Cinnamyl anthranilate causes coinduction of hepatic microsomal and peroxisomal enzymes in mouse but not rat

Cinnamyl anthranilate is a synthetic food flavoring and fragrance agent, formerly used at low levels. Although it is not genotoxic, very high doses have been shown to cause liver tumors in mice but not rats. In this report we characterize hepatic changes brought about by cinnamyl anthranilate in rats and mice. Groups of male CD1 mice and Fischer 344 rats received 0, 100, or 1000 mg/kg cinnamyl anthranilate by intraperitoneal injection daily for 3 days. After euthanization on the 4th day, plasma lipids and relative liver weight, tissue DNA, the peroxisome marker CN(-)-insensitive palmitoyl-CoA oxidation, cytochrome P450, microsomal lauric acid hydroxylation, aminopyrine N-demethylase and ethoxyresorufin O-deethylase, bilirubin UDP-glucuronosyltransferase, microsomal and cytosolic epoxide hydrolase, and the peroxisome/mitochondria ratio in liver sections were monitored. In mice a pattern of change pointing to peroxisomal proliferation was seen at both doses of cinnamyl anthranilate, but in rats fewer and smaller changes were seen with little or no evidence of peroxisomal proliferation at the doses used. Groups of male CD1 mice were given 0-200 mg/kg cinnamyl anthranilate daily for 3 days. At doses of 20 mg/kg and above, there were dose-dependent increases in relative liver weight, total cytochrome P450, and CN(-)-insensitive palmitoyl-CoA oxidation. The hepatic effects of cinnamyl anthranilate are apparently due to the intact ester, since neither its expected metabolites alone nor an equimolar mixture of the hydrolysis products, cinnamyl alcohol and anthranilic acid, had any significant effect on the weight or marker enzyme content of mouse liver. The data are discussed in relation to the species specificity of the hepatocarcinogenicity of cinnamyl anthranilate and to metabolic studies in rats and mice. It is suggested that in mice, peroxisomal proliferation occurs only at doses high enough to prevent complete metabolic hydrolysis.     

New quinoline di-Mannich base compounds with greater antimalarial activity than chloroquine, amodiaquine, or pyronaridine

We have compared the ex vivo antimalarial activity of 12 new quinoline di-Mannich base compounds containing the 7-dichloroquinoline or 7-trifluoromethylquinoline nucleus with amodiaquine, chloroquine, and pyronaridine using the Saimiri-bioassay model. Each compound was administered orally (30 mg/kg of body weight) to three or more noninfected Saimiri sciureus monkeys, and serum samples were collected at various times after drug administration and serially diluted with drug-free (control) serum. In vitro activity against the multidrug-resistant K1 isolate of Plasmodium falciparum was determined in serum samples by measuring the maximum inhibitory dilution at which the treated monkey serum inhibited schizont maturation in vitro. Of the 12 Mannich bases tested, 8 were associated with levels of ex vivo antimalarial activity in serum greater than those of amodiaquine, chloroquine, or pyronaridine 1 to 7 days after drug administration. Further studies were carried out with four of these compounds, and the results showed that the areas under the serum drug concentration-time curves for the four compounds were between 7- and 26-fold greater than that obtained for pyronaridine. Activity against four multidrug-resistant strains of P. falciparum was also much greater in serum samples collected from monkeys after administration of these four compounds than in serum samples collected after administration of pyronaridine or chloroquine. These findings suggest that these four quinoline Mannich base compounds possess a very marked and prolonged antimalarial activity and that further studies should be performed to determine their value as antimalarial drugs.     

New NO donors with antithrombotic and vasodilating activities, Part 24. Hydrazine derivatives

Suitable hydrazines like phenylhydrazine (1), N,N-dimethyl hydrazine (4), and N,N-diphenyl hydrazine (5) can be oxidized by hydrogen peroxide at pH 7.4 and 37 degrees C to nitrosohydrogen and further to nitrite and nitrate. The extent of this property is correlated with platelet aggregation inhibiting and antithrombotic effects of these compounds, suggesting that an NO mediated mechanism might be involved. All hydrazines tested and two N-ethoxycarbonyl prodrugs exhibited antihypertensive effects which were not correlated to the above properties. This is especially pronounced in hydralazine (6) and dihydralazine (7) which induced a strong decrease in blood pressure but exhibit neither antiplatelet nor antithrombotic effects. This indicates that the mechanism of the antihypertensive activity is different from that of the antiplatelet activity.     

