Environmental Toxicology and Pharmacology 5 (1998) 145 153

The effect of 2,2%-substitution on the metabolism and toxicity of dapsone in vitro and in vivo
M.D. Tingle a,*, R. Mahmud 1,a, J.L. Maggs a, S. Hawley a, M.D. Coleman b, S.A. Ward a, B.K. Park a
a

Department of Pharmacology and Therapeutics, Uni6ersity of Li6erpool, P.O. Box 147, Li6erpool, L69 3BX, UK b Department of Pharmaceutical Sciences, Aston Uni6ersity, Aston Triangle, Birmingham, B4 7ET, UK Received 8 July 1997; received in revised form 13 November 1997; accepted 18 November 1997

Abstract The effect of 2,2%-substitution with uorine, methyl or triuoromethyl groups on the toxicity, metabolism and pharmacological activity of dapsone has been investigated in vitro and in vivo. There was marked inter-species variation in the bioactivation (N -hydroxylation) of the compounds, as determined by methemoglobin formation. However, the inclusion of uorine signicantly (P B 0.01) reduced methemoglobin formation compared with dapsone in all species studied. All three analogs resulted in signicantly (P B 0.001) less methemoglobinemia than dapsone when given either intraperitoneally or intravenously to the male Wistar rat. Rapid plasma clearance of the analogs through increased lipophilicity and enhanced N -glucuronidation may account for the low toxicity compared with dapsone. Although triuoromethyl substitution resulted in a loss of activity against respiratory burst in human neutrophils in an in vitro model, all three analogs retained pharmacological activity against Plasmodium berghei malaria in an in vivo mouse model. 1998 Elsevier Science B.V. All rights reserved. Keywords: Dapsone analogs; Toxicity; 2,2%-Substitution

1. Introduction Dapsone is used in the treatment of infectious diseases such as leprosy (Vadher and Lalljee, 1992) and malaria (Shanks et al., 1992), as well as for the prevention and treatment of Pneumocystis carinii and Toxo Abbre6iations: FDDS, 2,2%-uoro-4,4%-diaminodiphenyl sulfone; CH3DDS, 2,2%-methyl-4,4%-diaminodiphenyl sulfone; CF3DDS, 2,2%triuoromethyl-4,4%diaminodiphenyl sulfone; NADPH, nicotinamide adenine dinucleotide phosphate; DMSO, dimethylsulfoxide; UDPGA, uridine 5-diphosphoglucuronic acid; LCMS, liquid chromatography-mass spectrometry; HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; i.p., Intraperitoneal; i.v., Intravenous. * Corresponding author. Present address: Department of Pharmacology and Clinical Pharmacology, School of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Fax: + 64 9 3737556; e-mail: m.tingle@auckland.ac.nz 1 Present address: Drug Research Centre, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia. 1382-6689/98/$19.00 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 2 - 6 6 8 9 ( 9 7 ) 1 0 0 7 0 - 9

plasma gondii infections in HIV-positive patients (Girard et al., 1993; Jorde et al., 1993). The drug also has limited use in the treatment of inammatory disease, although it is the rst-line treatment for specic dermatological disorders such as dermatitis herpetiformis (Prussick et al., 1992). Ever since dapsone was rst used in animal studies (Raiziss et al., 1938), it has been associated with toxicity, which is due primarily to a hydroxylamine metabolite (Hjelm and DeVerdier, 1965). The N -hydroxylation of dapsone is catalysed by a range of enzymes (Hjelm and DeVerdier, 1965; Uehleke and Tabarelli, 1973; Uetrecht et al., 1988) to yield the hydroxylamine which is directly toxic to erythrocytes (Kramer et al., 1972; Glader and Conrad, 1973) and mononuclear leucocytes (Coleman et al., 1989) in vitro. Whilst the drug is effective in patients, administration of the drug is associated with dose-dependent toxicity

