Phenotyping with sulfasalazine - time dependence and relation to NAT2 pharmacogenetics.

OBJECTIVE: N-acetyltransferase 2 (NAT2) genotype-phenotype relation with sulfasalazine as probe drug by means of detailed genotype analysis and kinetic data evaluation. BACKGROUND: Though phenotype analysis of sulfasalazine metabolism has been described before, genotype investigations in this regard are scarce. The influence of different single point mutations on the metabolism of the sulfasalazine metabolite sulfapyridine (SP) should give more insight into the functionality of different alleles especially with those still under discussion. METHODS: In two bioavailability studies performed under comparable conditions with 24 healthy subjects of both genders equally distributed, plasma levels of SP and acetylsulfapyridine (Ac-SP) were determined after oral intake of enteric coated formulations of sulfasalazine (500 mg and 1,000 mg, respectively). The resulting metabolic ratios were calculated. NAT2 genotype was analyzed in parallel for all subjects deducing haplotype set as well as putative functional phenotype as (homozygous or heterozygous) rapid acetylator (RA) or slow acetylator (SA) and correlated with the PK results. RESULTS AND DISCUSSION: RA genotype in the overall study population was seen with 45.5% (including 6.8% homozygous wildtype *4/*4) and SA genotype with 54.5%. Compared to RA genotype, apparent terminal elimination half-life of SP as well as of Ac-SP was prolonged in the SA genotype population, C(max) and AUC values of SP were higher whereas average C(max) value of Ac-SP was lower (with AUC only some tendency to lower values). In general, phenotype-genotype correlation was good with only few exceptions. Strongest functional effect on enzyme activity was noticed in slow acetylators carrying the 341T > C mutation, followed by 590G > A mutation whereas the influence of 857G > A was considerably less pronounced. Homozygous 803A > G mutation (lysine > arginine shift) did not reveal enzyme activity reduction.

Reboxetine and cytochrome P450--comparison with paroxetine treatment in humans.

OBJECTIVES: The noradrenaline-selective antidepressant reboxetine in vitro is a weak inhibitor of both cytochrome P450 (CYP) 2D6 and CYP3A4. Thus, in this study the pharmacokinetics of reboxetine in relation to pharmacogenetics and the effects of reboxetine compared to paroxetine treatment on the CYP2D6 and CYP3A4 phenotype were analyzed in healthy control subjects. METHODS: Healthy male volunteers were treated with either 6 mg reboxetine (n = 26) or 30 mg paroxetine (n = 25). On Days 10/11 of treatment, serum concentrations of the antidepressants were measured and pharmacokinetic parameters calculated. Volunteers were phenotyped at the end of treatment and after at least 3 weeks washout (true phenotype) using 30 mg dextromethorphan (DM) hydrobromide given orally and measuring DM and metabolites in serum 2 h after intake. CYP2D6 and CYP2C19 genotypes were determined in parallel. RESULTS AND CONCLUSION: Reboxetine serum concentrations showed no correlation with the CYP2D6 genotype and the CYP2D6 phenotype, whereas paroxetine concentrations showed some dependence on CYP2D6. In contrast to in vitro investigations, indicating a major role of CYP3A4 in reboxetine metabolism, reboxetine concentrations in serum showed no correlation with the respective DM metabolic ratios. There was also no correlation between paroxetine concentrations and the CYP3A4 phenotype data. The CYP2C19 genotype (only heterozygosity) had no influence on reboxetine and paroxetine pharmacokinetics. There were only minor changes in the DM metabolite pattern on treatment with reboxetine and no evidence of enzyme inhibition was obtained. In contrast and as expected, paroxetine strongly inhibited CYP2D6. Thus, reboxetine treatment has no effect on the CYP2D6 genotype and no clinically relevant drug interactions involving CYP2D6 are anticipated.

In vitro investigations on the differential pro-oxidant and/or antioxidant properties of cyclosporin A and tacrolimus in human and rat liver microsomes.

OBJECTIVE: The use of cyclosporin A (CSA) and tacrolimus (TAC) in organ transplantation and in the therapy of immune disorders is often hampered by adverse effects, mainly nephro-, hepato- and neurotoxicity. For the development of these side effects, among others, an increased formation of reactive oxygen species, probably generated by the cytochrome P450 (CYP) system, has been accused. Since in this respect literature data are inconsistent, in the present study possible pro- and/or antioxidant effects of CSA and TAC and the involvement of the CYP system were re-evaluated in vitro. METHODS: Effects of CSA and TAC were examined on CYP mediated oxidase functions by stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) or luminol (LM) amplified chemiluminescence (CL) in liver microsomes of either untreated rats or of rats treated with beta-naphthoflavone (BNF), phenobarbital (PB) or dexamethasone (DEX) and in human liver microsomes. RESULTS: In rat liver microsomes, CSA displayed pro-oxidant properties (though only very slightly), whereas in human liver microsomes small antioxidant effects were seen. With TAC in both species the antioxidant capacity prevailed. Treatment of rats with BNF or DEX caused an increase in the pro-oxidant effects of CSA with respect to LPO or LM-CL, whereas in liver microsomes of DEX-treated rats H2O2 production and LC-CL were diminished. CONCLUSIONS: CSA seems to have both pro-oxidant and antioxidant properties, whereas with TAC mainly an antioxidant capacity was seen. The CYP system seems to be involved in the pro-oxidant influence of CSA. Whether pro-oxidant or antioxidant effects predominate may depend on the antioxidant capacity of a tissue and on the CYP isoforms mainly present.