Ethanol interactions with other cytochrome P450 substrates including drugs, xenobiotics, and carcinogens

Chronic ethanol abuse is associated with increased activity of the microsomal ethanol-oxidizing system. This effect is due primarily to induction by ethanol of a specific cytochrome P450 (CYP2E1) responsible for enhanced oxidation of ethanol and other P450 substrates and, consequently, for metabolic tolerance to these substances. Furthermore, cytochrome 450 induction increases the activation of numerous xenobiotics to toxic metabolites and of chemical carcinogens to reactive metabolites, thereby accelerating their adverse effects. Microsomal enzyme induction has been associated with increased reactive oxygen species production and enhanced lipid peroxidation, as well as with decreased enzymatic and nonenzymatic scavenger activity, providing another possible explanation for ethanol-mediated toxicity. Yet another effect of chronic alcohol abuse is chronic immune system activation, which is the mechanism underlying alcohol-related liver disease. The metabolism of steroids and vitamins is catalyzed by P450 and is altered in chronic alcoholics. This article reviews recent advances in the understanding of ethanol interactions with drugs, toxic agents, and carcinogens, as well as with steroids and vitamins.     

Sulfotransferase-mediated activation of 7,8,9,10-tetrahydro-7-ol, 7,8-dihydrodiol, and 7,8,9,10-tetraol derivatives of benzo[a]pyrene

Some hydroxymethyl-substituted polycyclic aromatic hydrocarbons have been shown to be converted to electrophilic, mutagenic, or tumorigenic sulfuric acid ester metabolites by cytosolic sulfotransferase activity in rodent liver. Likewise, certain types of aromatic compounds with a secondary alcoholic functional group at the benzylic position undergo metabolic activation through sulfonation. Enzymatic oxidation of benzo[a]pyrene produces such secondary alcohols as dihydrodiol and tetraol derivatives as primary metabolites. Sulfo conjugation of the benzylic hydroxy group of each of these metabolites is expected to generate an electrophilic sulfuric acid ester capable of covalently binding to DNA, which may contribute to mutagenesis and carcinogenesis by benzo[a]pyrene. Although the model benzo-ring secondary benzyl alcohol, 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, covalently bound to DNA and also exerted mutagenicity in the presence of rodent hepatic cytosols and 3'-phosphoadenosine 5'-phosphosulfate, no such sulfotransferase-dependent activation was observed with dihydrodiol or tetraol derivatives of benzo[a]pyrene. Thus, it seems likely that appearance of the adjacent non-benzylic hydroxy functional group(s) in latter metabolites hinders the benzylic sulfonation in these molecules.     

Model studies on calcium-containing quinoprotein alcohol dehydrogenases. Catalytic role of Ca2+ for the oxidation of alcohols by coenzyme PQQ (4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2, 7,9-tricarboxylic acid)

Mechanistic studies on the action of calcium-containing quinoprotein alcohol dehydrogenases have been performed by using a series of PQQ model compounds in anhydrous organic media. The PQQ model compounds are shown to form 1:1 complexes with a series of alkaline earth metal ions by spectroscopic methods and theoretical calculations. The site of coordination of the PQQ molecule to the metal ions in solution is indicated to be the same as in the case of enzymatic systems. It has also been found that Ca2+ binds to the quinone most strongly among the alkaline earth metal ions. Formation of the C-5 hemiacetal derivatives with methanol, ethanol, and 2-propanol is also investigated spectrophotometrically to show that the alcohol addition to the quinone is enhanced in the presence of the metal ions. In this case as well, Ca2+ shows the highest efficiency for the enhancement of the C-5 hemiacetal formation. Addition of a strong base such as DBU into an MeCN solution containing the Ca2+ complex of the PQQ model compounds and the alcohols leads to the redox reactions to afford reduced PQQ derivatives and the corresponding aldehydes. On the basis of detailed kinetic studies on the redox reactions, including structural effects of PQQ analogues and metal ion effects, we propose the addition-oxidative elimination mechanism through the C-5 hemiacetal intermediate.     

