Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

In vitro Antitumour Activity of Orsellinates

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative. and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out hi vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC(50) Values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 mu g/ml, against all the cell lines tested. The compound was more active (IC(50), = 11.4 mu g/mL) against B16-F10 cells than was cisplatin (12.5 mu g/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC(50) value higher than 50 mu g/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Influence of N-Hexane Inhalation on the Enantioselective Pharmacokinetics and Metabolism of Verapamil in Rats

Verapamil (VER) is commercialized as a racemic mixture of the (+)-(R)-VER and (-)-(S)-VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP-dependent pathways. N-hexane is a solvent that can alter the metabolism of CYP-dependent drugs. The present study investigated the influence of n-hexane (nose-only inhalation exposure chamber at concentrations of 88, 176, and 352 mg/m(3)) on the kinetic disposition of the (+)-(R)-VER, (-)-(S)-VER, (R)-NOR and (S)-NOR in rats treated with a single dose of racemic VER (10 mg/kg). VER and NOR enantiomers in rat plasma was analyzed by LC-MS/MS (m/z = 441.3 > 165.5 for the NOR and m/z 455.3 > 165.5 for the VER enantiomers) using a Chiralpak (R) AD column. Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was enantioselective in control rats, with higher plasma proportions of the (-)-(S)-VER eutomer (AUC(0-infinity) = 250.8 vs. 120.4 ng/ml/h; P <= 0.05, Wilcoxon test). The (S)-NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC(0-infinity) ratio of 1.5. The pharmacokinetic parameters AUC(0-infinity), Cl/F, Vd/F, and t(1/2) obtained for VER and NOR enantiomers were not altered by nose-only exposure to n-hexane at concentrations of 88, 176, or 352 mg/m(3) (P > 0.05, Kruskal-Wallis test). However, the verapamil kinetic disposition was not enantioselective for the animals exposed to n-hexane at concentrations equal to or higher than the TLV-TWA. This finding is relevant considering that the (-)-(S)-VER eutomer is 10-20 times more potent than R-(+)-VER in terms of its chronotropic effect on atrioventricular conduction in rats and humans. Chirality 22:29-34, 2010. (C) 2009 Wiley-Liss, Inc.

[beta]-Hydroxy-F128b-Methylbutyrate (HMB) Supplementation and the Promotion of Muscle Growth and Strength

[beta]-Hydroxy [beta]-methylbutyrate (HMB), a metabolite of the essential amino acid leucine, is one of the latest dietary supplements promoted to enhance gains in strength and lean body mass associated with resistance training. Unlike anabolic hormones that induce muscle hypertrophy by increasing muscle protein synthesis, HMB is claimed to influence strength and lean body mass by acting as an anticatabolic agent, minimising protein breakdown and damage to cells that may occur with intense exercise. Research on HMB has recently tested this hypothesis, under the assumption that it may be the active compound associated with the anticatabolic effects of leucine and its metabolites. While much of the available literature is preliminary in nature and not without methodological concern, there is support for the claims made regarding HMB supplementation, at least in young, previously untrained individuals. A mechanism by which this may occur is unknown, but research undertaken to date suggests there may be a reduction in skeletal muscle damage, although this has not been assessed directly. The response of resistance trained and older individuals to HMB administration is less clear. While the results of research conducted to date appear encouraging, caution must be taken when interpreting outcomes as most manuscripts are presented in abstract form only, not having to withstand the rigors of peer review. Of the literature reviewed relating to HMB administration during resistance training, only 2 papers are full manuscripts appearing in peer reviewed journals. The remaining 8 papers are published as abstracts only, making it difficult to critically review the research. There is clearly a need for more tightly controlled, longer duration studies to verify if HMB enhances strength and muscular hypertrophy development associated with resistance training across a range of groups, including resistance trained individuals.

