Stereospecificity of flobufen metabolism in guinea pigs in vitro and in vivo: phase I of biotransformation

Flobufen (F) is an original nonsteroidal antiinflammatory drug that exists in two enantiomeric forms. Its biotransformation was investigated in male guinea pigs because of the similarities shown in a preliminary F metabolic study between guinea pig and man. Stereospecificity of the respective enzymes was studied in vitro, using microsomes and cytosol, and in vivo, in urine and feces. Rac-F, R-F, and S-F served as substrates. The amount of 4-dihydroflobufen stereoisomers (DHF) and other metabolites (M-17203 and UM-2) was determined by chiral HPLC using an R,R-ULMO column. It was observed that F reductases were distributed differently in microsomes and cytosol. The microsomal fraction showed higher activity and different stereospecificity in rac-F, R-F, and S-F reduction compared to cytosol. (2R;4S)-DHF was the principle metabolite in microsomes and (2S;4S)-DHF was the principle metabolite in cytosol. In vivo experiments revealed the excretion of a main metabolite UM-2 in addition to other metabolites M-17203 and DHF stereoisomers. UM-2 was predominantly excreted after S-F administration. Stereoselectivity of DHF stereoisomers excretion was different in urine and in feces. The absence of UM-2 and M-17203 in microsomes and cytosol and their presence in urine and feces showed that both could arise in some other extrahepatic tissue or cell compartment or that their formation depends on liver cell integrity.     

In vivo and in vitro oxidative biotransformation of dimethylformamide in rat

In rats and in humans, dimethylformamide (DMF) is mainly metabolized into N-hydroxymethyl-N-methylformamide (DMF-OH). The in vitro oxidation of DMF by rat liver microsomes is decreased in the presence of catalase and superoxide dismutase. The radical scavengers, dimethylsulfoxide (DMSO), tertiary butyl alcohol (t-butanol), aminopyrine, hydroquinone and trichloroacetonitrile reduce the oxidation of DMF to DMF-OH in vitro and in vivo. Conversely, DMF inhibits the demethylation of DMSO, t-butanol and aminopyrine. The addition of iron-EDTA to the incubation system induces the production of N-methylformamide (NMF) from DMF. These results support the hypothesis that the metabolic pathway leading from DMF to DMF-OH and NMF involves hydroxyl radicals. Superoxide radical and hydrogen peroxide take part in the metabolic process. DMF is preferentially metabolized into DMF-OH. NMF appears mainly when the production of hydroxyl radicals is stimulated, the methyl group being recovered as formic acid.     

Polyunsaturated phosphatidylcholine and lipolysis: metabolic interactions in vitro and in vivo.

Polyunsaturated phosphatidylcholine (EPL) is known to have a number of effects on lipid metabolism. We tested the drug on rat adipose tissue in vitro: stimulated lipolysis was inhibited without any effect on cyclic AMP level. Administration of EPL in rats enhanced the basal lipolysis and inhibited the hormone stimulated process. In fasting rats, the high plasma FFA level was further increased by EPL, while no modification on total esterified fatty acids, glucose and cholesterol contents was found. These effects could be due to the activity of EPL on various enzymes, for instance lipoprotein lipase, or to a general effect on membranes.     

Characterization of the in vivo and in vitro metabolic profile of PAC-1 using liquid chromatography-mass spectrometry

In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers--a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer--were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C(18) column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed.     

Novel heterocyclic glucocorticoids: in vitro profile and in vivo efficacy

A series of novel ligands for the glucocorticoid receptor containing two heterocycles were synthesized. These compounds were investigated for a dissociative profile using transrepression and transactivation assays. Several compounds were tested in vivo and showed the ability to reduce inflammation in a mouse.     

Relationships betweenin vitro andin vivo biotransformation of drugs in humans and animals: pharmaco-toxicological consequences

Given the crucial role played by hepatocytes in the detoxification/toxification processes of drugs, these cells have been increasingly used during the last decade in various pharmaco-toxicological areas. The majority of these studies have, however, dealt with animal cells, although examples of failures in the extrapolation of the data to man are frequent. This drawback, together with the ethical considerations in performingin vivo experiments, makes the application of the human hepatocyte model critical in the preclinical evaluation of new compounds. However, before making extensive use of these promising tools for prospective pharmaceutical research, one must ensure that they can generate data that correlate well with those obtainedin vivo. This is only possible through extensive studies on drugs showing a variety of phase I and phase II metabolic pathways in hepatocytes from different species, including man, and comparison within vivo data. Providing this validation step is undertaken, the use of such systems in drug research and development may greatly enhance the rational design of safe and effective drugs, allowing savings in time, cost and test materials as well as minimizing the use of animals.Abbreviations AMT 3-amino-3-deoxythymidine - AZT 3-azido-3-deoxythymidine - GAMT 5-glucuronyl-3-amino-3-deoxythymidine - GAZT 3-azido-3-deoxy-5-O--d-glucopyranuronosylthymidine - HIV human immunodeficiency virus - MAO monoamine oxidase     

Pituitary and brain D2 receptor density measured in vitro and in vivo in EEDQ treated male rats.

