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1. Metabolic degradation of the tetrahydro-oxazine ring of 2-(2-ethoxyphenoxymethyl)-2,3,5,6-tetrahydro-1,4-oxazine (I.C.I. 58,834) gives rise to one- or two-carbon fragments which are utilized by endogenous metabolic pathways. 2. Evidence of this in dogs is shown by the 14C-labelled residues in tissues, 14C-labelled material in blood which has a half-life of three weeks, and elimination of [14C]urea in urine. 3. The same phenomenon occurs in rat, mouse and man, but to a smaller extent than in the dog. 4. Intravenous administration of [14C]ethanolamine to a dog gave rise to residual 14C blood levels with a half-life comparable to that produced by metabolic incorporation of 14C from 14C-I.C.I. 58,834.

The kinetics of xenobiotic biotransformation was measured in single, isolated, perifused liver cells with microspectrofluorometry using highly sensitive photodetection systems. A program of further applications to biochemical toxicological problems is presented.

Bacteria, actinomycetes, yeasts, and fungi were screened for their ability to modify the structure of ansamitocins, a group of antitumor ansamycin antibiotics. Many strains, mostly actinomycetes, were found to convert ansamitocin P-3 to one or more products. These products, compounds A, B, C, and D, were prepared using Bacillus megaterium IFO 12108, Streptomyces coelicolor IFO 3807, Streptomyces castaneus IFO 13670 and Streptomyces minutiscleroticus IFO 13361, and were identified as 20-O-demethylansamitocin P-3, maytansinol, 15-hydroxyansamitocin P-3 and N-demethylansamitocin P-3, respectively. Other maytansinoids also underwent these microbial conversions.

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)

A rapid method for isolation of cells from the small intestine of the rat resulted in a preparation where 95--100% of the cells excluded NADH or trypan blue. Isolated intestinal cells catalyzed the cytochrome P-450 dependent metabolism of benzo(a)pyrene, harmine, ethoxyresorufin and ethoxycoumarin. Isolation of intestinal cells 24 hours after a single oral dose of 3-methylcholanthrene resulted in 25--45-fold increases in benzo(a)pyrene, ethoxycoumarin and ethoxyresorufin metabolism, whereas the rate of demethylation of harmine was doubled. Harmine metabolism led to the formation of harmol which was subsequently conjugated with glucuronic acid. Very little sulphate conjugate was detected. Intestinal cells catalyzed glucuronidation of 1- and 2-naphthol at a linear rate for up to one hour. Glucuronidation of 1- and 2-naphthol was saturated at 50 muM, whereas a concentration of 800 muM was necessary for saturation of harmol glucuronidation. Intestinal cells metabolized paracetamol to the glucuronide, sulphate, glutathione and cysteine conjugates. The latter two are evidence of cytochrome-P-450-dependent metabolic activation of paracetamol by intestinal cells.

Liver microsomes from 3-methylcholanthrene-pretreated Long-Evans rats catalyzed the 2- and the 4-hydroxylation of biphenyl and the O-deethylation of ethoxyresorufin, sustained by either NADPH or cumene hydroperoxide. In contrast, the liver microsomes from corn oil- or phenobarbital-pretreated rats catalyzed the NADPH- or cumene hydroperoxide-sustained 4-hydroxylation of biphenyl, but the rates of 2-hydroxylation or ethoxyresorufin deethylation were negligible. A monooxygenase system reconstituted with partially purified NADPH-cytochrome c reductase and cytochrome P-448 catalyzed NADPH-supported biphenyl 2- and 4-hydroxylation and ethoxyresorufin deethylation. A monooxygenase system reconstituted with the reductase and cytochrome P-450 catalyzed NADPH-supported biphenyl 4-hydroxylation but exhibited negligible 2-hydroxylation or ethoxyresorufin deethylation activites. Solubilized cytochrome P-448 catalyzed biphenyl 2- and 4-hydroxylation and ethoxyresorufin deethylation sustained by cumene hydroperoxide in the absence of both NADPH and NADPH-cytochrome c reductase, whereas solubilized cytochrome P-450, under the same conditions, catalyzed only biphenyl 4-hydroxylation. It is concluded that the patterns of biphenyl hydroxylation and ethoxyresorufin deethylation observed with live- microsomes from untreated or inducer-treated rats are due largely to the inherent enzymic specificities of their cytochromes P-450 and P-448.