Investigation of the antinociceptive efficacy and relative potency of extended duration injectable 3-acylmorphine-6-sulfate prodrugs in rats

This investigation was designed to examine the antinociceptive activity in rats of 3-O-acyl prodrugs of M6S relative to the parent drug, after intravenous and intramuscular injection, using the tail flick latency test of antinociception. M6S, 3-acetylmorphine-6-sulfate (3AcM6S), 3-propionylmorphine-6-sulfate (3PrM6S), 3-butanoylmorphine-6-sulfate (3BuM6S) and 3-heptanoylmorphine-6-sulfate (3HpM6S) were administered by the IV route in a dose of 4.10 mu mol/kg. Relatively high levels of antinociception (>40% Maximum Possible Effect) were achieved following administration of M6S, 3AcM6S and 3PrM6S, whereas insignificant antinociception (<20%MPE) was achieved following administration of 3BuM6S or 3HpM6S. Although the mean duration of action for 3AcM6S (6 h) was longer than for M6S or 3PrM6S (4 h), the mean area (+/- S.E.M.) under the degree of antinociception versus time curve (AUG) for 3AcM6S (151.6 +/- 6.9%MPE h) was not significantly different (p <0.05) from that for M6S (120.8 +/- 32.7%MPE h) or for 3PrM6S (106.0 +/- 21.3%MPE h). The mean ED50 (range) doses for M6S, 3AcM6S and 3PrM6S were calculated to be 4.16 (3.61-4.48), 4.32 (3.55-5.09) and 4.54 (4.21-4.79) mu mol/kg, respectively. Preliminary studies were conducted on potential long-acting formulations containing 8 x ED50 doses of M6S and the 3-acetyl and 3-propionyl esters suspended in soybean oil. These showed that 3PrM6S gave a greater AUC (mean + S.E.M.) (1087.4 +/- 97.4%MPE h) and longer duration of action (20 h) than did M6S (613.1 +/- 155.9%MPE h; 10 h duration) or 3AcM6S (379.3 + 114.2%MPE h: 8 h duration). Further studies are needed to more fully investigate these findings. (C) 1998 Elsevier Science B.V. All rights reserved.

Speciation of arsenic metabolites in the urine of occupational workers and experimental rats using an optimised hydride cold-trapping method

A hydride cold-trapping technique was developed and optimised for the measurement of urinary arsenic metabolites. The analytical precision of the method was found to be 6.1, 4.0 and 4.8% (n = 5) for inorganic arsenic (As-i), monomethylarsonate (MMA) and dimethylarsinate (DMA), respectively, with recoveries close to 100%, The detection limits were 1.0, 1.3 and 3 ng for As-i, MMA and DMA, respectively. The method was then used to analyse urine samples obtained from three groups of workers for occupational exposure in three companies where copper chrome arsenate was used for timber treatment. The results were compared with those for a normal control group of laboratory workers. Arsenic and its metabolites were also measured in experimental rats given 5 mg As kg(-1) body mass by oral gavage in the form of sodium arsenite, calcium arsenite or sodium arsenate. Occupational workers showed a significantly higher excretion of As-i, Up to two fold increases of urinary As-i excretion in rats compared with control rats were also observed in animals dosed with various forms of arsenicals. The method is suitable for the measurement of arsenic metabolites in urine of both humans and experimental animals.

PACAP stimulates dopamine neuronal activity in the medial basal hypothalamus and inhibits prolactin

In sheep intracerebroventricular injection of PACAP (10 nmol) significantly (P < 0.01) stimulated the levels of the dopamine metabolite DOPAC within the medial basal hypothalamus las measured by in vivo microdialysis) and this effect was temporally correlated with a significant (P < 0.05) suppression in peripheral prolactin concentrations. This result is in accord with the hypothesis that PACAP suppresses prolactin secretion from the anterior pituitary gland by stimulating dopamine release from tuberoinfundibular dopaminergic neurons. (C) 1998 Elsevier Science B.V.

Dragmacidins: New protein phosphatase inhibitors from a southern Australian deep-water marine sponge, Spongosorites sp.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

The excitatory behavioral and antianalgesic pharmacology of normorphine-3-glucuronide after intracerebroventricular administration to rats

In the adult male Sprague-Dawley rat, a species commonly used to study tolerance to the antinociceptive effects of morphine, approximate to 10% of the morphine dose is metabolized to normorphine-3-glucuronide (NM3G). In contrast, NM3G is a relatively minor metabolite of morphine in human urine reportedly accounting for approximate to 1% of the morphine dose. To date, the pharmacology of NM3G has been poorly characterized. Therefore, our studies were designed to determine whether the intrinsic pharmacology of NM3G is similar to that of morphine-3-glucuronide (M3G), the major metabolite of morphine, which has been shown to be a potent central nervous system (CNS) excitant and to attenuate the intrinsic antinociceptive effects of morphine in rats. The CNS excitatory potency of NM3G was found to be approximately half that of M3G, inducing convulsions in rats at intracerebroventricular (i.c.v.) doses of greater than or equal to 16.8 nmol. When administered before morphine (70 nmol i.c.v.), NM3G (8.9 nmol i.c.v.) attenuated antinociception for up to 2 hr, but when administered after morphine, no significant attenuation of morphine antinociception was observed. Thus, after i.c.v. administration, NM3G like M3G, is a potent CNS excitant and antianalgesic in the rat. NM3G may therefore play a role in the development of tolerance to the antinociceptive effects of morphine in the rat as has been proposed previously for M3G.

