Michael R. Franklin

A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda

Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is due to reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Previous studies have demonstrated that protein levels of URO-D do not change when catalytic activity is reduced, suggesting that an inhibitor of URO-D is generated in hepatocytes. Here, we describe the identification and characterization of an inhibitor of URO-D in liver cytosolic extracts from two murine models of PCT: wild-type mice treated with iron, δ-aminolevulinic acid, and polychlorinated biphenyls; and mice with one null allele of Uro-d and two null alleles of the hemochromatosis gene (Uro-d+/−, Hfe−/−) that develop PCT with no treatments. In both models, we identified an inhibitor of recombinant human URO-D (rhURO-D). The inhibitor was characterized by solid-phase extraction, chromatography, UV-visible spectroscopy, and mass spectroscopy and proved to be uroporphomethene, a compound in which one bridge carbon in the uroporphyrinogen macrocycle is oxidized. We synthesized uroporphomethene by photooxidation of enzymatically generated uroporphyrinogen I or III. Both uroporphomethenes inhibited rhURO-D, but the III isomer porphomethene was a more potent inhibitor. Finally, we detected an inhibitor of rhURO-D in cytosolic extracts of liver biopsy samples of patients with PCT. These studies define the mechanism underlying clinical expression of the PCT phenotype, namely oxidation of uroporphyrinogen to uroporphomethene, a competitive inhibitor of URO-D. The oxidation reaction is iron-dependent.

The anticonvulsant profile of rufinamide (CGP 33101) in rodent seizure models

Purpose: To evaluate the anticonvulsant profile and behavioral toxicity of rufinamide in animal seizure models compared to the established antiepileptic drugs (AEDs): phenytoin, phenobarbital, valproate, and ethosuximide, or vehicle.

Cytochrome P-450 metabolic-intermediate complex formation and induction by macrolide antibiotics; a new class of agents

1. By several criteria, macrolide antibiotics constitute a new class of nitrogenous cytochrome P-450 metabolic-intermediate complex-forming compounds. 2. Macrolide antibiotic metabolic-intermediate complexes are only formed in livers induced with phenobarbital or with the macrolide antibiotics themselves. The extent of metabolic-intermediate complex formation in microsomes from phenobarbital-induced rats is lower than that seen for members of the amphetamine and SKF 525-A classes of compounds. 3. Cytochrome P-450 induced by macrolide antibiotics, of which troleandomycin is the most potent, is extensively sequestered as a metabolic intermediate complex in vivo. 4. Cytochrome P-450 induced by troleandomycin differs, using several criteria, from those induced by phenobarbital, beta-naphthoflavone or SKF 525-A, and those present in uninduced rats.

Complexes of Metabolites of Amphetamines with Hepatic Cytochrome P-450

Abstract1. Amphetamine, methamphetamine and many derivatives, in the presence of NADPH, O2 and hepatic microsomes, form complexes with cytochrome P-450, detectable by their absorbance at 455 nm.2. Para-hydroxylated amphetamines, the main metabolic products of amphetamine and methamphetamine in rats, and β-hydroxylated derivatives do not exhibit this spectral formation under similar conditions.3. The relationship between the amphetamines and other compounds known to exhibit this spectrum is discussed and indicates that an intermediate formed during an hydroxylation reaction may be responsible for the 455 nm absorbance.

A novel group of ovarian toxicants: the psoralens.

