the whole genome of Mycobacterium tuberculosis (MTB) was recently elucidated and increasing knowledge on various mycobacterial virulence genes will promote the progression in the identification of genes that code for new drug targets. Using such findings on MTB genome, drug development using quantitative structure-activity relationship may be possible in the near future.

Characteristics of Suppressor Macrophages Induced by Mycobacterial and Protozoal Infections in relation to Alternatively Activated M2 Macrophages

In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of certain types of suppressor macrophage populations. During the course of Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare complex (MAC) infections, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This paper describes the immunological properties of M1- and M2-type macrophages generated in tumor-bearing animals and those generated in hosts with certain microbial infections. In addition, this paper highlights the immunological and molecular biological characteristics of suppressor macrophages generated in hosts with mycobacterial infections, especially MAC infection.

Recent advances in antituberculous drug development and novel drug targets

TB, especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and the novel development of effective anti-tuberculous drugs is urgently needed. Newly elucidated, critical information on the entire genome of Mycobacterium tuberculosis (MTB) and advances in knowledge regarding various mycobacterial virulence genes are promoting progression in the identification of genes that code for new drug targets. With this background, this review deals with the following areas: first, the future development of new anti-tuberculous drugs is discussed according to the potential pharmacological targets of MTB; and second, a review of the present development status of new anti-tuberculous drugs is conducted, particularly focusing on some promising new anti-tuberculous agents, such as nitroimidazoles, diarylquinolines and oxazolidinones.

Development of new antituberculous drugs based on bacterial virulence factors interfering with host cytokine networks.

The worldwide increase in the prevalence of tuberculosis (TB), especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and new effective drugs are urgently needed. Information on the genome of Mycobacterium tuberculosis (MTB) and various mycobacterial virulence genes is leading to the identification of genes that code for new drug targets. Mycobacterium tuberculosis (MTB) is resistant to the antimicrobial mechanisms of host macrophages and can survive and replicate in macrophages for long periods, resulting in a persistent infection. Mycobacterial virulence factors suppress macrophage bactericidal functions partly via their downregulatory effects on the host antimicrobial cytokine networks, consisting of proinflammatory, immunopotentiating, and Th1-inducing cytokines. Thus, for the development of unique drugs that exhibit antimycobacterial action through novel mechanisms, it is reasonable to search for targets among bacterial genes encoding virulence factors which interfere with the host cytokine responses protective to mycobacterial pathogens. In this review, we discuss the profiles of cytokine networks related to host resistance to mycobacteria, including the mechanisms of downregulation of host antimycobacterial immunity due to immunosuppressive cytokines, which are occasionally induced in the advanced stages of TB. We also highlight the development of antituberculous drugs based on bacterial virulence factors interfering with the host antimycobacterial cytokine network.

The role of B7 molecules in the cell contact‐mediated suppression of T cell mitogenesis by immunosuppressive macrophages induced with mycobacterial infection

We found previously that immunosuppressive macrophages (Mφs) induced by Mycobacterium intracellulare infection (MI‐Mφs) transmitted their suppressor signals to target T cells through cell contact with target T cells. In this study, we examined what kinds of Mφ surface molecules are required for such cell–to–cell interaction. First, it was found that a B7‐1‐like molecule (B7–1LM) recognizable with one of three test clones of anti‐B7‐1 monoclonal antibodies (mAbs) was required for expression of the Mφ suppressor activity. Neither anti‐B7‐2, anti‐ICAM‐1, nor anti‐VCAM‐1u2003mAb blocked the Mφ suppressor activity. Second, MI‐Mφs increased the expression of B7–1LM in parallel with the acquisition of the suppressor activity. Moreover, MI‐Mφs bound with target T cells in a B7–1LM‐dependent fashion. Third, mAb blocking of CTLA‐4 on target T cells did not reduce the suppressor activity of MI‐Mφs, suggesting the role of a putative molecule on target T cells other than CTLA‐4 as the receptor for B7–1LM of MI‐Mφs. Fourth, concanavalin A (Con A) stimulation of MI‐Mφs was needed for effective cell contact with target T cells and subsequent expression of the suppressor activity of MI‐Mφs. Fifth, the Con A‐induced increase in the suppressor activity of MI‐Mφs was inhibited by KN‐62 but not by herbimycin A, H‐7, nor H‐88, indicating that Con A‐induced up‐regulation of MI‐Mφ function is mediated by calmodulin‐dependent protein kinase II or ATP/P2Z receptors, but independent of protein tyrosine kinase, protein kinase C, and protein kinase A. These findings indicate that a B7/CTLA‐4‐independent mechanism is needed for the transmission of the suppressor signals from MI‐Mφs to target T cells.

