M. D. Coleman

An Investigation into the Haematological Toxicity of Structural Analogues of Dapsone In-vivo and In-vitro

Abstract— With microsomes prepared from a single human liver, 4,4′‐diaminodiphenyl sulphone (DDS), 4‐acetyl‐4‐aminodiphenyl sulphone (MADDS), 4‐acetyl‐4‐aminodiphenyl thioether (MADDT) and 4,4′‐diacetyldiphenyl thioether (DADDT) caused significantly greater methaemoglobin formation compared with control. In‐vitro in the rat, the pattern of toxicity was slightly different: DADDT was not haemotoxic, whilst 3,4′‐diaminodiphenyl sulphone (3,4′DDS) and 3,3′‐diaminodiphenyl sulphone (3,3′DDS) as well as DDS, MADDS and MADDT were significantly greater than control. 4,4′ Acetyl diphenyl sulphone (DADDS), 4,4′ diaminodiphenyl thioether (DDT), 4,4′‐diaminodiphenyl ether (DDE) and 4,4′ diamino‐octofluorodiphenyl sulphone (F8DDS) did not cause significant methaemoglobinaemia in either human or rat liver microsomes. DDS, MADDS, and MADDT were not significantly different in haemotoxicity generation in‐vitro in the presence of human microsomes. In the rat in‐vitro, DDS, MADDS, and 3,4′DDS did not differ significantly in red cell toxicity, and were the most potent methaemoglobin formers. The 3,3′ DDS and MADDT derivatives were both significantly less toxic compared with DDS. None of the compounds tested caused haemoglobin oxidation in the absence of NADPH in‐vitro. In the whole rat, DDS, MADDS and MADDT caused significantly higher levels of methaemoglobin compared with control. None of the remaining compounds caused methaemoglobin formation which was significantly greater than control. DDS and MADDS were the most potent methaemoglobin formers tested, in‐vivo and in‐vitro. The 3,3′ and 3,4′DDS analogues caused no detectable haemotoxicity in‐vivo. However, the plasma elimination of the 3,4′ analogue was much more rapid compared with that of DDS. Overall, there was no correlation between log k0 and increasing haemotoxicity. The use of the two‐compartment system together with in‐vivo studies may be applied to the evaluation of the structural features required for bioactivation of candidate antiparasitic compounds to haemotoxic metabolites by cytochrome P450 enzymes.

Gonadal influence on the metabolism and haematological toxicity of dapsone in the rat.

Abstract— Administration of dapsone (33 mg kg−1) to intact rats resulted in a marked elevation of methaemoglobin levels in male (435.0 ± 105.2% met Hb h) compared with female rats (59.0 ± 17.2% met Hb h). However, the clearance of dapsone was significantly faster in males compared with females. Female rats showed very low levels of methaemoglobin which were accompanied by significantly higher blood concentrations of parent drug. Clearance of dapsone in castrated animals was less than one‐third of that of the intact sham‐operated males (252.2 ± 67.2 vs 81.4 ± 33.0 mL h−1). Likewise, clearance of dapsone in ovarectomized rats was approximately half that of intact females. There were no significant differences in the disposition of dapsone between the ovarectomized (AUC, 431.0 ± 31.7 μg h mL−1; t 1/2, 15.62 ± 1.8 h) and castrated (AUC, 450.6 ± 150.9 μg h mL−1; t 1/2, 17.6 ± 7.9 h) animals. However, methaemoglobin levels in castrated males, although less than a third of those of intact males, significantly exceeded those of ovarectomized animals. There was no significant difference between the four groups of animals with respect to red cell sensitivity to the methaemoglobin‐forming capacity of the toxic metabolite of dapsone, the hydroxylamine. Metabolic conversion of dapsone to the hydroxylamine in the presence of NADPH was 7.6 ± 1.5% for liver microsomes from intact males and was significantly greater (P < 0.05) than the corresponding values for liver microsomes from castrated rats (5.3 ± 0.59%). Conversion of dapsone to dapsone‐NOH by liver microsomes from intact females and ovarectomized animals was below 1 % in both cases. This study illustrates the androgenic control of N‐hydroxylation in the rat.

