An investigation of the role of metabolism in dapsone-induced methaemoglobinaemia using a two compartment in vitro test system.

1. We have utilized a two compartment system in which two teflon chambers are separated by a semi-permeable membrane in order to investigate the role of metabolism in dapsone-induced methaemoglobinaemia. Compartment A contained a drug metabolizing system (microsomes prepared from human liver +/- NADPH), whilst compartment B contained target cells (human red cells). 2. Incubation of dapsone (1-100 microM) with human liver microsomes (2 mg protein) and NADPH (1 mM) in compartment A (final volume 500 microliters) led to a concentration-dependent increase in the methaemoglobinaemia (15.4-18.9% at 100 microM) compared with control (2.3 +/- 0.4%) detected in the red cells within compartment B. In the absence of NADPH dapsone had no effect. 3. Of the putative dapsone metabolites investigated, only dapsone-hydroxylamine caused methaemoglobin formation in the absence of NADPH (40.6 +/- 6.3% with 100 microM). However, methaemoglobin was also detected when monoacetyl-dapsone, 4-amino-4'-nitro-diphenylsulphone and 4-aminoacetyl-4'-nitro-diphenylsulphone were incubated with human liver microsomes in the presence of NADPH. 4 Dapsone-dependent methaemoglobin formation was inhibited by addition of ketoconazole (1-1000 microM) to compartment A, with IC50 values of 285 and 806 microM for the two liver microsomal samples studied. In contrast, methaemoglobin formation was not inhibited by cimetidine or a number of drugs pharmacologically-related to dapsone. The presence of glutathione or ascorbate (500 microM) did not alter the level of methaemoglobin observed.     

The effect of preincubation with cimetidine on the N-hydroxylation of dapsone by human liver microsomes.

We have examined the ability of cimetidine to inhibit the oxidative metabolism and hence haemotoxicity of dapsone in vitro, using a two compartment system in which two Teflon chambers are separated by a semi-permeable membrane. Compartment A contained a drug metabolizing system (microsomes prepared from human or rat liver +/- NADPH), whilst compartment B contained human red cells. Preincubation (30 min) of human liver microsomes with cimetidine (0-1000 microM) and NADPH prior to the addition of dapsone (100 microM) and NADPH (1 mM) resulted in a concentration-dependent decrease in the concentrations of dapsone hydroxylamine (from 179 +/- 47 to 40 +/- 6 ng) in compartment B. This reduction of hydroxylamine metabolite was reflected in the concentration-dependent reduction in methaemoglobin measured (from 7.1 +/- 0.7 to 3.5 +/- 1.5%) in parallel experiments. Preincubation of microsomes with cimetidine in the absence of NADPH had no effect. The effect of cimetidine pretreatment on dapsone-dependent methaemoglobin was confirmed using microsomes prepared from a further three sources of human liver, as well as from rat liver.     

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

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 micrograms h mL-1; t1/2, 15.62 +/- 1.8 h) and castrated (AUC, 450.6 +/- 150.9 micrograms h mL-1; t1/2, 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 less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of dapsone-induced methaemoglobinaemia by cimetidine in the presence of trimethoprim in the rat.

Administration of dapsone in combination with trimethoprim and cimetidine to male rats resulted in a marked decrease (P less than 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 less than 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 micrograms min mL-1) in the presence of dapsone compared with trimethoprim alone (2122 +/- 552 micrograms 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 micrograms min mL-1) compared with trimethoprim alone (2122 +/- 552 micrograms min mL-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of cimetidine as a selective inhibitor of dapsone N-hydroxylation in man.