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towards erythrocytes (methaemoglobinaemia) in all individuals (Zuidema et al., 1986), and in rarer instances with dose-independent reactions such as sulphone syndrome (Kroman et al., 1982) and agranulocytosis (Cockburn et al., 1993). The incidence of adverse reactions to dapsone appears to be higher in HIV-positive patients compared with other patient groups, resulting in therapy being discontinued in up to 55% of patients (Martin et al., 1992). Several groups have therefore tried to develop less toxic analogues of dapsone that retain pharmacological activity (Popoff et al., 1971b; Wiese et al., 1987; Saxena et al., 1989). The intrinsic activity of dapsone as an anti-bacterial lies in the nucleophilicity of the 4-NH2C6H4-SO2 moiety (de Benedetti et al., 1987). As a dihydropteroate synthase inhibitor, dapsone activity is enhanced by electron-releasing substituents that transmit electronic effects through the aromatic ring onto the sulphone group, resulting in high negative charge density on the oxygens of the sulphone group which mimic the electrostatic charge of the carboxyl group of para -aminobenzoic acid (de Benedetti et al., 1985, 1987). Dapsone is effective in several inammatory disorders due to inhibition of myeloperoxidase (Bozeman et al., 1992) and neutrophil adherence (Booth et al., 1992). However, although many analogues of dapsone have been screened for pharmacological activity, there are only a few reports on the structure toxicity and structuremetabolism relationships of these compounds (Coleman et al., 1996, 1997). Recently, we have shown that the haemotoxicity of dapsone analogues and simple aniline derivatives is related to the electron-withdrawing nature of the 4-substituent, as represented by the Hammett constant, |p, and the lowest unoccupied molecular orbital (LUMO) (Mahmud et al., 1997). The aim of this study was to investigate the effect of ring-substitution on the metabolism and toxicity of dapsone, whilst maintaining the diaminodiphenylsulphone structure. Three substituents were investigated (Fig. 1): uorine, which although highly electronegative, is a similar size to hydrogen; the electropositive methyl group, which is larger than hydrogen; and the triuoromethyl group, which is of a similar size to the methyl group, but it is electronegative. Both the metabolism and the toxicity of the compounds have been investigated in vitro and in vivo and compared with dapsone. Furthermore, the pharmacodynamic ac-

tivity of the compounds has been assessed in terms of anti-Plasmodium activity in vivo plus their ability to inhibit the respiratory burst of human neutrophils in vitro.

2. Materials and methods

2.1. Materials
Dapsone (4,4%-diaminodiphenyl sulphone, DDS), reduced nicotinamide adenine dinucleotide phosphate (NADPH; tetrasodium salt), uridine-5%-diphosphoglucuronic acid (UDPGA), brij 58, potassium ferricyanide, lucigenin and zymosan were purchased from Sigma (UK). Sodium sulphide, 3,4-diuoronitrobenzene, analar tin, m -chloroperbenzoic acid and chromium trioxide were purchased from Aldrich (Dorset, UK). 2Chloro-5-nitrotoluene, 2-chloro-5-nitrobenzotriuoride and potassium ethyl xanthate were purchased from Lancaster Synthesis (Lancaster, UK). Acetic anhydride was purchased from May and Baker (Dagenham, UK). All other reagents were obtained from FSA Laboratory Supplies (Loughborough, UK). Microsomes were prepared from a single human liver and animal livers by the method of Gill et al., (1995). The human (male, age 56) liver was histologically normal and was obtained from a kidney transplant donor. Ethical consent for the use of human livers was obtained from the local ethics committee. Isolated rat hepatocytes were kindly provided by Dr J. Dixon, Department of Anatomy and Cell Biology, University of Liverpool.