Inhibition of dapsone-induced methaemoglobinaemia in the rat isolated perfused liver.

Abstract— We have investigated the disposition of dapsone (DDS, 1 mg) in the rat isolated perfused liver in the absence and the presence of cimetidine (3 mg). After the addition of DDS alone to the liver there was a monoexponential decline of parent drug concentrations and rapid formation of DDS‐NOH (within 10 min) which coincided with methaemoglobin formation (11.7 ± 3.0%, mean ± s.d.) which reached a maximum (22.6 ± 9.2%) at 1 h. The appearance of monoacetyl DDS (MADDS) was not apparent until 30–45 min. Addition of cimetidine resulted in major changes in the pharmacokinetics of DDS and its metabolites. The AUC of DDS in the presence of cimetidine (1018.8 ± 267.8 μg min mL−1) was almost three‐fold higher than control (345.0 ± 68.1 μg min mL−1, P < 0.01). The half‐life of DDS was also prolonged by cimetidine compared with control (117.0 ± 48.2 min vs 51.2 ± 22.9, P < 0.05). The clearance of DDS (3.0 ± 0.55 mL min−1) was greatly reduced in the presence of cimetidine (1.03 ± 0.26 mL min−1 P < 0.01). The AUC0.3 h for DDS‐NOH (28.3 ± 21.2 μg min mL−1) was significantly reduced by cimetidine (8.1 ± 3.40 μg min mL−1, P < 0.01). In contrast, there was a marked increase in the AUC0.3 h for MADDS (32.7 ± 25.8 μg min mL−1) in the presence of cimetidine (166.0 ± 26.5 μg min mL−1 P < 0.01). The methaemoglobinaemia associated with DDS was reduced to below 5% by cimetidine. Hence, a shift in hepatic metabolism from bioactivation (N‐hydroxylation) to detoxication (N‐acetylation) caused by cimetidine, was associated with a fall in methaemoglobinaemia. These data suggest that the combination of DDS with a cytochrome P450 inhibitor might reduce the risk to benefit ratio of DDS.

The disposition of pyrimethamine in the isolated perfused rat liver

We have investigated the disposition of pyrimethamine base in the isolated perfused rat liver (IPRL) preparation after the administration of pyrimethamine (0.5 mg, 5 microCi). In the first half hour of the study, pyrimethamine underwent marked hepatic uptake, thereafter perfusate plasma drug levels declined monoexponentially with a half life (t 1/2) of 3.0 +/- 1.0 hr. Area under the perfusate plasma concentration/time curve (AUC)0----infinity was 6.9 +/- 1.9 microgram/hr/ml. Pyrimethamine was found to be a low clearance compound (78.4 +/- 25.3 ml/hr identical to 8.6% of liver perfusate flow) with a large volume of distribution (267.5 +/- 55.3 ml) in the IPRL. The combined AUCS(0----5hr) for pyrimethamine (AUC 4.8 +/- 0.5 microgram/hr/ml) and pyrimethamine 3-N-oxide (AUC0----5hr 0.9 +/- 0.6 microgram/hr/ml) accounted for 57% of the total AUC0----5hr of [14C] radioactivity (10.0 +/- 2.6 micrograms/hr/ml). This indicates the presence of metabolites of pyrimethamine as yet unidentified in the perfusate. Biliary excretion of [14C] during the course of the IPRL preparations was extensive (29.0 +/- 10.3%) though only a small proportion was due to pyrimethamine and the 3-N-oxide metabolite. The majority of radioactivity in the bile was attributable to highly polar, but unidentified metabolites of pyrimethamine. At the conclusion of each experiment (5 hr), a significant proportion of [14C] radioactivity was recovered from the livers (22.9 +/- 5.3%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged pyrimethamine, with trace levels of the 3-N-oxide metabolite. Sub-cellular fractionation of the homogenized livers revealed the most pronounced localisation of pyrimethamine to be in the lipid rich 10,000 g pellet (13.0 +/- 2.6%), the remainder being distributed equally between the 105,000 g pellet and supernatant. Neither pyrimethamine, [14C] radioactivity, nor pyrimethamine 3-N-oxide were extensively taken up by red cells throughout the study. Therefore, the large volume of distribution (267.5 +/- 55.3 ml) underlines the extent of pyrimethamine localisation in the liver.

