[Guideline for the treatment of Hansen's disease in Japan].

Ad hoc committee of Japanese Leprosy Association recommends standard treatment protocol of leprosy in Japan, which is a modification of World Health Organization's multidrug therapy (WHO/MDT, 1997). For paucibacillary (PB) leprosy, 6 months treatment by rifampicin and dapsone (MDT/PB) is enough. However, for high bacterial load multibacillary (MB) leprosy, 12 months treatment seems insufficient. Thus, (A) For MB with bacterial index (BI) > or = 3 before treatment, 2 years treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. (A-1) When satisfactory decrease of BI (BI value decrease > or = 2 steps, or final BI < 3) is obtained after completion of 2 years MDT/MB, maintenance therapy by dapsone and clofazimine is recommended until BI negativity and loss of active lesions. (A-2) When BI decrease is not satisfactory (BI value decrease < 2 steps, or final BI > or = 3), MDT/MB should be continued until BI negativity and loss of active lesions. (B) For MB with BI < 3 or fresh MB (less than 6 months after the onset of the disease) with BI > or = 3, 1 year treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. (B-1) When BI become negative and active lesion is lost within one year, no maintenance therapy is necessary. (B-2) When BI is still positive or active lesion is remaining, additional therapy with MDT/MB for one more year is recommended. Brief summary of diagnosis, purpose of therapy, character of drugs, and prevention of deformity is also described.

[Chemoprophylaxis against Mycobacterium avium complex infection induced in mice].

M. avium complex (MAC) is one of the important causative agents of opportunistic infections among AIDS patients. Recent evidence showed that the entry of infection is through the gastrointestinal tract. In the present study, we compared the prophylactic effect of some antimicrobials against MAC infection induced in mice. Different groups of beige mice were fed with pellets containing 0.0067% (10 mg/kg) of KRM-1648, rifabutin (RFB) and clarithromycin (CAM). Seven days after the administration of drugs, the mice were infected with M. intracellular N-241 (5 x 10(8) CFU) orally, five times, every other day and killed one and 126 days after the last infection. The effect of drug was evaluated using the frequency and severity of gross lung lesions in the mice and by the total CFU recovered from the lungs and spleen. MAC infection was not likely to be established since there was no macroscopic evidence of lesion in organs and the recovery of cultures from lungs and spleen tested was negative, in 3 of 10 mice in the control group, 2 of 9 in the CAM group, 4 of 9 in the RFB group and 4 of 10 in the KRM group. These mice were excluded from the analysis of the study results. Thus, we examined 7 mice in the control group, 7 in the CAM group, 5 in the RFB group, and 6 in the KRM group. Tubercle-like lesions were observed in the lungs of all 7 mice in the control group (severity: 3+ in 5 mice; 4+ in 2 mice), in 5 of 7 mice (71%) in the CAM group (severity: 2+ in 1 mouse; 3+ in 4 mice), and in 4 of 5 mice (80%) in the RFB group (severity: 1+ in 1 mouse; 2+ in 1 mouse; 4+ in 2 mice), while only slight lesions (severity: 1+) were observed in 4 of 6 mice (67%) in the KRM group. There was no macroscopic evidence of lesion in spleen, liver and kidneys. The log CFU was determined at the next day of the completion of the last infection. The log CFU of the lungs was 2.49 and 2.28 in the control group and the CAM group, respectively. The bacteria were not recovered either from the lungs in the RFB and KRM groups, nor from the spleen in all the groups. The order of efficacy of the drugs on the basis of the CFUs recovered from the lungs and spleen in each group determined 126 days after the completion of the last infection was as follows; KRM > CAM > RFB in the lungs and KRM > CAM [symbol: see text] RFB in the spleen, although there was no significant difference among the three drugs (P < 0.05). However, the significantly preferable effect was obtained in the three drug groups as compared with the control group.

Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene.

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

[The control leprous peripheral neuropathy and chemotherapy].