A murine model of bacteremic Haemophilus influenzae type b pneumonia was used to evaluate the therapeutic efficacies of the quinolone antimicrobial agents enoxacin and ofloxacin compared with those of ampicillin and chloramphenicol. Ampicillin-susceptible (AS) and ampicillin-resistant (AR) challenge strains were employed. Treatment with enoxacin or ofloxacin produced intrapulmonary killing of H. influenzae that was superior to that achieved with ampicillin (P less than 0.01 to P less than 0.001 for both AS and AR strains). Ofloxacin and enoxacin also provided killing greater than that with chloramphenicol for the AS strain (P less than 0.01 to P less than 0.001). For the AR strain, ofloxacin provided killing greater than that obtained with chloramphenicol (P less than 0.001). Survival from AS strain pneumonia was 60% in enoxacin-treated and 78% in ofloxacin-treated animals compared with 41% for chloramphenicol-treated and 23% for ampicillin-treated groups. We conclude that enoxacin and ofloxacin may be effective antimicrobial agents in treating either AS or AR strains causing H. influenzae pneumonia.

Twelve healthy male volunteers received 14 oral doses of ofloxacin (300 mg each), and the concentrations of the unchanged drug were measured at various times in serum and urine over a period of 15 days. Serum and urine ofloxacin concentrations were determined in specific assays using high-pressure liquid chromatography (HPLC); urine levels were also determined by means of a microbiological assay. A washout period of 72 hours followed the first and 14th doses, allowing comparison of serum pharmacokinetics at the beginning and end of the multiple-dose regimen. Doses 2 to 14 were administered at 12-hour intervals. Maximum serum concentration (Cmax), concentration at 12 hours after dosing (C12), and area under the serum concentration-time curve (0 to 72 hours) were all 1.3 to 1.7 times greater after the 14th dose than after the initial dose. A 1.6-fold increase in C12 was already evident after the third dose; C12 remained more or less constant thereafter. Thus it is concluded that ofloxacin rapidly attained steady-state serum levels under a multiple-dose regimen, at levels only slightly above those following a single dose. High serum Cmax levels (4.6 and 5.9 micrograms/ml after the first and 14th doses, respectively) and long serum half-lives (6.0 and 7.3 hours after the first and last doses, respectively) indicated long-lasting, clinically relevant serum ofloxacin concentrations after oral administration. Serum ofloxacin levels 24 hours after the last dose were in the range of 0.3 to 0.7 microgram/ml, above the minimal inhibitory concentration (MIC90) for most bacterial strains. Cumulative urinary recovery remained high throughout the study. After 14 doses of ofloxacin (total, 4.2 gm), 88% of the unchanged drug was recovered in the urine; urinary concentrations remained above 1 microgram/ml, far above the MIC90 for most bacterial strains, for at least 108 hours after the final dose. High renal clearance values relative to total clearance (98%) confirmed the importance of the renal elimination route. Determinations of ofloxacin in urine using a microbiological assay were in close agreement with the HPLC determinations for all samples obtained throughout the study. Thus no detectable appearance of active metabolites occurred under the multiple-dose regimen. Ofloxacin was well tolerated; mild, probably drug-induced side effects were reported by three subjects, but they did not require any countermeasures.

