Studies on the treatment of experimental tuberculosis of the guinea pig with intermittent doses of isoniazid

Tubercle (1973) 54,211

STUDIES ON THE TREATMENT OF EXPERIMENTAL TUBERCULOSIS OF THE GUINEA PIG WITH INTERMlTlYENTDOSES OF ISONIAZID By JEAN M. DICKINSON,V. R. ABER and D. A. MITCHEON from the Medical Research Council’s Unitjiw Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School. London W12 OHS

SUMMARY Guinea-pigs with established experimental tuberculosis were treated for 6 weeks with isoniazid at mean dosages of 1,2 and 4 mg/kg/day. At each dosage level, groups were given doses at intervals ranging from 1-14 days and the response to treatment was assessed from the amount of macroscopic disease in the organs and from viable counts on the spleen. The efficacy of treatment was constant as the interval between doses was increased from l-4 days but decreased considerably with wider intervals. When the size of the dose given at intervals of 7 or 10 days was increased either as a single large dose or as multiple smaller doses within a 24-hour period, the response was best with total doses of 20-30 mg/kg isoniazid and could not be improved with higher dosage. The best responses obtainable were never as good with doses at 7-day intervals as with twice-weekly doses and were even less good with lo-day intervals. These findings suggest that the observed failure of human rapid inactivators to respond as well as slow inactivators to once-weekly chemotherapy of isoniazid might be prevented by substituting for the usual dose of 15 mg/kg isoniazid a dose of a delayed release preparation of the drug sufficient to double the area under the isoniazid serum curve.

Des cobayes avec une tuberculose experimentale avancee ont tte trait& pendant six semaines avecde l’lsoniazide a la dose moyenne de 1,2 et 4 mg/kg jour. Pour chacune de ces doses, des groupes d’animaux recevaient leur traitement a des intervalles allant de 1 a 14 jours. L’effet du traitement a CtCCtabli sur l’importance des lesions macroscopiques dans les organes et sur les comptes d’unitb viables dans la rate. L’efficacite du traitement a Cte constante tandis que l’intervalle entre chaque prise augmentait de 1 a 4 jours mais il a diminue considerablement pour les intervalles plus grands. Lorsque l’importance de la dose de medicament don&e a intervalle de 7 a 10 jours Ctait accrue, soit que la dose ait Ctt importante et unique, soit que la dose ait CtC rtpartie en plus petites prises au tours des 24 heures, les meilleurs resultats ont CtCobtenus avec des doses de 20 a 30 mg/kg d’Isoniazide et n’ont pas ttC ameliores avec des doses plus fortes. Les meilleurs r&hats obtenus avec des prises de medicament a des intervalles de 7 jours n’ont jamais 6th aussi bons qu’avec une administration deux fois par semaine et ils ttaient encore moins bons avec des intervalles de 10 jours. Ces rtsultats suggerent que les Cchecs observes parmi les sujets humains inactivateurs rapides qui ne repondent pas aussi bien que les inactivateurs lents a une chimiotherapie par I’Isoniazide une fois par semaine pourraient &tre prevenus en remplacant la

212

DICKINSON

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OTHliHS

dose habituelle de 15 mg/kg. d’lsoniazide par une dose d’une preparation retard de la drogue suffisante pour doubler la surface circonscrite par la courbe du taux strique d’lsoniazide. ___-.__ _I_____ Un grupo de cobayos inoculados con tuberculosis fueron tratados durante 6 semanas con isoniazida a las dosis de I, 2 y 4 mg/Kg/dia. Para cada dosis se establecieron grupos que recib’an la droga con intervalos entre 1 y 14 dias, evaluandose la respuesta terapeutica a traves de las lesiones macroscopicas visibles y el conteo bacilar en el bazo. La eficacia terapeutica fue constante mientras 10s intervalos oscilaron entre 1 y 4 dias, pero disminuyo francamente con intervalos mayores. Para 10s intervalos entre 7 y 10 dias, el aumento de la dosis, en forma de una sola dosis grande o de varias dosis pequeiias dentro de las 24 horas, la respuesta fue mas eficaz con dosis totales diarias de 20 a 30 mg/Kg de isoniazida y no mejoro con dosis mayores. Las mejores respuestas no eran nunca superiores con las dosis cada 7 d’as que con dosis bisemanales, y eran sun peores cuando 10sintervalos eran de 10 dias. Estos hallazgos sugieren que 10s fracasos abservados en 10s inactivadores rapidos frente a 10s inactivadores lentos en 10s esquemas de una vez por semana de isoniazida pueden prevenirse reemplazando la dosis corriente de 15 mg/Kg de isoniazida por una preparacidn de liberation retardada suficiente coma para duplicar el area de la curva serica de la isoniazida.