Clarithromycin(CAM), Roxithromycin(RXM), Minocycline(MINO) and Fosfomycin(FOM) has anti-inflammatory action and immunomodulatory activity, while the anti-mycobacterium leprae activity is shown. CAM and RXM suppress the rat carrageenin edema, and MINO suppresses the rat adjvant arthritis. There is the immunosuppression on adrenocorticosteroid while the inflammatory cytokine is suppressed. CAM, MINO, FOM suppresses the inflammatory cytokine, while it has the immunomodulatory activity. Fusidic acid(FA) suppresses the inflammatory cytokine with the action of being similar to cyclosporin A, and it has the immunomodulatory activity. New macrolides derivatives, CAM and RXM showed the inflammatory regulation, and MINO showed the anti-inflammatory activity with FA. The combination chemotherapy can be enforced, while peripheral neuropathy is prevented by the control of the leprosy reaction.

The possible role of interleukin (IL)-12 and interferon-gamma-inducing factor/IL-18 in protection against experimental Mycobacterium leprae infection in mice.

Cell-mediated immunity participates in host defense against mycobacterial infection. Both interleukin 12 (IL-12) and interferon-gamma-inducing factor (IGIF/IL-18), produced mainly by macrophages, play a critical role in expression of cell-mediated immunity. To investigate the role of IL-12 and IGIF/IL-18 in vivo, we examined cytokine profile, bacterial growth, and the potential benefit of cytokine therapy in susceptible and resistant mice infected with Mycobacterium leprae. The early expression of IL-12 p40 and IGIF/IL-18 at the site of inoculation was found in resistant mice 3-72 h after the infection, but not in susceptible mice. Both strains of mice did not show expression of IFN-gamma and IL-4. IL-12 administration resulted in a significant reduction of bacterial counts in mice with established M. leprae infection. The results imply that susceptible mice exhibit decreased expression of type 1 helper T (Th1) response without reciprocal increased Th2 response and show responsiveness to exogenous IL-12. IL-12 therapy may be a possible rationale for treatment of M. leprae infection.

Pathological investigation of armadillos infected with Mycobacterium leprae.

An infection experiment with M. leprae was carried out using 20 nine-banded armadillos. As a result, the development of leprous lesions and a marked multiplication of AFB were confirmed in a high rate of 13 out of 15 cases (86.8%) in the inoculated groups. These changes were found to be progressing at post mortem of one case even with the shortest life period for 7.5 months and were very serious in one case with the longest life period for 33 months, suggesting the continuation of symptoms, though it is an expression neglecting the individual difference in susceptibility to leprosy. Among infected viscera with AFB, the most conspicuous lesions were found in the liver and spleen. The developed lesions were found in the lung, stomach and kidney which had been never seen in HD in human cases, and so, which may characterize armadillos' leprosy. The change in the peripheral nerve was not so severe when compared with that in HD in human cases. This difference will remain as a future pathological problem to be solved.

Inhibition of the multiplication of Mycobacterium leprae in nude mice by intermittent administration of a new rifamycin derivative, 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) combined with sparfloxacin.

Inhibition of the multiplication of Mycobacterium leprae in the footpads of nude mice by the oral administration of sparfloxacin, a new quinolone, and 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648), selected from a series of newly synthesized benzoxazinorifamycins, was studied. When the 2 drugs were administered alternately at intervals of 3 or 4 days, (i.e., each drug was administered once weekly), or simultaneously once weekly, between 3 and 5 months after inoculation of nude mice with M. leprae, 10 mg sparfloxacin and 0.6 mg KRM-1648 per kg bodyweight were sufficient to prevent multiplication of the organisms. Only partial inhibition of multiplication was achieved by alternate administration of 5 mg sparfloxacin and 0.3 mg KRM-1648 per kg, as was the case for 20 mg sparfloxacin per kg or 1 mg KRM-1648, each drug administered alone once weekly. The addition to these 2 drugs of dapsone, administered in the diet in a concentration of 0.001 g per 100 g, enhanced their effect. The potential usefulness of multidrug regimens including these compounds is considered.

Bactericidal action at low doses of a new rifamycin derivative, 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl) benzoxazinorifamycin (KRM-1648) on Mycobacterium leprae inoculated into footpads of nude mice.