2.2. Synthesis of 2,2 %-substituted DDS analogues

2,2%-Methyl-4,4%-dinitrodiphenylsulphide and 2,2%triuoromethyl 4,4%-dinitrodiphenylsulphide were prepared using potassium ethyl xanthate and 2-chloro-5-nitrotoluene or 2-chloro-5-nitrobenzotriuoride (Price and Stacy, 1946). Sulphides were oxidised to sulphones using chromium trioxide (Shriner et al., 1930). The nitro groups were reduced to amines by a modication of the method of Amstutz using tin and concentrated hydrochloric acid (Amstutz, 1950). 2,2%Fluoro-4 amino-4%nitrodiphenylsulphide was synthesised by coupling 3,4-diuoronitrobenzene with sodium sulphide under reux. The nitro group was reduced to an amine with tin/hydrochloric acid then the amino groups protected with acetic anhydride. The sulphide was oxidised to a sulphone by m -chloroperbenzoic acid and the amide hydrolysed in hydrochloric acid. All products were pure by TLC (petroleum ether:ethyl acetate 1:3 v/v) plus HPLC and their identities conrmed by NMR and LC mass spectrometry.

Fig. 1. Structure of dapsone and 2,2%-substituted analogues.

M.D. Tingle et al. / En6ironmental Toxicology and Pharmacology 5 (1998) 145153 Table 1 Calculated physicochemical parameters determined for dapsone and 2,2%-substituted analogues Analogue Log P a NH2 DDS FDDS CH3DDS CF3DDS
a b

147

HOMO (eV)b NHOH 0 0.998 0.503 1.146 NH2 8.88 9.06 9.05 9.86 NHOH 9.10 9.17 9.50 9.57 NO 9.24 10.05 9.46 9.73

LUMO (eV)b NH2 0.25 0.47 0.42 1.27 NHOH 0.51 0.45 0.76 0.45 NO 1.30 1.39 1.52 1.73

MR

0.886 1.389 1.884 2.032

6.81 7.89 6.99 7.98

Values determined using Medchem v3.54. Values determined using Mopac v6.

2.3. Determination of physicochemical parameters

Calculated values of molar refractivity (MR) and the logarithm of the octanol water partition coefcient (log P ) were obtained from the Medchem (v3.54) software. The energies of the lowest unoccupied and highest occupied molecular orbitals (LUMO and HOMO, respectively) were calculated using the AM1 Hamiltonian (Dewar et al., 1985) in the Mopac v6.0 software. The compounds had been modelled previously in the Cosmic molecular modelling package (Vinter et al., 1987). Full geometry optimisation was carried out with MOPAC. These calculations were carried out for the amine (LUMONH2 and HOMONH2), the hydroxylamine metabolite (LUMONHOH and HOMONHOH) and the nitroso metabolite (LUMONO and HOMONO). The calculations were carried out with one amine and either one hydroxylamine or nitroso group. Values are shown in Table 1.

of 20 mM MgCl2, and brij 58 (detergent:protein ratio = 0.15) in 50 mM TrisHCl in the presence or absence of 5 mM UDPGA (Miners et al., 1990). The nal incubation volume was 250 v l. After 3 h, the reactions were stopped by the addition of methanol (250 v l) and protein was precipitated overnight at 20C. An aliquot (50 v l) of supernatant was assayed for novel peaks by HPLC (Tingle et al., 1997). Further studies were performed using isolated hepatocytes (5 106 cells) in Williams E. medium (5 ml) at 37C with oxygenation. After 3 h, an aliquot (2 ml) was removed and an equal volume of methanol added. The supernatant was then assayed by HPLC.

2.6. Toxicity of dapsone analogues in 6i6o

Male Wistar rats (200280 g; n = 4/group) were administered the analogue (100 v mol/kg) in DMSO (1 ml/kg, i.p.) and blood samples ( : 150 v l) were obtained from the tail artery at 0, 1, 2, 3, 5, 8 and 24 h. For intravenous studies, male Wistar rats (n = 4/group) were anaesthetised with urethane (70 mg/kg) in saline (1 ml/kg i.p.) and appropriate polypropylene cannulae inserted into the trachea, carotid artery and jugular vein. Animals were administered analogues (100 v mol/ kg) in DMSO (1 ml/kg) via the jugular vein and blood samples obtained from the carotid artery at 0, 15, 30, 45, 60, 120, 180, 240 and 300 min. All blood samples were assayed immediately for methaemoglobin and plasma was prepared and stored at 20C until analysed for drug level by HPLC as follows. An aliquot of plasma (50 v l) was spiked with internal standard (3,3%-diaminodiphenylsulphone, 1 v g) then extracted with ethyl acetate (2 1 ml). The organic layers were combined, solvent removed under a stream of nitrogen and the residue re-dissolved in methanol (100 v l). An aliquot of this solution (50 v l) was then injected onto an octadecyl-bonded silica column (Spherisorb 5 v m ODS2, 25 0.46 cm) and compounds eluted with a mobile phase of 20 mM ammonium formate, pH 3.5:acetonitrile (75:25 v/v) owing at 1 ml/min. The eluant was monitored at 254 nm.

