Assessment of bronchial effects following topical administration of butylamino-phenoxy-propanol-acetate, an oculoselective beta-adrenoceptor blocker in asthmatic subjects.

1. Butylamino-phenoxy-propanol-acetate (BPPA) is a new topical oculoselective beta-adrenoceptor blocker for the reduction of intraocular pressure (IOP) in man. Its potency on the airways of normal subjects was identical with that of placebo. A study was carried out to determine the potential of BPPA to cause bronchoconstriction in mild asthmatics (FEV1 greater than or equal to 60% predicted) with normal IOP. 2. Twelve nonsmoking outpatients who bronchoconstricted to 0.25 or 0.50% of timolol eye drops (fall in FEV1 23.33 +/- 1.20% (mean +/- s.e. mean), range 16-30) were investigated in this double-masked, randomized, 3-period, crossover study. On three different occasions six incremental concentrations of BPPA (range: 0.1-2%; maximum cumulative concentration 4%), timolol (0.1-1%; 2%), and placebo were administered bilaterally until bronchoconstriction (decrease in FEV1 greater than or equal to 20% and in specific airway conductance (sGaw) greater than or equal to 35% simultaneously) or the maximum cumulative concentration was reached. 3. Airway response was measured as change in FEV1 and sGaw and dose-response curves to timolol, BPPA and placebo were performed. IOP was measured 3 h after the highest concentration of each study day. 4. Timolol caused dose-dependent falls in FEV1 and sGaw as well as clinical symptoms of respiratory distress in all subjects. The median cumulative concentrations of timolol required to decrease FEV1 by 20% and sGaw by 35% were 0.98% and 1.53%. Neither placebo (P greater than 0.05) nor BPPA (P greater than 0.05) caused a significant change in sGaw. A fall in FEV1 by 20% not accompanied by a simultaneous fall in sGaw by 35% was found in four subjects following BPPA and in five subjects following placebo.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of systemic effects of different ophthalmic beta-blockers in healthy volunteers.

Systemic beta-blockade after single doses of ophthalmic beta-blockers (one drop in each eye) was investigated in healthy volunteers in two randomized, double-blind, crossover, placebo-controlled studies. beta-Blockade was evaluated by displacement of the bronchodilator (specific airway conductance), positive chronotropic (heart rate), and tremorogenic (finger tremor amplitude) dose-response curve for inhaled isoproterenol. In study 1, 0.5% betaxolol, 0.6% metipranolol, and 0.5% timolol were tested in 16 subjects. Compared with placebo, all beta-blockers resulted in a significant systemic beta-blockade (p greater than 0.05); the increasing order of potency was betaxolol, metipranolol, and timolol. In study 2, 2% butylamino-phenoxy-propanol-acetate (BPPA; a noncardioselective but topically oculoselective drug) and 1% timolol were investigated in 12 subjects. Placebo and BPPA showed no differences (p greater than 0.05), whereas timolol resulted in a significant beta-blockade (p less than 0.05). Topical oculoselectivity is an important aspect of drug safety of beta-blocking eyedrops. Measure of tremor is appropriate to evaluate beta 2-blockade.

Norfloxacin does not alter warfarin's disposition or anticoagulant effect.

Drug interactions related to inhibition of hepatic drug metabolism have been identified for some fluoroquinolone antibiotics. This study was designed to investigate whether the fluoroquinolone norfloxacin at the usual clinical dosage interacts with the anticoagulant agent warfarin. Ten healthy male subjects were administered a single oral dose of 30 mg warfarin sodium alone or during multiple-dose treatment with norfloxacin, 400 mg bid, in a randomized, crossover fashion. Plasma warfarin concentrations and prothrombin times were measured for 6 days after each of the two warfarin doses. The pharmacokinetic parameters of warfarin were comparable in the absence and presence of norfloxacin, including no significant differences in warfarin's elimination half-life, apparent total clearance, apparent volume of distribution, or peak plasma concentration. Norfloxacin also had no significant effect on the anticoagulant effect of warfarin, as assessed by the area under the prothrombin time versus time curve and the maximum response for prothrombin time. The lack of pharmacokinetic or pharmacodynamic interaction observed in this study suggests that a clinically important interaction of norfloxacin and warfarin is unlikely to occur in patients requiring both drugs.