Among a series of newly-synthesized benzoxazinorifamycins, 2 of the 3'-hydroxy-5'-(4-alkyl-1-piperazinyl) derivatives, named KRM-1648 and KRM-2312, whose respective alkyl residues are isobutyl and isopropyl, were examined for efficacy against nude mouse-model leprosy. KRM-1648 completely inhibited the growth of leprosy bacilli inoculated into nude mouse footpads, even 6 months after the medication had been stopped, when given orally at a daily dose of 0.6 mg/kg, 5 or 6 times weekly, during 3-5 months postinoculation. In comparison, the growth inhibition by KRM-2312 was incomplete under the same conditions, though it was still stronger than that by rifampicin. Complete growth inhibition by KRM-1648 was also observed when it was given orally at a dose of 1 or 3 mg/kg twice weekly during the same period. In contrast, the growth inhibition by rifampicin was only slight at 1 mg/kg and partial at 3 mg/kg under the same condition.

Activity of sparfloxacin against Mycobacterium leprae inoculated into footpads of nude mice.

The antileprosy activity of a new quinolone, sparfloxacin, was examined in the nude mouse footpad model. By serial dosing (once a day, 5 or 6 times per week, during the 3rd-5th months postinoculation), 10 mg/kg of sparfloxacin displayed bactericidal-type activity and bacteriostatic activity was present at daily doses of 5 and 2 mg/kg. By intermittent dosing (once a day, twice weekly at daily doses of 10 and 20 mg/kg or once weekly at a daily dose of 30 mg/kg, during the 3rd-5th months postinoculation), sparfloxacin markedly inhibited the growth of leprosy bacilli with slight remultiplication at later stages. Sparfloxacin seems to be worth studying clinically as a novel antileprosy drug.

Studies on the development of novel anti-leprous chemotherapeutics using nude mice with special reference to a new quinolone carboxylic acid, AT-4140.

In order to develop a novel drug for antileprosy chemotherapy, the inhibitory effects of three synthesized compounds, a supplied antituberculous one and three quinolone carboxylic acids were examined on the growth of leprosy bacilli inoculated into the footpads of nude mice. Amongst them, a new quinolone carboxylic acid, AT-4140 whose chemical structure was 5-amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(cis-3, 5-dimethyl-1-piperazinyl)-4-oxoquinoline-3-carboxylic acid strongly inhibited the growth of leprosy bacilli at doses of 15 and 30 mg/kg. Whereas, the effect of Ofloxacin used as a positive control was limited at the same doses.

The inhibitory effects by combined doses of DDS and several immunostimulants on the growth of leprosy bacilli inoculated into footpads of hybrid nude mice, Jcl:AF-nu.

The inhibitory effects by the combined doses of bacteriostatic DDS and several immunostimulants on the growth of leprosy bacilli (LB) inoculated into the footpads of nude mice were examined, using a strain of hybrid nude mice named Jcl:AF-nu, which is resistible to infections more than strain BALB/c-nu. The results found were: 1. LB could proliferate up to 10(9) level in the footpads of this strain of hybrid nude mice. 2. The combined dose of muramyl dipeptide (MDP) or a water-soluble lipoidal amine, CP-46665 and DDS mixed with chow in the content ratio of 0.005% completely inhibited the growth of LB. Whereas, the growth inhibition by DDS alone was only partial. 3. A derivative of MDP, muroctasin could partially inhibit the growth of LB without combined dose of DDS. 4. The combined dose of an antitumor beta-1, 3-glucan named ATSO could not enhance the partial growth inhibition due to the dose of DDS through 0.005%-DDS chow. Based on these results, the possibility of Jcl:AF-nu mice as a favorable animal model for examining the synergic inhibitory effect on the growth of LB due to the combined dose of an antileprous chemotherapeutic and an immunostimulant was discussed.

Determination of three main antileprosy drugs and their main metabolites in serum by high-performance liquid chromatography.

The simultaneous analysis of main antileprosy drugs such as 4,4'-diaminodiphenyl sulfone (DDS), clofazimine, rifampicin and their main metabolites in serum was examined by high-performance liquid chromatography using a muBondapak C18 column. When the drugs dissoluted from serum were developed by tetrahydrofuran-0.5% acetic acid (40:60), clofazimine and rifampicins could be analyzed separately. Apart from the mutual separation of water-soluble conjugates of DDS, the individual analysis of DDS, its main liposoluble metabolite and a few related sulfone compounds is possible when the drugs are first developed by acetonitrile-water (20:80). By the use of tetrahydrofuran-water (50:50) containing PIC B-5, the rapid measurement of clofazimine isolated from the other compounds is also possible.