Para-nitrophenol glucuronidation and sulfation in rat and human liver slices.

Para-nitrophenol (PNP) is a well-known substrate for both phase I (hydroxylation at cytochrome P450) and phase II reactions (glucuronidation and sulfation). HPLC separation of PNP conjugates has already been described, but not for respective studies with liver slices, which nowadays have proven to be a suitable model for metabolic studies. Therefore we adapted an HPLC method for the simultaneous measurement of PNP glucuronidation (PNP-G) and sulfation (PNP-S) in this in vitro system. Both activities are substantially maintained over an incubation period of 24 h. PNP-G activity, however, seems to be better preserved, as indicated by stable values for PNP-G but reduced PNP-S values after 48 h liver slice preincubation. 24 h exposure of the slices to beta-naphthoflavone or phenobarbital does not change PNP-G or PNP-S activities.

Effects of 28-day mechanical and chewing stress on content of bound and diffusible ions in muscles of mastication.

Type I and type II muscle fibres have different ion concentrations. Muscles adapt to chronic stress by changing of fibre types and remodelling of the myosin heavy chains in the muscle fibres. The present investigation on ionic change during muscular contraction was carried out on 10-week-old pigs (6 treated animals, 6 controls) over a 28-day period. Six pigs received acrylic build-ups to induce mechanical advancement of the lower jaw and chronic chewing stress. Muscle tissue was taken from the masseter (M1, M2, M3), temporal (TP1, TP2), medial pterygoid (PM) and geniohyoid (GH) muscles by a standardized method. Eighty-four muscle samples were used for histological fibre differentiation with mATPase. Energy-dispersive X-ray microanalysis of muscles was carried out in an environmental scanning electron microscope. Endurance stress in the stressed muscles was seen as an increase of type I fibres (P < 0.001). This histological change and ionic alterations were measured in the anterior region of the masseter (M1 and M2) and in the posterior region of the temporal muscle (TP2). Smaller changes were found in the medial pterygoid muscle. We measured in this muscles increases in potassium, sulphur, chloride (P < 0.05) and even larger increases in phosphate (up to 1.5 mmol/g to 2.3 mmol/g, P < 0.001) and sodium (3-fold, P < 0.001). The results reveal the effects of chronic stress on muscle fibres and ion concentration in the muscle. Chronic stress resulted in an increase of type I fibres and increased ion concentration in the same muscle region. These are considered to be indicators of more efficient contraction. The changes in ion concentration are an important factor in muscle contraction.

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin.

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.

Is there a beneficial effect of the calcium channel blocker diltiazem on cyclosporine A nephrotoxicity in rats?

To investigate whether or not there is a beneficial effect of diltiazem (D) on cyclosporine A (CsA) nephrotoxicity, renal function, CsA blood levels, and effects of CsA on biotransformation in the liver and on lipid peroxidation were characterized in rats. A single administration of D (60 mg/kg b.wt.) reduced urinary volume (UV), GFR and excretion of Na+ and K+, whereas a single dose of CsA (60 mg/kg b.wt.) alone had no respective effects. P-aminohippurate excretion was almost equal in all groups. Lower doses of D (and CsA) were without effects. After repeated CsA treatment a retardation in body weight gain was seen, with little effect of a co-administration with D hereon. In all tests, thymus mass was reduced by CsA, the weight of spleen, liver, adrenal glands, and kidney were not generally affected by any of the treatments. Furthermore, after repeated administration of CsA and/or D, urinary volume, GFR and Na+ excretion were reduced by CsA, too. Electrolyte concentrations in plasma showed no evident changes by any of the treatments for Na+ and Ca2+. After long time treatment, CsA and CsA + D quite similarly led to higher K+ but lower Mg2+ concentrations in plasma. Only with 7 days highest dosage treatment PAH excretion was reduced significantly by CsA and CsA + D treatment. Surprisingly, CsA levels measured in blood and in kidney tissue, showed lower values after co-administration with D compared to CsA treatment alone. This could be caused by higher activities of monooxygenase functions revealed after pretreatment with D alone. Reduced glutathione (GSH) contents in kidney were elevated in CsA and CsA + D treated groups. In general no significant differences were to be observed concerning lipid peroxidation and stimulated H2O2 formation. Altogether evident protective effects of diltiazem on CsA nephrotoxicity in rats could not be proven.

Evaluation of a possible cocataractogenic potential of Ofloxacin.

Ofloxacin, a new antibiotic of the group of quinolones, was tested for its possible cocataractogenic potential. Two cataract models of the rat, the naphthalene cataract and the true diabetic cataract were used, cataract development in-vivo was monitored with the slit lamp microscope and documented with the Scheimpflug camera Topcon SL-45. After a six weeks observation and treatment period, where Ofloxacin was dosed with 20 mg/kg body weight daily with a stomach tube, the animals were sacrificed and their lenses used for biochemical analyses. Although the results of Scheimpflug photography showed that the combination of the 2 cataract models together with the Ofloxacin treatment lead to higher densities in the cortical layer, a marked cocataractogenic potential could not be demonstrated. Also the biochemical data made it evident, that Ofloxacin has no obvious cocataractogenic potential, neither single nor in combination with 1 or 2 cataract models.