Spectral properties of the cytochrome P-450 substrates, methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (MeOPx, EtOPx, PeOPx, and BzOPx, respectively) were investigated from 350 to 600 nm in ethanol and aqueous buffer. In ethanol, each alkoxyphenoxazone displayed a lambda max at 460 nm and a shoulder around 390 nm. Extinction coefficients (EmM) in ethanol were calculated as MeOPx, 20.5; EtOPx, 20.4; PeOPx, 24.7; and BzOPx, 22.4. In aqueous buffer, only MeOPx obeyed the Lambert-Beer law (lambda max = 480 nm, EmM = 22.1). Three substrates, EtOPx, PeOPx, and BzOPx, displayed anomalous behavior in aqueous solution, wherein the lambda max shifted to lower wavelengths (480-430 nm) and EmM (apparent) decreased as the alkoxyphenoxazone concentration increased. This behavior was dependent on the side chain, and the concentrations at which the spectral changes took place were estimated as: BzOPx, 2 microM; PeOPx, 5 microM; EtOPx, 17 microM; and MeOPx, greater than 20 microM. The blue shift and decreased EmM (apparent) observed for PeOPx at high concentration in aqueous buffer was reversed at high temperature. Unlike EtOPx, PeOPx, and BzOPx, and like MeOPx, hydroxyphenoxazone (resorufin) and unsubstituted phenoxazone obeyed the Lambert-Beer law in aqueous buffer and ethanol. The data suggest that the pentoxy and benzyloxy substituents facilitated a self-association process among the phenoxazones in aqueous solution. The data further show that aqueous solutions should be avoided when spectral data are used to determine alkoxyphenoxazone concentrations.

Ofloxacin was administered to 17 patients suffering from a pulmonary disorder necessitating surgery. The daily dosage employed was 200 mg b.i.d. during the 48 hours preceding the operation and 200 mg one hour before the operation. During surgery, blood samples and specimens of healthy and diseased lung tissue were taken simultaneously. The ofloxacin levels were determined by HPLC. The mean values of the tissue concentration/plasma concentration ratio were as follows: 3.5 +/- 0.4 for the healthy tissue and 3.9 +/- 0.4 for the diseased tissue. These values reflected good penetration of ofloxacin into both the healthy and atelectatic pulmonary parenchyma.

The effects of the topical application of glucocorticoid steroid creams used in clinical practice, on the activity of drug metabolising enzymes in the skin of adult hairless mice has been investigated. Treatment with hydrocortisone and fluandrenolone had no effect on the activity of ethoxycoumarin O'dealkylase (EOD) in the skin whereas all other fluorinated synthetic glucocorticoids tested, significantly induced cutaneous EOD activity. Fluincinolone acetonide, Fluincinonide , and Betamethasone Valertate increased enzyme activity 2-3-fold, and clobetasol propionate induced enzyme activity 6-fold. The ability of each steroid preparation to induce enzyme activity was related to its clinical potency, and induction of enzyme activity by clobetasol propionate was maximal at 0.05%, the concentration used in clinical practice. The effects of clobetasol propionate on cutaneous ethoxycoumarin and ethoxyresorufin dealkylase activities were different from those produced by 3 methycholanthrene , indicating that glucocorticoids may represent a class of inducing agents with different properties from the polycyclic hydrocarbons.

The authors evaluated the tissue levels of ofloxacin in 63 patients with urogenital diseases, in comparison to the minimum inhibitory concentrations 80% (MIC80) of some strains. The drug was administered in a single dose (300 mg) about 3 hours before surgery. After washing carefully, the tissue-levels were measured by using the HPLC method. In the normal renal parenchyma of 8 patients, the mean value was 22.10 micrograms/g of tissue; 19.23 micrograms/g in 13 patients with deranged excretory pathways; 10.62 micrograms/g in 25 patients with benign prostatic hypertrophy; 10.90 micrograms/g in 14 deferent duct patients. These results indicate that the drug is promptly distributed in various tissues of the urinary-genital apparatus at high concentrations.

