ANTiMICROBIAL AGzNTS AND CHzMOTHERAPY, Nov. 1975, p. 561-557 Copyright X) 1975 American Society for Microbiology
Vol. 8, No. 5
Printed in U-SA.
Minimal Inhibitory Concentration of Dapsone for Mycobacterium leprae in Rats J. H. PETERS,* G. R. GORDON, J. F. MURRAY, JR., A. H. FIELDSTEEL, AND L. LEVY Life Sciences Division, Stanford Research Institute, Menlo Park, California 94025,* and the Leprosy Research Unit, Public Health Service Hospital, San Francisco, California 94118
Received for publication 16 July 1975
To define the minimal inhibitory concentration (MIC) of dapsone (DDS) for Mycobacterium leprae in rats, we determined the relationship between dietary and plasma levels of DDS in uninfected male and female Lewis rats. This knowledge was applied to the design of experiments using rats inoculated in the footpads with M. leprae. The MIC for DDS in male and female rats, respectively, was 1.5 to 4.0 ng and 1.8 to 3.0 ng of DDS/ml of plasma, even though the sexes exhibited markedly different concentrations of DDS when receiving the same dietary level of DDS. These values for the MIC of DDS for M. leprae in rats are nearly identical to the previously determined MIC of DDS for M. leprae in mice.
The establishment of the mouse footpad infection with Mycobacterium leprae as a tool for leprosy research (27) opened up avenues of investigation that had been blocked since 1873. In that year, Hansen (3, 10) first reported that human leprosy was caused by M. leprae. Despite many attempts in the intervening years, no one has successfully cultivated this bacterium in vitro. All our present knowledge of the sensitivity or resistance of M. leprae to drugs has been derived from the application of the mouse footpad test system (25, 29). Of the several drugs known to be active against M. leprae infections, dapsone (4,4'-diaminodiphenyl sulfone, DDS) is the most thoroughly studied and the most widely used for the treatment of leprosy in man (28). It is extremely potent. The estimated minimal inhibitory concentration (MIC) against M. leprae in the mouse footpad test system ranges from 1 to 10 ng per ml of plasma (2, 20). From chemotherapeutic studies in leprosy patients using very low doses of DDS (2) or its repository form, N,N'-diacetyl DDS (19, 20, 22, 23), we can estimate that the MIC of DDS for M. leprae in man is c30 ng per ml of plasma Although the infection of immunologically normal mice with M. leprae has been a valuable tool, this infection does not provide a model of human lepromatous leprosy suitable for chemotherapeutic studies. Multiplication of M. leprae in the footpads of these mice is selflimiting, reaching a plateau of about 106 bacteria 4 to 6 months after infection with the usual inoculum of 5 x 103 bacteria (24, 27). Also, these rodents differ both qualitatively and quantitatively from man in the disposition of DDS (7, 15).
Other animals reported to be susceptible to infection with M. leprae are rats (6, 11) and the nine-banded armadillo (13, 14). Attempts to infect monkeys have been unsuccessful (3, 12). Recent studies in neonatally thymectomized rats have suggested that M. leprae multiply to greater numbers in these animals than in intact mice (4, 5). In addition, other studies (16) have demonstrated that the disposition of DDS in rats is more like that in man than in mice. Thus, a rat footpad test system may provide certain advantages over that of mice for research. The current studies were undertaken to establish the relationship between dietary levels of DDS and plasma concentrations of the drug in normal male and female Lewis rats receiving DDS in the diet and to determine the MIC of DDS for M. leprae in infected rats of both sexes. The plasma levels of monoacetyl DDS (MADDS), a DDS metabolite in rats and man (7, 16), also were measured. MATERIALS AND METHODS Short-term studies in normal rats. Lewis rats approximately 12 weeks old were purchased from Simonsen Laboratories, Inc., Gilroy, Calif. The average weight of the male rats was 257 g (range, 235 to 272 g); that of the female rats was 174 g (range, 164 to 190 g). The DDS used (K & K Laboratories, Irvine, Calif.) was found to contain 94% DDS by microbore column chromatography (G. R. Gordon, D. C. Ghoul, and J. Peters, J. Pharm. Sci., in press). Diets containing DDS (0.05, 1.5, 5.0, 15, and 50 x 10-5 g%) were prepared by adding appropriate aliquots of a solution of DDS in 95% ethanol to granular rat chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill.) in a liquid-solid, twin-shell blender (Patterson-Kelly Co., East Stroudsburg, Pa.). The 551
