Tubercle, Lond., (19701, 51. 95

RIFAMPICIN IN THE TREATMENT OF EXPERIMENTAL TUBERCULOSIS IN MICE: STERILIZATION OF TUBERCLE BACILLI IN THE TISSUES

By JOHN BATTEN

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

SUMMARY Groups of mice were infected intravenously with Mycobacterium tuberculosis and treated with rifampicin alone or with rifampicin and isoniazid.

After completion of daily treatment for four months with rifampicin at 40mg./kg. in combination with isoniazid 25mg./kg. no tubercle bacilli could be cultivated from the lungs and spleens up to three months later, even when high doses of cortisone were given during the second or third month.

When rifampicin was given alone daily in the same dose, tubercle bacilli revived in only one animal out of 10, two months after the completion of treatment. On the other hand, when rifampicin (120mg./kg.) was given twice weekly there was a significantly greater revival of tubercle bacilli.

The implications of this persistent sterilization of tubercle bacilli for the treatment of human tuberculosis are discussed.

Des groupes de souris ont CtC infect& par voie intraveineuse avec Mycobacterium tubercu- losis et trait& par la rifampicine seule ou par rifampicine et isoniazide.

A la fin d’un traitement quotidien de quatre mois par rifampicine 40 mg/kg associCe B l’isoniazide 25 mg/kg, aucun bacille tuberculeux n’a pu Ctre cultivC & partir des poumons et des rates ainsi que pendant les trois mois suivants m&me quand de fortes doses de cortisone ont CtC do&es au tours des deuxieme et troisibme mois.

Lorsque la rifampicine a ttC donnCe seule quotidiennement g la meme dose, les bacilles tuberculeux ont rCapparu chez seulement un animal sur 10, deux mois apr&s la fin du traitments. D’autre part, lorsque la rifampicine (120 mg/kg) a CtC don&e deux fois par semaine il y eut significativement plus de rdapparition de bacilles tuberculeux.

Les implications de cette stCrilisation durable de bacilles tuberculeux pour le traitement de la tuberculose humaine sont discutCes.

RESUMEN

Varios grupos de ratones fueron infectados por via intravenosa con Mycobacterium Tuberculosis y tratados con rifampicina sola o con rifampicina e isoniazida.

Desputs de haber completado un period0 de cuatro meses de tratamiento diario con rifampicina (40 mg/kg), en combinaci6n con isoniazida (25 mg/kg), 10s cultivos de1 pulm6n y de1 bazo fueron negativos hasta tres meses despds. a6n cuando al Segundo y tercer mes se dieron altas dosis de cortisona.

96 HATTEN

Cuando se suministrd rifampicina sola a la misma dosis. dos meses despues del tratamiento se aislaron bacilos en un animal cada diez. Por otra parte, cuando se die rifampicina (120 mg/kg) dos veces por semana hubo abundancia de bacilos.

Se discuten las implicancias de esta esterilizacion persistente de bacilos tuberculosos para el tratamiento de la enfermedad en el hombre.

ZUSAMMENFASSUNG Gruppen von Mgusen wurden intravenijs mit Mycobacterium tuberculosis infiziert und mit Rifampicin allein oder mit Rifampicin und lsoniazid behandelt.

Nach AbschluP der taglichen Behandlung iiber vier Monate mit 40 mg/kg Rifampicin in Kombination mit 25 mg/kg Isoniazid konnten aus Lunge und Milz keine Tuberkulose- bakterien geziichtet werden bis drei Monate danach, selbst wenn im zweiten und dritten Monat hohe Dosen von Cortison gegeben wurden.

Wenn Rifampicin in der gleichen Dosis allein verordnet wurde, konnten Tuberkulose- bakterien zwei Monate nach Beendigung der Behandlung nur bei jedem zehnten Tier isoliert werden. Wenn dagegen 120 mg/kg Rifampicin zweimal wiichentlich verabfolgt wurde, konnten signifikant haufiger Tuberkulosebakterien gefunden werden.

Die Folgerungen aus der anhaltenden Sterilisierung tuberkuliiser Ver&rderungen und ihre Bedeutung fi.ir die Behandlung der Tuberkulose des Menschen werden diskutiert.

Introduction In a previous paper (Batten, 1969) rifampicin was reported to sterilize tubercle bacilli uniformly

in the lungs and spleens of mice after four months treatment (i.e. no tubercle bacilli could be recovered from the tissues). This sterilization followed daily treatment with rifampicin at 40mg./kg. alone or with isoniazid at 25mg./kg., or twice weekly with rifampicin alone at 120mg./kg. The duration of this sterilization and its modification with cortisone has now been studied.