Phenoxazinone synthase is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The mechanism of this complex 6-electron oxidation was determined by using a variety of substituted 2-aminophenols, designed to block the reaction at intermediate stages. Thus, with 3,5-di-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the o-quinone imine 17; with 5-tert-butyl-2-aminophenol (19) as substrate, the reaction was blocked at the p-quinone imine 20; and with 5-methyl-2-aminophenol (21) as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone 22. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate 4 that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct 5 to the p-quinone imine 6 was followed by a second conjugate addition and a final 2-electron oxidation to give the product, 2-aminophenoxazinone. The role of the enzyme in the catalysis of each of these steps was examined. It was found that the second conjugate addition generated a racemic center at C4a, suggesting that this reaction did not occur at the active site. A deuterium isotope effect on the cleavage of the C4-H bond of 2-aminophenol suggested that partial dissociation of an intermediate from the enzyme occurred after the first conjugate addition. It is proposed that 2-aminophenoxazinone synthesis proceeds via a sequence of three consecutive 2-electron aminophenol oxidations and that the aminophenol moiety is regenerated during the reaction sequence by facile tautomerization reactions. Thus, what initially appears to be an impressively complex mechanism may, in fact, be ingeniously simple.

Ten patients (mean age 63 years) undergoing alloarthroplastic hip joint replacement received 400 mg ofloxacin orally before anaesthesia. Venous blood samples were obtained before administration of the drug and then at intervals of up to 24 h. Bone samples were taken from femoral head and femoral shaft. Antibiotic concentrations were assessed by means of HPLC. The maximum serum concentration was 3.45 mg/l on average, after 90 min, and the maximum average bone concentration was 1.22 mg/l after 270 min. The half-life of ofloxacin in bone was shorter than that in serum. The concentrations measured in bone were superior to the minimal inhibitory concentrations reported for most pathogens of bone and joint infections. Therefore ofloxacin appears to be a valuable contribution to the therapeutic options of oral treatment for bone and joint infections.

Pharmacokinetics and gastric secretion suppressing properties of a new drug, trithiozine (TR), were studied in man. Its availability, if taken orally, is prompt and very good; its blood plasma level lasts for over 5 hr. In basal conditions volume of gastric juice is suppressed by TR as it is by propantheline bromide (PPB). In contradistinction to PPB, TR reduces basal acid output. After histamine stimulation TR is not as effective as PPB in reducing both volume and acid output. TR did not appear to affect the plasma gastrin level. Lack of typical anticholinergic side-effects such as dryness in mouth, vision blurring, etc., on the one hand, and antisecretory activity limited to basal conditions on the other, seem to offer a new modification of the old approach to therapy of gastroduodenal ulcer. Clinical trials seem justified.

The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed 'PB1c' and 'PB2c'. These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from the major phenobarbital-inducible proteins. PB1a and PB1b differed in Mr (52,700 and 52,900), absorption spectra and papain-proteolysis fragments. However, they had identical N-terminal sequences. PB2a, PB2b and PB2d had apparent Mr values of 52,900, 52,900 and 50,800. PB2a and PB2b had different N-terminal sequences and, after digestion with papain, gave different papain-proteolysis fragments. The N-terminal sequence of PB2b was similar to, but not identical with, that of pregnenolone-16 alpha-carbonitrile-inducible P-450 species, and PB2b was the protein most closely related to PB2c. The extent of immunocross-reactivity among the forms was stronger within, than between, the PB1 and PB2 groups. Even structurally similar forms were functionally diverse, exhibiting large differences in metabolic specificity in the dealkylation of a series of alkoxyresorufins.

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)

Ofloxacin has been evaluated in an in vitro model where microorganisms were exposed to the varying concentrations met in human serum after the oral administration of a 200 mg dose. The recently isolated strains were selected with minimum inhibitory concentrations (MICs) representative of the MICs 90% of ofloxacin against the species considered. Results showed that ofloxacin sterilized the bacterial cultures of S. aureus, P. morganii and E. coli, with 99.99% of killing still evident for P. aeruginosa after 12 hours of exposure. All the reference compounds (amoxicillin 1 g b.i.d., cefaclor 500 mg t.i.d., rifampicin 450 mg b.i.d.) showed, in the same test, worse results than ofloxacin.