2.4. Metabolism and toxicity of dapsone analogues in 6itro

Analogues (100 v M) in dimethylsulphoxide (DMSO) (10 v l) were incubated with washed human erythrocytes, NADPH (omitted from controls) and liver microsomes (1 mg of protein) prepared from male Wistar rats. After a 1 h incubation, the methaemoglobin content of the cells was determined (Harrison and Jollow, 1986). In parallel incubations, the metabolism of the analogues in the absence of erythrocytes was determined by LC mass spectrometry following precipitation of protein with methanol (Tingle et al., 1995). Species variation in the bioactivation of the analogues was investigated by measuring methaemoglobin formation in the presence of liver microsomes prepared from human, male Wistar rat, male and female CD1 mouse or male hamster.

2.5. N -glucuronidation of analogues in 6itro

Analogues (100 v M) in DMSO (10 v l) were incubated with rat liver microsomes (2 mg) in the presence

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Table 2 Toxicity (methaemoglobin formation) and metabolism of dapsone and 2,2%-substituted analogues in vitro in the presence of rat liver preparations Analogue % Methaemoglobin (Microsomes) % N -hydroxylation (Microsomes) % N -glucuronidation

(Microsomes) DDS FDDS CH3DDS CF3DDS 44.8 9 0.5 8.2 9 4.7** 17.5 9 0.7** 7.5 9 0.4** 22.0 9 1.3 22.6 9 1.4 28.2 9 1.7 9.0 9 0.5* NDa ND 70.1 9 1.4 ND

(Hepatocytes) ND ND 42.0 9 8.2 28.5 9 13.6

Values shown are the mean 9 S.D. (n = 4). ND, no novel peak corresponding to a glucuronide detected. * PB0.01; ** PB0.001 compared with dapsone.
a

2.7. Metabolism of dapsone analogues in 6i6o

Male Wistar rats (200 280 g; n = 4/group) were anaesthetised with urethane (70 mg/kg) in saline (1 ml/kg i.p.). Appropriate polypropylene cannulae were inserted into the trachea, carotid artery and bile duct and the penis was ligated. Animals were administered analogues (100 v mol/kg) in DMSO (1 ml/kg) via the carotid artery and bile collected in 30 min fractions for 300 min. After this time, urine was collected from the bladder. Analysis of bile and urine (10 v l) was by reverse-phase HPLC linked to mass spectrometry as described previously (Tingle et al., 1995).

method of Coleman et al. (1997). Compounds (0.110 mM) were dissolved in DMSO and an aliquot (1 v l) added to samples of whole blood (100 v l) and pre-incubated for 30 min at 37C prior to addition of 5 mg/ml zymosan (300 v l) and 0.5 mM lucigenin (100 v l). The respiratory burst was measured at various time points, to a maximum of 15 min, using a Bio-Orbit 1253 Luminometer (Labtech International, Sussex, UK) at 425 nm, and results are expressed as relative light units (rlu).

2.10. Statistical analysis

Results in the text are given as mean 9 S.D. Values were compared using Students t -test for non-paired data. Where necessary, data was analysed by ANOVA with Bonferronis correction. A difference was deemed signicant when P B 0.05. The area under the curves (AUC(0 24)) were calculated by the linear trapezoidal rule using the Topt program (Schering, Germany). The IC50 value, corresponding to dose required to reduce the number of infected erythrocytes by 50% compared with control (vehicle-treated) animals, was calculated using the Grat program (v3.01, Erithacus Software, UK).

