Clinical Trial of Ofloxacin Alone and in Combination with Dapsone ...

Vol. 38, No. 4

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1994, p. 662-667

0066-4804/94/$04.00+0 Copyright X) 1994, American Society for Microbiology

Clinical Trial of Ofloxacin Alone and in Combination with Dapsone plus Clofazimine for Treatment of Lepromatous Leprosy BAOHONG JI,l* EVELYN G. PERANI,' CORINNE PETINOM,' LAURENT N'DELI,2 AND JACQUES H. GROSSET1 Faculte de Medecine Pitie-Salpetriere, Paris, France,' and Institut Raoul Follereau, Adzope, Cote d'Ivoire2 Received 18 October 1993/Returned for modification 23 January 1994/Accepted 2 February 1994

Twenty-four patients with newly diagnosed lepromatous leprosy were allocated randomly to three groups and treated for 56 days with 400 mg of ofloxacin daily, 800 mg of ofloxacin daily, or 400 mg of ofloxacin, 100 mg of dapsone, and 50 mg of clofazimine daily plus 300 mg of clofazimine once every 28 days. The patients in all three groups demonstrated remarkable clinical improvements, accompanied by rapid decline of the morphological index in skin smears during treatment. More than 99, >99.99, and >99.99%o of the viable Mycobacterium leprae organisms had been killed by 14, 28, and 56 days of treatment, respectively, as measured by inoculation into the footpads of immunocompetent and nude mice of organisms recovered from skin biopsy specimens obtained before and during treatment. Mild to moderate elevations of the serum glutamic pyruvic transaminase level were observed in four patients, all after 28 days of treatment, which returned to normal after the trial had been completed. Clinical improvement, bactericidal activity, and hepatotoxicity did not differ significantly among the three groups. Ofloxacin displayed powerful bactericidal activity against M. kprae in leprosy patients and may be an important component of new multidrug regimens for the treatment of leprosy. Its optimal dosage appears to be 400 mg daily, and combination with dapsone and clofazimine does not enhance its activity. human leprosy. The third objective was to determine the usefulness of combining ofloxacin with dapsone and clofazimine in a new multidrug regimen. The bactericidal activities of the treatments were monitored by inoculation into mouse footpads of organisms recovered from skin biopsy specimens taken before and at intervals during treatment (3, 5). Because we already knew that 22 doses of ofloxacin alone killed about 99.99% of the viable organisms, nearly the limit of sensitivity of the technique even with nude mice (3, 5), the duration of the trial was set at 56 days.

Development and implementation of multidrug therapy (13) are the most important achievements in the history of leprosy control (12). However, the duration of the treatment is too long, especially the minimal duration for multibacillary leprosy therapy, which is 2 years, which causes serious operational difficulties in certain areas (5). Of the three components of the multidrug regimen for multibacillary leprosy, rifampin is the only drug that displays powerful bactericidal activity against Mycobacterium leprae; the other two drugs, dapsone and clofazimine, show only a modest degree of bactericidal activity. Without the introduction of new, more powerful bactericidal drugs to the regimens, it is unlikely that the duration may be

MATERIALS AND METHODS

substantially shortened (5). Among the major commercially available fluoroquinolones, ofloxacin is by far the most active compound against M. leprae (2). A clinical trial with patients with lepromatous leprosy demonstrated that 400 mg of ofloxacin daily was highly effective, producing very remarkable clinical improvement and killing an average of 99.99% (4 orders of magnitude) of M. leprae organisms after only 22 doses of treatment (3). This observation represented the first lead to an important new antileprosy drug since the introduction of rifampin more than

Patients. Twenty-four newly diagnosed leprosy patients (19 men and 5 women) with a mean age of 38 years (range, 18 to