The minimal inhibitory concentrations (MIC) of ofloxacin against 400 isolates cultured from the urine of urological patients with complicated urinary tract infections (UTI) resulted in an inhibition of 94% (99%) of the gram-negative strains at a concentration of 1 mg/l (2 mg/l) and an inhibition of 59% (100%) of the gram-positive strains at a concentration of 1 mg/l (4 mg/l). According to the MIC 90% values, the corresponding grade of activity was as follows: ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enoxacin, pipemidic acid, enoxacin and nalidixic acid. After an oral dose of 400 mg of ofloxacin, the mean peak serum concentration of ten patients was 5.5 mg/l. The mean renal excretion during 24 hours was 41%. In ten patients undergoing transurethral resection of the prostate, the median serum concentration was 1.87 mg/l and the median prostatic adenoma tissue concentration 1.20 mg/kg 14.5 to 19 hours after oral administration of 400 mg ofloxacin. Due to the in vitro activity and the concentrations obtained in serum, urine and tissue, ofloxacin appears to be well suited for treatment of complicated UTI.

Rats were exposed for 3 days by inhalation to 2000 ppm of a xylene mixture, or the individual constituents, o-xylene, m-xylene, p-xylene and ethylbenzene. All solvents increased hepatic cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity, although p-xylene did not increase the cytochrome P-450 content as much as the other compounds, showing the importance of the substitution pattern. Increases were observed in the in vitro O-deethylation of 7-ethoxyresorufin and in the hydroxylation of n-hexane and benzo[a]pyrene. The metabolite profiles obtained with these substrates and the results of gel electrophoresis in the presence of sodium dodecyl sulfate indicate that the induction is of the phenobarbital type. In kidney microsomes an increased concentration of cytochrome P-450 was obtained following exposure to a xylene mixture or to o- or m-xylene. The O-deethylation of 7-ethoxyresorufin was increased by exposure to all solvents. In lung microsomes xylene and xylene isomers but not ethylbenzene caused a decrease in cytochrome P-450 content and a reduction in n-hexane hydroxylation. However, the O-deethylation of 7-ethoxyresorufin was not affected. In general the effect of the xylene mixture reflected the content of the dominating component m-xylene. The ability of xylene and xylene isomers to modify the metabolism of other potentially toxic substances in liver, kidney and lung microsomes suggests the possibility of synergistic toxic responses.

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O2 and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450s were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450PB-B greatly inhibited the reaction (greater than 90%), while antibodies to P-450PB-C or P-450PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9-75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced approximately 140-fold at this dose and approximately 4-fold by a dose of phenobarbital as low as 0.9 mg/kg.

After oral administration of a single dose of ofloxacin (100, 300 or 600 mg) to 13 healthy male volunteers in an open, randomized crossover study, concentrations of the unchanged drug were estimated at various times in serum and urine, over 28 hours and 48 hours, respectively. Each dose was followed by a wash-out period of 1 week. Ofloxacin concentrations were determined using both high pressure liquid chromatography (HPLC) and a microbiological assay. The measurements obtained were compared by linear distribution independent regression, and were found to be equivalent, indicating no major metabolism of ofloxacin. Maximum serum concentrations (Cmax) of ofloxacin after administration of 100, 300 or 600 mg were, respectively, 1.0, 3.4 and 6.9 mg/ml (HPLC, median values). A linear relationship between Cmax and dose was demonstrated within the range tested (coefficient of correlation r = 0.88). The same applied to AUC0-28 (r = 0.98) and to urinary recovery of the drug (r = 0.98). Time to reach Cmax varied between 0.5 and 1.1 hours (median values), indicating rapid absorption of the drug. Biological half-life (t1/2 beta) was determined by fitting a two-compartment open model to the date: t1/2 beta was in the range 5.6 to 6.4 hours (HPLC, median values) and was not relevantly dose-dependent. Urinary concentrations of ofloxacin remained above 1 microgram/ml, i.e. above the minimum inhibitory concentrations (MIC90) for most bacterial strains at all dosages tested, for at least 36 hours after drug administration. General tolerability was good; no side-effects were reported.

