294 Tubercle, Lo&., (1969), 50, 294

RIFAMPICIN IN TREATMENT OF EXPERIMENTAL. TUBERCULOSIS IN MICE

By JOHN BATTEN

from the Brompton Hospital. Fulham Road, London, S. W.3

SUMMARY Groups of mice were infected intravenously with M. tuberculosis and treated with rifampi- tin (40 mg./kg.) alone, isoniazid (25 mg./kg.) alone or both drugs together.

Neither rifampicin nor isoniazid given daily for 12 weeks reduced the populations of tubercle bacilli in lungs and spleen below detectable levels in all the animals, whether treatment was started immediately or delayed for three weeks. On the other hand, when both drugs were given together for the same period the lungs and spleen of all but one of 30 animals appeared to be sterilized of tubercle bacilli.

When 40 mg./kg. of rifampicin was given daily or 120 mg./kg. twice weekly for four months no tubercle bacilli could be recovered from lungs or spleen in the 10 mice treated in each group.

Des groupes de souris ont CtC infect& avec M. tuberculosis par voie intraveineuse puis trait& par rifampicine (40 mg./kg.) seule, isoniazide (25 mg./kg.) seul ou les deux drogues ensembles.

Ni la rifampicine, ni l’isoniazide donnC tous les jours pendant 12 semaines n’ont rCduit les populations de bacilles tuberculeux dans les poumons et la rate au dessous d’un niveau dCcelable chez tous les animaux, que le traitement soit commencC immkdiatement, ou aprtts un dClai de 3 semaines aprb l’injection.

D’autre part, quand les deux drogues ont et6 donnCes en association pendant la m&me pCriode, la sterilisation des poumons et des rates a ttC obtenue chez 30 animaux sauf un.

Avec les doses de 40 mg./kg. de rifampicine par jour ou 120 mg./kg. deux fois par semaine pendant quatre mois, aucun bacille tuberculeux n’a pu Qtre mis en Cvidence dans les poumons et les rates de 10 souris traitCes dans chaque groupe.

RESUMEN Varios grupos de ratones, inoculados por via intravenosa, con Mycobacterium tubercu- losis, fueron tratados con rifampicina (40 mg/Kg) sola, con isoniazida (25 mg/Kg) sola y con ambas drogas juntas.

Ni la rifampicina ni la isoniazida solas suministradas diariamente durante 12 semanas redujeron la poblaci6n bacilar en el pulm6n y en el bazo a cifras muy bajas en todos 10s animales, tanto en 10s tratamientos iniciados de inmediato coma en aquellos iniciados al cabo de tres semanas de la inoculaci6n. Por otro lado, cuando se dieron ambas drogas combinadas, durante igual lapso, el pulmon y el bazo de 10s 30 animales, con excepcidn de 1, estaban completamente libres de bacilos.

RIFAMPICIN IN MOUSE TUBERCULIN 295

Se die 40 mg/Kg de rifampicina diariamente y 120 mg/Kg, dos veces por semana, durante 4 meses, a dos grupos de 10 ratones cada uno; la esterilizacion era completa en el pulmon y en el bazo en todos 10s animales.

ZUSAMMENFASSUNG

Gruppen von Mhsen wurden intraveniis mit M. tuberculosis infiziert und behandelt mit Rifampicin (40 mg/kg) allein, Isoniazid (25 mg/kg) allein oder beiden Medikamenten zusammen.

Weder Rifampicin noch Isoniazid allein vermochten bei taglicher Gabe tiber 12 Wochen die Zahl der Tuberkulosebakterien in der Lunge und Milz bei allen Tieren unter die Nachweisgrenze zu senken, gleichgtiltig, ob die Behandlung sofort oder mit einer Ver- zbgerung von drei Wochen eingeleitet wurde. Andererseits erwiesen sich-mit einer Ausnahme-Lunge und Milz bei allen 30 Tieren als steril, wenn die Medikamente gemeinsam tiber die gleiche Zeit gegeben wurden.

Wenn 40 mg/kg Rifampicin taglich oder 120 mg/kg zweimal wochentlich tiber vier Monate verabfolgt wurde, so konnten keine Tuberkulosebakterien aux der Lunge oder Milz der 10 in jeder Gruppe behandelten Tiere nachgewiesen werden. *

Introduction

The high degree of antituberculosis activity of rifampicin in murine tuberculosis was demonstrated by Grumbach & Rist (1967). Further evidence of this was provided by Verbist & Gyselen (1968) and recently by Grumbach (1969). The latter showed that rifampicin at 25 mg./kg. alone and with isoniazid at 25 mg./kg. given for three or four months was unable to reduce the numbers of tubercle bacilli below detectable levels in the lungs of all mice so treated, though it did so in a very high proportion after four months treatment.

The effect of rifampicin 40 mg./kg. given for three or four months on the populations of tubercle bacilli in the lungs and spleens of mice has been studied and is here reported.

Materials and Methods

Groups of 20 g. white male Schneider mice were infected intravenously with about lo6 organisms obtained from a seven day culture of the virulent ICI strain of M. tuberculosis. At required times after infection three to ten mice in each group were killed, the lungs and spleens homogenised and cultivated by the quantitative technique originally described by Fenner, Martin & Pierce (1949) and extended for the study of antituberculosis drug activity by McCune & Tompsett (1956). Rifampicin 40 mg./kg. with or without isoniazid 25 mg./kg. were given by mouth (gavage) either from the day of infection or after an interval of three weeks. Rifampicin was given either daily for six days a week or twice weekly (120 mg./kg.-equivalent to a daily dose of 40 mg./kg.).

