60, 3^ Correspondence^ 485

3. RIDLEY, D. S. and JOPLING, W. H. Classificationof leprosy according to immunity; a five-group sys-tem. Int. J. Lcpr. 34 (1966) 255-273.

4. SHETTY, V. P. and ANTIA, N. H. Degeneration andregeneration of unmyelinated fibres in experimentalleprous neuropathy. Int. J. Lepr. 49 (1981) 324-330.

5. SIIETTY, V. P., ANTIA, N. H. and JACOBS, J. M. Thepathology of early leprous neuropathy. J. Neurol.Sci. 88 (1988) 115-131.

6. TURK, L. J. and NARAYAN, R. B. The origin, mor-phology and function of epithelioid cells. Immu-nobiology 161 (1982) 274-282.

Clarithromycin at Very Low Levels and onIntermittent Administration Inhibits the

Growth of M. leprae in Mice

To THE EDITOR:Clarithromycin was found previously to

inhibit the metabolic activity of Mycobac-terium leprae in cell-free and tissue culturesystems (2). Furthermore, it had been de-termined that when Al. leprae-infected micewere fed a diet containing clarithromycin[0.01% (4) and 0.1% ( 5) and 25 mg/kg bygavage five times weekly ( 7)], bactericidalactivity for Al. leprae was consistently ob-served. In none of these studies was clarith-romycin administered at a dietary concen-tration which was found ineffective and,therefore, we initiated these current studiesto define clarithromycin's minimal inhibi-tory dietary, serum, and tissue concentra-tions for Al. leprae in infected mice. TheWorld Health Organization ( 14) has for thepast decade advocated monthly rifampin inthe treatment of leprosy. Since a companionantimicrobial agent which has proven ac-tivity on intermittent administration mightoffer particular utility in the practical deliv-ery of effective therapy to leprosy patientsin endemic countries, in these studies wealso determined the efficacy of intermittentclarithromycin administration in this mousemodel system.

Eight-week-old, female, BALB/c wean-ling mice (Jackson Laboratories, Bar Har-bor, Maine, U.S.A.) were infected in bothhind foot pads with 5000 Al. leprae. The M.leprae isolate utilized in this study originallyhad been obtained from a lepromatous lep-rosy patient, maintained in mouse passagein our laboratory for the previous 10 years,and was harvested from the foot pads ofmice near the time of its peak multiplica-

tion. From the time of infection, groups ofmice were continuously fed diet containingvarious concentrations of clarithromycin[0% (control), 0.0001%, 0.001%, 0.005%,0.01%, 0.05%, 0.1°4 Also, groups of micewere treated with 0.1% in diet 3 days weekly(M, W, F), 1 day weekly, and 1 day monthly.At 7, 9, and 11 months after infection, thenumber ofM. leprae from the foot pad poolsof two mice (four feet) from each group weredetermined by standard microscopic pro-cedures ( 12). When the number of M. lepraeper foot pad was found to be 10', bacillarygrowth was considered to have occurred ( 11 ).

At 7 months, Al. leprae grew to 2 x 10 5/foot pad in the untreated control mice, andremained at approximately that level at the9- and 11-month harvest intervals (The Ta-ble). Clarithromycin 0.0001% in the diet didnot inhibit Al. leprae multiplication (TheTable). However, clarithromycin 0.001%,0.005%, 0.01%, 0.05%, and 0.1% each pre-vented Al. leprae multiplication at all threeharvest intervals (The Table). Clarithro-mycin 0.1% in the diet 3 days weekly andeven 1 day weekly, also, consistently inhib-ited the growth of AI. leprae, while multi-plication of Al. leprae in mice treated onlyonce monthly was inhibited both at 7 and9 months, but not at the 11-month interval(The Table).

Also, in these studies, mice were fed 2weeks of each of the test diets, and clarith-romycin concentrations in the mouse serum(five mice) and foot pads (one mouse) weredetermined by an agar disk diffusion meth-od employing Micrococcus htteus ATCC93418 (3) (Abbott Laboratories, North Chi-cago, Illinois, U.S.A.). In the untreated con-

486^ International Journal of Leprosy^ 1992

THE TABLE. Activity of clarithrotnycin against M. leprae-inlected mice.