Labelled 2-oxo-2H-1,3-benzoxazine-3(4H)-acetamide (caroxazone), has been synthesized by condensing N-(2-hydroxylbenzyl/glycinamide with 14C phosgene. Metabolic studies were performed administering the labelled drug to man and recovering metabolites from the urines. Beside the unchanged drug, five metabolites were identified and confirmed by synthesis, namely (3,4-dihydro-3-carboxamidomethyl-2-oxo-2H-1,3-benzoxazin-4-yl)urea (IX), N-carboxamidomethyl o-hydroxymethylphenyl carbamate (V), 4-methoxy-2-oxo-2H-1,3-benzoxazine-3(4H)acetamide (VIIIa), 2-oxo-2H-1,3-benzoxazine-3(4H)acetic acid (III) and 4-hydroxy-2-oxo-2H-1,3-benzoxazine-3(4H)acetamide (IV).

Fly ash from either peat- or coal-fired power plants is unable to block the inducer activity of benzo[a]pyrene in the gastrointestinal tract. Activated charcoal retains adsorbed polycyclic aromatic hydrocarbons during passage through the gut so that no induction of polysubstrate monooxygenase occurs in the mucosa.

As a substrate for human liver microsomes, ethoxyresorufin appears to be metabolised by a group of cytochrome P450 isoenzymes which are inducible by cigarette smoking. Kinetic studies in microsomes from four human livers indicate that only one enzyme component is involved over the full substrate range. Ethoxyresorufin O-deethylation activity at both high and low substrate concentrations correlated with the high affinity component of ethoxycoumarin O-deethylation and of diphenyloxazole metabolism.

Deprivation of pups from mother and sibs for 3 min daily from day 5 to day 41 of life reduced activities of 4 hepatic mixed function oxidases (MFO) expressed per mg protein in male rats compared to unhandled control rats. These decreases, though generally small, 22.4% and under, reached statistical significance for the substrates aminopyrine, benzphetamine and ethoxycoumarin. This handling procedure did not consistently affect the inductive response to phenobarbital. Previously ignored as a source of variability in response to xenobiotics, "handling" appears from these results to merit further investigation as such a factor in uninduced rats. Differences among rats in "handling" could contribute to large day-to-day variations in their metabolism of xenobiotics.

The antimicrobial activity of ofloxacin was tested against 15 standard strains and 37 clinical and environmental strains of Legionella pneumophila by agar dilution susceptibility studies with a new growth medium. The ofloxacin MICs were inoculum dependent and ranged from 0.03 to 0.125 microgram/ml. The antibacterial activities of other agents tested relative to ofloxacin were rifampin greater than ofloxacin greater than josamycin greater than pipemidic acid. Ofloxacin, at concentrations equal to or greater than 0.05 microgram/ml, inhibited the growth of L. pneumophila grown in human monocytes. The therapeutic efficacy of ofloxacin in experimental guinea pig L. pneumophila pneumonia was greater than that observed with erythromycin or josamycin therapy; it was less effective than was rifampin. Ofloxacin was very active against intracellular L. pneumophila in these experiments and should be studied in the therapy of human Legionnaires disease.