1. The N-hydroxylation of dapsone is thought to be responsible for the methaemoglobinaemia and haemolysis associated with this drug. We wished to investigate the effect of concurrent administration of cimetidine (400 mg three times per day) on the disposition of a single dose (100 mg) of dapsone in seven healthy volunteers in order to inhibit selectively N-hydroxylation. 2. The AUC of dapsone (31.0 +/- 7.2 micrograms ml-1 h) was significantly increased (P less than 0.001) in the presence of cimetidine (43.3 +/- 8.8 micrograms ml-1 h). 3. Peak methaemoglobin levels observed after dapsone administration (2.5 +/- 0.6%) were significantly (P less than 0.05) reduced in the presence of cimetidine (0.98 +/- 0.35%). 4. The percentage of the dose excreted in urine as the glucuronide of dapsone hydroxylamine was significantly (P less than 0.05) reduced in the presence of cimetidine (34.2 +/- 9.3 vs 23.1 +/- 4.2%). 5. Concurrent cimetidine therapy might reduce some of the haematological side-effects of dapsone.     

Studies on the toxicity and efficacy of some ester analogues of dapsone in vitro using rat and human tissues

The toxicity and efficacy of a series of 13 anti-tubercular sulphone esters has been evaluated using human and rat tissues. The toxicity studies involved comparison of the esters' ability to generate rat microsomally mediated NADPH-dependent methaemoglobin with that of dapsone. All the compounds formed significantly less methaemoglobin in the 1 compartment studies compared with dapsone itself. The ethyl, propyl, 3-methyl-butyl cyclopentyl esters and the carboxy parent derivative all yielded less than 5% of the methaemoglobin generated by dapsone. The 3-nitro benzoic acid ethyl and propyl esters generated 30 and 25% of dapsone's methaemoglobin formation. A similar effect was seen in the 2 compartment system, except for the butyl ester, which yielded similar haemoglobin oxidation to dapsone. The low toxicity ethyl and propyl esters, were also low in toxicity using human liver microsomes, producing less than 30% of the dapsone mediated methaemoglobin. All the compounds except the benzoic acid parent were superior to dapsone in terms of suppression of human neutrophil respiratory burst using a lucigenin-based chemiluminescence assay. The most potent derivatives were the phenyl, propyl and 3-nitro benzoic acid ethyl esters, which were between two- and threefold more potent compared with dapsone in arresting the respiratory burst. Overall, the ethyl ester showed the best combination of low toxicity in the rat and human microsomal systems and its IC(50) was approximately 40% lower than that of dapsone in neutrophil respiratory burst inhibition. These compounds indicate some promise for future development in their superior anti-inflammatory capability and lower toxicity compared with the parent sulphone, dapsone.     

Indomethacin and cognitive function in healthy elderly volunteers.

1. The aim of this randomised, double-blind four way crossover study was to assess the interaction between the new calcium antagonist, lacidipine and atenolol, in patients with mild to moderate hypertension. 2. Sitting blood pressure at 4 h post-dosing with lacidipine (4 mg) and atenolol (100 mg) alone was significantly lower compared with placebo (137/89 +/- 3/3 mmHg; 142/89 +/- 5/3 mmHg; and 154/98 +/- 5/3 mmHg respectively; P < 0.001). Co-administration of both drugs produced a significant additive effect compared with atenolol and lacidipine alone (124/80 +/- 4/2 mmHg; P < 0.002). 3. Heart rate on treatment with lacidipine alone was significantly greater at 4 h compared with placebo (86 +/- 1 beats min-1 and 74 +/- 2 beats min-1 respectively; P < 0.001). When both drugs were used in combination, there was a significant decrease in pulse rate compared with lacidipine alone (58 +/- 1 beats min-1 and 86 +/- 1 beats min-1 respectively; P < 0.001). 4. Home blood pressure recordings confirmed the statistically significant reduction in blood pressure on co-dosing (120/82 +/- 10/2 mmHg) compared with lacidipine (140/92 +/- 5/3 mmHg) and atenolol (146/90 +/- 6/3 mmHg) given alone (P < 0.05). 5. Lacidipine alone produced a significant exercise tachycardia compared with atenolol alone and the atenolol/lacidipine combination (97 +/- 8 beats min-1; 65 +/- 4 beats min-1 and 75 +/- 7 beats min-1 respectively; P < 0.001). Exercise tolerance was not adversely affected by the co-administration of both lacidipine and atenolol.     