Human liver debrisoquine 4-hydroxylase: test for specificity toward various monooxygenase substrates and model of the active site.

Polyclonal antibodies raised toward a debrisoquine 4-hydroxylating cytochrome P-450 species purified from rat liver (P-450UT-A) were used to determine which monooxygenase reactions are linked to debrisoquine hydroxylation in human liver. Anti P-450UT-A did not inhibit the oxidation of dimethylnitrosamine, morphine, diazepam, vinylidene chloride, trichloroethylene, benzo(a)pyrene and its 7.8-dihydrodiol, but was inhibitory for the hydroxylation of debrisoquine, (+/-)-bufuralol, lasiocarpine and monocrotaline. A model interpreting the substrate specificity of the human liver enzyme is presented.

Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation.

Nine forms of cytochrome P-450 have been purified to electrophoretic homogeneity from human-liver microsomes. These include the enzymes involved in debrisoquine 4-hydroxylation, phenacetin O-deethylation and mephenytoin 4-hydroxylation, three reactions which are characterized by genetic polymorphism in humans. Evidence for the involvement of the above enzymes comes from reconstituted immunochemical inhibition studies with human-liver microsomes. These and other lines of evidence are consonant with the view that different forms of cytochrome P-450 are involved in the three reactions. The debrisoquine 4-hydroxylase has been studied most extensively in terms of its substrate specificity. In addition, an analogous rat enzyme shows some homology and serves as a useful model. The use of antibodies raised to the rat-liver enzyme in immuno-inhibition studies with human-liver microsomes provides a means of determining the extent to which this enzyme participates in other reactions. Translation of rat-liver mRNA in vitro yields the intact debrisoquine 4-hydroxylase; studies with human mRNA suggest a lower frequency than in rats. The basis for impaired catalytic activity in phenotypically poor human metabolizers appears to be an altered enzyme in all three cases, as opposed to a decreased level of a single enzyme. Using antibody screening of fusion proteins expressed in a cDNA library, it has been possible to isolate cDNA probes for all three of these cytochromes P-450 for use in screening individuals and ultimately determining the basis of these polymorphisms.

Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism.

Two forms of cytochrome P-450 were purified to apparent homogeneity from several different preparations of human liver microsomes. One form, designated P-450DB, had relatively high catalytic activity towards the drugs debrisoquine, sparteine, bufuralol (both the (+)- and (-)-isomers), encainide, and propranolol and appears to be the enzyme involved in the polymorphic distribution of oxidative activities towards these substrates in humans. The other form, designated P-450PA, had relatively high phenacetin O-deethylase activity and appears to be involved in the variation of this activity among humans. Polyclonal antibodies raised to the two enzymes were specific for the antigens as judged by immunoelectrophoresis and immuno-inhibition studies. The two enzymes and their activities were distinguished by chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, immuno-inhibition studies, and steady-state kinetic assays. Immunochemical studies suggest that each form represents only a small fraction of the total cytochrome P-450 in human liver microsomes. These biochemical studies provide a basis for better understanding the mechanisms which underlie genetic polymorphisms involving P-450 cytochromes in humans.

Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling.