Metabolism and pharmacokinetics of barnidipine hydrochloride, a calcium channel blocker, in man following oral administration of its sustained release formulation

1. The metabolism and pharmacokinetics of barnidipine hydrochloride, a 1, 4-dihydropyridine calcium antagonist were evaluated following single oral administration of a sustained release formulation (SR) capsule comprising of quick and slow release pellets to healthy male volunteers. 2. Various metabolites were identified and quantitated by newly established GC-MS analytical methods. Major metabolites were the hydrolyzed product of the benzyl-pyrrolidinyl ester (M-3) in plasma and its oxidized pyridine product (M-4) in plasma and urine. The pyridine form of unchanged barnidipine and the N-debenzylated product were observed as minor metabolites. Therefore, the primary metabolic pathways in man are (a) hydrolysis of the benzylpyrrolidine ester, (b) N-debenzylation, and (c) oxidation of the dihydropyridine ring. 3. When the SR and normal capsules were administered at a dose of 10 mg to six subjects in a crossover design, AUC 0-infinity of unchanged drug, M-3 and 4 in each subject receiving the SR were 97 +/- 15, 85 +/- 31 and 76 +/- 21% respectively of those subjects receiving the normal formulation. The sum of the excretion of urinary metabolites for the SR formulation was 65 +/- 6% of that for the normal formulation. These data suggest that the absorption of the SR formulation is slightly reduced but that its bioavailability is comparable to that of the normal formulation.     

Identification of hepatic metabolites of two highly carcinogenic polycyclic aza-aromatic compounds, 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine

The hepatic microsomal metabolites of the highly carcinogenic dimethylbenzacridines, 7,9-dimethylbenz[c]acridine (7,9-DMBAC), and 7,10-dimethylbenz[c]acridine (7,10-DMBAC) were obtained with preparations from 3-methylcholanthrene-pretreated rats. Metabolites were separated by reversed-phase HPLC and characterized using UV spectral data and chemical ionization-mass spectrometry after trimethylsilylation and GC. Comparisons with products formed in the presence of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide and with those formed from the three synthetic alcohol derivatives of each parent compound, aided the assignment of firm or tentative structures to 16 products from 7,9-DMBAC found in 22 reversed-phase chromatographic peaks, and for 17 products of 7,10-DMBAC found in 19 chromatographic peaks. The more abundant metabolites were derived from oxidation of the methyl groups. Other metabolites were dihydrodiols, epoxides, phenols and secondary metabolites. The 9-methyl group prevented dihydrodiol formation at the 8,9-position from 7,9-DMBAC, and for each carcinogen, the 3,4-dihydrodiol was formed. As well, 3,4-dihydrodiols of methyl oxidized compounds were found.     

Cytotoxic activities of Mannich bases of chalcones and related compounds

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.     

The cell surface of Trypanosoma musculi bloodstream forms. II. Lectin and immunologic studies

Report is given on a metabolic investigation with non-radioactive and 14C-labelled methiothepin(1-[10',11'-dihydro-8'-(methylthio)-dibenzo mean value of b,f thiepin-10'yl]-4-methyl-piperazine) in rat, dog, and man. After i.p. and oral administration of the drug to the rat, the metabolites of methiothepin were excreted fecally. In the same species, a considerable biliary secretion of the compounds has been demonstrated. In dog and in man, excreted metabolites have been found both in urine and feces after oral application of the drug. The biotransformation of methiothepin within the species investigated proceeds via hydroxylation, sulfoxidation, O-methylation, N-demethylation, N-oxidation and formation of conjugates. The large number of metabolites is due to the various sites of action within the molecule, that are accessible to in vivo oxidation. Of a large number of isolated positionally isomeric compounds, merely the basal structures could be clarified. Possibly the mode of biotransformation to which methiothepin is subjected in the organism, proves determinant for the way of excretion. In the rat, all metabolites are hydroxylated and reach the intestinal tract as conjugates with the bile. In dog and man, however, non-hydroxylated, sulfoxidized metabolites were likewise found, which were excreted mainly renally in both species.     

Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway

In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.     

Spectrofluorometric determination of substituted tetrahydrocarbazoles by a methylene blue sensitized photolytic reaction

A spectrofluorometric method was developed for the determination of 6-chloro-9-[2-(2-methyl-5-pyridyl)ethyl]-1,2,3,4-tetrahydrocarbazole-2-methanol hydrochloride and its carboxylic acid analog in blood and urine. It involves extraction of both compounds at neutral pH, either from blood into ethyl acetate (the residue of which is dissolved in either) or from urine directly into ether. Both the alcohol and the acid are separated from each other by selective extraction into acid or base, respectively, and then reextracted into either from the respective aqueous medium by appropriate pH adjustment. The residues of the ether extracts containing the compounds are dissolved separately in 0.25 N NH4OH. Methylene blue is added to all samples, which are then exposed to UV energy for 15 min to produce the fluorophores. The fluorescence of the solutions is read at 370 nm, with excitation at 340 nm. The linear range of quantitation of both compounds is 0.02-10 mug/each/ml of final solution. The method was applied to the determination of blood levels and urinary excretion of the alcohol and its acid metabolite in a dog.     