ZUSAMMENFASSUNG

Meerschweinchen mit experimentell erzeugter Tuberkulose wurden 6 Wochen mit Isoniazid in mittleren Dosen von I,2 und 4 mg/kg/Tag behandelt. In jedem Dosisbereich wurden gruppenweise die Dosen in Intervallen von l-14 Tagen verabfolgt. Der Behandlungserfolg wurde anhand des Befalls der inneren Organe sowie der Zahl der lebenden Erreger in der Milz bestimmt. Die Behandlung war bis zu einem maximalen Interval1 von 4 Tagen zwischen den einzelnen Dosen von gleicher Wirksamkeit; danach kam es zu einem zunehmenden Wirkungsverlust. Wenn bei Intervallen von 7 oder IO Tagen die Dosis erhiiht wird, entweder in Form einer groi3en Einzeldosis oder in mehreren kleinen Dosen innerhalb von 24 Stunden, so war das Ergebnis bei Gesamtdosen von 20-30 mg/kg Isoniazid am gtinstigsten und lief3 sich such durch weitere Steigerung der Dosis nicht verbessern. Auch die optimalen Ergebnisse waren beim 7-Tage-Interval1 niemals so gut wie bei zweimaliger Gabe pro Woche und beim IO-Tage-Interval1 noch schlechter. Nach diesen Befunden miiDte sich das beobachtete schlechtere Ansprechen von schnellen Inaktivierern (im Vergleich zu langsamen Inaktivierern) auf die einmalige Medikamentengabe pro Woche dadurch verhindern lassen, dai3 man die iibliche Dosis von 15 mg/kg Isoniazid in Form eines Retard-Prsparates gibt, aus dem Isoniazid so langsam freigesetzt wird, dai3 sich der Bereich unter der Isoniazid-Serum-Kurve verdoppelt. Introduction

Twice-weekly high dosage isoniazid and streptomycin has been shown to be a highly effective regimen in the treatment of pulmonary tuberculosis, irrespective of whether the patient is a slow or rapid inactivator of isoniazid (Tuberculosis Chemotherapy Centre, Madras, 1964, 1970; WHO Collaborating Centre for Tuberculosis Chemotherapy, Prague, 1971). However, treatment was less satisfactory if the doses were given at intervals of a week and the efficacy of the once-weekly regimen was substantially lower in rapid than in slow inactivators (Tuberculosis Chemotherapy

