Metabolization of porphyrinogenic agents in brain: involvement of the phase I drug metabolizing system. A comparative study in liver and kidney.

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochrome P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that delta-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic delta-aminolevulinic acid administration. Acute delta-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic delta-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and delta-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ.

Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital.

A comparative study of uranyl nitrate and cisplatin-induced renal failure in rat.

Renal dysfunction can have substantial effects on the pharmacokinetics and pharmacodynamics of drugs. A wide variety of animal models have been developed in an attempt to mimic conditions seen in human renal failure. In reality, no single animal model would be completely satisfactory because the etiology and development of renal failure are diverse. During recent years injection of uranyl nitrate has been found to be the most effective and easiest method to produce renal dysfunction in laboratory animals. Changes over the last 10 years in government regulations on the production and use of radioactive substances make the compound less available. There is, therefore, a need for a more accessible compound comparable to uranyl nitrate as an inducer of renal failure. The present study compares the effects of another known nephrotoxin, cisplatin, with uranyl nitrate in the rat. Cisplatin was chosen because of its ability to produce kidney damage and its identical site and mechanism of action on the kidneys as uranyl nitrate. In the present study, rats were given different i.v. doses of uranyl nitrate or cisplatin dissolved in 0.9% of saline solution. The effects of nephrotoxins were evaluated on the basis of changes in body weight, creatinine and blood urea nitrogen (BUN) concentrations. It was found that the degree of renal damage produced by uranyl nitrate and cisplatin is a function of the administered dose. With increasing dose there is evidence of more severe kidney damage, as measured by substantially increased plasma concentrations of creatinine and BUN. The time required to return to normal creatinine and BUN concentrations was also a function of dose. Furthermore, plasma alanine aminotransferase (ALT) activity was measured as an index of hepatocellular damage. The ALT test showed that a single dose does not affect the liver function. From dose-response curves a dose of 4 mg/kg body weight of uranyl nitrate or cisplatin was chosen to produce acute renal failure in animals for pharmacokinetic study of barbital. Barbital (100 mg/kg) was administered on the fifth day (the day of maximum renal dysfunction) to uranyl nitrate, cisplatin-treated and control rats. The elimination rate constant (k), elimination half life (t1/2), volume of distribution at steady state (Vss), total (CLt) and renal clearance (CLr) were significantly different in treated groups of rats from control, however no such difference was detected between uranyl nitrate and cisplatin-treated group of rats. In short, cisplatin is comparable to uranyl nitrate in producing renal failure in the rat and can be considered a suitable alternative.

Transport of various substances through human enamel and dentine.

The penetration of 14C-labelled alcohols (methanol, ethanol, n-butanol), 14C-labelled carbonic acids (formic, acidic, propionic, valerianic, octanoic, malonic, succinic and lactic acid), 14C-drugs (procain, barbital), and 14C-sugars (saccharose, xylose) into about 800 human deciduous or permanent teeth, both healthy and carious, was investigated. Dental enamel up to the cemento-enamel junction was incubated at a pH-value of 5.0 or 6.8 for 1 or 24 hours. For measurement of radioactivity, the dentine of the root was obtained by trepanation. Between intact and carious permanent teeth only slight differences were observed in case of the diffusion of methanol and ethanol (2% of the incubation medium), while n-butanol penetrated the dentine to an extent of 4.2% at a pH of 5.0. The monocarbonic acids penetrated the enamel of healthy teeth within 24 hours to an extent of 6.6-19.2% of the content of the incubation medium, while the dicarbonic (succinic and malonic) acids reached amounts of 3.6 and 9.2%, and the percentage of lactid acid which penetrated the enamel reached 2.9%, respectively. Under all conditions tested, saccharose penetration was higher in carious than in healthy teeth (3.8 vs 6.5%). The highest uptake was found in experiments with barbital; it was more pronounced in deciduous than permanent teeth (16.2 vs 12.4%). The data could be of interest in the therapy of inflammatory and other processes of the pulp.

Comparative pharmacodynamics of hepatic cytochrome P450 2B induction by 5,5-diphenyl- and 5,5-diethyl-substituted barbiturates and hydantoins in the male F344/NCr rat.

To explore further the structural requirements for ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of CYP2B induction by 5,5-diphenylbarbituric acid, phenytoin (5,5-diphenylhydantoin), barbital (5,5-diethylbarbituric acid) and 5,5-diethylhydantoin were investigated in the male F344/NCr rat. Steady-state total (free plus protein-bound) serum drug concentration, measured after 14 days of administration of the compounds in the diet, was used as an approximation of intrahepatocellular drug concentration. The serum concentrations associated with half-maximal hepatic CYP2B induction (EC50 values) were 6 to 11 microM and 15 to 18 microM for the diphenyl-substituted barbiturate and hydantoin, respectively, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values for the diethyl-substituted barbiturate and hydantoin were 16 to 20 microM and > or = 500 microM, respectively. The magnitudes of the maximal CYP2B induction responses elicited by the diphenyl-substituted congeners and by barbital were 94 to 122% of the responses resulting from phenobarbital itself. In contrast, the maximum induction responses elicited by 5,5-diethylhydantoin were only 24% as great as those elicited by phenobarbital. The finding of a CYP2B inducer with a potency value 2 to 3 orders of magnitude lower than those for certain other prototype CYP2B inducers is suggestive but not proof of receptor mediation in the induction process.

Effects of flunarizine and diltiazem on physical dependence on barbital in rats.

The effects of flunarizine and diltiazem both on development of physical dependence on barbital and on barbital withdrawal signs in rats were examined using the drug-admixed food (DAF) method. Rats were chronically treated with barbital or barbital in combination with flunarizine (fixed at 1.5 mg/g of food) or diltiazem (fixed at 0.75 mg/g of food)-admixed food on the schedule of gradually increasing doses of barbital. Motor incoordination during the treatment was potentiated by coadministration of flunarizine, but not by coadministration of diltiazem. After the termination of drug treatment, the body weight loss and withdrawal scores were significantly suppressed in the group coadministered flunarizine, but not in that coadministered diltiazem. There were no significant differences in plasma barbital levels after the withdrawal between groups. In the substitution test, flunarizine (20 and 40 mg/kg, IP) significantly suppressed the body weight loss and withdrawal scores after the withdrawal, but diltiazem (20 mg/kg, IP) did not. These results indicated that flunarizine suppressed both the development of physical dependence on barbital and barbital withdrawal signs, mainly according to the suppression of convulsions, but not diltiazem, which is known to poorly penetrate into the brain. Therefore, the present findings suggest that central calcium channels may be involved in both the development of physical dependence on barbital and the appearance of barbital withdrawal signs.