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prepared diets were stored at 5 C; the feeders in the rat cages were replenished weekly. The rats were caged individually and provided food and water ad libitum. Two of each sex were fed one of the diets containing DDS for 26 days. After the first day, we determined the amount of food consumed daily during the subsequent 3 days by male rats and during the subsequent 10 days by female rats. Thereafter, in both sexes, the daily food consumption by each rat was calculated from the quantity of food consumed over 2- to 3-day periods. For male rats, 10 measurements of diet consumption were made, and for female rats, 16 measurements. The measurements of daily food intake were converted to grams per kilograms per day for each animal. No consistent changes in the daily food intake during the 26-day study were noted in any of the animals receiving any of the five DDS-containing diets. Finally, the observations on the two rats of each sex on each diet were combined to assess possible differences in food consumption between sexes and among dietary levels of DDS within sexes. Also, dosages of DDS received by each group were calculated from the mean consumption of the various DDS-containing diets. For the determination of plasma levels of DDS and MADDS, we collected blood samples (1.0 ml) from each rat by heart puncture (nonterminally), using a syringe wetted with 0.1 ml of saline containing 33 U of heparin; these samples were taken at 8:00 a.m. and 4:00 p.m. on days 5, 12, and 19 and at 8:00 a.m. on day 26. Plasma was prepared immediately by centrifugation and stored frozen before analysis. Long-term studies in rats infected with M. leprae to determine the MIC of DDS. Twenty Lewis rats of each sex (4 to 6 weeks old) were inoculated in each hind footpad with 5 x 103 M. leprae of a strain originally isolated from a patient with lepromatous leprosy by C. C. Shepard (Center for Disease Control, Atlanta, Ga.) and subsequently maintained in mouse passage in our laboratories. Multiplication of this strain of M. leprae in mice was consistently inhibited by administration of a diet containing 10-4 g% DDS (L. Levy, unpublished data). The inoculated rats were divided into 10 groups of four each, and one group of each sex was fed a drug-free diet. The other four groups of each sex were fed DDS-containing diets selected from the results of the short-term feeding trial to provide plasma levels in both sexes that would bracket the MIC for DDS in mice of 1 to 10 ng per ml of plasma (2, 20), taking into account the sex difference noted in the shortterm study. The four groups of male rats were fed diets containing 1.5, 5.0, 15, and 50 x 10-5 g% DDS; the four groups of female rats were fed diets containing 0.5, 1.5, 5.0, and 15 x 10-i g% DDS. These diets were fed to male rats for 140 days and to female rats for 141 days after inoculation. Thereafter, all rats received drug-free diets for the remainder of the study. In male rats, the number of M. leprae in the hind footpads was determined on day 140 in one rat from all groups; on days 202 and 240 in one rat from each group that had previously received dietary DDS;
ANTIMICROB. AGENTS CHEMOTHER. and on day 260 from one rat that had received no DDS throughout the study. In female rats, the number ofM. leprae in the hind footpads was determined on days 141 and 239 in one rat from all groups and on days 199 and 331 in one rat from each group that had previously received dietary DDS. The methods for processing the footpads and for counting M. kprae were described previously (27, 30). For the determination of plasma levels of DDS and MADDS, we obtained blood samples (1.0 ml) nonterminally at 8:00 a.m. as described above on days 62 and 93 from the male rats and on days 63 and 92 from the female rats. Also, blood was obtained from the rat ofeach group sacrificed for bacteriologic assessment on days 140 or 141. In studies of both the infected and uninfected rats, we determined plasma levels of DDS and MADDS by a chromatographicfluorometric procedure (17). The practical limit of sensitivity of these analyses was 0.5 ng of DDS or MADDS per ml because plasma volumes of 0.5 ml were analyzed.