INTERMITTENT

ISONIAZID

213

Centre, Madras, 1970). When the once-weekly regimen was preceded by one month of daily treatment with isoniazid and streptomycin, a favourable response was obtained in 95 per cent of slow inactivators, but in only 76 per cent of rapid inactivators. Thus, if it were possible to improve the response to isoniazid in rapid inactivators, once-weekly chemotherapy with streptomycin and isoniazid would become a practical possibility. In a further study at Madras, patients were treated with once-weekly streptomycin and isoniazid at two dose sizes (Tuberculosis Chemotherapy Centre, Madras, 1973a). The serum isoniazid curves in slow inactivators had peak concentrations 28 per cent higher than in rapid inactivators, but in slow inactivators the area under the curve (the exposure) was 2.4 times as great and the duration of the coverage period during which bacteriostatic concentrations were maintained was twice as long. The response of the patients was related to the exposure to isoniazid and to the coverage period but not to the peak concentration. These findings indicated the possibility of obtaining effective once-weekly treatment by using a delayed-release preparation of isoniazid, that could give an increased exposure and coverage period in rapid inactivators without causing high and toxic peak concentrations of the drug in slow inactivators (Ellard and others, 1972; 1973). The half-life of isoniazid is similar in the guinea-pig and in human rapid inactivators. Furthermore, previous experiments .on the efficacy of intermittent isoniazid in experimental tuberculosis of the guinea-pig have yielded results analogous to those in human rapid inactivators (Dickinson, Ellard & Mitchison, 1968). This paper describes fresh studies in the guinea-pig to investigate relationships between therapeutic efficacy, the rhythm of dosage and the shape of the serum isoniazid curve following a dose of isoniazid. The shape of the serum concentration curve was altered by giving two or more doses closely following each other, thus increasing the exposure and prolonging the coverage period but not increasing the peak concentrations substantially. In this way it was possible to simulate in a very approximate fashion the difference between human rapid and slow inactivators and to obtain results that could aid the development of slow-release preparations for intermittent chemotherapy. Methods

Guinea-pigs were inoculated

intramuscularly

with 1 mg (moist weight) of M~cobacterium

tuberculosis, strain 175. Three weeks after infection treatment was started with isoniazid in 1 per

cent methyl cellulose administered by gastric intubation. After 6 weeks of treatment, surviving animals were sacrificed and examined in a random order. The extent of disease visible to the naked eye was assessed in these animals, and in any that had died previously, and was expressed as the root-index of disease (Dickinson and Mitchison, 1966; 1970). Counts of viable organisms in spleens were done in 7HlO oleic acid-albumin medium by the method of Selkon & Mitchison (1957). Sensitivity tests to isoniazid were set-up on cultures from two spleens in each experimental group as described in Section II of Canetti and others (1969). The guinea-pigs were arranged in four experiments. In each of these, a group was sacrificed at three weeks to confirm the presence of moderate disease before treatment was started. The remaining animals were divided as follows: Experiment 1 contained 155 guinea-pigs divided into 31 groups, each of 5 animals. Five groups were given a mean daily dosage of 1 mg isoniazid per kg body weight per day, the first group receiving 1 mg/kg in a single daily dose, the second group 2 mg/kg every 2 days, the third group 4 mg/kg every 4 days, the fourth group 7 mg/kg every 7 days and the fifth group 10 mg/kg every 10 days. Correspondingly, 5 groups were given a mean dosage of 2 mg/kg and 5 groups a mean dosage of 4 mg/kg, the 5 groups at each dosage level being given single doses at intervals of 1, 2, 4, 7 or 10 days. A further set of 15 groups received an identical dosage pattern except that each dose of isoniazid was divided into two portions with an interval of 4 hours between them. Finally, a control group received no treatment.

214

DICKINSON

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Experiment 2 contained 143 guinea-pigs divided into 31 groups, each of 4-5 animals. The arrangement of the groups was the same as in experiment 1, except that the intervals between doses (whether single or divided) were I, 3-4, 7, IO-11 and 14 days. Doses were omitted on the Sunday of each week of treatment. Experiment 3 contained 98 guinea-pigs divided into 14 groups, each containing 7 animals. One group received no treatment (untreated controls) and another received 40 mg isoniazid per kg every 3-4 days (treated controls). Three groups were given isoniazid every 7 days, either as a single dose of 10 mg/kg, or as two doses of 10 mg/kg separated by 6 hours or in three doses of 10 mg/kg each, the second and third doses at 6 and at 24 hours after the first dose. A further three groups received 1,2 or 3 doses of 20 mg/kg every 7 days. The remaining 6 groups were arranged in the same manner except that doses of 15 mg/kg or 30 mg/kg were given every 10-I I days. Experiment 4 contained 121 guinea-pigs divided into 11 groups, each of 11 animals. The design was similar to experiment 3 and included the same untreated and treated controls. Doses of 7.5, 15 or 30 mg/kg were given once, twice or three times at intervals of 7 days. Isoniazid concentration in guinea-pig serum Blood was obtained from the lateral ear vein and the concentration of isoniazid in the serum was measured fluorimetrically by the method of Ellard, Gammon & Wallace (1972).