Inhibition of dapsone-induced methaemoglobinaemia by cimetidine in the rat during chronic dapsone administration

Abstract— Dapsone undergoes N‐acetylation to monoacetyl dapsone as well as N‐hydroxylation to a hydroxylamine which is responsible for the haemotoxicity (i.e. methaemoglobinaemia; Met Hb) of the drug. Since dapsone is always given chronically, we have investigated the ability of cimetidine to inhibit Met Hb formation caused by repeated dapsone administration. The drug was given (i.p.) to four groups (n = 6 per group) of male Wistar rats, 300–360 g. Group I received 10 mg kg−1 at 1, 24, 48 and 72 h. Group II received 10 mg kg−1 at 1, 8, 24, 32, 48, 56, 72 and 80 h. Groups III and IV received the drug as for groups I and II, respectively, as well as cimetidine (50 mg kg−1) 1 h before each dose of dapsone. Twice daily dapsone administration (Group II) resulted in a significantly greater (P < 0.05) Met Hb AUC (757 ± 135 vs 584 ± 115% Met Hb h), dapsone AUC (140 ± 17.5 vs 113 ± 130 μg h mL−1) and monoacetyl dapsone AUC (48.2 ± 18.3 vs 10.8 ± 4.6 μg h mL−1) compared with a single daily dapsone dose (group I). The administration of cimetidine before the once daily dose of dapsone (group III) resulted in a significant (P < 0.05) fall in Met Hb (302 ± 179 vs 584 ± 115% Met Hb h) and an increase in both the dapsone (151 ± 22.2 vs 113 ± 13.0 μg h mL−1) and monoacetyl dapsone AUC values (33.6 ± 5.8 vs 10.8 ± 4.0 μg h mL−1) compared with a single daily dose of dapsone (group I). Administration of cimetidine before the twice daily dose of dapsone (group IV) resulted in no significant change in Met Hb or monoacetyl dapsone levels, despite a marked increase in the AUC after dapsone compared with control (303 ± 53.2 vs 140 ± 17.5 μg h mL−1 P < 0.05; group II). The administration of a single dose of monoacetyl dapsone alone resulted in rapid production of methaemoglobinaemia (17.1 ± 7.2%) at 1 h; however, prior administration of cimetidine did not significantly affect methaemoglobin levels over 24 h (287.6 ± 77.9 vs 316.4 ± 120.2% Met Hb h). These studies indicate that although cimetidine may reduce Met Hb formation during chronic dapsone administration, dose reduction of dapsone is required to avoid haemotoxicity because of the increased accumulation of both parent drug and its monoacetyl metabolite.

The sustained release of pyrimethamine base or pyrimethamine pamoate from a biodegradable injectable depot preparation in mice