RESULTS Studies in uninfected rats. Table 1 presents the mean consumption by the two sexes of the various DDS-containing diets, the dosages of DDS, and the percentages of the dosages received by the male relative to the female rats. As shown in column 2, no differences were noted in the mean food consumption by male rats receiving a 100-fold range of DDS-containing diets. In female rats (column 3), mean food consumption was significantly higher (P < 0.05) only in those animals receiving 1.5 x 10-5 g% DDS compared with those receiving 15 x 10-5 g% DDS. That a significant difference was found in only one of ten possible comparisons in this group suggests that the dietary level of DDS had little effect on the amount of food consumed by both the female and male rats. However, the female rats consumed significantly greater amounts of the three lower DDS diets than did the male rats. The dosages of DDS received by the two sexes shown in columns 4 and 5 reflect these small differences in food intake. The differences between the three lower dosages of DDS received by the two sexes were not substantial, as shown by the calculated percentages listed in column 6. At the two higher levels of DDS in the diet, no differences were noted between the sexes in any regard. As described in the Materials and Methods section, we obtained plasma at 8:00 a.m. and 4:00 p.m. on days 5, 12, and 19 and at 8:00 a.m. on day 26 of this study. No marked or consistent differences were observed in the plasma levels of DDS or MADDS at these times. (For male rats, in only one of the ten possible comparisons was the mean of samples collected at 4:00 p.m. significantly different from the mean of the corresponding samples collected at 8:00 a.m. In
DAPSONE FOR M. LEPRAE IN RATS
VOL. 8, 1975
553
TABLE 1. Dosages of DDS in rats receiving dietary DDS DDS diet level (xlO-' g%)
0.5 1.5 5 15 50
Diet consumed (mean g/kg per day + SE)a Male
74.4 70.9 70.4 72.1 74.5
+ ± ± ± ±
2.11 1.59 3.37 1.79 2.30
Female
85.0 83.3 81.3 76.3 77.2
± ± ± ± ±
3.93b 2.46b
2.18b 1.97 3.81
DDS dosage
(mg/kg per day
1O-3)
Male
Female
Male dosage/ female dosage (%)
3.7 10.6 35.2 108 372
4.2 12.5 40.6 114 386
88.1 84.8 86.7 94.7 96.4
x
a Mean of the average food consumed daily by two rats of each sex as described in the text. SE, Standard error. b Significantly greater (P < 0.02) than the corresponding values for male rats.
female rats, only two of the means of the 4:00 p.m. observations were significantly different from those at 8:00 a.m. Raw data are available from the senior author.) For brevity and because the later studies of infected rats involved only single blood samplings at 8:00 a.m., Table 2 presents only the mean values of DDS and MADDS for the samples collected at 8:00 a.m. on the various days. In both sexes consuming the diet containing 0.5 x 10-5 g% DDS, we found plasma levels of DDS and MADDS at or below the practical limit of sensitivity of the assay procedure in the majority of cases (line 1 of Table 2). However, at all higher DDS diet levels, DDS was easily measured in plasma of female rats; they consistently exhibited three- to fourfold higher levels than did male rats on the same diets. Also, female rats exhibited markedly higher levels of MADDS at the four higher DDS diets than did the male rats. In fact, the MADDS levels were disproportionately higher than the DDS levels, as shown by calculations of the percentage of acetylation of DDS (MADDS [as DDS equivalents] x 102/DDS + MADDS) in the two sexes receiving the various diets. This calculation yielded 17 and 18% acetylation by the male rats at the two higher dietary levels of DDS and 29 to 32% acetylation in the female rats consuming the four higher levels of dietary DDS. Clearly, female rats exhibit markedly different levels of both DDS and its metabolite, MADDS, than male rats. These differences were not attributable to the slightly higher dosages of DDS received by the female rats, as shown in Table 1. To assess rigorously the relationship between dietary and plasma DDS, we have shown in Fig. 1 the regression of plasma levels of DDS on the dietary DDS levels in both sexes. The mean values at the various blood sampling times for each rat receiving dietary DDS were plotted to show the observations made on each animal. However, the regression lines were calculated