The effect of the alkylating compound N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (20 mg/kg, 24 h) on dopamine D2 receptor density in rat pituitary and brain was measured using in vitro and in vivo radioligand binding techniques. In the in vitro radioligand binding experiments EEDQ was found to reduce the density (Bmax) of [3H]-spiperone binding sites in the striatum by 86% while in the pituitary the corresponding decrease was only 37%. The affinity (KD) of the remaining striatal and pituitary D2 receptors was not different in EEDQ treated animals as compared to controls. When D2 receptor density was measured in vivo the effect of EEDQ was less pronounced. Thus, in rats given EEDQ the specific binding of either of the two D2 ligands [3H]-raclopride or [3H]-spiperone (administered in a single dose) in striatum and in the limbic forebrain was reduced by 45-62%; moreover, no significant decrease in pituitary D2 receptor density was observed. The data are discussed in relation to the finding (presented in a separate paper) that the same dose of EEDQ that failed to influence pituitary D2 receptor density as measured in vivo effectively antagonizes the prolactin decreasing effect of the partial D2 agonist (-)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine [(-)-3-PPP].     

Genetic effects of isoniazid and the relationship to in vivo and in vitro biotransformation

The mutagenic activity of isoniazid, N-acetyl-isoniazid and hydrazine dihydrochloride was investigated in S. typhimurium. Isoniazid was found to possess a weak mutagenic activity only in repair-deficient strains TA1535 and TA100 as well as in the plasmid-containing strain TA92 (10-30 mg/plate) in the Ames test without metabolic activation. Addition of microsomal enzymes by S9 mix decreased this direct mutagenic activity. In contrast, preincubation of isoniazid with crude liver homogenate from mice, rats or Syrian golden hamsters for 4 h prior to plating with bacteria liberated a mutagenic compound which is equally active in both repair-deficient and repair wild-type strains (0.5-5 mg/plate). This activation pathway is independent of NADPH, is heat-sensitive and is operative only in a total liver homogenate in suspension. The highest capacity for mutagenic activation was achieved with liver homogenate from hamsters, followed by that from mice and rats. Furthermore, this mutagenic activation is paralleled by formation of hydrazine, as demonstrated in colorimetric measurements with p-dimethylaminobenzaldehyde. N-Acetyl-isoniazid is without mutagenic activity under similar conditions, and liberation of hydrazine was never detected. This means that, besides having a weak direct genetic activity, isoniazid is a promutagen, and formation of hydrazine is the first step in metabolic activation. It is concluded that the genotoxic properties of isoniazid in mammals are primarily determined by the pharmacokinetic behavior of the ultimate reactive metabolite. This result must be taken into consideration in risk assessment performed for mutagenic and carcinogenic properties of isoniazid in man.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.     

Ethanol potentiates in vivo hepatotoxicity of endosulfan in adult male rats.

In an attempt to evaluate the effect and interaction of ethanol on endosulfan-induced hepatotoxicity in vivo to adult male rats, both, endosulfan (7.5 mg/kg body wt) and ethanol (1.5 g/kg body wt) were studied separately as well as in combination after a chronic oral exposure of 30 days. When fed separately, both the agents were found to induce microsomal mixed function oxidase (MFO) system in treated animals. A simultaneous induction in the activity of cytosolic GSH-s-transferase was found to be associated with significantly induced ascorbate-induced microsomal lipid peroxidation. Both endosulfan and ethanol showed increasing trends in the activities of reducing equivalent (NADPH)-generating enzymes in liver. The activity of hepatic alcohol dehydrogenase was, however, found to be relatively unaffected. When ethanol was administered in combination with endosulfan, the observed effects on the activities of major drug metabolizing enzymes, microsomal lipid peroxidation and NADPH generation were further pronounced. Findings demonstrated the MFO inducing capability of both endosulfan and ethanol, and showed further that chronic ethanol ingestion might potentiate the in vivo hepatotoxicity of endosulfan if administered in combination.     

Correlation between in vivo and in vitro metabolic measurements. Maximum capacity for urea synthesis.

Estimations of enzyme activity in vivo have been or can often only be done at unphysiological conditions. A main biochemical goal is to correlate in vivo and in vitro measurements. A possible approach to this problem is presented based on forcing metabolic activity in vivo to the maximum for a certain metabolic sequence. Since the urea synthesis system, including maximal rates of enzyme activities, is well known, we have compared in vitro maximum rates for the individual enzymes of urea synthesis with in vivo rates as judged by urea levels in blood of rats given large amounts of protein. The excellent agreement found between the calculated maximum activities from in vitro measurements to the time needed to metabolize a protein overload is presented and comments made on its significance and on the importance of maintaining protein intake at moderate levels, for the capacity of the urea system is limited. Since the intake of large quantities of protein increases the urea level in blood and in other tissues and since high urea levels are somewhat deleterious "per se" and particularly due to equilibrium with cyanate, ingestion of excessive amounts of protein is at best expensive and possibly hazardous.     