The pharmacokinetics and mass fate in mice, of pyrimethamine (425 mg kg−1 s.o.) administered subcutaneously either as the base (BASE) or the pamoate salt (PAM) in an injectable oil mixture (benzyl benzoate‐peanut oil 50:50 v/v) have been evaluated. Maximum measured plasma pyrimethamine levels after BASE were attained within 24 h, and were twice as high as after PAM. 25% of animals dosed with BASE died; among the survivors plasma drug levels fell rapidly below the minimum inhibitory concentration (MIC) for Plasmodium berghei (100–200 ng ml−1) by 5 weeks. In contrast, no mice dosed with PAM died and plasma levels were sustained above the MIC for 13 weeks, drug still being detectable in plasma after four months. Overall, there was no significant difference between areas under the curve from zero time to the time of the final sampling of pyrimethamine following PAM or BASE. The rapid initial elimination of 14C‐radioactivity (2ṁ64 ± 0ṁ47% dose day−1 over 4 weeks) seen after dosage with [14C]BASE reflected the plasma disposition of pyrimethamine in the mice dosed with BASE. 90% of the excreted 14C was eliminated by one month by which time less than 1% (0ṁ03 ± 0ṁ02%) of the [14C]BASE was recovered from the injection site. Both BASE and [14C]BASE studies suggest that exhaustion of this preparation occurred by 7 weeks. Excretion of 14C‐radioactivity after [14C]PAM was gradual and sustained with a low mean daily rate, that was maintained throughout the study i.e. 1ṁ21 ± 0ṁ17% day−1 (4 weeks), 0ṁ88 ± 0ṁ28% day−1 (8 weeks), 0ṁ5 ± 0ṁ31% day−1 (12 weeks), 0ṁ42 ± 0ṁ27% day−1 (16 weeks). At the end of the study, 7ṁ11 ± 1ṁ90% of the [14C]PAM remained in the dose site. No visible tissue reactions were observed to either preparation. Overall recovery of radioactivity was 85ṁ00 ± 11ṁ31% for both [14C]PAM and [14C]BASE. These studies indicate that the PAM preparation is worthy of further long term evaluation.

Pyrimethamine pharmacokinetics and its tissue localization in mice: effect of dose size

The plasma pharmacokinetics and mass fate of [14 C]pyrimethamine were investigated in the mouse, following dosage with 12.5, 25, 50, and 75 mg kg−1 (i.p.). Peak plasma concentrations of pyrimethamine were reached between 1 and 2 h and then declined monoexponentially. The mean values for AUC 0 → 30 h increased linearly in relation to the administered dose of pyrimethamine (r = 0.979, P < 0.001). The mean values for intraperitoneal clearance and half‐life were not significantly different between dose groups, indicating that the plasma pharmacokinetics of pyrimethamine were independent of dose. The percentage of the administered dose excreted in urine as pyrimethamine (1.3−3.5%) and 14 C‐radioactivity (21.7−29.1%) did not change with increasing dose. In contrast, the cumulative percentage of the dose excreted as 14 C‐radioactivity in faeces (16.7−22.8%) after the three highest doses 25, 50 and 75 mg kg−1 was significantly less than that seen with the lowest dose of 12.5 mg (50.3%). This suggests extensive biliary excretion of radioactivity, and that the capacity of this process may have been exceeded with the highest doses. Seven days after the administration of each of the three highest doses, a significantly greater percentage of [14 C]pyrimethamine was localized in the soft tissues; i.e. heart, lung and kidney (7.8−13.8%), gut (5.4−9.4%) and particularly the liver (25.0−27.9%) when compared with the lowest dose of the drug (1.2, 1.0, 0.3% respectively). Following each dose, between 85 and 97% of the administered radioactivity was accounted for. These studies indicate, that with higher doses of pyrimethamine, the parent drug and/or metabolites may accumulate in soft tissue, particularly the liver, but without appreciable effects on the plasma disposition and urinary excretion of the drug.