The psoralens are naturally occurring metabolites found in many crop plants; synthetic forms of 5‐methoxypsoralen (bergapten) and 8‐methoxypsoralen (xanthotoxin) are widely used in skin photochemotherapy. Our previous research documented that dietary bergapten and xanthotoxin reduced birthrates in female rats when males and females were exposed to these chemicals. The present study was designed to determine the cause of this reduced birthrate and whether this resulted from direct impact on the females. The study demonstrates that bergapten and xanthotoxin administered, either alone or in combination to female rats (mated to undosed males), significantly reduced the number of implantation sites, pups, and corpora lutea in dosed females compared with control animals. Additionally, full uterine weight and empty uterine weight were significantly reduced. These compounds also significantly reduced circulating estrogen levels in a dose‐dependent manner. Interestingly, the psoralens significantly induced mRNAs of liver enzymes typically induced by polycyclic aromatic hydrocarbons, CYP1A1 and UGT1A6; the higher the dose, the greater the induction. UGT 2B1 mRNA, typically induced by phenobarbital‐like compounds, was not significantly affected. Thus, enhanced oxidative metabolism and conjugation of estrogens in psoralen‐treated animals may provide a partial explanation for the effects observed. These findings are also consistent with psoralen‐induced reduction in ovarian follicular function and ovulation.

Separation of four glucuronides in a single sample by high-pressure liquid chromatography and its use in the determination of UDP glucuronosyltransferase activity toward four aglycones

A single-solvent, reverse-phase HPLC system that separates glucuronides has been developed, and its applicability to the separation of glucuronides of four substrates (1-naphthol, morphine, testosterone, and estrone), representative of different UDP-glucuronosyltransferase enzymes, has been demonstrated. The combined supernatants from four separate microsomal incubations from which the protein has been precipitated can be used directly for the quantitation of the glucuronides formed. Variations in the four enzyme activities, upon treatment of rats with microsomal enzyme-inducing agents, after liver trauma and in the Gunn strain of rats have been investigated.

Nanosilver Particle Effects on Drug Metabolism In Vitro

Nanosilver particles are present in consumer and health care products. Their effects on human microsomal cytochrome P450 (P450) activities and induction in luciferase reporter-engineered Caco-2 (MDR1.C) and HepG2 (DPX2 and 1A2DRE) cells have been investigated. The LD50 values were ∼4 μg silver/ml for HepG2 and 5 μg/ml for Caco-2 cells. At silver concentrations that showed no decreased cell viability (<1 μg silver/ml), the pregnane X receptor (PXR)-driven 4.5-fold induction response of MDR1.C cells to 50 μM omeprazole was unaffected. In DPX2 cells, the PXR-driven 5.5- and 6.5-fold induction responses to omeprazole and 10 μM rifampicin were attenuated to 4- and 3.5-fold, respectively. Nanosilver particles alone showed no induction. In 1A2DRE cells, the aryl hydrocarbon receptor-driven 5.5-fold induction response to omeprazole was attenuated to 4-fold. In 1A2DRE cells, nanosilver alone elicited slight induction at 1 μg/ml. The inhibition of human P450-selective activities by nanosilver particles in vitro was proportional to the silver/microsomal protein ratio. At a fixed (0.5 mg/ml) protein concentration, P450-selective activities differed in sensitivity (IC50 value). Coumarin 7-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation exhibited the highest IC50 values (33.5 and 31.9 μM, respectively) and S-mephenytoin 4-hydroxylation exhibited the lowest (6.4 μM). Other IC50 values were, in ascending order, 8.0 to 9.3 μM (testosterone 6β-hydroxylation, 7-benzyloxyquinoline debenzylation, and diclofenac 4-hydroxylation), 16.0 μM (chlorzoxazone 6-hydroxylation), 21.2 μM [7-methoxy-4-(aminomethyl)-coumarin O-demethylation], and 24.4 μM (7-methoxyresorufin O-demethylation). An investigation of 70 μM nanosilver particles showed that microsomal NADPH cytochrome c reductase activities were inhibited <12%. From our in vitro observations, we extrapolated that nanosilver particles reaching the liver may be a potential source of drug-drug interactions.