\

10

\ 5 TWO DOSES

z

;\ z

Y

P N 4

$ v, a 3 ::

VI

0

2

I I I

1

0.5

\

I 0.2

0

1

2

3

4

5

6

7

8

HOURS FIG. 1 Serum concentrations of isoniazid in groups of 3 guinea-pigs given one or two oral doses each of 14 mg/kg body weight isoniazid.

215

INTERMITTENT ISONIAZID

SERUMCONCENTRATIONS OF ISONIAZID The concentration of isoniazid was measured in the pooled serum from groups of 3-4 guinea-pigs at intervals after either a single oral dose of 14 mg isoniazid/kg body weight or two doses of 14 mg/kg separated by 4 hours. The peak concentration attained after the second dose was only slightly greater than after the first dose, but the coverage period, over which a concentration of 0.2 pg/ml isoniazid was maintained, was extended from about 9 hours to about 138 hours (Fig. 1). Peak serum concentrations averaged 10.1 pg/ml isoniazid in the guinea-pig, very similar to the peak concentration of 9.6 pg/ml in human rapid inactivators after a dose of 15.4 mg/kg (Tuberculosis Chemotherapy Centre, Madras, 1970). Furthermore, the half-life of isoniazid in the guinea-pig was 77 minutes, and in human rapid inactivators has recently been estimated as 80 minutes by Tiitinen (1969) and 83 minutes by Scott and others (1969). MORTALITY The results on 11 guinea-pigs which died before 40 days after infection were excluded from analysis. Death on or after 40 days occurred in 17 guinea-pigs, of which 6 were probably due to tuberculosis. The deaths were fairly evenly distributed throughout the groups in each experiment. SINGL.EAND DIVIDEDDOSES Root-indices of‘disease

The means of the root-indices of disease in each experimental group are set out in Table I for experiment 1 and in Table 11for experiment 2. The mean root-index in the untreated control group of experiment 1 was l-08, a value within the range 1*08-1.18obtained in previous similar experiments (Dickinson & Mitchison, 1966, 1970; Dickinson, Ellard & Mitchison, 1968). However, the corresponding root-index in experiment 2 was only 0.96, suggesting that this experiment might be less capable of distinguishing differences in the efficacy of the various treatment regimens than experiment 1. In both experiments, as the mean daily dose of isoniazid was increased from 1 mg/kg to 4 mg/kg there was only a slight fall in the mean root-index (Tables I and II, right-hand column); the association was not statistically significant. Considering the interval between doses, the mean root-indices TABLEL-ROOT-INDICESOF DISEASE IN GUINEA-PIGS TREATED WITHI~~NIAZID(EXPT. 1)

Dose

Mean daily dose (mg/kg)

Interval between doses (days) -________1

-~~~__

2

4

7

10

Mean root-index of disease

1.08 0.69 0.53 0.57 0.81 O-89 0.64 2 0.47* 0.61 0.58 0.74 0.813 0.58 4 0.48 0.57 0.47 0.59 0.77 _______ ~~0.63 Mean 0.53 0.57 0.54 0.71 0.82 ___-~0.56 1 0.49 0.59 044 0.73 0.57 Divided 0.56 2 0.41 0.54* 0.52 0.59* 0.75* 0.56 4 0.49 0.57 0.51 0.64 0.61 ---___0.56 Mean 0.46 0.57 0.49 0.65 0.64 -.. ~~____~~__~~~_~_ _~_... ~~~. _. * Result on one guinea-pigin each group of 5 excludedfor death before day 40.

Single

0

-

1

064

216

DICKINSON

AND

OTHERS

0.8

0.5

0.4

1

Root-indices

2

4 8 INTERVAL BETWEEN DOSES (DAYS)

16

FIG. 2. of disease related to interval between doses of isoniazid.