The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp

2D-NMR spectroscopic data is reported for the haliclonacyclamines A - D (1)-(4) and for two bismethiodide adducts (5) and (6). The structures of two new alkaloids, haliclonacyclamines C (3) and D (4), which are the 15,16-dihydro analogues of the haliclonacyclamines A (1) and B (2) are described. Revised assignments deduced by 2D-INADEQUATE spectroscopy are presented for (1) and (2). The alkene substituent in the C,, spacer group of (2) and (4) is positioned between C27-C28 by NMR, and confirmed by x-ray structural analysis for (2). Metabolite (3) has a C25-C26 double bond. (C) 1998 Elsevier Science Ltd. All rights reserved.

A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation

Light-microscopic and electron-microscopic studies of the tropical marine sponge Haliclona sp. (Or der: Haplosclerida Family: Haliclonidae) from Heron Island, Great Barrier Reef, have revealed that this sponge is characterized by the presence of dinoflagellates and by nematocysts. The dinoflagellates are 7-10 mu m in size, intracellular, and contain a pyrenoid with a single stalk, whereas the single chloroplast is branched, curved, and lacks grana. Mitochondria are present, and the nucleus is oval and has distinct chromosomal structure. The dinoflagellates are morphologically similar to Symbiodinium microadriaticum, the common intracellular symbiont of corals, although more detailed biochemical and molecular studies are required to provide a precise taxonomic assignment. The major sponge cell types found in Haliclona sp, are spongocytes, choanocytes, and archaeocytes; groups of dinoflagellates are enclosed within large vacuoles in the archaeocytes. The occurrence of dinoflagellates in marine sponges has previously been thought to be restricted to a small group of sponges including the excavating hadromerid sponges; the dinoflagellates in these sponges are usually referred to as symbionts. The role of the dinoflagellates present in Haliclona sp. as a genuine symbiotic partner requires experimental investigation. The sponge grows on coral substrates, from which it may acquire the nematocysts, and shows features, such as mucus production, which are typical of some excavating sponges. The cytotoxic alkaloids, haliclonacyclamines A and B, associated with Haliclona sp. are shown by Percoll density gradient fractionation to be localized within the sponge cells rather than the dinoflagellates. The ability to synthesize bioactive compounds such as the haliclonacyclamines may help Haliclona sp. to preserve its remarkable ecological niche.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences; In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

A H-1 MRS study of probable Alzheimer's disease and normal aging: Implications for longitudinal monitoring of dementia progression

In order to evaluate the capability of H-1 MRS to monitor longitudinal changes in subjects with probable Alzheimer's disease (AD), the temporal stability of the metabolite measures N-acetylaspartate and N-acetylas-partylglutamate (NA), total Creatine (Cr), myo-Inositol (mI), total Choline (Chol), NA/Cr, mI/Cr, Chol/Cr and NA/mI were investigated in a cohort of normal older adults. Only the metabolite measures NA, mi, Cr, NA/Cr, mI/Cr, and NA/mI were found to be stable after a mean interval of 260 days. Relative and absolute metabolite measures from a cohort of patients with probable AD were subsequently compared with data from a sample of normal older adult control subjects, and correlated with mental status and the degree of atrophy in the localized voxel. Concentrations of NA, NA/Cr, and NA/mI were significantly reduced in the AD group with concomitant significant increases in mi and mI/Cr. There were no differences between the two groups in measures of Cr, Chol, or Chol/Cr. Significant correlations between mental status as measured by the Mini-Mental State Examination and NA/mI, mI/Cr and NA were found. These metabolite measures were also significantly correlated with the extent of atrophy (as measured by CSF and GM composition) in the spectroscopy voxel. (C) 1999 Elsevier Science Inc.

Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a P-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 Angstrom N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba(67,95)]HIVPR and [Lys(7),Ile(33),Aba(67,95)]- HIVPR used in this work were shown to have very similar crystal structures.