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone ‘Compound 1’ caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1’s MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols.     

Bioactivation of dapsone to a cytotoxic metabolite: in vitro use of a novel two compartment system which contains human tissues.

1. A two compartment system, comprising two adjacent teflon chambers separated by a semi-permeable membrane, has been devised with which to investigate the generation of drug metabolites that are toxic to human cells in vitro. 2. Compartment A contained a drug-metabolising system (human liver microsomes +/- NADPH) and compartment B contained target cells (human mononuclear leucocytes). The semi-permeable membrane retained protein (m.w. greater than 10,000) but allowed equilibration (within 1 h) of drug and drug metabolites, during which time cells remained viable. 3. Incubation of dapsone (100 microM) with human microsomal protein (2 mg ml-1) and NADPH (1 mM) in compartment A caused cell death (8.7 +/- 1.8%) in compartment B, which was reduced significantly (P less than 0.05) by the addition of glutathione (500 microM). Dapsone in the absence of NADPH was not cytotoxic. 4. Chemical analysis showed the presence of dapsone hydroxylamine as the only stable metabolite in both compartment A (5.2 +/- 0.4% incubated drug) and compartment B (3.5 +/- 0.5%). 5. Irreversible binding of dapsone to cells was significantly (P less than 0.05) reduced by omission of NADPH (85 +/- 13 pmol/10(6) cells) or addition of glutathione (103 +/- 9) compared with control values (153 +/- 51).     

Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30–120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy.     

The involvement of ATP-sensitive potassium channels and adenosine in the regulation of coronary flow in the isolated perfused rat heart.

1. The roles of adenosine 5'-triphosphate (ATP)-sensitive potassium channels (KATP) and endogenous adenosine in the regulation of coronary flow have been assessed in the isolated, buffer-perfused heart of the rat. 2. In the presence of glibenclamide 10 microM there was a significant (P < 0.001) reduction in coronary flow from a baseline value of 8.78 +/- 0.76 ml min-1 g-1 to 3.89 +/- 0.59 ml min-1 g-1. This change was accompanied by a significant (P < 0.01) reduction in cardiac mechanical performance as shown by the decrease in the pressure-rate product from 21,487 +/- 2,577 mmHg min-1 to 6,950 +/- 1,104 mmHg min-1. 3. The non-selective adenosine antagonist 8-phenyltheophylline (10 microM) also caused a significant (P < 0.001) reduction in coronary flow from a basal value of 10.4 +/- 0.6 ml min-1 g-1 to 6.32 +/- 0.60 ml min-1 g-1. The subsequent addition of glibenclamide, in the presence of 8-phenyltheophylline, brought about a further significant (P < 0.001) reduction in coronary flow to 3.05 +/- 0.55 ml min-1 g-1 and this value was similar to that in the presence of glibenclamide alone. 4. In hearts perfused under constant flow conditions, exogenous adenosine caused dose-related reductions in coronary perfusion pressure described by a maximum reduction in pressure of 30.7 +/- 3.9 mmHg and an ED50 of 977 +/- 813 pmol. Addition of glibenclamide caused a significant (P < 0.01) increase in coronary perfusion pressure of 44.7 +/- 7.2 mmHg and a significant (P < 0.05) rightward shift of the dose-response curve for the depressor effects of adenosine (ED50 = 13.5 +/- 3.8 nmol), with a depression (P < 0.05) of the maximum (16.3 +/- 2.4 mmHg). 5. In conclusion, both KATP and endogenous adenosine make major contributions towards coronary vascular tone and the regulation of coronary flow in the rat isolated heart. Furthermore, in the coronary vasculature a significant proportion of the vasodilator action of adenosine is mediated through the activation of KATP.     