Selective changes in cytochrome P-450 and UDP-glucuronosyltransferase subpopulations following partial hepatectomy in rats

Following 65% hepatectomy in adult male rats, the cytochrome P-450 in the residual liver decreased continuously from 1.0 to approximately 0.5 nmol/mg protein over a period of 2 to 3 days, and then recovered slowly to presurgery values by 14 days. In contrast UDP-glucuronosyltransferase activity (per mg protein) declined (25 to 40% depending on the aglycone used to assay the enzyme) for only 1 day, with a rapid recovery to presurgery values by 5 days. Transferase activity toward estrone overshot control values during recovery and was elevated above its presurgery value at 7 days. The changes in drug-metabolizing enzymes were accompanied by a surge in mitosis which peaked at 2 days. The extent of the decline in cytochrome P-450 and the increase in the mitotic index were proportional to the amount of liver removed. The decline in cytochrome P-450 was selective for cytochrome P-450 which elutes in the void volume upon anion-exchange high-pressure liquid chromatography of solubilized microsomes. Accompanying the decline in cytochrome P-450 in this eluate was a decline in protein(s) with a molecular weight of 51,500. The decline in cytochrome P-450 also caused disproportionate decreases in microsomal ethylmorphine N-demethylase activity and metabolic-intermediate complex formation from norbenzphetamine and SKF 525-A, but not in p-nitroanisole O-demethylase activity, metabolic-intermediate complex formation from isosafrole or metyrapone binding to cytochrome P-450.

Drug metabolizing enzyme induction by benzoquinolines, acridine, and quinacrine; Tricyclic aromatic molecules containing a single heterocyclic nitrogen

Rats were treated with nitrogen-containing phenanthrene (3,4-, 5,6-, or 7,8-benzoquinoline) or anthracene (acridine or quinacrine) derivatives at a dose of 75 mg/kg, daily for 3 days. The hepatic drug metabolizing enzyme response ranged from no induction (quinacrine) through low (5,6-benzoquinoline), intermediate (acridine), and high (3,4-benzoquinoline) magnitude increases of only phase II enzymes, to induction of both phase I and phase II enzymes (7,8-benzoquinoline). The phase I enzyme response of 7,8-benzoquinoline was an induction of CYP1A. All three benzoquinolines, but neither anthracene derivative, elevated NAD(P)H quinone oxidoreductase activity. A similar pattern but of lesser magnitude was seen with glutathione S-transferase activity. 3,4-Benzoquinoline was the only agent to significantly increase microsomal epoxide hydrolase activity (2,3-fold). Both 3,4- and 7,8-benzoquinoline increased UDP-glucuronosyltransferase activity toward 4-nitrophenol (40% and 70%, respectively), but only the 3,4-isomer increased activity toward morphine (75%), diclofenac (75%), and testosterone (23%), and only the 7,8-isomer increased activity toward chloramphenicol (105%). 3,4-Benzoquinoline elevated the hepatic mRNA concentration of UGT2B1 but not UGT1*6. Acridine treatment increased UDP-glucuronosyltransferase activity toward morphine (47%), 1-naphthol (28%), testosterone (19%), and estrone (19%). Quinacrine failed to elevate any UDP-glucuronosyltransferase activity and depressed activities toward testosterone and estrone by 20%. This study shows that some tricyclic aromatic compounds containing a single heterocyclic nitrogen atom have the potential for use as chemoprotective agents based upon their ability to selectively induce only phase II enzymes.

High magnitude hepatic cytochrome P-450 induction by an N-substituted imidazole antimycotic, clotrimazole

A 4-fold induction of hepatic microsomal cytochrome P-450 following 3 days of treatment of rats with clotrimazole (75 mg/kg), a potent monooxygenase inhibitor, greatly exceeded that evident from similar phenobarbital and dexamethasone treatment. The clotrimazole-induced microsomes exhibited a pattern of monooxygenase activities similar to that seen in microsomes from both phenobarbital- and dexamethasone-treated animals. Precautions were necessary to determine both monooxygenase activities and the full amount of cytochrome P-450 present in microsomes because of interference by residual clotrimazole in the microsomes.