Clarithromycinconcentration

in diet

Months after foot pad infection

No. M. leprete per foot pad

7 9 I^1

Control 2.42 x 10' 1.13^x 10' 1.01^x 10'0.0001•. 6.59 x 10' 8.36 x 10' 1.87 x 10'0.001%• 2.63 x 1W < 8.24 x 1W 7.02 x 10'0.005%" < 6.6^x 1W • < 9.79 x 1W < 6.69 x 10'0.01%' < 9.86 x 10' < 6.02 x 1W < 6.33 x 10'0.05%' < 8.76 x 10' < 8.22 x 10' < 6 69 x 10'0.1%' < 6.69 x 10' < 8.88 x 10' < 6.69 x 10'

Three days per week 0.1% 2.66 x 104 <^1.15 x Hp 6.33 x 10'One day per week 0.1% 9.34 x 10' < 2.4^x 10' 1.07 x 1WOne day per month 0.1% 6.13^x 10' 1.66 x 10' 1.07x 10'

Mice fed continuously; numbers of M. leprae per foot pad were obtained from a pool of four feet.

trol mice and those treated with 0.0001%,0.001%, 0.005%, and 0.01%, clarithromy-cin serum levels were consistently < 0.025pg per ml and foot pad concentrations <0.7 pg/g. Clarithromycin 0.05% in the dietresulted in serum levels of 0.16 ± 0.11 pg/ml and a foot pad level of 1.7 pg/g, whileclarithromycin 0.1% in diet resulted in se-rum levels of 0.36 ± 0.23 pg/m1 and footpad level of 3.3 pg/g.

These studies have demonstrated that lowdietary, serum, and tissue levels of clarith-romycin inhibit the growth of M. leprac inmice. Previously, it was found that 0.01%and 0.1% dietary clarithromycin preventedthe multiplication of Al. leprae in mousefoot pads. In this study, 0.001% dietary ,cla-rithromycin and all higher tested concen-trations (0.005%, 0.01%, 0.05% and 0.1%)consistently prevented the growth ofM. lep-rae in infected mice. Furthermore, clarith-romycin was found effective on intermittentadministration, consistently when admin-istered in the diet 1 day and 3 days weeklyand even partially active when adminis-tered 1 day monthly. Of the three new can-didate antimicrobials that appear promisingfor the therapy of leprosy, minocyclinc ( 6 )and now clarithromycin, but not fluoro-quinolones ( 10), are active against M. leprae-infected mice when administered less fre-quently than three times per week.

Previously we found that against M. lep-rae-infected mice, dietary erythromycin(0.06%) and azithromycin (0.1%) were con-sistently inactive. On the other hand, cla-rithromycin (0.1%) and roxithromycin(0.1%) were consistently active and, in fact,

bactericidal, clarithromycin being foundmore active than roxithromycin. Franzblauand Hastings ( 4) found that while clarith-romycin (0.01%) was bactericidal for .11.leprac, erythromycin (0.01%) and roxith-romycin (0.01%) were inactive. These cur-rent studies demonstrate that a tenfold low-er concentration of clarithromycin than hadbeen studied previously, 0.001% clarithro-mycin, has activity for M. leprae. Thus, itwould appear that, as had been found pre-viously for both M. aviton ( 2•') and .11. che-lonaen, clarithromycin is superior to othermacrolides in its activity for Al. leprae.

The levels of clarithromycin found in themouse serum and foot pad with 0.1% ad-ministered in the diet were oldie same orderof magnitude ( 4 . 5 ), albeit somewhat lowerthan the levels generated by that same dietin previous studies. Because of the lack ofsufficient sensitivity of the bioassay, theminimal inhibitory serum concentrationagainst M. leprae could not be determined.However, it was certainly remarkably low,< 25 ng/ml. Assuming the same linear re-lationship between dietary concentrationand resultant serum levels obtained be-tween dietary concentrations of 0.01% and0.001% as was found at 0.1% and 0.05%,the minimal inhibitory serum concentra-tion for clarithromycin against Al. lepraewould be < 2.5 ng/ml, approximately thatfound previously only for dapsone againstAl. leprae, 3 ng/ml (s). Because a small 400-mg human dose of clarithromycin results ina peak serum level of 1.1 pg per ml and inmice the highest dietary concentration test-ed, 0.1% dietary clarithromycin (100-fold

60, 3^ Correspondence^ 487

greater than minimal effective dietary con-centration), results in serum levels of ap-proximately threefold less, there is everyreason to believe that the pharmacokineticsin man would result in clarithromycin beingequally effective in the treatment of humandisease.

In conclusion, the very low dietary, se-rum, and tissue concentrations required toinhibit M. leprae found in these studies, andthe fact that clarithromycin has been foundconsistently bactericidal against M. lepraein Mice, lend optimism to the applicationof clarithromycin in the treatment of lep-rosy. Because clarithromycin is active onintermittent administration in mice and alsoat levels easily obtained in man, there areprospects that clarithromycin might, as withrifampin, be applied in some intermittentschedule for the treatment of lepromatousleprosy patients.

—Robert H. Gelber, M.D.Lydia P. Murray

Patricia SiuMabel Tsang

Medical Research Institute of CaliforniaPacific Medical Center

2200 Webster StreetSan Francisco, California 94115 andG WL Hansen's Disease CenterCarville, Louisiana 70721, U.S.A.

Acknowledgment. These studies were supported bya grant from Abbott Laboratories and contract #9007-02 from the GWL Hansen's Disease Center in Carville,Louisiana. We want to thank Roger Hill for his assis-tance in the preparation of this manuscript.

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