from all observations of each sex on each diet. The correlation coefficients for the regression lines for the female and male rats were 0.9930 and 0.9852, respectively. The slopes of the two lines were not significantly different (8). This graph clearly demonstrates a direct linear relationship between DDS in the diet and that in the plasma, although this relationship differs quantitatively between the sexes. From this information, we selected diets to be used for the determination of the MIC of DDS for M. leprae. Studies in infected rats. To learn if longterm feeding of DDS-containing diets produced plasma levels of DDS and MADDS different from those of the short-term study, we measured these levels in infected male rats at 62 and 93 days and in female rats at 63 and 92 days after inoculation with M. leprae and the start of feeding with the various diets. Comparison of the levels of DDS and MADDS in the two sexes at 62 and 63 days (Table 3) with those found in the short-term study (Table 2) showed that only the mean DDS level of 2.0 ng/ml in male rats receiving 5.0 x 10-5 go for 62 days was significantly different from the corresponding mean value of 1.4 ng of DDS/ml in the short-term study. No other comparison in either sex demonstrated a significant difference. In addition, the levels found at 93 and 92 days in the male and female rats, respectively, were not consistently different from either the 62- or 63-day values or those of the short-term study. Therefore, for brevity, the 92- and 93-day values have not been presented. From these comparisons, we conclude that continued feeding of these DDS diets for long periods has no influence on plasma levels of DDS and MADDS. The DDS and MADDS levels found in the male rats sacrificed for bacteriologic assessment on day 140 are shown in Table 3. These levels are also very similar to the levels observed in groups of rats bled at the earlier times. As shown in this table, large numbers of M.
554
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PETERS ET AL.
TABLE 2. Mean plasma levels of DDS and MADDS in rats receiving dietary DDS Mean (+SE) plasma level (nglml)a
DDS diet level
MADDS MADDS
DDS DDS
(x10-5 g%)
Female
Male
0.5 1.5 5 15 50
Vol. 8, No. 5
Printed in U-SA.
Minimal Inhibitory Concentration of Dapsone for Mycobacterium leprae in Rats J. H. PETERS,* G. R. GORDON, J. F. MURRAY, JR., A. H. FIELDSTEEL, AND L. LEVY Life Sciences Division, Stanford Research Institute, Menlo Park, California 94025,* and the Leprosy Research Unit, Public Health Service Hospital, San Francisco, California 94118
Received for publication 16 July 1975
To define the minimal inhibitory concentration (MIC) of dapsone (DDS) for Mycobacterium leprae in rats, we determined the relationship between dietary and plasma levels of DDS in uninfected male and female Lewis rats. This knowledge was applied to the design of experiments using rats inoculated in the footpads with M. leprae. The MIC for DDS in male and female rats, respectively, was 1.5 to 4.0 ng and 1.8 to 3.0 ng of DDS/ml of plasma, even though the sexes exhibited markedly different concentrations of DDS when receiving the same dietary level of DDS. These values for the MIC of DDS for M. leprae in rats are nearly identical to the previously determined MIC of DDS for M. leprae in mice.
The establishment of the mouse footpad infection with Mycobacterium leprae as a tool for leprosy research (27) opened up avenues of investigation that had been blocked since 1873. In that year, Hansen (3, 10) first reported that human leprosy was caused by M. leprae. Despite many attempts in the intervening years, no one has successfully cultivated this bacterium in vitro. All our present knowledge of the sensitivity or resistance of M. leprae to drugs has been derived from the application of the mouse footpad test system (25, 29). Of the several drugs known to be active against M. leprae infections, dapsone (4,4'-diaminodiphenyl sulfone, DDS) is the most thoroughly studied and the most widely used for the treatment of leprosy in man (28). It is extremely potent. The estimated minimal inhibitory concentration (MIC) against M. leprae in the mouse footpad test system ranges from 1 to 10 ng per ml of plasma (2, 20). From chemotherapeutic studies in leprosy patients using very low doses of DDS (2) or its repository form, N,N'-diacetyl DDS (19, 20, 22, 23), we can estimate that the MIC of DDS for M. leprae in man is c30 ng per ml of plasma Although the infection of immunologically normal mice with M. leprae has been a valuable tool, this infection does not provide a model of human lepromatous leprosy suitable for chemotherapeutic studies. Multiplication of M. leprae in the footpads of these mice is selflimiting, reaching a plateau of about 106 bacteria 4 to 6 months after infection with the usual inoculum of 5 x 103 bacteria (24, 27). Also, these rodents differ both qualitatively and quantitatively from man in the disposition of DDS (7, 15).