Materials and Methods Groups of 20g. 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 and Pierce (1949) and extended for the study of antituberculosis drug activity by McCune and Tompsett (1956). Rifampicin with or without isoniazid given by mouth (gavage), was started three weeks after infection. Rifampicin was given either daily for six days a week in a dose of 4Omg./kg. or twice weekly in a dose of 120mg./kg.--equivalent to the daily dose of 40mg./kg. Isoniazid was given daily in a dose of 25mg./kg. or twice weekly in a dose of 75 mg./kg. Some animals received cortisone (lmg. per day) during either the second or third month after treatment with anti-tuberculosis drugs had ended.

Serial dilutions of the tissue homogenates were cultivated in Middlebrooks 7H10 medium. Concentration of the homogenates was achieved by minimal dilution when measuring the volume of the organs by fluid displacement. As approximately the expected proportions of colonies were obtained from the serial dilutions, there was no evidence that tissue constituents were inhibiting bacterial growth. The drug susceptibility of M. tuberculosis cultivated from the tissues of animals that had completed treatment was studied by the resistance ratio method using H.37Rv and a wild strain as controls.

RIFAMPICIN IN MOUSE TUBERCULOSIS 97

rare I.-NIJMBER OF MICE FROM WHICH M. TUBERCULOSIS WERE CULTIVATED AFTER TREATMENT FOR FOIJR MONTHS WITH RIFAMPICIN ALONE OR WITH ISXNIAZID

Regimen*

--___- RIF 40 daily

RIF 120 twice-weekly

RIF 40 daily - INH 25 daily -

Months after end of treatment __-

I 2 3

No IV0 cortisone Cortisone? Total cortisone Cortisone Total

__~ O/IO l/IO 015 l/l5 019 015 o/14

__- O/IO l/l0 215 3115 3112 215 5117

~_

O/l0 O/IO Oil O/II O/l5 O/5 o/20

* RIF 40-rifampicin 4Omg./kg; RIF 120-rifampicin 120mgJkg.; INH 25-isoniazid 25mg./kg. -i Cortisone-lmg./day during the second or third month. All the strains isolated were sensitive.

Results In Table I is recorded the number of mice from which M. tuberculosis were cultivated after

treatment for four months with rifampicin at 40mg./kg. alone or in combination with isoniazid at 25mg./kg. From none of the animals given rifampicin and isoniazid daily were M. tuberculosis cultivated up to three months after completion of treatment. Furthermore, no growth was obtained even when cortisone was given in a high dose of lmg. per day during the second (one animal) or third month (five animals) after the end of the chemotherapy.

From only one animal after the end of treatment could tubercle bacilli be recovered from the tissues when rifampicin had been given daily alone. When, however, rifampicin was given twice- weekly, organisms were recovered from a larger number of mice in both the cortisone-treated and non-cortisone treated groups at three months. Although the difference at three months is just not significant (P=O.O7), the difference between the combined totals at two and three months (namely 1 of 29 and 8 of 32) is significant at the 3 % level. All the tubercle bacilli recovered during this period of observation were sensitive to rifampicin.

In Table II is recorded the number of animals from which M. tuberculosis were recovered after

TABLE H-NUMBER OF MICE FROM WHICH M. TUBERCULOSIS WERE CULTIVATED AFTER TREATMENT FOR THREE MONTHS WITH RIFAMPICIN AND ISONIAZID TOGETHER

--___

Months after end of treatment

Regimen 0 I 2 3 ___._

No cortisone Cortisone* Total

/

RIF l/IO 2110 3/10 1 l/l0 2115 INH 40 > 25 daily / - I--

RIF I INH 75 twice

120 > weekly ~

3/10 2/10 ~/IO j 4110 215 6115 I

,._~~~~_ -____ _.-. * Cortisone 1 mg./day during the third month.

H

98 BATTl:N

only three months treatment with rifampicin and isoniazid given daily or twice weekly. Tubercle bacilli revived in a considerable proportion of animals in both groups when the drug combination had been given for this shorter period of time. Tubercle bacilli were also cultivated in a small proportion of animals even at the end of treatment and at the end of the first month after completion of treatment. Once again all the organisms recovered in this experiment were drug susceptible. Concentrated homogenates of lung and spleen from three animals in either group which had completed three months after chemotherapy were inoculated into guinea pigs and subsequently no evidence of bacterial growth was obtained.

Discussion

Rifampicin was previously reported at 40mg./kg. to be unique in its anti-tuberculosis activity in mice, for it uniformly sterilized tubercle bacilli in lungs and spleens when given alone daily or twice weekly (at equivalent daily dose). Isoniazid occasionally sterilizes tubercle bacilli in lungs but not spleens after three months treatment (McCune and Tompsett, 1956). Tsoniazid in combination with pyrazinamide is reported to sterilize lungs and spleens of mice uniformly after three months treatment, (McCune and others, 1966). Revival of bacilli, however, follows, so that organisms were cultivated from the tissues of thirty per cent of animals after a further three months without treatment. This proportion rose to almost one hundred per cent when cortisone was given to mice at a high dose of lmg. per day during the third month after the completion of isoniazid and pyrazinamide treatment. All the tubercle bacilli recovered in these experiments after the completion of treatment were drug susceptible.