Data obtained from human studies in vivo show that the dispositions of the tricyclic antidepressant drugs desmethylimipramine (DMI) and nortriptyline are related to the debrisoquine hydroxylation phenotype. To obtain insight into the enzymic mechanisms behind this, the metabolism of debrisoquine and antidepressant drugs by human liver preparations have been studied. The 2-hydroxylation of DMI in vitro correlates with the 4-hydroxylation of debrisoquine among various livers (rs = 0.90). Debrisoquine inhibits DMI hydroxylation competitively, and DMI inhibits debrisoquine hydroxylation, suggesting that DMI hydroxylation is catalysed by the debrisoquine hydroxylase in human liver. By monitoring the hydroxylation of DMI in various fractions during separation and purification of cytochrome P-450 from human liver microsomes we have purified a cytochrome P-450 which efficiently hydroxylates this drug. The apparently electrophoretically homogeneous enzyme had a molecular weight of 51,500 and hydroxylated DMI and debrisoquine at rates of up to 0.95 and 0.45 nmol/min . nmol P-450, respectively. This is probably the major debrisoquine hydroxylating cytochrome P-450 in man. Nortriptyline 10-hydroxylation correlates strongly (r = 0.96) with debrisoquine hydroxylation in human liver microsomes. Nortriptyline inhibits DMI-hydroxylation competitively, and the drug also inhibits the 4-hydroxylation of debrisoquine. Thus it is probable that nortriptyline is hydroxylated by debrisoquine hydroxylase. Imipramine N-demethylation did not correlate significantly (P greater than 0.1) with debrisoquine hydroxylation among microsomes from nine livers. However, if a liver from a subject, which was a poor metabolizer of debrisoquine in vivo, was included, a correlation was obtained (r = 0.79, P less than 0.01, N = 10). Imipramine demethylation also correlated with DMI-hydroxylation only if the 'poor metabolizer' liver was included (r = 0.75, P less than 0.05, N = 10). Debrisoquine inhibited imipramine demethylation competitively. The data indicate that imipramine can interact with debrisoquine- and DMI-hydroxylase, but it is uncertain if this enzyme plays an important quantitative role in its demethylation. Ethoxyresorufin O-deethylation correlated with DMI hydroxylation (r = 0.80) in human liver preparations, and DMI inhibited the former reaction in what is probably a mixed competitive-non-competitive inhibition. Liver preparations from a subject who was a poor oxidizer of debrisoquine both in vivo and in vitro had unusually low capacity to metabolize ethoxyresorufin. Thus ethoxyresorufin, at least partly, seems to interact with an enzyme that can metabolize DMI in human liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Using an agar well diffusion assay, the concentrations of ofloxacin were measured in serum and skin blister fluid after an oral dose of 300 mg given with or without a standardized meal. The apparent lag time was longer when the drug was taken with food than in the fasting state and the serum peak concentrations occurred later; the rates of absorption being 1.72 +/- 1.12/h (S.D.) and 3.40 +/- 1.56/h respectively. The mean peak concentrations of each individual in serum and blister fluid were 3.8 +/- 1.6 mg/l and 1.7 +/- 0.5 mg/l, respectively, when the drug was taken with food, compared with 4.2 +/- 1.9 mg/l and 2.1 +/- 0.7 mg/l in the fasting state. The rate of penetration into blister fluid was not influenced by the intake of food. The elimination half-life was 9.0 +/- 6.2 h in serum and 13.5 +/- 7.8 h in blister fluid when the volunteers were fasting. The corresponding values in the non-fasting state were 6.3 +/- 5.5 h and 10.6 +/- 4.6 h. The ratio of the area under the concentration versus time curve (AUC0-infinity) for blister fluid and serum was 1.20 +/- 0.70 when ofloxacin was given without food and 1.03 +/- 0.34 when given with the meal.

Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.

As part of a systematic investigation of the penetration of antibiotics into human bronchial mucus, we assayed ofloxacin concentrations following ingestion of a single dose. 25 patients with acute superinfection of a chronic lower respiratory tract disease were studied. Each patient had single drug therapy with ofloxacin in a daily dosage of 200 mg taken in the morning on an empty stomach. Patients were divided into five groups according to the time interval between ingestion of ofloxacin and collection of samples (bronchial mucus and serum): 1 hour, 3 hours, 6 hours, 12 hours or 24 hours. Duplicate determinations of ofloxacin on individual samples were done using a microbiologic method. Mean serum concentrations were 1.85, 1.64, 1.32, 0.75 and 0.20 mg/l respectively, with a half-life of 6.7 hours; the corresponding concentrations in mucus were 1.83, 1.51, 1.20, 0.66 and 0.19. These results demonstrate ofloxacin's outstanding penetration into bronchial mucus.

A novel enzymic activity, responsible for the conversion of mannopine to agropine by lactonization, has been identified in Agrobacterium strains. This activity is encoded by octopine-type and agropine-type Ti or Ri plasmids, and is inducible by mannopine and agropine. In crude extracts it is stable for long periods and can be used for preparative synthesis of agropine from mannopine. The physiological role of this activity is not understood. However, it is probably involved in degradation of opines of the agropine family since it is always associated with agropine utilization in wild-type strains.