Experiment

1.

in experiment 1 were similar for doses given at intervals of 1, 2 and 4 days, but increased sharply when the interval was prolonged to 7 and 10 days. A similar trend was evident, though less clearly, in experiment 2. The relation between the mean root-index and the interval between doses is shown in Figs. 2 and 3. In experiment 1, the overall mean root-index was O-63 for all guinea-pigs treated with a single dose and 0.56 for those given the same dose divided into two parts; the difference is significant (P
The viable counts on the spleens are summarized in Table III and IV. The mean counts for the groups which received treatment were, in each experiment, lower than for the untreated controls. However, the difference was smaller in experiment 2 than in experiment 1, again suggesting that experiment 2 had a lower ability to discriminate between the different treatment groups. In both experiments, there was a tendency for spleen counts to rise as the interval between doses was

INTERMITTENT

217

ISONIAZID

TABLEII.-ROOT-INDICES OF DISEASEIN GUINEA-PIGS TREATEDWITH WNIAZID

Mean daily dose (mglkg)

Dose

1

Single

I i Divided

0 1 2

4 Mean 1 2 4

I

Mean

Interval between doses (days) .-,-.,1 I 3.5 , 7 : 10.5 / 14 ._ -I__.-! _i0.60 0.52 j 0.56 0.80 0.72 053 0.54 ) 0.67 0.74 0.81 064 0.66 0.52* 0.50 1064 _’ _‘. 0’73 -._-8 _. --, 0.51 060 0.69 0.55 0.70* 0.45 0.57 0.57 0.72 0.80 0.42 0.49 0.64 0.70 0.54 _‘. ____ __-1 I 0.46 / 0.55 0.63 0.66 0.68

(EXPT. 2)

Mean root-index qf disease

-I

0.96

i j

064 0.66 0.59

1

0.63

/

0.61 0.62 0.56

’

0.60

I * Result on one guinea-pig in each group of 4 excluded for death before day 40.

TABLE III.-VIABLE

GJUNTS ON SPLEENS(EXPT. 1)

Interval between doses (days)

Mean

Dose

Single Divided Mean

3.30 1 3.99

4.10 j 4.31 ’4.07 !

j

I Untreated controls:

TABLE IV.-VIABLE

6.47

CCXJNTSON SPLEENS(EXPT. 2)

Interval between doses (days)

Mean

count WdWspleen) Single

Divided Mean

4.70 4.53 4.34 4.99 ________ 4.52 4.76

5.09 5.24

5.04 5.11

5.16

5.08

5.40 1 4.97 I -__ 5.19 1

I Untreated controls:

5.77

4.95 4.93

~-

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DICKINSON

AND

OTHERS

0.9

Single Dose

0.8

Divided Dose

0.5

0.4 1

2 4 INTERVAL BETWEEN DOSES (DAYS)

8

16

FIG.3 Root-indices of disease related to interval between doses of isoniazid. Experiment 2.

increased (P ~0.025 and P
SPACED INTERVALS

Root-indices of disease In experiment 3, basic doses of 10 mg/kg at 7-day or 15 mg/kg at 10*5-day intervals were increased either by repeating the dose twice or three times within a 24-hour period, or by doubling the size of the single dose (Table V). When the basic dose was 10 mg/kg every 7 days, the root-index of disease decreased from 0.86 with a single dose to 0.65 with two doses and then to 0.55 with three doses (i.e. a total of 30 mg/kg weekly). Giving a single 20 mg/kg dose decreased the root-index to

INTERMITTENT

219

ISONIAZID

TABLEV.-ROOT-INDICESOFDISEASE RELATED TOSIZEANDMULTIPLICITY OFWIDELYSPACED DOSS OFISONIAZID (EXPT. 3) .~

--~

--_

~ -....

I Number

of

doses

Intervals

; ~

between

.,7 10 mglkg

1

j

20 mgikg 060 0.57 0.63

Mean

0.69

P*

< O+lOl

060 0.3

~

10.5

L_

0.86 0.65 0.55

2 3

doses (days)

15 mgikg ____--

i I / !