THE DISPOSITION OF SURAMIN IN THE ISOLATED PERFUSED RAT LIVER

We have investigated the disposition of suramin in the isolated perfused rat liver preparation (IPRL) after the administration of suramin (18 mg, 8 muCi). At 30 min post drug administration, almost 100% of the [14C]radioactivity and unchanged suramin were located in the perfusate plasma. During the course of the study, the elimination of suramin from the IPRL was barely perceptible. The AUC0-5 hr of suramin (730.6 +/- 86.2 micrograms hr/ml) corresponded to that of [14C] radioactivity (815.1 +/- 105.5 micrograms ml/hr) at 5 hr, indicating a lack of perfusate suramin metabolites. At 5 hr only a small proportion of [14C] radioactivity was recovered from the livers (2.5 +/- 1.1%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged suramin. Sub-cellular fractionation of the homogenised livers revealed suramin to be distributed in the liposomal rich tissue fractions (10,000 g pellet, 1.6 +/- 0.8%; 105,000 g supernatant, 1.1 +/- 0.35%). Biliary excretion of [14C] radioactivity was low (2.1 +/- 0.7%), however, none could be accounted for as unchanged suramin. Previously undetected metabolites of suramin may have accounted for the unidentified biliary radioactivity.

Inhibition of Dapsone-induced Methaemoglobinaemia by Cimetidine in the Presence of Trimethoprim in the Rat

Abstract— Administration of dapsone in combination with trimethoprim and cimetidine to male rats resulted in a marked decrease (P < 0·05) in measured methaemoglobin levels (46·2±24% Met Hb h) compared with administration of dapsone alone (124·5 ± 24·4% Met Hb h). The elimination half‐life of dapsone (814 ± 351 min) was more than doubled in the presence of trimethoprim and cimetidine compared with control (355 ± 160 min, P < 0·05). However, there were no significant differences in AUC and clearance when dapsone was administered in combination with trimethoprim and cimetidine compared with dapsone alone. Co‐administration of trimethoprim with dapsone in the absence of cimetidine did not affect either methaemoglobin formation, AUCs, half‐lives, or clearance values of dapsone compared with control. There was a threefold increase in the AUC of trimethoprim (6296 ± 2249 μg min mL−1) in the presence of dapsone compared with trimethoprim alone (2122 ± 552 μg min mL−1). There was also a corresponding decrease in the clearance of trimethoprim in the presence of dapsone compared with control (19·1±6·9 vs 60·8 ± 21·0 mL min−1). However, there was no change in the elimination half‐life of trimethoprim between the two experimental groups (273 ± 120 vs 292 ± 54 min). The AUC of trimethoprim increased more than threefold in the presence of cimetidine (7100 ± 1501 μg min mL−1) compared with trimethoprim alone (2122 ± 552 μg min mL−1). There was also a corresponding reduction in the clearance of trimethoprim in the presence of cimetidine (61·2 ± 21·2 vs 17·8 ± 9·3 mL min−1) compared with control. However, there was no significant change in the elimination half‐life of trimethoprim after the administration of cimetidine (273 ± 136 vs 215 ± 109 min). Administration of either trimethoprim or cimetidine alone did not cause methaemoglobin levels to exceed control values. The administration of trimethoprim with dapsone and cimetidine resulted in a significant increase in AUC (2122 ± 552 vs 5744 ± 3289 μg min mL−1), a fall in clearance (17·8 ± 9·3 vs 60·8 ± 21 mL min−1), but no change in half‐life (252 ± 134 vs 273 ± 136 h) of trimethoprim. The co‐administration of cimetidine significantly reduced dapsone‐mediated methaemoglobin formation in the presence of trimethoprim, whilst the AUC of trimethoprim was significantly increased in the presence of both cimetidine and dapsone.

The pharmacokinetics of pyrimethamine in the rat: effect of mefloquine

The pharmacokinetics and tissue distribution of pyrimethamine have been determined in the rat. Following administration of pyrimethamine alone, drug concentrations declined biexponentially. By contrast, in the presence of mefloquine, the decline in pyrimethamine concentration more closely fitted a monoexponential pattern and the AUC0‐6h, for pyrimethamine was significantly reduced. Significantly more pyrimethamine was recovered from the livers but less from the lungs of the mefloquine‐dosed rats compared with control. This study outlines a potentially clinically relevant drug interaction.