A significant population of humans (5 to 10%) are phenotypic poor metabolizers of debrisoquine. We have isolated the cytochrome P-450 isozyme from rat liver responsible for this activity and have shown that antibodies raised against the protein are able to inhibit this catalytic activity in human liver microsomes (Distlerath, L. M., and Guengerich, F. P., Proc. Natl. Acad. Sci. USA, 81: 7348-7352, 1984). These antibodies were utilized to determine which metabolic transformations are linked to debrisoquine 4-hydroxylation in human liver microsomes using techniques of immunochemical inhibition. The antibodies almost completely inhibited debrisoquine 4-hydroxylation and bufuralol 1'-hydroxylation in microsomes prepared from several different human livers. The oxidation of the pyrrolizidine alkaloids lasiocarpine and monocrotaline were inhibited by roughly one-third. The antibodies did not inhibit N,N-dimethylnitrosamine N-demethylation, oxidation of vinylidene chloride to 2,2-chloroacetaldehyde, oxidation of trichloroethylene to chloral, N-oxidation of azoprocarbazine, morphine N-demethylation, diazepam N-demethylation, oxidation of benzo(a)pyrene to alkali-soluble metabolites, oxidation of benzo(a)pyrene 7,8-dihydrodiol to products covalently bound to DNA, the N- and ring-oxidation of 1- and 2-naphthylamine and 2-aminofluorene, or the conversion of aflatoxin B1 to DNA adducts or aflatoxin Q1. Studies with space-filling models of the drugs the metabolism of which is associated with debrisoquine 4-hydroxylase in the literature indicated that all can be fitted to a general structure in which a basic nitrogen is about 5 A away from the site of carbon hydroxylation and a hydrophobic domain is near the site of hydroxylation. These results may be useful in predicting which chemicals may or may not be metabolized in an atypical manner by a segment of the human population.

Metabolic activation of 3-(2-chloroethoxy)-1,2-dichloropropene: a mutagen structurally related to diallate, triallate, and sulfallate.

3-(2-Chloroethoxy)-1,2-dichloropropene (CP), a Salmonella promutagen that was recently isolated from a sample of residue organics previously concentrated from drinking water, is structurally related to three other chlorinated promutagens, the S-chloroallyl thiocarbamate herbicides diallate, triallate, and sulfallate. These four chloroallyl ether compounds were found to be similar with respect to strain specificity, potency, and requirement for specific metabolic activation. The 9,000g supernatant (S9) fractions from polychlorinated biphenyl Aroclor 1254- or phenobarbital-induced rats metabolized the four chloroallyl ethers to mutagenic products, whereas S9 from 3-methylcholanthrene-induced or uninduced rats did not. The metabolic activation of CP, diallate, and triallate to mutagens was catalyzed by the 100,000g microsomal pellet of S9 alone, but the activation of sulfallate to mutagenic metabolites required both microsomal and cytosolic fractions of S9. Direct-acting (minus S9) mutagenic metabolites of diallate and triallate could be extracted into methylene chloride from S9 incubation mixtures. Incubations containing S9 and either sulfallate or CP did not yield methylene chloride-extractable metabolites with direct-acting mutagenic activity. On the basis of these results and those from previous studies on the metabolism of diallate, triallate, and sulfallate, a tentative model for the metabolic activation of CP is proposed in which this chloroallyl ether undergoes alpha-carbon hydroxylation to form multiple mutagenic products.

Characterization of a human liver cytochrome P-450 involved in the oxidation of debrisoquine and other drugs by using antibodies raised to the analogous rat enzyme.

Debrisoquine 4-hydroxylase activity is a prototype for genetic polymorphism in oxidative drug metabolism in humans; approximately 10% of Caucasian populations exhibit the poor metabolizer phenotype, and the clearance of at least 14 other drugs has been shown to be deficient in patients exhibiting this phenotype. Antibodies prepared to a cytochrome P-450 shown to be responsible for debrisoquine 4-hydroxylation in rats were found to inhibit the oxidation of debrisoquine and sparteine, encainide, and propranolol, three other drugs suggested to be associated with this phenotype, in human liver microsomes. The antibodies did not inhibit the oxidation of seven other cytochrome P-450 substrates. The antibodies recognized a single polypeptide of Mr51,000 after combined sodium dodecyl sulfate/polyacrylamide electrophoresis and immunochemical staining of human liver microsomes. The intensity of this band was significantly correlated with debrisoquine 4-hydroxylase activity when liver microsomes from 44 organ donors were examined. Immunoprecipitation of in vitro translation products of total liver RNA revealed major electrophoretic bands corresponding to the cytochrome P-450 in rats and humans. The level of translatable mRNA coding for the debrisoquine-hydroxylating cytochrome P-450 was an order of magnitude less in human liver than in rat liver. The availability of these antibodies provides a biochemical basis for further basic and clinical studies on the role of a particular cytochrome P-450 polymorphism in humans.

Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism.

Genetic polymorphism in oxidative drug metabolism is perhaps best exemplified in the case of debrisoquine 4-hydroxylase activity, where the incidence of deficient metabolism ranges from 1% to 30% in various populations and this defect is also linked to an impaired ability to metabolize a number of other drugs effectively. Sprague-Dawley (SD) rats possess this activity, but females of the DA strain do not, although total cytochrome P-450 (P-450) levels are similar. We have purified, by using debrisoquine 4-hydroxylase activity as an assay, a minor P-450 to electrophoretic homogeneity from male SD rats and designate this as P-450UT-H. P-450UT-H differs from eight other purified rat liver P-450s as judged by peptide mapping and immunochemical analysis and thus appears to be isozymic with these other P-450s. P-450UT-H exhibited considerably more debrisoquine 4-hydroxylase activity than any of the other purified P-450s and, on a total P-450 basis, more than total microsomal P-450. Antibodies raised against P-450UT-H specifically recognized P-450UT-H and inhibited more than 90% of the debrisoquine hydroxylase activity present in SD rat liver microsomes. The level of P-450UT-H in SD rat liver microsomes accounted for less than 10% of the total P-450, as judged by immunochemical quantitation. These assays also indicated that the level of P-450UT-H in female DA rat liver microsomes is only about 5% of that in male or female SD rat liver microsomes, consonant with the view that deficiency of this form of P-450 is responsible for the defective debrisoquine 4-hydroxylase activity in the former animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens.

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.

Effects of metyrapone on microsomal-dependent Salmonella mutagenesis. Studies with chloroallyl ethers and model compounds.

Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone, MTP) is used as an inhibitor of cytochrome P-450 enzymes, particularly those induced by phenobarbital (PB). We examined the effects of MTP on the microsomal dependent mutagenesis of a newly isolated promutagen, 3-(2-chloroethoxy)-1,2-dichloropropene (CP), three S-chloroallyl thiocarbamate herbicides, and four model promutagens aflatoxin B1 (AFB), 2-acetylaminofluorene (2AAF), 2-aminoanthracene (2AA) and benzo[a]pyrene (BP). Salmonella tester strains TA98, TA100 and TA1535 and liver microsomal preparations (S9) from rats induced with PB or Aroclor 1254 (PCB) were employed. For statistical analysis, mutagenesis data were transformed and subjected to two-way analysis of variance. Metyrapone alone was not mutagenic in the absence or presence of S9. In a dose-dependent manner, MTP inhibited mutagenesis of AFB for strains TA98 and TA100 and enhanced mutagenesis of 2AAF, 2AA and BP for these strains. 3-(2-Chloroethoxy)-1, 2-dichloropropene and the herbicides diallate, triallate and sulfallate are all chloroallyl ethers. They are similar in their mutagenesis for Salmonella with respect to strain specificity, mutagenic potency, and requirement for activation by specifically-induced microsomes. Metyrapone inhibited the mutagenesis of CP, triallate and sulfallate for strain TA100 in the presence of either PB- or PCB-induced S9, and had no apparent effect on diallate mutagenesis; the same results were obtained for TA1535 with PCB-induced S9. On this basis, the mutagenic activation of diallate appears to be different from that of the other chloroallyl ethers tested. Our results indicate that MTP can inhibit as well as enhance microsomal dependent mutagenesis for Salmonella. We conclude that MTP may be a useful tool in characterizing pathways for promutagen activation.