Characterization of tachykinin receptors mediating plasma extravasation and vasodilatation in normal and acutely inflamed knee joints of the rat.

1. Inflammatory actions of tachykinins in normal rat knee joints were compared with those of animals with acutely inflamed joints induced by intra-articular injection of 2% carrageenan. Plasma protein extravasation in rat knee joints, measured by protein micro-turbidimetry, was induced by intra-articular perfusion of selective tachykinin receptor agonists. Changes in joint blood flow, measured by laser Doppler perfusion imaging, were produced by topical applications of selective tachykinin receptor agonists to the joint capsule. 2. Carrageenan-injected rat knee joints showed significantly higher (P < 0.001) basal plasma extravasation (56 +/- 4 micrograms ml-1, n = 5) than normal rat knee joints (10 +/- 4 micrograms ml-1, n = 6). Intra-articular perfusion of the selective neurokinin1 (NK1) receptor agonist [Sar9, Met(O2)11]-substance P (0.8 nmol min-1) for 60 min elevated the basal plasma extravasation to 90 +/- 17 micrograms ml-1 (n = 6, P < 0.001) in normal joints, and to 150 +/- 14 micrograms ml-1 (n = 5, P < 0.001) in inflamed joints. Perfusion of the selective NK1 receptor antagonist N2-[(4R)-4-hydroxy-1-(1-methyl-1H- indol-3-yl)carbonyl-L-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)- L-alaninamide (FK888; 0.8 nmol min-1) for 20 min followed by co-perfusion with the NK1 receptor agonist (0.8 nmol min-1) produced complete inhibition of the NK1 receptor agonist-induced plasma extravasation in the two groups of animals (for both groups; n = 3, P < 0.001). 3. Intra-articular perfusion of the selective NK receptor agonist [Nle10]-neurokinin A4-10 (0.8 nmol min-1) and the selective NK3 receptor agonist [MePhe7]-neurokinin B (0.8 nmol min1) produced no increase in plasma extravasation in normal or in inflamed rat knee joints (n = 4 and 11, P > 0.05). 4. Topical bolus applications of the NK1 receptor agonist [Sar9, Met(O2)11]-substance P onto normal joint capsules produced dose-dependent vasodilatation expressed as a voltage increase from control level. The maximum increase in blood flow was 2.05-0.21 V from a basal voltage of 3.42 +/- 0.07 V (n = 13, P < 0.001). To a much lesser extent, administration of the NK2 receptor agonist [Nle10]-neurokinin A4-10 also produced dose-dependent vasodilatation with maximum increase of 0.46 +/- 0.08 V from a basal level of 3.38 +/- 0.1 V (n = 7, P < 0.01). Animals with acutely inflamed joints showed enhanced vasodilator responses to the NK1 and NK2 receptor agonists (for both: P vs non-inflamed joints < 0.001). Thus, the NK1 and NK2 receptor agonists produced maximum increases of 2.56 +/- 0.19 V (basal level = 5.84 +/- 0.07 V; n = 7, P < 0.001) and 1.97 +/- 0.26 V (basal level = 6.31 +/- 0.23 V; n = 11, P < 0.001), respectively. The NK3 receptor agonist [MePhe7]-neurokinin B produced no change in blood flow in normal or in inflamed rat knee joints (n = 7 and 5, P > 0.05). 5. Bolus administration of the NK1 receptor antagonist FK888 (10 pmol) alone followed 5 min later by another dose of 10 pmol FK888 (i.e. total dose of 2 x 10 pmol) applied together with the NK1 receptor selective agonist [Sar9, Met(O2)11]-substance P produced partial, but significant inhibition of the NK1 receptor agonist-induced vasodilatation in both normal (maximum response reduced by 51.9 +/- 5.4%; n = 6, P < 0.001) and inflamed rat knee joints (maximum response reduced by 49.3 +/- 6.1%; n = 5, P < 0.001). The NK2 receptor agonist [Nle10]-neurokinin A4-10-induced vasodilator responses in inflamed joints were not affected by this treatment (n = 6, P > 0.05). However, with two higher doses of FK888 (both 1 nmol), the NK1 and the NK2 receptor agonist-induced vasodilator responses were abolished in the two groups of animals (n = 6-8, P < 0.005). 6. Administration of two doses of the selective NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) -butyl]benzamide (SR48968;...     