2.8. Anti -malarial acti6ity against Plasmodium berghei in 6i6o

The in vivo activity of the analogues was assessed by the 4-day test (Peters, 1970) in P. berghei -infected male CD1 mice. Mice (25 30 g, n = 6/group) were inoculated with 107 parasitised erythrocytes (N strain of P. berghei ) and 4 h later dosed with analogue (0, 50, 100, 200 or 400 v mol/kg) in DMSO (100 v l, i.p.). Animals received further doses of the drug 24, 48 and 72 h later. Thin blood lms were prepared from each mouse 24 h after the nal dose. The lms were xed with methanol and stained with 10% w/v Giemsa solution. The parasitaemia values were determined as percentage of infected erythrocytes. The activity was also determined after a single dose (Barlin and Tan, 1985). Mice were infected with 106 parasites and the infection monitored until 20 50% of erythrocytes were infected, at which point the animals were administered a single dose (0 400 v mol/kg) of analogue. The parasitaemia was monitored daily until the death of the animal.

3. Results

3.1. Toxicity of analogues in 6itro

The in vitro bioactivation of the analogues in the presence of male rat liver microsomes resulted in NADPH-dependent methaemoglobin formation and metabolite (hydroxylamine) formation, as shown in Table 2. All compounds were converted to a single metabolite that was identied by LC-mass spectrometry as M + 16, corresponding to a hydroxylamine. No absolute quantication of the detected metabolite was attempted due to a lack of standards, but results are expressed as the relative peak area calculated by integration.

2.9. Inhibition of respiratory burst in human neutrophils in 6itro

The ability of dapsone and analogues to inhibit respiratory burst in neutrophils was determined by the

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149

Fig. 2. Species variation in the bioactivation of dapsone and 2,2%-substituted analogues in vitro, as determined by methaemoglobin formation. Values shown are mean 9 S.D. (n = 4). * P B 0.05; ** P B 0.01; *** P B 0.001 compared with dapsone.

The species variation in the bioactivation of three 2,2%-substituted analogues and dapsone, as measured by methaemoglobin formation is shown in Fig. 2. All four compounds underwent signicant (P B 0.05) NADPHdependent bioactivation in the presence of microsomes prepared from the livers of all species studied. 2,2%uoro-4,4%-diaminodiphenyl sulphone (FDDS) was signicantly (P B 0.01) less toxic than dapsone in all experiments, whereas 2,2%-triuoromethyl-4,4%-diaminodiphenyl sulphone (CF3DDS), was signicantly (P B 0.001) less toxic only in the presence of rat, hamster and human liver microsomes. 2,2%-methyl-4,4%-diaminodiphenyl sulphone (CH3DDS) showed signicantly lower toxicity than dapsone in the presence of rat (P B 0.001), hamster (P B 0.001) and human (P B 0.05) liver microsomes, whereas in the presence of male and female mice liver microsomes CH3DDS was signicantly more toxic compared with DDS (P B 0.001 and P B 0.01 respectively).

the detected metabolite was attempted due to a lack of standards, but results are expressed as the relative peak area calculated by integration.

3.3. Toxicity of the analogues in 6i6o

The i.p. administration of DDS (100 v mol/kg) to male Wistar rats resulted in a time-dependent increase in methaemoglobinaemia (Fig. 3a) which reached a maximum of 29.1 9 9.25% 1 h after administration. The mean area under the curve value for methaemoglobinaemia (AUC(0 24h)) was 348 9 112% metHb/h. The mean AUC(0 24h) of the FDDS analogue was approximately one third that of DDS, at 98.2 9 36% metHb/h (a). No signicant differences in methaemoglobinaemia compared with control (0 h) was observed with CH3DDS or CF3DDS (Fig. 3a) analogues, therefore no AUC(0 24h) values could be determined for these analogues. Dapsone could be detected in the plasma of animals administered the compound, with the mean area under the curve value, AUC(0 24h) = 4675 9 1337 nmol/ml per h. The administration of FDDS also resulted in a comparable mean AUC(0 24h) = 3588 9 1167 nmol/ml per h. However, neither CH3DDS nor CF3DDS could be detected in the plasma of animals even 1 h after administration of compound, hence it was not possible to determine their respective AUC(0 24h) values. Following intravenous administration of the compounds (100 v mol/kg), signicant (P B 0.001) methaemo-