Stereospecificity of the in vitro and in vivo blockade of beta-receptors by FM 24, a slowly reversible ligand

Exo FM 24 (1-(2-exo-bicyclo[2,2,1]hept-2-ylphenoxy)-3[(1-methylethyl) amino]-2-propanol hydrocloride, a long lasting β-blocker is a mixture of four enantiomers. Exo FM 24 and its endo derivative were 5 to 8 times more potent after preincubation on [³H]DHA binding to rat brain membranes. Similar results were obtained with the four enantiomers, the order of potency being (αS,2S) > (αR,2S) > (αS,2R) > (αR,2R). These four enantiomers behave as competitive antagonists when no preincubation is performed but blocked β-receptors in a non competitive manner after preincubation. Under conditions in which the effect of (S,R) propranolol was completely reversed (7 cycles of washing), the effect of the two 2 S enantiomers was not reversed whereas the effect of the two 2R enantiomers was partially reversed. The potency and duration of blockade of β-receptors, as measured by the in vivo binding of [¹²⁵I] hydroxybenzylpindolol to mouse brain, heart and lung, correlated very well with the in vitro results. The potency and duration of exo FM 24 appeared to be the mean of its four enantiomers. It is proposed that the exo FM 24 formed a reversible complex with β-receptors which is slowly transformed to a non competitive slowly reversible complex which corresponds to the two 2R enantiomers, and to a non competitive irreversible complex which corresponds to the two 2S enantiomers.     

Characterization of the reproductive effects of the anorexigenic VGF-derived peptide TLQP-21: in vivo and in vitro studies in male rats

VGF (nonacronymic) is a 68-kDa protein encoded by the homonymous gene, which is expressed abundantly at the hypothalamus and has been involved in the control of metabolism and body weight homeostasis. Different active peptide fragments are generated from VGF, including TLQP-21. Circumstantial evidence has suggested that VGF might also participate in the control of reproduction. Yet its mechanisms of action and the eventual role of specific VGF-derived peptides on the hypothalamic-pituitary-gonadal (HPG) axis remain unknown. Herein we report a series of studies on the reproductive effects of TLQP-21 as evaluated in male rats by a combination of in vivo and in vitro analyses. Central administration of TLQP-21 induced acute gonadotropin responses in pubertal and adult male rats, likely via stimulation of GnRH secretion, as documented by static incubations of hypothalamic tissue. In addition, in pubertal (but not adult) males, TLQP-21 stimulated LH secretion directly at the pituitary level. Repeated central administration of TLQP-21 to pubertal males subjected to chronic undernutrition was able to ameliorate the hypogonadotropic state induced by food deprivation. In contrast, chronic administration of TLQP-21 to fed males at puberty resulted in partial desensitization and puberty delay. Finally, in adult (but not pubertal) males, TLQP-21 enhanced hCG-stimulated testosterone secretion by testicular tissue in vitro. In summary, our data are the first to document a complex and multifaceted mode of action of TLQP-21 at different levels of the male HPG axis with predominant stimulatory effects, thus providing a tenable basis for the (direct) reproductive role of this VGF-derived peptide.     

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.     

Studies on biotransformation of lysozyme. III. Comparative studies on biotransformation of exogenous and endogenous lysozyme in rats.

Exogenous hen lysozyme or endogenous rat lysozyme labeled with 131I was intravenously injected to rats with the same dosage, respectively, and the uptake and degradation of injected 131I-labeled rat lysozyme in liver and kidney were studied in comparison with those of 131I-labeled hen lysozyme. 1. Although the serum levels of both enzymes injected were almost indentical during the first 6 h, the liver uptake of 131I-labeled hen lysozyme was 2.2-fold more than that of 131I-labeled rat lysozyme at the peak time of 5 min after injection. The uptake and clearance of 131I-labeled rat lysozyme in the kidney were exclusively slow as compared with those of 131I-labeled hen lysozyme. 2. The intracellular distribution in the liver and kidney were examined by the differential centrifugation after injection of each lysozyme. The protein-bound radioactivity of each subcellular fraction was found to be the highest in the 12 000 X g (10 min) fraction in the liver and the 19 600 X g (20 min) fraction in the kidney. The relative specific activity of 12 000 X g fraction of the liver after injection increased with the time lapse. On the other hand, the relative specific activity of 105 000 X g (1 h) fraction of the liver attained a maximum within 5 min after injection and thereafter decreased. It was assumed that the mechanism of the uptake of injected 131I-labeled rat lysozyme in the liver and kidney was similar to that of 131I-labeled hen lysozyme. 3. The degradation of exogenous or endogenous lysozyme in subcellular particles was examined. From the effect of pH, activator and inhibitor on the degradation, the proteolytic enzyme to degrade the injected 131I-labeled hen lysozyme was indicated to be mainly cathepsin BL, with the optimal pH of about 5.0, and the injected 131I-labeled rate lysozyme was mainly degraded by cathepsin D, with the optimal pH of about 3.5 The in vitro degradation of exogenous and endogenous lysozymes showed a tendency similar to the in vivo clearance from the liver and kidney.