60 years) who demonstrated large bacterial populations and active skin lesions were selected; 20 were classified as polar lepromatous and 4 were classified as borderline lepromatous by histopathological examination of skin biopsies. During the first visit to our clinic, all denied previous antileprosy treatment and analyses of urine for dapsone were negative. The patients were allocated randomly to one of three groups; the groups were comparable with regard to pretreatment characteristics, including sex, age, classification of the disease, severity of skin lesions, mean bacterial index (BI) and morphological index (MI) in skin smears, and proportion of viable M. leprae organisms in the bacterial population. Treatment. The patients in group A were treated with 400 mg of ofloxacin once daily, those in group B were treated with 800 mg of ofloxacin once daily, and those in group C were treated with 400 mg of ofloxacin, 100 mg of dapsone, and 50 mg of clofazimine once daily, together with 300 mg of clofazimine once every 28 days for 56 days. In other words, the patients of group C were treated with ofloxacin plus the standard dosages of dapsone and clofazimine employed in the current multidrug regimen for multibacillary leprosy (13). The

20 years ago. The current trial was carried out with several objectives in mind. First, because the number of spontaneously arising rifampin-resistant mutants in an untreated lepromatous patient is estimated to be no more than 104 (3, 5), it was necessary to confirm the killing of 99.99% of M. leprae by a short course of ofloxacin treatment. Second, because the activity of ofloxacin against M. leprae (2) and Mycobacterium tuberculosis (11) in mice is dose related, it was important to compare the therapeutic effects of and adverse reactions to 400 mg of ofloxacin daily with those of 800 mg of ofloxacin daily in the treatment of * Corresponding author. 662

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patients were hospitalized during the trial, and all drugs were administered under supervision. Examinations before and during the trial. The clinical, bacteriological and other laboratory examinations have been described at length elsewhere (3, 7). In brief, the following examinations were performed for each patient before starting treatment. A thorough physical examination (especially that for leprosy) was performed, photographs of skin lesions were taken, the lepromin test was performed, chest X rays were taken, skin smears from six sites for measurement of BI and MI were taken, tests of hepatic and renal function were performed, and skin biopsies from two distinct, active lesions for measurement of the proportion of viable M. leprae organisms by inoculation of the mouse footpad were taken. Of the two specimens, only the organisms from the lesion demonstrating the greater concentration of M. leprae, in terms of the logarithm1o of the number of acid-fast bacilli (AFB) per milligram of tissue (3), or the higher MI were selected for mouse inoculation. Except for the lepromin test and chest X ray, the examinations were repeated regularly during the trial. The skin smears were taken from the same six sites as before treatment, and the skin biopsy was taken from the pretreatment site that had provided M. leprae for mouse inoculation. Compared with pretreatment manifestations, the clinical responses, on the bases of the amelioration of nasal obstruction, regression of infiltration, and flattening or disappearance of nodules, lepromas, or plaques, were scored as no change, definite improvement, and marked improvement (3, 6, 7). The proportion of viable M. leprae organisms in the bacterial population was estimated by inoculation of serially diluted suspensions prepared from skin biopsy specimens into the hind footpads of mice. Four dilutions of the organisms recovered from the pretreatment (day 0) specimens were inoculated into footpads of immunocompetent (normal) mice at inocula of 5 X 103, 5 x 1O2, 5 x 10', and5 x 100 organisms per footpad. From the specimens obtained on day 14 of the trial, 0.03 ml of undiluted tissue suspension (i.e., the maximal available number of organisms) was inoculated into each hind footpad of a congenitally athymic (nude) mouse (3, 6, 7), and two dilutions (5 x103 and5 x 102 organisms per footpad) were inoculated into normal mice. The M. leprae recovered from specimens obtained on day 28 or 56 of the trial were inoculated with the maximal available number of organisms into the footpads of nude mice and were inoculated with one dilution (5 x103 organisms per footpad) into normal mice. Ten mice were inoculated per dilution, and the footpads were harvested 12 months after inoculation for counting of AFB by Shepard's method (10). Under the conditions prevalent in our laboratory, detection of a single bacillus in the course of examining 40 oil-immersion fields indicates that the suspension contains 1.9 x 104 AFB per footpad. In normal mice, the organisms are considered to have multiplied (i.e., the inoculum included viable organisms) 210 if 5AFB per footpad are harvested. In nude mice, the criterion of multiplication was originally defined as an at least twofold increase in the number of AFB per footpad over that inoculated, but for the reason explained below, this criterion has since been abandoned in favor of the same criterion as that employed for normal mice. Statistical analysis. The proportion of viable organisms in an inoculum was calculated by the Spearman-Karber method (9) from the results of multiplication ofM. leprae in the mouse footpads that had been inoculated with several dilutions of the organisms. If only normal mice are inoculated and the maximal inoculum is x5 i03 AFB per footpad (3, 5), a proportion of viable M. leprae organisms as low as 0.006% may be measured;