3.2. N -glucuronidation of the analogues in 6itro

Following incubation of dapsone and the 2,2%-substituted analogues with rat liver microsomes and the co-factor UDPGA, it was possible to detect the formation of a glucuronide only with CH3DDS. No glucuronide was detected in the absence of the UDPGA. Following incubation of the analogues with isolated hepatocytes, glucuronides of CH3DDS and CF3DDS were detected (Table 2). No absolute quantication of

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M.D. Tingle et al. / En6ironmental Toxicology and Pharmacology 5 (1998) 145153 Table 3 Activity of dapsone and 2,2%-substituted analogues against P. berghei in the mouse in vivo and inhibition of respiratory burst in human neutrophils in vitro expressed as percentage of control Analogue P. berghei (100 v mol/kg) 54.4 9 2.5 23.8 9 3.7* 28.8 9 2.3* 33.8 9 4.2* Respiratory burst (100 v M) 78.3 9 3.7** 87.4 9 5.3** 82.9 9 5.1** 98.2 9 3.9

globinaemia was detected at all time points after dosing with dapsone (Fig. 3b; AUC(0 24) = 66.8 9 22.4% metHb/h). After administration of FDDS, signicant (P B 0.05) methaemoglobinaemia could also be detected, although the AUC(0 24) (16.8 9 10.6% metHb/h) was signicantly (P B 0.01) lower than that of dapsone. A small but signicant (P B 0.05) increase in methaemoglobin levels were detected 15 and 30 min after administration of CF3DDS and CH3DDS (Fig. 3b). The plasma AUC(0 24h) was 5197 9 854 nmol/ml per h for dapsone and 127 9 18 nmol/ml per h for FDDS. No FDDS could be detected in the plasma of animals after 120 min. Only trace levels of CH3DDS and CF3DDS could be detected in the plasma of animals, even 15 min after administration of compound.

DDS FDDS CH3DDS CF3DDS

* PB0.001 compared with dapsone; ** PB0.05 compared with solvent control.

3.4. The metabolism of dapsone analogues in the male Wistar rat

Following administration of FDDS, several metabolites were identied in the bile. There were some traces

of free hydroxylamine, N -glucuronide of the parent compound (m/z 478 corresponding to [M + NH4] + ) and O -glucuronide of the hydroxylamine form (m/z 494 corresponding to [M + NH4] + ) as well as the unchanged parent compound (m/z 302 corresponding to [M + NH4] + ). Analysis in the urine showed the unchanged parent compound, N -glucuronide of the parent compound and the O -glucuronide of the hydroxylamine. In the bile of animals administered CH3DDS, an O -glucuronide of the hydroxylamine (m/z 486 corresponding to [M + NH4] + ), a N -glucuronide of the parent compound (m/z 470 corresponding to [M + NH4] + ) and the unchanged parent compound were identied. In the urine, only the parent compound and its glucuronide were identied. After administration of CF3DDS, major peaks in bile were characterised as the hydroxylamine O -glucuronide (m/z 594 corresponding to [M + NH4] + ) and the N glucuronide of the parent compound (m/z 578 corresponding to [M + NH4] + ), whereas only the glucuronide of the parent compound was identied in urine. Due to a lack of authentic standards, it was not possible to quantify accurately the formation of these metabolites. The metabolism of DDS in the rat has been reported previously (Tingle et al., 1997).