Modulation of vasodilatation to levcromakalim by adenosine analogues in the rabbit ear: an explanation for hypoxic augmentation.

1. We have used a rabbit isolated ear, buffered-perfused preparation to investigate the effects of adenosine analogues on the vasodilatation to the potassium channel opener, levcromakalim (the active (-)-enantiomer of cromakalim). We have examined the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 antagonist, on vasodilatation to levcromakalim under hypoxic conditions and also following inhibition of nitric oxide synthesis. 2. Levcromakalim relaxed preconstricted preparations with an EC50 = 369 +/- 48 nM and maximum relaxation of tone (Rmax) = 81.0 +/- 3.2%. In the presence of 1 microM N6-cyclohexyladenosine (CHA) a selective adenosine A1 agonist, there was a significant (P < 0.01) leftward shift in the concentration-response curve with an EC50 = 194 +/- 54 nM and Rmax = 93.2 +/- 2.0%. Conversely, the presence of CHA did not influence vasodilatation to either pinacidil or sodium nitroprusside. 3. Hypoxia also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 134 +/- 22 nM), and this enhancement was completely reversed (EC50 = 380 +/- 107 nM, P < 0.01) by pretreatment of the preparations with 5 microM DPCPX, a selective A1 adenosine antagonist. However, under normoxic conditions DPCPX did not influence vasodilatation to levcromakalim. 4. Inhibition of nitric oxide synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME) caused a significant (P < 0.001) leftward shift in the concentration-response curve to levcromakalim (EC50 = 73.0 +/- 7.6 nM). Pretreatment of preparations with DPCPX partially reversed the increase in potency found in the absence of nitric oxide synthesis (EC50 = 153 +/- 18 nM, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse.

1. Mice were sensitized by 7 intraperitoneal injections of ovalbumin without adjuvant (10 micrograms in 0.5 ml of sterile saline) on alternate days and after 3 weeks exposed to either ovalbumin (2 mg ml-1 in sterile saline) or saline aerosol for 5 min on 8 consecutive days. One day before the first challenge, animals were injected intraperitoneally on a daily basis with vehicle (0.25 ml sterile saline), dexamethasone (0.5 mg kg-1) or metyrapone (30 mg kg-1). 2. In vehicle-treated ovalbumin-sensitized animals ovalbumin challenge induced a significant increase of airway responsiveness to metacholine both in vitro (27%, P < 0.05) and in vivo (40%, P < 0.05) compared to saline-challenged mice. Virtually no eosinophils could be detected after saline challenge, whereas the numbers of eosinophils were significantly increased (P < 0.01) at both 3 and 24 h after the last ovalbumin challenge (5.48 +/- 3.8 x 10(3) and 9.13 +/- 1.7 x 10(3) cells, respectively). Furthermore, a significant increase in ovalbumin-specific immunoglobulin E level (583 +/- 103 units ml-1, P < 0.05) was observed after ovalbumin challenge compared to saline challenge (201 +/- 38 units ml-1). 3. Plasma corticosterone level was significantly reduced (-92%, P < 0.001) after treatment with metyrapone. Treatment with metyrapone significantly increased eosinophil infiltration (17.4 +/- 9.93 x 10(3) and 18.7 +/- 2.57 x 10(3) cells, P < 0.05 at 3 h and 24 h, respectively) and potentiated airway hyperresponsiveness to methacholine compared to vehicle-treated ovalbumin-challenged animals. Dexamethasone inhibited both in vitro and in vivo hyperresponsiveness as well as antigen-induced infiltration of eosinophils (0, P < 0.05 and 0.7 +/- 0.33 x 10(3) cells, P < 0.05 at 3 h and 24 h, respectively). Metyrapone as well as dexamethasone did not affect the increase in ovalbumin-specific immunoglobulin E levels after ovalbumin challenge (565 +/- 70 units/ml-1; P < 0.05; 552 +/- 48 units ml-1, P < 0.05 respectively). 4. From these data it can be concluded that exogenously applied corticosteroids can inhibit eosinophil infiltration as well as airway hyperresponsiveness. Vise versa, endogenously produced corticosteroids play a down-regulating role on the induction of both eosinophil infiltration and airway hyperresponsiveness.     