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the minimal proportion that can be measured may be reduced to between 0.0001% and 0.00001% if both normal and nude mice are inoculated and larger numbers of organisms are inoculated into the footpads of the nude mice (3, 6). Other results were analyzed and compared by Student's t test and Fisher's exact probability calculation. Differences were considered significant at the 95% level of confidence. RESULTS Clinical responses. As shown in Table 1, definite improvement occurred in more than half of the patients by day 14, all patients showed either definite or marked improvement by day 28, and the great majority of patients showed marked improvement by day 56. Many of the nodules, lepromas, or diffuse infiltrations present before treatment had resolved significantly, and some of the smaller lesions had disappeared by the end of the trial. The clinical responses did not differ significantly among the three groups at any interval (P > 0.05). Changes of BI and MI in skin smears. As is also shown in Table 1, the mean BI among the patients of all three groups appeared to have decreased by an average of 0.2 U during the 56 days of the trial; however, the values at the end of the trial did not differ significantly from the pretreatment values (P > 0.05). On the other hand, the mean MI decreased considerably among the patients of all three groups (P < 0.01); the mean MI on day 14 was smaller than that on day 0 (P < 0.05), had decreased to the baseline between day 14 and day 28 (P < 0.01), and remained at the same low level until the end of the trial. No significant difference in the reduction of MI could be detected among the three groups (P > 0.05). Bactericidal activities of the treatments against M. keprae. On the basis of the results of mouse footpad inoculation, the number of cases in which viable organisms had been included in the inocula of various concentrations, the calculations of the proportions of viable organisms, and the percentage of viable organisms killed by the treatments are summarized in Table 2. After 14 days of treatment, the number of cases in which viable organisms were included in the inoculum may have been lower among the patients of group B than among the patients of the other two groups, although the difference was not statistically significant (P > 0.05); the numbers were virtually the same among the three groups by days 28 and 56. The proportions of viable organisms in the pretreatment skin biopsy specimens varied widely, ranging from 0.014 to 27%, but the distributions were similar among the three groups (P > 0.05), with median values slightly above 1%. The proportions of viable organisms had decreased significantly between day 0 and day 14 (P < 0.01) and between day 14 and day 28 (P < 0.05 or P < 0.01); because the proportions had reached the lower limit of measurement on day 28, they were not significantly different from those measured on day 56. Calculation of the proportions of viable organisms revealed that >99 or >99.9% of the viable M. leprae organisms present at the beginning of treatment had been killed by 14 days of treatment, and >99.99% killing had occurred among the patients of all three groups after treatment for 28 or 56 days. No significant difference in bactericidal activity was discerned among the three groups (P > 0.05). Because, as shown in Table 2, the results did not differ significantly among the three groups, they have been combined and are further' summarized in Table 3 to facilitate further analysis. Before treatment, theM. leprae were observed to have multiplied in more than 57% of the inoculated footpads of normal mice. Among the 240 mice that had been inoculated with each dilution, multiplication was observed in 208 (86.7%), 167 (69.6%), 120 (50.0%), and 55 (22.9%) of the mice that had