3.5. The anti -malarial acti6ity of dapsone analogues in 6i6o

All four compounds tested were active in vivo against P. berghei in the 4-day mouse model, with IC50 values 132.2 (DDS), 19.1 (FDDS), 9.9 (CH3DDS), and 19.1 (CF3DDS) mmol/kg. At 100 v mol/kg, all of the analogues were signicantly (P B 0.001) more active than DDS (Table 3). No drug or metabolite of any analogues could be detected in the plasma of animals 24 h after the last dose. In the single dose study in pre-infected animals, no compound had any effect on the increasing parasitaemia and hence survival of animals compared with solvent control (data not shown).

Fig. 3. Methaemoglobin levels in male Wistar rats following administration of dapsone and 2,2%-substituted analogues (100 v mol/kg) administered intraperitoneally (A) or intravenously (B). Values shown are mean 9 S.D. (n = 4). * P B 0.05; ** P B 0.01; *** P B 0.001 compared with dapsone.

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3.6. Inhibition of respiratory burst in human neutrophils in 6itro

Inhibition of respiratory burst in neutrophils was observed with dapsone, FDDS and CH3DDS, but this did not exceed 50%, so no IC50 values could be calculated. Results (Table 3) are therefore expressed as percentages of control stimulation (495.7 9 34.0 rlu) in the presence of vehicle alone at the highest concentration studied (100 v M).

4. Discussion Dapsone and its 2,2%-substituted analogues were bioactivated in vitro by liver preparations, which resulted in methaemoglobin formation in erythrocytes. The haemotoxicity of dapsone is known to be mediated by a hydroxylamine (Hjelm and DeVerdier, 1965; Uehleke and Tabarelli, 1973), and metabolites with M + 16, corresponding to a hydroxylamine, were identied for each compound by LC-mass spectrometry. Substitution at the 2,2%-positions in the molecule had a varied effect on the toxicity of dapsone in vitro. Substitution with uorine signicantly reduced the methaemoglobin formation in all species studied, whereas substitution with triuoromethyl groups decreased toxicity in the presence of rat, hamster and human liver microsomes, but not male or female mouse preparations. Substitution with methyl groups decreased toxicity with rat, hamster and human liver microsomes, but increased toxicity in the presence of male and female mouse liver microsomes. It has been shown previously (Mahmud et al., 1997) that the methaemoglobin forming ability of dapsone and 4-substituted analogues in vitro is related to the ability of the 4-substituent to be electron-withdrawing, as described by the Hammett constant |p, and the LUMO for the hydroxylamine and nitroso metabolites. Interestingly, however, methaemoglobin formation for the 2,2%-substituted analogues does not appear to follow this pattern. The decrease in toxicity observed with the electron-withdrawing uorine and triuoromethyl groups may be a consequence of the decreased charge density on the amino group which would reduce the ability of cytochrome P450 to N -hydroxylate the compounds (Koymans et al., 1993; Yin et al., 1995). In contrast, the electron-donating methyl group would increase the electron density on the nitrogen compared with dapsone so that it would be expected to be more vulnerable to N -hydroxylation. The varied effect of substitution with methyl groups suggests that other factors, for example size, which dictate the compounds accessibility to the active site of cytochrome P450 isoform(s), may be important for this series of analogues. Thus the variation in the effect of