Reactivity of haloketenes and halothioketenes with nucleobases: chemical characterization of reaction products

Halothioketenes and haloketenes are postulated as intermediates in haloolefin bioactivation. Little is known about the interactions of these reactive intermediates with macromolecules such as DNA. DNA binding, however, may be relevant in the toxicity of the parent olefins since they or their proximate metabolites are genotoxic. This prompted us to elucidate the structures and properties of potential DNA adducts formed. Adenine, cytosine, guanine, and thymine were reacted with chloro- and dichlorothioketene, chloro- and dichloroketene, and chloro- and dichloroacyl chloride. While thymine did not react, adenine and cytosine formed stable DNA base adducts with all reaction partners as demonstrated by HPLC analysis. Guanine yielded only products with chloroketene and chloroacetyl chloride. The pH-dependent UV spectra, 1H and 13C NMR, FT-IR, and elemental analysis showed (i) nucleophilic attack of the exocyclic amino groups of the DNA bases yielded haloacyl (thio)amides with all reactants as clearly demonstrated by the FT-IR spectra; (ii) the sulfur in the initial thioamides seems to be rapidly exchanged with oxygen; (iii) the acyl chlorides form identical products but in lower yields as compared to the haloketenes. Reactions of the nucleosides with haloketenes showed the formation of similar nucleoside adducts upon HPLC and MS analysis. Beside the modification of the base moieties, additional peaks in the reaction mixtures analyzed suggested acylation of the deoxyribose hydroxyl groups. In aqueous solutions at pH 7 N6-(chloroacetyl)adenine, N4-(chloroacetyl)cytosine, and N2-(chloroacetyl)guanine are not stable and cleaved to the original base or form 1,N6-acetyladenine, 3,N4-acetylcytosine, 1,N2-acetylguanine, and N2,3-acetylguanine. Under the same conditions, N6-(dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine were completely hydrolyzed to adenine and cytosine, respectively. All haloacyl DNA base adducts proved to be stable at pH 5 but were rapidly degraded at neutral or alkaline pH. The compounds with an additional five-membered ring remained unchanged after 1 week at room temperature. All synthesized DNA base adducts except N2-(chloroacetyl)guanine and 1,N2-acetylguanine were fluorescent. The characterized compounds, especially the etheno (epsilon) base adduct-related derivatives, may represent potential DNA adducts formed as a consequence of haloolefin bioactivation.     

Bioactivation of a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, via glucuronidation. LC/MS/MS characterization of the GSH-glucuronide diconjugates

The hepatotoxicity of the anticonvulsant drug valproic acid may be associated with the formation of potentially reactive metabolites, one of which is (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene VPA). This report describes the characterization of new GSH-related conjugates of this diene. Bile samples collected from male Sprague-Dawley rats dosed ip with (E)-2,4-diene VPA (100 mg/kg) were analyzed by LC/MS/MS. Initial Q1 parent in scanning indicated that the daughter ions m/z 162 and 123 could be derived from the ions at m/z 624 and 480, respectively. Subsequent collision-induced dissociation (CID) of these parent ions revealed a common neutral loss of 176 Da which is diagnostic for glucuronides. A similar neutral loss of 176 Da was observed in daughter ion spectra of the biliary metabolites arising from [2H7]-4-ene VPA dosed ip to rats, where the ion fragments containing the VPA portion were 7 amu higher than those derived from the unlabeled drug. CID of the ion at m/z 624 also gave fragments characteristics for GSH conjugates such as the loss of glycine and glutamate moieties. Based on the MS data, the metabolites were assigned the diconjugate structures 1-O-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-beta-D-glucur onide (5-GS-3-ene VPA-glucuronide I, MH+, 624) and the corresponding 5-NAC-3-ene VPA-glucuronide (MH+, 480). Further proof of structural identity was obtained from 1H NMR of HPLC-purified metabolites. The amount of biliary 5-GS-3-ene VPA-glucuronide I was 7-fold greater than the corresponding 5-GS-3-ene VPA, the sum of the two metabolites accounting for 6.6% of the dose. Incubation of 1-O-(2-propyl-2,4-pentadienoyl)-beta-D-glucuronide (2,4-diene VPA-glucuronide) with GSH in the presence or absence of GST enzyme led to the formation of 5-GS-3-ene VPA-glucuronide I which was readily detected by LC/MS/MS, suggesting that in vivo the diconjugate may arise from the reaction of GSH with 2,4-diene VPA-glucuronide. To our knowledge, this is the first recorded instance in which glucuronide formation activates a drug to further conjugate with GSH via a Michael addition reaction.     

Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: potential relevance to the pathogenesis of Parkinson's disease

We have proposed that a very early step in the pathogenesis of idiopathic Parkinson's disease is the elevated translocation of L-cysteine into neuromelanin-pigmented dopaminergic neurons in the substantia nigra. This influx of L-cysteine was proposed to divert the normal neuromelanin pathway by scavenging dopamine-o-quinone, formed by autoxidation of cytoplasmic dopamine, to give initially 5-S-cysteinyldopamine, which is further oxidized to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). In a recent report, it was demonstrated that DHBT-1 evokes inhibition of complex I respiration when incubated with intact rat brain mitochondria and a time-dependent irreversible inhibition of NADH-coenzyme Q1 (CoQ1) reductase when incubated with mitochondrial membranes. In this study, it is established that the time dependence of NADH-CoQ1 reductase inhibition reflects the oxidation of DHBT-1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an o-quinone imine intermediate that rearranges to 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (BT-1) and decarboxylates to 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine (BT-2), which are further catalytically oxidized to o-quinone imine intermediates. The electrophilic o-quinone imine intermediates formed in these mitochondria-catalyzed oxidations of DHBT-1, BT-1, and BT-2 are proposed to bind covalently to key sulfhydryl residues at the complex I site, thus evoking irreversible inhibition of NADH-CoQ1 reductase. Evidence for this mechanism derives from the fact that greater than equimolar concentrations of glutathione completely block inhibition of NADH-CoQ1 reductase by DHBT-1, BT-1, and BT-2 by scavenging their electrophilic o-quinone imine metabolites to form glutathionyl conjugates. The results of this investigation may provide insights into the irreversible loss of glutathione and decreased mitochondrial complex I activity, which are both anatomically specific to the substantia nigra and exclusive to Parkinson's disease.     

A lignan and four terpenoids from Brucea javanica that induce differentiation with cultured HL-60 promyelocytic leukemia cells

A novel lignan, guaiacylglycerol-beta-O-6'-(2-methoxy)cinnamyl alcohol either, three known simaroubolides, brusatol, dehydrobrusatol, yadanziolide C, and the known terpenoid, blumenol A, were obtained as active compounds from an ethyl acetate-soluble extract of Brucea javanica, using a bioassay based on the induction of cell differentiation with human promyelocytic leukemia (HL-60) cells. Also obtained were the known coumarinolignan, cleomiscosin A, and the known quassinoid glycoside, bruceoside B, which were inactive in the HL-60 cell test system. The structure of the new lignan was determined by a combination of 1D and 2D NMR techniques.     

Cocaine and its metabolites constrict cerebral arterioles in newborn pigs

We examined the effect of cocaine and several of its metabolites on cerebral arterioles in newborn pigs and evaluated the sympathomimetic properties of each of the compounds as a vasoactive mechanism. After piglets were equipped with closed cranial windows, compounds were suffused over the brain surface and pial arteriolar diameter (base line, approximately 100 microns) was recorded. Cocaine, cocaethylene, norcocaine, ecogonine, benzoylecgonine and ecgonine methylester each caused a dose-dependent (10(-8) M to 10(-4) M) decrease in pial arteriolar diameter: maximum percent reductions in diameter induced by each compound (10(-4) M) were, respectively, 12 +/- 1, 12 +/- 2, 11 +/- 1, 7 +/- 1, 7 +/- 2 and 5 +/- 1. In analyzing the dose-response curves, cocaethylene was the most potent vasoconstrictor, followed by cocaine, norcocaine and then ecogonine, benzoylecgonine and ecgonine methylester. Cerebral vasoconstriction induced by topically applied norepinephrine was enhanced by cocaine, norcocaine and cocaethylene, but not by the other three metabolites. Topical application of phentolamine failed to block vasoconstriction elicited by cocaine or its metabolites, although it did block vasoconstriction elicited by norepinephrine. These observations indicate that cocaine and its metabolites constrict the immature cerebrovasculature by a non-sympathomimetic mechanism.     