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TABLE 1. Clinical responses and changes in the BI and MI from skin smears in three groups of patientsa Response to treatment in: Parameter and date Group A

Group B

Group C

4 4 0

1 7 0

3 5 0

0 3 5

0 3 5

0 6 2

0 2 6

0 2 6

0 3 5

5.04 ± 0.45 4.88 ± 0.59 4.85 ± 0.64 4.83 ± 0.41

5.19 ± 0.31 5.15 ± 0.34 5.04 ± 0.47 4.95 ± 0.42

5.17 ± 0.24 5.19 ± 0.24 5.06 ± 0.12 4.98 ± 0.11

Score of clinical response Day 14 No change Definite improvement Marked improvement Day 28 No change Definite improvement Marked improvement Day 56 No change Definite improvement Marked improvement BI

Day 0 Day 14 Day 28 Day 56 MI (%) Day 0 Day 14 Day 28 Day 56

9.4 5.3 1.8 0.6

± ± ± ±

4.4 1.5 1.3 0.5

8.5 ± 4.7 3.7 ± 2.2 1.0 ± 1.1 1.1 ± 1.4

8.6 4.2 1.5 1.2

± ± ± ±

4.2 1.7 1.4 1.3

a Groups A to C (n = 8 for each group), respectively, were treated with 400 mg Of ofloxacin daily; 800 mg of ofloxacin daily; and 400 mg of ofloxacin, 100 mg of dapsone, and 50 mg of clofazimine daily plus 300 mg of clofazimine once every 28 days. Values for BI and MI are means ± standard deviations.

been inoculated with 5 x 103, 5 x 102, 5 x 10', and 5 x 100 M. leprae organisms per footpad, respectively. After 14 days of treatment, however, the percentage of normal mice in which multiplication had occurred had decreased to approximately 2%; multiplication was observed in only 7 (2.9%) and 3 (1.3%) of the 240 normal mice that had been inoculated with 5 x 10 and 5 x 102 organisms per footpad, respectively. The percentage of footpads in which multiplication had occurred was very significantly higher among the nude mice (P < 0.01), because unequivocal multiplication (the number of AFB per footpad had increased more than twofold over that inoculated) was observed in 72 (34.3%) footpads. At the same time, very few organisms (3.0 x 105 AFB per footpad but fewer than twice the number inoculated. The organisms from each nude mouse footpad

were subinoculated into 5 to 10 normal mice, in most cases between 1.0 x 103 to 5.0 x 103 organisms per footpad, and harvests were performed 12 months later. Except for one subinoculation, in which M. leprae multiplied in only a portion of the footpads, multiplication was observed in all footpads that had been subinoculated with organisms from 15 nude mouse footpads that had yielded 2105 AFB per footpad. On the other hand, multiplication was observed- only in one of the three subinoculations with organisms harvested from the nude mouse footpads that had yielded 99.99% of the viable M. leprae organisms present before treatment by 14 and 28 days, respectively. Therefore, these results confirmed our earlier observation (3). In the treatment of multibacillary leprosy, one should eliminate all of the drug-resistant mutants before stopping multidrug therapy. Employing the current multidrug regimen (13), the rifampin-resistant mutants, which should respond to other antimicrobial agents in the same manner as do rifampinsusceptible organisms, can be killed only by dapsone and clofazimine. However, because these latter drugs display only weak bactericidal effects, treatment must be continued for at least 2 years to ensure elimination of the rifampin-resistant mutants (5). As already described, (i) the total number of rifampin-resistant mutants of M. leprae in an untreated lepromatous patient has been estimated to be no greater than 104 (3, 5), (ii) killing of >99.99% of the viable M. leprae organisms present before treatment within 28 days of ofloxacin treatment

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TABLE 3. Numbers of inoculated footpads of normal and nude mice in terms of AFB per footpad at harvest No. (%) of inoculated footpads with given result from:

Normal micea

Nude miceb

Duration of treatment (days) Total

0