substitution may reect the fact that subtle changes in the molecule have altered the binding of the substrate to cytochrome P450 enzymes, which are subject to species variation in expression (Gonzalez, 1990), rather than merely a change in the intrinsic ability of the compounds to undergo oxidation. Furthermore, data for methaemoglobin formation and N -hydroxylation of the analogues in the presence of rat liver microsomes suggests that the hydroxylamines have different intrinsic toxicity towards erythrocytes compared with dapsone, as has been seen previously for procainamide hydroxylamine and phenylhydroxylamine (Tingle and Park, 1993; Mahmud et al., 1997), possibly due to the changes in the electronic properties of the molecule. Initial in vivo studies in the rat revealed that FDDS underwent signicant bioactivation in vivo when administered intraperitoneally, resulting in methaemoglobinaemia, although to a signicantly lesser extent than dapsone itself. In contrast, no signicant methaemoglobinaemia was detected at any time point following i.p. administration of CH3DDS or CF3DDS. Analysis of plasma revealed that no CH3DDS or CF3DDS could be detected at any time point, suggesting that either the compounds were not dispersed from the site of injection or they were cleared very rapidly from the plasma. However, following intravenous administration of the compounds, a similar toxicity prole was obtained, suggesting that a lack of dispersion was not the reason for the decreased toxicity associated with these compounds. Analysis of the plasma revealed that CH3DDS and CF3DDS were cleared very rapidly from plasma, with B 1% dose/ml after 15 min. Dapsone is known to accumulate in fatty tissues (Chatterjee and Poddar, 1957), thus the rapid clearance of the analogues from plasma may be due to rapid tissue accumulation as a consequence of the greatly increased lipophilicity. The rapid clearance from plasma may also have been a consequence of enhanced clearance through metabolism, again promoted by increased lipophilicity. Analysis of bile and urine from animals administered the compounds intravenously revealed that all three analogues underwent N -hydroxylation in vivo. Following administration of FDDS both free and conjugated parent compound and hydroxylamine were detected, as was seen with dapsone (Tingle et al., 1997). In contrast, although glucuronides of CH3DDS and CF3DDS hydroxylamine were detected, no free hydroxylamines could be. Furthermore, no free CF3DDS could be detected in bile or urine, suggesting that these analogues and their respective hydroxylamines are good substrates for glucuronidation. Experiments with rat liver microsomes and isolated hepatocytes conrmed a previous study that has shown dapsone to be a very poor substrate for N -glucuronidation in vitro (Ebner and Burchell, 1993). However, glucuronides of CF3DDS and CH3DDS were formed

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with in the presence of isolated hepatocytes. These results suggest that 2,2%-substitution with either CH3 or CF3 groups enhances glucuronidation, possibly due to the increase in lipophilicity, a factor known to be important in glucuronidation (Kim, 1991). Indeed, the log P values for CH3DDS and CF3DDS (1.88 and 2.03) and their respective hydroxylamines (1.83 and 1.99) are very close to the optimum lipophilicity for UDPGT reactions, a log P of 2 (Kim, 1991). The rate of glucuronidation for CH3DDS may be enhanced further because of the greater electron density on the nitrogen, which would favour the SN2 reaction of glucuronyl transfer (Timbrell, 1991). Importantly, all three analogues of dapsone were active against Plasmodium both in vitro and in vivo. A previous study has investigated the effect of 2,2%-substitution on the in vitro activity of dihydropteroate synthase from P. berghei, Mycobacterium lufu and Escherichia coli and found that CH3DDS and CF3DDS were more active than dapsone itself (Wiese et al., 1987). However, in vivo, substitution with electronwithdrawing or electron-donating groups decreased activity against P. berghei in mice (Popoff et al., 1971a). There is no data in the literature for FDDS. However, it must be noted that the animal used for in vivo studies, the mouse, does not appear to N -hydroxylate or N -glucuronidate dapsone in vivo (Tingle et al., 1997). Hence the rapid clearance of the analogues from plasma seen in the rat, which resembles man more closely than the mouse for dapsone metabolism, may not occur in the mouse. Interestingly, substitution of dapsone with either uorine or methyl groups, but not triuoromethyl groups, retained the ability to inhibit respiratory burst in vitro, although none of the compounds were potent inhibitors. In conclusion, 2,2%-substitution has profound effects on the metabolism, and hence toxicity, of dapsone. Substitution with uorine resulted in a marked decrease in the AUC(0-24) for methaemoglobin, with only a 25% reduction in the AUC(0 24) for drug in plasma following i.p. administration. In contrast, substitution with either methyl or triuoromethyl groups appeared to abolish methaemoglobinaemia in vivo in rat; however, the drugs were also cleared from plasma extremely rapidly, thus it may be difcult to achieve therapeutic concentrations of such compounds in vivo. Hence 2,2%-substitution may be a viable route to retain or improve sulphone efcacy, whilst reducing the toxicity of the compounds, although profound effects on the pharmacokinetics of the compounds must be considered.

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