Methyl conjugation in uraemia: catechol-O-methyltransferase.

1 Erythrocyte (RBC) catechol-9-methyltransferase (COMT) activity is significantly higher in erythrocytes from uraemic patients on maintenance haemodialysis, 18.7 +/- 1.4 units/ml RBC (mean +/- s.e. mean, n = 22) than in the blood of randomly selected subjects, 12.0 +/- 0.2 units/ml (mean +/- s.e. mean, n = 557, P < 0.001). 2 Uraemic plasma contains larger quantities of endogenous methyl acceptors than does normal plasma, and it reversibly inhibits RBC lysate COMT activity to a greater degree than does normal plasma. 3 There are large individual variations in the degree of inhibition of RBC COMT activity plasma from patients with renal failure. Inhibition varied from 10-43% when 40 microliters plasma from each of 19 randomly selected uraemic patients was tested, and there as a direct correlation between the inhibition of COMT by plasma from an individual uraemic patient and its content of endogenous methyl acceptors (r = 0.64, n = 19, P < 0.01). 4 Kinetic studies with pooled uraemic plasma demonstrate that inhibition of COMT by uraemic plasma is uncompetitive with respect to both the catechol substrate and the methyl donor for the reaction, S-adenosyl-L-methionine. 5 Plasma from uraemic patients does not inhibit partially purified rat liver COMT, an observation which suggests that the inhibition is not due to a direct effect on COMT but requires the presence of other constituents of the RBC lysate, perhaps other methyltransferase enzymes.     

Deficient nitric oxide responsible for reduced nerve blood flow in diabetic rats: effects of L-NAME, L-arginine, sodium nitroprusside and evening primrose oil.

1. This study examined the potential role of impaired nitric oxide production and response in the development of endoneurial ischaemia in experimental diabetes. Rats were anaesthetized (Na pentobarbitone 45 mg kg-1, diazepam 2 mg kg-1) for measurement of sciatic nerve laser Doppler flux and systemic arterial pressure. Drugs were administered into the sciatic endoneurium via a microinjector attached to a glass micropipette. 2. In two separate studies comparing diabetic rats (streptozotocin-induced; 8-10 wk duration) with controls, nerve Doppler flux in diabetic rats (Study 1, 116.6 +/- 40.4 and Study 2, 90.1 +/- 34.7 (s.d.) in arbitrary units) was about half that measured in controls (219.6 +/- 52.4 and 212.8 +/- 95.5 respectively; P < 0.005 for both). There were no significant differences between the two in systemic arterial pressure. 3. Inhibition of nitric oxide production by microinjection of 1 nmol L-NAME into the endoneurium halved flux in controls (to 126.3 +/- 41.3 in Study 1 and 102.1 +/- 38.9 in Study 2; both P < 0.001), with no significant effect in diabetic rats, indicating markedly diminished tonic nitric oxide production in the latter. D-NAME was without effect on nerve Doppler flux. 4. L-Arginine (100 nmol), injected after L-NAME, markedly increased flux in controls (by 65.8% (P < 0.03) and 97.8% (P < 0.01) in the two studies) and by proportionally similar amounts in diabetic rats [75.8% (P < 0.001) and 60.2% (P < 0.02)]. The nitro-donor, sodium nitroprusside (SNP; 10 nmol) had similar effects to L-arginine in both groups (increases of 66.0% in controls and 77.5% in diabetics; both P < 0.002). 5. A second diabetic group, treated with evening primrose oil performed exactly like control rats in respect of responses to L-NAME, L-arginine and SNP. 6. These findings implicate deficient nitric oxide in nerve ischaemia of diabetes and suggest correction thereof as a mechanism of action of evening primrose oil.     