Other animals reported to be susceptible to infection with M. leprae are rats (6, 11) and the nine-banded armadillo (13, 14). Attempts to infect monkeys have been unsuccessful (3, 12). Recent studies in neonatally thymectomized rats have suggested that M. leprae multiply to greater numbers in these animals than in intact mice (4, 5). In addition, other studies (16) have demonstrated that the disposition of DDS in rats is more like that in man than in mice. Thus, a rat footpad test system may provide certain advantages over that of mice for research. The current studies were undertaken to establish the relationship between dietary levels of DDS and plasma concentrations of the drug in normal male and female Lewis rats receiving DDS in the diet and to determine the MIC of DDS for M. leprae in infected rats of both sexes. The plasma levels of monoacetyl DDS (MADDS), a DDS metabolite in rats and man (7, 16), also were measured. MATERIALS AND METHODS Short-term studies in normal rats. Lewis rats approximately 12 weeks old were purchased from Simonsen Laboratories, Inc., Gilroy, Calif. The average weight of the male rats was 257 g (range, 235 to 272 g); that of the female rats was 174 g (range, 164 to 190 g). The DDS used (K & K Laboratories, Irvine, Calif.) was found to contain 94% DDS by microbore column chromatography (G. R. Gordon, D. C. Ghoul, and J. Peters, J. Pharm. Sci., in press). Diets containing DDS (0.05, 1.5, 5.0, 15, and 50 x 10-5 g%) were prepared by adding appropriate aliquots of a solution of DDS in 95% ethanol to granular rat chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill.) in a liquid-solid, twin-shell blender (Patterson-Kelly Co., East Stroudsburg, Pa.). The 551
552
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prepared diets were stored at 5 C; the feeders in the rat cages were replenished weekly. The rats were caged individually and provided food and water ad libitum. Two of each sex were fed one of the diets containing DDS for 26 days. After the first day, we determined the amount of food consumed daily during the subsequent 3 days by male rats and during the subsequent 10 days by female rats. Thereafter, in both sexes, the daily food consumption by each rat was calculated from the quantity of food consumed over 2- to 3-day periods. For male rats, 10 measurements of diet consumption were made, and for female rats, 16 measurements. The measurements of daily food intake were converted to grams per kilograms per day for each animal. No consistent changes in the daily food intake during the 26-day study were noted in any of the animals receiving any of the five DDS-containing diets. Finally, the observations on the two rats of each sex on each diet were combined to assess possible differences in food consumption between sexes and among dietary levels of DDS within sexes. Also, dosages of DDS received by each group were calculated from the mean consumption of the various DDS-containing diets. For the determination of plasma levels of DDS and MADDS, we collected blood samples (1.0 ml) from each rat by heart puncture (nonterminally), using a syringe wetted with 0.1 ml of saline containing 33 U of heparin; these samples were taken at 8:00 a.m. and 4:00 p.m. on days 5, 12, and 19 and at 8:00 a.m. on day 26. Plasma was prepared immediately by centrifugation and stored frozen before analysis. Long-term studies in rats infected with M. leprae to determine the MIC of DDS. Twenty Lewis rats of each sex (4 to 6 weeks old) were inoculated in each hind footpad with 5 x 103 M. leprae of a strain originally isolated from a patient with lepromatous leprosy by C. C. Shepard (Center for Disease Control, Atlanta, Ga.) and subsequently maintained in mouse passage in our laboratories. Multiplication of this strain of M. leprae in mice was consistently inhibited by administration of a diet containing 10-4 g% DDS (L. Levy, unpublished data). The inoculated rats were divided into 10 groups of four each, and one group of each sex was fed a drug-free diet. The other four groups of each sex were fed DDS-containing diets selected from the results of the short-term feeding trial to provide