Comparative in vitro and in vivo antimicrobial activities of sitafloxacin, gatifloxacin and moxifloxacin against Mycobacterium avium

Moxifloxacin exhibits therapeutic activity against Mycobacterium avium infection in mice. Since not only moxifloxacin but also another 8-methoxy quinolone, gatifloxacin, and a C-8-chloro quinolone, sitafloxacin, show favourable antimycobacterial activity in vitro, their anti-M. avium activities were compared in vivo. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and mutant prevention concentrations (MPCs) of the test quinolones for M. avium were determined by microdilution in 7HSF broth. Antimicrobial activity against intracellular bacteria was measured using Mono Mac 6 human macrophages. Therapeutic efficacy of the quinolones when administered subcutaneously with or without clarithromycin plus ethambutol was assessed using mice intravenously infected with M. avium in terms of changes in bacterial loads in the lungs and spleen following infection. Based on the MICs, MBCs and MPCs, the in vitro activities of sitafloxacin and moxifloxacin were greater than that of gatifloxacin. Moxifloxacin exhibited the strongest activity against intramacrophage M. avium. When each test quinolone was administered alone to infected mice, sitafloxacin and gatifloxacin exhibited greater therapeutic efficacy than moxifloxacin based on intrapulmonary bacterial elimination. However, moxifloxacin exerted greater activity in killing bacteria in the spleen. Moxifloxacin and sitafloxacin exhibited combined effects on intrapulmonary bacterial elimination when administered to mice in combination with clarithromycin plus ethambutol. Sitafloxacin exerted the most marked combined effects in bacterial killing in the spleen. Levofloxacin displayed the lowest in vitro and in vivo activities amongst the tested quinolones. Taken together, these findings indicate that sitafloxacin and moxifloxacin exhibit favourable activities against M. avium in vitro and in vivo.

Present status and future prospects of chemotherapeutics for intractable infections due to Mycobacterium avium complex.

Mycobacterium avium-intracellulare complex (MAC) infections are frequently encountered in immunocompromised hosts, especially AIDS patients, although nodular-bronchiectasis type MAC infections without predisposing conditions are steadily increasing, particularly in Japan. Clinical control of MAC infection is difficult, since it responds poorly to available antimycobacterial regimens because of the intrinsic resistance of MAC organisms to common antituberculosis drugs, although some antimycobacterial drugs, such as macrolides (clarithromycin, azithromycin), ethambutol, clofazimine and rifamycins (especially rifabutin), are fairly or modestly effective in controlling AIDS-associated MAC bacteremia. In addition, treatment of pulmonary MAC infections is also difficult, even with available antimycobacterial multi-drug therapies. The insufficient efficacy of the majority of ordinary antimycobacterial drugs in treating MAC diseases is principally due to the low susceptibility of MAC to the majority of ordinary antimycobacterial drugs, the extremely wide range of susceptibilities of MAC isolates to most antimicrobial drugs, and the fact that polyclonal MAC infections are occasionally seen in AIDS patients. Therefore, the development of new antimicrobials and administration protocols that are safe and potently effective against MAC infections is urgently needed. However, despite the gradual but steady increase in the incidence of MAC infections, and thereby the urgent call for new drug development, new drugs that are truly useful for the treatment of refractory MAC diseases continue to elude us. In this review article, the following topics will be described: (1) the present status of the existing anti-MAC drugs, with special reference to the recent findings of the in vitro and in vivo anti-MAC activities of macrolides and other moderately useful drugs, such as new rifamycins, clofazimine and some fluoroquinolones, and (2) the present status and future prospects of the development of new promising antimicrobials with anti-MAC activity.