Tubercle bacilli apparently previously sterilized by four months’ daily treatment with rifampicin 40mg./kg. regrew in only one animal out of 10 two months after treatment stopped and cortisone did not appear to accelerate this rate of re-growth. There was a significantly greater revival of tubercle bacilli after completion of twice-weekly rifampicin at a dose of 120mg./kg. (equivalent to a daily dose of 40mgJkg.): tubercle bacilli were cultivated from three out of twelve animals three months after the end of treatment and from two out of five animals given cortisone during the last month of the three month period of treatment. Thus, while daily and twice weekly treatment sterilized tubercle bacilli in all animals after four months’ therapy, the intermittent regimen appeared to be less effective in preventing subsequent regrowth of the organism.

The addition of isoniazid at 25mg./kg. to the daily rifampicin regimen during four months treatment abolished revival of tubercle bacilli in the lungs and spleens of all the thirty five animals so treated. Furthermore, cortisone for one month during the second or third months after the end of treatment did not impair this effect.

Rifampicin is therefore unique in two respects: (i) It is able to sterilize tubercle bacilli after four months’ treatment when given alone; (ii) In combination with isoniazid for four months it sterilizes tubercle bacilli in such a way that subsequent growth of the bacilli is inhibited for at least three months after completion of treatment. The duration of this combined treatment is important, for three months’ treatment did not uniformly sterilize tubercle bacilli and revival occurred in a considerable proportion of animals, even during the first month after stopping treatment. Even less effective was this combination given twice-weekly instead of daily, although the differences were not significant.

Grumbach and Rist (1967) reported that the proportion of mutants resistant to rifampicin in a given population of tubercle bacilli was of the order of 1 in 10s. In these experiments treatment was started three weeks after infection, when the populations were of the order of 10’ in both lung and spleen. It is unlikely, therefore, that resistant mutants would be present. If they had been. they would probably have emerged in the post-treatment phase of the experiments.

These experiments may have implications for the treatment of human tuberculosis, for rifampicin is so powerful in its antituberculosis effect that it might allow a reduction in the duration of treat-

RIFAMPICIN IN MOUSE TUBERCULOSIS 99

ment when used in combination, for example, with isoniazid or ethambutol. The following observations may be made:-

1. The absolute level of daily dose may be important for reduction to 25mg./kg. (Grumbach, 1969) marginally impairs the effectiveness of rifampicin.

2. Increasing the interval between doses may impair the long-term chemotherapeutic effective- ness. Reports of the effectiveness of twice-weekly and even once-weekly treatment are impressive (Verbist and Gyselen, 1968). But evidence is here reported that post-treatment revival of tubercle bacilli is enhanced by increasing the intervals between doses.

As Canetti (1968) indicated, tuberculosis in man and mouse are not comparable after completion of chemotherapy. In fact, murine tuberculosis is a much more stringent test for post-treatment relapse. If, therefore, reduction in duration of treatment of human tuberculosis is to be considered, such factors as the efficacy of drug combinations including rifampicin in prevention of post- treatment growth of tubercle bacilli in murine tuberculosis may well prove to be relevant and important.

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 equipment.

REFERENCES BATTEN, J. (1969). Rifampicin in treatment of experimental tuberculosis in mice. Tubercie, 50, 294. CANETTI, G. (1968). Experiments on long-term intermittent chemotherapy in advanced tuberculosis in mice. Tubercle,

49, (March suppl.). p. 70. FENNER, F., MARTIN, S. P., & PIERCE, C. H. (1949). The enumeration of viable tubercle bacilli in cultures and

infected tissues. Annals of the New York Academy of Sciences, 52, 751. GRUMBACH, F., & RUT, N. (1967). ActivitC antituberculeuse exp&imentale de la Rifampicine, d&iv6 de la Rifamycine,

SV. Revue de la Tuberculose et de Pneumologie, 31, 749. GRUMBACH, F. (1969). Experimental ‘in vivo’ studies of new antituberculosis drugs; capreomycin, ethambutol and

rifampicin. Tuber& 50, (March suppl.). p. 12. MCCUNE, K. M., 8t TO~SETT, R. (1956). The fate of Mycobacterium tuberculosis in mouse tissues as determined by

n-ncroblal enumeration technique: 1. Persistence of drug-susceptibie tubercle bacilli in the tissues despite pro- longed antimicrobial therapy. Journal of Experimental Medicine, 104, 737.

MCCUNE, .R. M., FELDMANN, F. M., LAMBERT, H. P., & MCDERMOTT W. (1966). Microbial persistence. I. The capacity of tubercle bacilli to survive sterilisation in mouse tissues. Journal of Experimental Medicine, 123, 445.

VERBIST, L.. & GYSELEN, A. (1968). Antituberculous activity of Rifampin in vitro and in vivo and the concentrations attained in human blood. American Review of Respiratory Disease, 98, 923.