Results

In Fig. 1 are shown the changes in populations of tubercle bacilli in the lungs of mice given isoniazid alone 25 mg./kg., rifampicin 40 mg./kg. alone or both drugs together daily from the start of infection for 12 weeks. These are compared with the changes resulting from the same treat- ment started three weeks after infection when the populations had reached a high level of IO7 in the lungs.

In Fig. 2 are shown the simultaneous changes in the spleen.

296 TUBERCLE

Rifampicin, like isoniazid, reduced populations below detectable levels in only a proportion of animals whether treatment started immediately or was delayed three weeks. In both tissues how- ever, in this experiment, rifampicin and isoniazid together reduced populations below detectable levels in 10 out of 10 animals with immediate treatment and 10 out of 10 animals when treatment was delayed three weeks. Altogether 30 animals have been treated with the combination of rifam- picin (40 mg./kg.) and isoniazid (25 mg./kg.) and the lungs and spleens of all but one of the animals

Weeks r

3 6 12 15 24

9- Rx ??_---- ____------

------___ ____ ____

O~~ome/k -fly

.m25me RrF4omm k t

__-- Limit of -ity

FIG. 1

The changes in populations of M. tuberculosis (ICI strain) in lungs of mice treated with rifampicin and isoniazid.

Each symbol above the limit of detectability represents the mean value obtained from at least three animals-each symbol below the limit represents each animal from which no

organisms could be cultivated. Rx - indicates when treatment started INH - isoniazid RIF - rifampicin

have apparently been sterilized of tubercle bacilli. In this animal less than ten viable units of tubercle bacilli were grown from the lungs, and none from the spleen.

Note however that, in Figs. 1 and 2, when rifampicin was given alone for 21 weeks in the delayed treatment group, no tubercle bacilli could be recovered from either tissue in 6 out of 6 mice. In another experiment, rifampicin (40 mg./kg.) was given alone daily or twice weekly for four months. Treatment was started three weeks after infection. The results are shown in Fig. 3. Tubercle bacilli were not grown from the lungs and spleens of the 10 animals treated daily or the 10 animals treated twice weekly (in two animals the cultures were contaminated). A further 10 mice were treated with rifampicin and isoniazid together for four months and no tubercle bacilli were recovered from the lungs or spleen in any of them.

Weeks I

3 6 12 15 24 Rx

RX

I

0 Control 0 INH 25 mn/k

- - - Limit of &ectability FIG. 2

The changes in populations of M. tuberculosis (ICI strain) in spleens of mice treated with rifampicin and isoniazid.

I I At end of_ treatment I

RIF 40 Daily 4 mths.

RIF 40 L Twice Weekly 4 mths.

tt- S

FIG. 3 LQ&” viable units of M. tabercuiosis (ICI strain) in lungs and spleens of mice after

treatment with rifampicin for 4 months. c - indicates that cultures were so contaminated that the number of colonies of

M. tuberculosis wuld not be counted.

s” I% Rif 40 - tSuqicin, 40 mg&g.

298 TUBERCLE

Discussion

Rifampicin given alone daily or twice weekly at 40 mg./kg. for 21 weeks to mice infected intra- venously with large numbers of tubercle bacilli was able to reduce the numbers of these organisms to below detectable levels in the lungs and spleens of a/Z animals so far treated, even when that treat- ment has been delayed three weeks so that the populations, especially in the lungs, were at a very high level. Furthermore, in the spleen, nearly all the tubercle bacilli are intracellular. It appears that rifampicin is able to exert its full antituberculosis activity within the host cell. Such a remarkable degree of activity has been achieved by no other drug when given alone. It was also achieved when rifampicin was given in combination with isoniazid. This consistent sterilization of the tissues prob- ably depends on an adequate dose, for Grumbach (1969) did not achieve such consistent sterilization in the lungs of her mice treated with rifampicin at 2.5 mg./kg.

Tsoniazid 25 mg./kg. and pyrazinamide 2 g./kg. given together for three months consistently sterilized tubercle bacilli in mouse tissues (McCune and others, 1966). This, however, was a tem- porary phenomenon and tubercle bacilli revived after a latent period in increasing numbers and this revival was accelerated by cortisone. The duration of sterilization of tubercle bacilli in mice by rifampicin and its modification by cortisone is at present under study and will be reported later.

It is concluded that rifampicin exerts uniquely powerful antituberculosis activity in mouse tissues when given alone daily or twice weekly at a mean daily dose of 40 mg./kg.

I am indebted to the Board of Governors of the Brompton Hospital for a grant in support of this work, to Mrs. M. Chadwick for her technical help and to Lepetit for providing equipmenr.

REFERENCES FENNER, F., MARTIN, S. P., & PIERCE, C. H. (1949). Enumeration of viable tubercle bacilli in cultures of infected

tissues. Ann. N.Y. Acad. Sci., 52,,751. GRUMBACH, F. & RET, N. (1967). Actlvitb antituberculeuse expkimentale de la Rifampicine, d&iv6 de la Rifamycine.

SV. Revue Tuberc. Pneumol., 31, 749. GRUMBACH, F. (1969). Experimental ‘in vivo’ studies of new antituberculosis drugs: capreomycin, ethambutol and

rifampicin. Tube&e Land., 50, 12 (March Supplement). MCCUNE, R. M. & TOMPSETT, R. (1956). Fate of Mycobacterium tuberculosis in mouse tissues as determined by the

microbial enumeration technique. J. Exp. Med., 104, 737. MCCUNE? R. M., FELDMANN, F. M., LAMBERT, H. P., & MCDERMOTT (1966). The capacity of tubercle bacilli to

survive sterilisation in mouse tissues. J. Exp. Med., 123,445. VERBIST, L. & GYSELEN, A. (1968). Antituberculous activity of Rifampicin in vitro and in vivo and the concentrations

attained in human blood. Am. Rev. resp. Dis., 98, 923.