Variability in the stereoselective disposition of ibuprofen in patients with rheumatoid arthritis.

1. Patients suffering from rheumatoid arthritis received oral doses of 600 mg racemic ibuprofen (n = 25; RAC) or 400 mg (S)-ibuprofen (n = 25; S-IBU) in a double-blind, randomized parallel-group study. 2. The pharmacokinetic parameters of (S)-ibuprofen were not statistically different between treatments (P > 0.05). Comparing (S)- and (R)-ibuprofen within the group receiving the racemate significantly higher Cmax (20.3 +/- 5.3 vs 17.7 +/- 4.4 micrograms ml-1; P < 0.02; 95% confidence interval for differences (CI): 0.5-4.6), AUC (86.2 +/- 23.5 vs 67.6 +/- 26.6 micrograms ml-1 h; P < 0.001; CI: 9.5-27.6), mean residence time (4.5 +/- 1.1 vs 4.1 +/- 1.2 h; P < 0.01; CI: 0.1-0.6) and renal clearance (0.8 +/- 0.6 vs 0.0 +/- 0.0 ml min-1; P < 0.001; CI: 0.5-1.0) values were observed for the (S)-enantiomer. 3. No difference was found (P > 0.05) between treatments in the percentage of the dose recovered in the urine as (R)- or (S)-ibuprofen plus metabolites (S-IBU: 80.2 +/- 8.47 vs RAC: 74.1 +/- 14.0%). 4. Interindividual variation in the pharmacokinetics of (S)-ibuprofen following administration of the racemate was similar to that following the administration of the single isomer suggesting that chiral inversion is not a major factor contributing to variability in the disposition of this drug.     

Contribution of the renin-angiotensin system to short-term blood pressure variability during blockade of nitric oxide synthesis in the rat.

1. The aim of this study was to investigate, by use of spectral analysis, (1) the blood pressure (BP) variability changes in the conscious rat during blockade of nitric oxide (NO) synthesis by the L-arginine analogue NG-nitro-L-arginine methyl ester (L-NAME); (2) the involvement of the renin-angiotensin system in these modifications, by use of the angiotensin II AT1-receptor antagonist losartan. 2. Blockade of NO synthesis was achieved by infusion for 1 h of a low-dose (10 micrograms kg-1 min-1, i.v., n = 10) and high-dose (100 micrograms kg-1 min-1, i.v., n = 10) of L-NAME. The same treatment was applied in two further groups (2 x n = 10) after a bolus dose of losartan (10 mg kg-1, i.v.). 3. Thirty minutes after the start of the infusion of low-dose L-NAME, systolic BP (SBP) increased (+10 +/- 3 mmHg, P < 0.01), with the effect being more pronounced 5 min after the end of L-NAME administration (+20 +/- 4 mmHg, P < 0.001). With high-dose L-NAME, SBP increased immediately (5 min: +8 +/- 2 mmHg, P < 0.05) and reached a maximum after 40 min (+53 +/- 4 mmHg, P < 0.001); a bradycardia was observed (60 min: -44 +/- 13 beats min-1, P < 0.01). 4. Low-dose L-NAME increased the low-frequency component (LF: 0.02-0.2 Hz) of SBP variability (50 min: 6.7 +/- 1.7 mmHg2 vs 3.4 +/- 0.5 mmHg2, P < 0.05), whereas the high dose of L-NAME not only increased the LF component (40 min: 11.7 +/- 2 mmHg2 vs 2.7 +/- 0.5 mmHg2, P < 0.001) but also decreased the mind frequency (MF: 0.2-0.6 Hz) component (60 min: 1.14 +/- 0.3 mmHg2 vs 1.7 +/- 0.1 mmHg2, P < 0.05) of SBP. 5. Losartan did not modify BP levels but had a tachycardic effect (+45 beats min-1). Moreover, losartan increased MF oscillations of SBP (4.26 +/- 0.49 mmHg2 vs 2.43 +/- 0.25 mmHg2, P < 0.001), prevented the BP rise provoked by the low-dose of L-NAME and delayed the BP rise provoked by the high-dose of L-NAME. Losartan also prevented the amplification of the LF oscillations of SBP induced by L-NAME; the decrease of the MF oscillations of SBP induced by L-NAME was reinforced after losartan. 6. We conclude that the renin-angiotensin system is involved in the increase in variability of SBP in the LF range which resulted from the withdrawal of the vasodilating influence of NO. We propose that NO may counterbalance LF oscillations provoked by the activity of the renin-angiotensin system.     