plasma levels in both sexes that would bracket the MIC for DDS in mice of 1 to 10 ng per ml of plasma (2, 20), taking into account the sex difference noted in the shortterm study. The four groups of male rats were fed diets containing 1.5, 5.0, 15, and 50 x 10-5 g% DDS; the four groups of female rats were fed diets containing 0.5, 1.5, 5.0, and 15 x 10-i g% DDS. These diets were fed to male rats for 140 days and to female rats for 141 days after inoculation. Thereafter, all rats received drug-free diets for the remainder of the study. In male rats, the number of M. leprae in the hind footpads was determined on day 140 in one rat from all groups; on days 202 and 240 in one rat from each group that had previously received dietary DDS;
ANTIMICROB. AGENTS CHEMOTHER. and on day 260 from one rat that had received no DDS throughout the study. In female rats, the number ofM. leprae in the hind footpads was determined on days 141 and 239 in one rat from all groups and on days 199 and 331 in one rat from each group that had previously received dietary DDS. The methods for processing the footpads and for counting M. kprae were described previously (27, 30). For the determination of plasma levels of DDS and MADDS, we obtained blood samples (1.0 ml) nonterminally at 8:00 a.m. as described above on days 62 and 93 from the male rats and on days 63 and 92 from the female rats. Also, blood was obtained from the rat ofeach group sacrificed for bacteriologic assessment on days 140 or 141. In studies of both the infected and uninfected rats, we determined plasma levels of DDS and MADDS by a chromatographicfluorometric procedure (17). The practical limit of sensitivity of these analyses was 0.5 ng of DDS or MADDS per ml because plasma volumes of 0.5 ml were analyzed.
RESULTS Studies in uninfected rats. Table 1 presents the mean consumption by the two sexes of the various DDS-containing diets, the dosages of DDS, and the percentages of the dosages received by the male relative to the female rats. As shown in column 2, no differences were noted in the mean food consumption by male rats receiving a 100-fold range of DDS-containing diets. In female rats (column 3), mean food consumption was significantly higher (P < 0.05) only in those animals receiving 1.5 x 10-5 g% DDS compared with those receiving 15 x 10-5 g% DDS. That a significant difference was found in only one of ten possible comparisons in this group suggests that the dietary level of DDS had little effect on the amount of food consumed by both the female and male rats. However, the female rats consumed significantly greater amounts of the three lower DDS diets than did the male rats. The dosages of DDS received by the two sexes shown in columns 4 and 5 reflect these small differences in food intake. The differences between the three lower dosages of DDS received by the two sexes were not substantial, as shown by the calculated percentages listed in column 6. At the two higher levels of DDS in the diet, no differences were noted between the sexes in any regard. As described in the Materials and Methods section, we obtained plasma at 8:00 a.m. and 4:00 p.m. on days 5, 12, and 19 and at 8:00 a.m. on day 26 of this study. No marked or consistent differences were observed in the plasma levels of DDS or MADDS at these times. (For male rats, in only one of the ten possible comparisons was the mean of samples collected at 4:00 p.m. significantly different from the mean of the corresponding samples collected at 8:00 a.m. In
DAPSONE FOR M. LEPRAE IN RATS
VOL. 8, 1975
553
TABLE 1. Dosages of DDS in rats receiving dietary DDS DDS diet level (xlO-' g%)
0.5 1.5 5 15 50
Diet consumed (mean g/kg per day + SE)a Male
74.4 70.9 70.4 72.1 74.5
+ ± ± ± ±
2.11 1.59 3.37 1.79 2.30
Female
85.0 83.3 81.3 76.3 77.2
± ± ± ± ±
3.93b 2.46b
2.18b 1.97 3.81
DDS dosage
(mg/kg per day
1O-3)
Male
Female
Male dosage/ female dosage (%)
3.7 10.6 35.2 108 372
4.2 12.5 40.6 114 386
88.1 84.8 86.7 94.7 96.4
x
a Mean of the average food consumed daily by two rats of each sex as described in the text. SE, Standard error. b Significantly greater (P < 0.02) than the corresponding values for male rats.