1 30 mgikg

0.78 0.70 0.68

0.72 0.75 0.65

0.72 0.1

0.71 0.2

~

Mean root-index of disease

’ ~

0.74 0.67 0.63

Treated controls: 0.49

Untreated controls: 1.21

* Probability for association between number of doses and root-index of disease.

0.60,

but when two or three doses of this size were administered, no further decrease in root-index occurred. Thus, in weekly chemotherapy, the best response obtainable was achieved with 20-30 mg/kg, whether given in successive doses of 10 mg/kg or as a single dose. The response was, however, never as good as with twice-weekly dosage since the minimal root-index of about 060 for weekly dosage was higher than the root-index of 0.49 obtained in the treated controls. In the guineapigs treated at intervals of 10.5 days, the minimal root-index appeared to be about 0.70, and was obtained with dosages of about 30 mg/kg given either divided or singly. The best response in these animals was less good than in those treated weekly. In experiment 4, basic doses of 7.5, 15 or 30 mg/kg were given only at 7-day intervals (Table VI). As in experiment 3, a minimal root-index of about 0.60 was obtained with a dosage of 20-30 mg/kg, TABLEVI.-ROOT-INDICESOFDISEASE RELATED TO SIZEAND MULTIPLICITY OF WEEKLYDOSESOFTSONIAZID (EXPT. 4)

Dose

Number sf doses

) 7.5 mg/kg

Mean P*

0.71 0.01

1 15 mg!kg

I 30 mglkg

j

;

Untreated controls: 1.08 *Probability for association-between

size

0.64 0.1

0.60 0.6

i

Mean root-index of disease

~

Treated controls : 0.49

number of doses and root-index of disease.

given in successive small doses or in a single dose. Any further increase in dosage, as by giving two or three doses of 30 mg/kg each, produced no additional fall in the root-index. Again, the treated

controls yielded a root-index of 0.49, lower than in any of the groups treated at weekly intervals.

220

DICKINSON

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OTHERS

Spleen counts The results of the viable counts on spleens (Tables VII and VIII) in experiments 3 and 4 support the conclusions drawn from the root-indices of disease. In experiment 3, a minimal viable count of about 4.3 log,, vu/spleen was obtained with a dose of about 30 mg/kg given every 7 days, but a higher dose may have been necessary when given every IO.5 days. The mean spleen counts were higher in guinea-pigs treated every 10.5 days than in those treated weekly (P
COUNTS ON SPLEENSRELATED TO SIZE AND MULTIPLICITY OF WIDELY SPACED DOSESOF ISONIAZID(EXPT. 3)

IntervaI between doses (days)

___-

AJumber 1 of doses

10 wk

-_____ 1

10.5

,

5.08 4.41 4.21

471 407 4.28

)

457

4.35

/ (logI

Mean vu/spleen)

1 15 mg/kg

Ixzl

(

2 3 Mean

---

7

’

I

1 30 mg/kg

5.68 4.83 4.65

5.04 482 4.58

5.05

i

5.13 4.53 4.43

4.81

I ..___

Untreated controls:

._

-.

Treated controls:

6.88

3.07.

TABLE VIII.-VIABLE COUNTS ON SPLEENS RELATED TO SIZE AND MULTIPLICITY OF WEEKLY DOSES OF ISONIAZID(EXPT. 4) I

Number

of doses -___

individual dose size (mg/kg)

__--

iT

1

2 3 Mean

1

ci_130-i

(loglO~~~lee~t)

___-

I : /

5.24 4.59 4.80

:

4.93 5.32 4.88

1 5.17 ’ 4.46 ’ 4.82

4.90

I

504

)

Untreated controls:

656

~_

5.12

----

4,79 4.85

4.82 Treated controls:

3.78

Discussion The results of the first two experiments together with a similar result reported in an earlier publication (Dickinson, Ellard & Mitchison, 1968) showed that the efficacy of treatment with isoniazid remained unchanged as the interval between doses was spaced out from 1 to 4 days but decreased rather abruptly as the interval was increased to 7 days or more. An explanation for