Cytostatic effects and some pharmacological properties of a new metabolic inhibitor "3-oxauracil" were studied. The cancerostatic effect was examined on 7 experimental tumors in mice and on two types of tumors in rats. After the i. p. application of 20 mg/kg, there was both a statistically significant decrease of tumor weight and increase of animals' survival time in NK lymphoma of mice. Significant changes in one of both parameters followed occured in all experimental tumors after the i. p. application but only in the Krebs ascitic carcinoma after the oral application of "3-oxauracil". The acute toxicity of the substance in water was 322 mg/kg i. p. and 850 mg/kg p. o. The ethanol solutions were more toxic. The distribution of the 3H- and 14C-labeled substance was followed up in blood, urine, liver, brain and kidney. After the p. o. application, the radioactivity peak was reached after 2 hr in blood and high radioactivity levels were found in kidney followed by brain and liver. 96 hr after the drug was applicated perorally, only 60% of radioactivity was found in urine.

Synthesis of several members of the 9-oxaergoline ring system is presented. Both the C/D cis and the C/D trans isomers of 4,6,6a,8,9,10a-hexahydro-7-ethyl-7H-indolo[3,4-gh] [1,4]benzoxazine were prepared, and the C/D trans isomer was resolved into its optical isomers. The enantiomer having the highest affinity for the [3H]apomorphine binding site, (-)-trans-6-ethyl-9-oxaergoline [(-)-6b], was shown to have the same absolute configuration as the natural ergolines, namely, 6aR, 10aR. In vivo and in vitro pharmacological evaluation shows these 9-oxaergolines to possess potent dopamine agonist properties.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.

The rates of metabolism of phenoxazone and a homologous series of its ethers (alkoxyresorufins) by liver and lung microsomes of rats exposed to cigarette smoke were compared with the metabolism in rats pretreated with 3-methylcholanthrene (3MC) or phenobarbitone (PB). The rate of resorufin production was dependent on the length of the ether side chain. Liver and lung microsomes from control rats differed in their activity profiles (rate of resorufin production plotted against side-chain length), showing highest activity with ethoxy- and benzyloxyresorufin respectively. 3MC and PB selectively induced hepatic microsomal resorufin production with only certain of the substrates and the two agents differed in their selectivity, inducing most greatly with ethoxy- and benzyloxyresorufin respectively. Pulmonary microsomal resorufin production was induced by 3MC with a substrate selectivity similar to that shown for liver, but PB suppressed pulmonary metabolism with all the substrates. A single, short exposure to cigarette smoke induced ethoxyresorufin O-deethylase activity transiently in liver and lung microsomes. Three consecutive daily short exposures to cigarette smoke caused a weak 3MC-like induction of liver microsomal alkoxyresorufin metabolism, but the effect on lung microsomes was like weak 3MC and PB inductions combined. It is concluded that cigarette smoke induces selected cytochrome P-450-linked alkoxyresorufin O-dealkylase activities to a similar extent in both lung and liver and that the effects of cigarette smoke are characteristic of both 3MC-type and non-3MC-type inducers.

The mixed-function-oxidase (MFO) activities, ethoxyresorufin and pentoxyphenoxazone O-dealkylase, of cultured Hooded-Lister(HL)-rat hepatocytes declined rapidly during 72 h of culture, whereas in Sprague-Dawley(SD)-rat hepatocytes the MFO activities increased between 24 and 72 h in culture. Cytochrome P-450 content declined at the same rate in both HL- and SD-rat hepatocyte cultures. NADPH:cytochrome c reductase and NADH:cytochrome b5 reductase were more stable in SD- than in HL-rat hepatocyte cultures. 16,16-Dimethylprostaglandins E2 and F2 alpha improved the maintenance of cytochrome P-450 content, MFO activity and NADPH:cytochrome c reductase in the HL-rat hepatocyte cultures. In SD-rat hepatocytes, the prostaglandins had no effect on cytochrome P-450 content or NADPH:cytochrome c reductase activity, whereas they prevented the increase observed in MFO activities between 24 and 72 h after culture.