female rats, only two of the means of the 4:00 p.m. observations were significantly different from those at 8:00 a.m. Raw data are available from the senior author.) For brevity and because the later studies of infected rats involved only single blood samplings at 8:00 a.m., Table 2 presents only the mean values of DDS and MADDS for the samples collected at 8:00 a.m. on the various days. In both sexes consuming the diet containing 0.5 x 10-5 g% DDS, we found plasma levels of DDS and MADDS at or below the practical limit of sensitivity of the assay procedure in the majority of cases (line 1 of Table 2). However, at all higher DDS diet levels, DDS was easily measured in plasma of female rats; they consistently exhibited three- to fourfold higher levels than did male rats on the same diets. Also, female rats exhibited markedly higher levels of MADDS at the four higher DDS diets than did the male rats. In fact, the MADDS levels were disproportionately higher than the DDS levels, as shown by calculations of the percentage of acetylation of DDS (MADDS [as DDS equivalents] x 102/DDS + MADDS) in the two sexes receiving the various diets. This calculation yielded 17 and 18% acetylation by the male rats at the two higher dietary levels of DDS and 29 to 32% acetylation in the female rats consuming the four higher levels of dietary DDS. Clearly, female rats exhibit markedly different levels of both DDS and its metabolite, MADDS, than male rats. These differences were not attributable to the slightly higher dosages of DDS received by the female rats, as shown in Table 1. To assess rigorously the relationship between dietary and plasma DDS, we have shown in Fig. 1 the regression of plasma levels of DDS on the dietary DDS levels in both sexes. The mean values at the various blood sampling times for each rat receiving dietary DDS were plotted to show the observations made on each animal. However, the regression lines were calculated
from all observations of each sex on each diet. The correlation coefficients for the regression lines for the female and male rats were 0.9930 and 0.9852, respectively. The slopes of the two lines were not significantly different (8). This graph clearly demonstrates a direct linear relationship between DDS in the diet and that in the plasma, although this relationship differs quantitatively between the sexes. From this information, we selected diets to be used for the determination of the MIC of DDS for M. leprae. Studies in infected rats. To learn if longterm feeding of DDS-containing diets produced plasma levels of DDS and MADDS different from those of the short-term study, we measured these levels in infected male rats at 62 and 93 days and in female rats at 63 and 92 days after inoculation with M. leprae and the start of feeding with the various diets. Comparison of the levels of DDS and MADDS in the two sexes at 62 and 63 days (Table 3) with those found in the short-term study (Table 2) showed that only the mean DDS level of 2.0 ng/ml in male rats receiving 5.0 x 10-5 go for 62 days was significantly different from the corresponding mean value of 1.4 ng of DDS/ml in the short-term study. No other comparison in either sex demonstrated a significant difference. In addition, the levels found at 93 and 92 days in the male and female rats, respectively, were not consistently different from either the 62- or 63-day values or those of the short-term study. Therefore, for brevity, the 92- and 93-day values have not been presented. From these comparisons, we conclude that continued feeding of these DDS diets for long periods has no influence on plasma levels of DDS and MADDS. The DDS and MADDS levels found in the male rats sacrificed for bacteriologic assessment on day 140 are shown in Table 3. These levels are also very similar to the levels observed in groups of rats bled at the earlier times. As shown in this table, large numbers of M.
554
ANTIMICROB. AGENTS CHZMOTHRR.
PETERS ET AL.
TABLE 2. Mean plasma levels of DDS and MADDS in rats receiving dietary DDS Mean (+SE) plasma level (nglml)a
DDS diet level
MADDS MADDS
DDS DDS
(x10-5 g%)
Female
Male
0.5 1.5 5 15 50