INTERMITTENT

ISONIAZlD

221

the loss of efficacy between the 4-day and the 7-day intervals is provided by the experiments of Awaness and Mitchison (1973) who measured the effects of pulsed exposures to isoniazid on cultures of M. tuberculosis by their bactericidal activity and by the length of the lag period between removal of isoniazid and the start of regrowth of the organisms. No bactericidal activity or appreciable lag period occurred when cultures were given short exposures to l-4 pg/ml isoniazid for 1-4 hours, but 5 such exposures given on successive days were bactericidal and were followed by lag periods averaging 4.9 days. Thus pulses of isoniazid have a cumulative effect, with the organism returning to their normal state about 5 days after exposure. Applying this concept to the guinea-pig model, the effects of doses given at intervals of l-4 days would tend to accumulate and the organisms would not have time to recover between them. When, however, the interval between doses was increased to 7 days, recovery might have occurred before the next dose. Now, Awaness and Mitchison (1973), as well as giving cultures 5 daily short pulses, also exposed their cultures to a single pulse lasting 5 times as long as the short pulse, so that organisms received the same total exposure to isoniazid but in a period of 5-20 hours instead of 5 days. The single long exposure was followed by a lag period, but in contrast to the 5 daily exposures, no bactericidal activity was evident. Thus to kill, the organisms must be under the influence of isoniazid (either in contact with the drug or recovering from its effects) for longer than 20 hours. It is now evident that if recovery of the organisms occurs between doses, as probably occurred in guinea-pigs dosed at intervals of 7 days or more, there would be little or no bactericidal effect, since there would never be time for bactericidal activity to start. This concept may explain the increase in bactericidal activity in murine tuberculosis gained by inserting a preliminary phase of daily dosage before intermittent treatment with isoniazid and streptomycin (Grumbach, 1965). In intermittent chemotherapy of human disease, an initial daily phase might prevent early deaths by more rapid killing of actively growing organisms. In man, a further disadvantage of recovery and multiplication of organisms between widely spaced doses is an increased liability to the emergence of drug-resistant strains (Tuberculosis Chemotherapy Centre, Madras, 1970). Resistance did not emerge in the guinea-pig, presumably because the period of treatment was too short and because of the low virulence of resistant mutants in the guinea-pig. Considering treatment given with doses at intervals of l-4 days there must be a minimal exposure (dose size) that maintains the cumulative effect of isoniazid throughout the treatment period. In vitro experiments with pulses given at intervals of 3 days (Bourgeois, Dubois-Verlitre & Ma&l, 1958) or 1 day (Armstrong, 1960; 1965) have shown that this minimal exposure can be expressed as the product of the exposure period and the isoniazid concentration, the antibacterial effect remaining the same whether a short exposure to a high concentration or a long exposure to a low concentration was given. This conclusion is consistent with the results of experiments 1 and 2, since the root-indices were similar whether isoniazid was given in a single or a divided dose at intervals of l-4 days. We have already noted that efficacy remained unchanged as the intervals between doses was increased from l-4 days, though it must be recalled that the size of the dose and hence the exposure was increased proportionately as the interval was spaced out so that the mean dosage over the treatment period remained constant. Thus, we can conclude that a minimal mean exposure appears to be necessary to maintain the cumulative effect of isoniazid and that neither the shape of the pulse nor the rhythm appreciably influences its size. In the guinea-pig this mean minimal exposure is produced by about 1 mg isoniazidlkg body weight/day, since reduction in the mean dose to 0.5 mg/kg/day resulted in lower efficacy in a previous similar experiment (Dickinson, Ellard 8z Mitchison, 1968). In experiments 1 and 2 there was no evidence of a reduction in the root-indices as the mean dosage of isoniazid was increased from 1 mg/kg/day to 4 mg/kg/day, so that an increase in the mean exposure above the minimal size appears to produce little additional benefit. This finding is consistent with conclusions from in vitro experiments that large increases in isoniazid concentrations result in only small increases in its bactericidal activity (Middlebrook, 1952; Mackaness & Smith, 1953; Singh & Mitchison, 1954; Peizer, Widelock & Klein, 1954; Hobby and Lenert, 1957).