Bioactivation of benzocaine to a methaemoglobin-forming metabolite by rat and human microsomes in vitro

Benzocaine-mediated methaemoglobin-generation was compared with that of dapsone in vitro. Direct incubation of benzocaine with washed human erythrocytes alone at up to 15 mM did not result in significant methaemoglobin formation (0.4 ± 0.1%). With rat microsomes, dapsone-dependent methaemoglobin formation was almost two-fold that of benzocaine at 30 min (56.5 ± 0.7% vs 31.6 ± 2.4% P < 0.005)). Benzocaine-mediated methaemoglobin formation was significantly reduced in the presence of DDC (diethyldithiocarbamate) at the 10 (P < 0.005) and 20 (P < 0.025) min time points. At 30 min, cimetidine reduced benzocaine-mediated methaemoglobin from 34.4 ± 8.7% to less than 3% (P < 0.005). The methaemoglobin forming capacity of dapsone was significantly inhibited at all three time points by both DDC (P < 0.005) and cimetidine (P < 0.005). Incubation of benzocaine with microsomes from five human livers showed that each liver produced methaemoglobin-forming metabolites. No inhibitory effect was seen with DDC, although cimetidine caused a significant reduction (32.8 ± 12.4% overall) in benzocaine-mediated methaemoglobin formation in the four livers tested.     

Protection of the aged substantia nigra of the rat against oxidative damage by (-)-deprenyl.

1. We have studied the effect of (-)-deprenyl on the oxidative damage that the rat substantia nigra suffers during aging. 2. (-)-Deprenyl (2 mg kg-1, three times a week) administered for two months, beginning at 22 months of age, produced a significant increase in tyrosine hydroxylase (TH) activity (2.67 +/- 0.40 and 3.64 +/- 0.38 nmol mg-1 protein h-1 in untreated aged rats and treated aged rats respectively, P < 0.05) and in TH amount (0.072 +/- 0.012 and 0.128 +/- 0.38 absorbance 405 nm in untreated aged and treated aged rats respectively, P < 0.05). 3. The proteins of aged rat substantia nigra showed a significant decrease of carbonyl groups in treated animals compared with saline-injected control rats (136.2 +/- 21.8 and 71.5 +/- 13.2 c.p.m. microgram-1 protein in untreated aged and treated aged rats respectively, P < 0.05). 4. The carbonyl groups measured in TH enzyme showed a statistically significant decrease (42.3%) after (-)-deprenyl treatment (471.4 +/- 73.0 and 271.9 +/- 50.00 c.p.m. in untreated aged and treated aged rats respectively, P < 0.001). 5. All these results suggest that oxidative damage produced during aging is prevented by (-)-deprenyl treatment and could explain the effect of this drug in Parkinson's disease (PD) and other degenerative diseases such as Alzheimer's disease.