Fate of etiproston, a synthetic analogue of PGF2 alpha, in cows

The pharmacokinetics and metabolic fate of labelled compounds were investigated after intramuscular administration of 3H-radiolabelled etiproston to nine cows. Elimination was rapid (t 1/2 beta = 2.8 h). Forty-eight h after administration 92.6% of the radioactivity had been eliminated, mainly via the urinary (66% at 48 h) and faecal routes (26% at 48 h). In comparison, little elimination in milk occurred (less than 0.034% dose/l by 24 h). Radioactivity at the injection site 48 h after administration was seen in one cow (< 4.68 x 10(-5%) dose/g). No radioactivity was detected in the tissues. Urinary metabolites were purified and isolated using XAD-2 extraction and preparative HPLC in reverse and normal phases. The main urinary metabolite, identified by mass spectrometry, was the tetranor acid derivative in equilibrium with its lactone form.     

Pharmacokinetics of active drug metabolites after oral administration of perillyl alcohol, an investigational antineoplastic agent, to the dog

The monocyclic terpene d-limonene, a major component in many citrus essential oils (1-3), has been used for many years as a flavoring agent, food additive, and fragrance (1, 2). It was recently demonstrated that limonene exhibits both chemopreventive and therapeutic effects against chemically induced mammary tumors in rats (4-10). Mechanistic studies revealed that limonene inhibits the posttranslational isoprenylation of 21-26 kDa cellular proteins implicated in cell growth and proliferation (11-13). Limonene is extensively metabolized by a variety of mammalian species (14-17). Its principal circulating metabolites identified in the rat, perillic acid and dihydroperillic acid, are also effective inhibitors of isoprenylation and cellular proliferation in vitro (17, 18). Furthermore, one of the metabolic precursors of these compounds, perillyl alcohol (16), is considerably more potent than limonene against the in vivo rat mammary tumor models (19). A preliminary report of an ongoing phase I clinical trial with limonene indicated that a single oral dose of 100 mg/kg is well tolerated (20). However, an extrapolation based upon the rat mammary tumor regression studies suggests that the minimum human dose requirement would be 1000 mg/kg/ day (6). The administration of such a large dose, which amounts to more than 80 ml of an oily volatile liquid, on a continuing basis may cause problems. Thus, perillyl alcohol is currently being developed as a clinical candidate at the National Cancer Institute because of its greater potency than limonene, which may enable potentially effective systemic concentrations of the active principals to be achieved at considerably lower doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

1- and 3-hydroxylations, in addition to 4-hydroxylation, of debrisoquine are catalyzed by cytochrome P450 2D6 in humans

Twenty-one healthy Swedish Caucasian volunteers, representing different groups with 0-13 functional cytochrome P450 (CYP) 2D6 genes, were given a single oral dose of 20 mg of debrisoquine. The hypothesis of further oxidation of the main metabolite, (S)-4-hydroxydebrisoquine, in subjects with multiple CYP2D6 genes was tested by screening the 0-8-hr urine samples for dihydroxylated metabolites of debrisoquine with protonated molecular ions at m/z 208, using LC/MS. Three peaks were detected in a subject with 13 functional CYP2D6 genes. One compound was identified as dihydroxylated debrisoquine (presumably with hydroxylation at position 4 plus one of the positions in the aromatic ring). This metabolite had not been previously demonstrated in humans and was detected only in this subject. The other two compounds, which were measurable in various amounts in all subjects investigated, were identified as 2-(guanidinomethyl)phenylacetic acid and 2-(guanidinoethyl)benzoic acid. They had been previously detected in the urine of humans, dogs, and rats. They were distinguished by acid-catalyzed deuterium exchange of the hydrogens at the alpha-position, with respect to the carboxylic acid group, of the former but not the latter acid. The acids are formed by 3- and 1-hydroxylation of debrisoquine, respectively, followed by ring opening to aldehydes, which are further oxidized to acids. Strong Spearman rank correlations between debrisoquine products of 1- or 3-hydroxydebrisoquine and debrisoquine/4-hydroxydebrisoquine ratios (rS = 0.97 and rS = 0.96, respectively), using the intensity of the peaks of the reconstructed ion-current chromatograms, clearly showed that both hydroxylation steps are catalyzed by CYP2D6. Because reference compounds for the two acids were not available, the absolute quantities could not be determined.