222

DICKINSON

AND

OTHERS

When the interval between doses was increased to 7 days or more, the results of experiments 3 and 4 suggested that, even if massive doses were given the results of treatment were never as good as when treatment was given twice-weekly, and, further, that the best results obtainable were less good with doses given at intervals of 10.5 days than with those at 7-day intervals. Presumably organisms were recovering between even the largest doses, though it may be that only a proportion of the viable population recovered. Thus, it seems probable that there is a maximal lag period which follows even very large pulses. In order to obtain the best therapeutic results with widely spaced intermittent chemotherapy, the size of the pulsed exposure attained with each dose should be sufficient to produce this maximal lag. Optimal results at both 7-day and 10*5-day intervals were achieved with doses of 20-30 mg/kg. Since the serum concentrations in the guinea-pig and in human rapid inactivators were very similar, the exposure to isoniazid obtainable with a dose of 15 mg/kg in man would only need to be doubled in rapid inactivators by the use of delayed release preparations to obtain optimal response. However, the exposure in slow inactivators is already 2.4 times as great as in rapid inactivators so that no benefit would be expected in slow inactivators from a further increase in exposure produced by a delayed release preparation. Although optimal results in experiments 3 and 4 were obtained either by giving single large doses of 20-30 mg/kg or by two doses of lo-15 mg/kg separated by 4 hours, the results of experiment 1, especially with doses at IO-day intervals, suggest that better results were obtained by giving a divided than a single dose. This advantage, which has not been clearly demonstrated, could arise because the longer period of coverage with the divided dose provided a better opportunity for bactericidal activity to start. Thus there is a suggestion that prolongation of the coverage period by the use of a delayed release preparations of isoniazid may be at least as important as increasing the exposure. The conclusion that the results of treatment with doses given at intervals of 1-4 days were not perceptibly improved by an increase in mean dosage above 1 mg/kg/day and were not influenced by the rhythm of dosage may appear to be at variance with the results of an earlier clinical study which showed that the response to isoniazid alone was better with a daily dosage of 8.7 mg/kg than of 4.5 mg/kg and was also better if given in a single daily dose than divided into two doses in the day (Tuberculosis Chemotherapy Centre, Madras, 1960). However, these differences in response were probably due to the inhibition by high peak concentrations of isoniazid resistant mutants with low degrees of resistance (Gangadharam and others, 1961: Selkon and others, 1964); such effects would not be demonstrated in the short term guinea-pig model, nor would they be observable in man if isoniazid were given with a second potent drug, such as streptomycin, which would by itself inhibit the resistant mutants. Nevertheless, should the second drug be less potent, some of the inhibition of mutants would depend on the brief exposures produced by the peak concentrations of isoniazid. In these circumstances, an increase in isoniazid dose size above the bare minimum should result, even in daily chemotherapy, in less frequent emergence of resistance, as probably occurred in a clinical study with thiacetazone and isoniazid (East African/British Medical Research Council, 1964). In twice-weekly chemotherapy, with a longer interval between exposures, not only would it be necessary to give the largest possible dose to inhibit resistant mutants, but a small difference in response might be expected between slow and rapid inactivators. Thus the size of the exposure effective against mutants, which is already critically small, would be larger in slow than in rapid inactivators since in slow inactivators peak concentrations are higher and the half-life of isoniazid is longer. The results of twice-weekly chemotherapy with isoniazid and PAS were found to be slightly better in slow than in rapid inactivators, though the difference did not attain statistical significance (Tuberculosis Chemotherapy Centre, Madras, 1973b). It remains to be seen whether further evidence of this difference will be seen in future studies of twice-weekly isoniazid and a second weak drug. We are grateful to Dr. G. A. Ellard and Miss P. T. Gammon for estimating isoniazid in guinea-pig serum and to Miss L. Carrol for technical assistance.

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ISONIAZID

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