Purification, Properties, and Cytotoxic Effect of a Bacteriocin from Mycobacterium smegmatis

The bacteriocin produced by Mycobacterium smegmatis ATCC 14468 was isolated, and a study was made of its chemical, physical, and biological properties. No appreciable bacteriocin activity was found in the culture supernatant fluids, but it was released in appreciable quantities after disruption of the cells. The material was purified 49-fold by means of chromatography on diethylaminoethyl-cellulose, ammonium sulfate fractionation, gel filtration on Sephadex G-200, and chromatography on diethylaminoethyl-Sephadex A-50. Its molecular weight was determined to be approximately 75,000 from the elution profile on Sephadex G-200 chromatography. The bacteriocin was resistant to deoxyribonuclease, ribonuclease, lipase, ultraviolet irradiation, and freeze-thawing, whereas it was relatively less thermostable and was sensitive to proteolytic enzymes. The lethal effect of the bacteriocin was demonstrated by the decrease in viable counts of the bacteriocin-sensitive indicator strain, M. diernhoferi ATCC 19340. The bacteriocin preparation inhibited the growth of HeLa-S3 cells.

Interaction of Antimycobacterial Drugs with the Anti-Mycobacterium avium Complex Effects of Antimicrobial Effectors, Reactive Oxygen Intermediates, Reactive Nitrogen Intermediates, and Free Fatty Acids Produced by Macrophages

ABSTRACT The profiles of the interaction of antimycobacterial drugs with macrophage (MΦ) antimicrobial mechanisms have yet to be elucidated in detail. We examined the effects of various antimycobacterial drugs on the anti-Mycobacterium avium complex (MAC) antimicrobial activity of reactive oxygen intermediates (ROIs), especially of an H2O2-halogen (H2O2-Fe2+-NaI)-mediated bactericidal system, reactive nitrogen intermediates (RNIs), and free fatty acids (FFAs), which are known as central antimicrobial effectors of host MΦs against mycobacterial pathogens. We have found that certain drugs, such as rifampin (RIF), rifabutin (RFB), isoniazid (INH), clofazimine (CLO), and some fluoroquinolones, strongly or moderately reduced the anti-MAC activity of the H2O2-Fe2+-NaI system, primarily by inhibiting the generation of hypohalite ions and in part by interfering with the halogenation reaction of bacterial cell components due to the H2O2-Fe2+-NaI system. This phenomenon is specific to the H2O2-Fe2+-NaI system, since these drugs did not reduce the anti-MAC activity of RNIs and FFAs. From the perspective of the chemotherapy of MAC infections, the present findings indicate an important possibility that certain antimycobacterial drugs, such as rifamycins (RIF and RFB), INH, CLO, and also some types of fluoroquinolones, may interfere with the ROI-mediated antimicrobial mechanisms of host MΦs against intracellular MAC organisms.

Correlation between variable-number tandem-repeat-based genotypes and drug susceptibility in Mycobacterium avium isolates

Little is known about the correlation between genotype and drug susceptibility in Mycobacterium avium (Mav) strains isolated from patients with Mav infections. To examine whether drug susceptibility profile of Mav is associated with genotype, we carried out variable-number tandem-repeat (VNTR) typing and drug susceptibility testing for Mav isolates from Japanese with nodular-bronchiectasis (NB)-type and cavitary disease (CA)-type diseases. We performed M. avium tandem repeat (MATR)-VNTR typing and drug susceptibility testing by the broth dilution method, using macrolides, rifamycins, ethambutol, isoniazid, aminoglycosides, and quinolones, for Mav isolates from patients with NB and CA-type diseases (NB-Mav and CA-Mav). Based on the VNTR genotyping, the Mav strains were grouped into three clusters. There was no difference with respect to the distribution of NB-Mav and CA-Mav among the clusters. We observed a strong association between VNTR genotype and susceptibility to quinolones (levofloxacin, moxifloxacin, gatifloxacin, sitafloxacin, and garenoxacin) and ethambutol. There was essentially no significant difference in drug susceptibility between NB- and CA-Mav strains, although NB-Mav was somewhat more resistant to fluoroquinolones, especially gatifloxacin, than CA-Mav. There was a significant association between VNTR genotype and susceptibility to quinolones and ethambutol in Mav isolates from Japanese patients.