Species differences in the metabolism of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid in vitro: implications for prediction of metabolic interactions in vivo

1. Mouse studies have indicated that the antitumour effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) are dramatically potentiated in combination with other drugs, and it has been proposed that optimization of the therapeutic potential of DMXAA would exploit combination therapy. The aim was to identify the most appropriate animal model for further investigations of the pharmacokinetics of possible DMXAA-drug combinations and their extrapolation to patients. 2. Qualitatively, the metabolic profile for DMXAA in liver microsomes was similar in mouse, rat, rabbit and humans, with glucuronidation and 6-methylhydroxylation the two major metabolic pathways. In all species, the intrinsic clearance by glucuronidation was at least 2-fold that due to hydroxylation. There was significant variability in the in vitro kinetic parameters (Km, Vmax), with the mouse being the least efficient DMXAA metabolizer compared with the other species. 3. Mouse, rat and rabbit renal microsomes exhibited DMXAA glucuronidation activity, but only the rabbit showed 6-methylhydroxylation. For the total in vitro CL(int) (Vmax/Km) by glucuronidation and 6-methylhydroxylation, the ratio of kidney:liver was 0.67, 0.03 and 0.34 in the mouse, rat and rabbit respectively. However, taking into account the liver and kidney weight difference, it is apparent that the in vivo renal metabolism would not be a major contributor to the overall elimination of DMXAA. 4. The inhibitory profile for liver DMXAA glucuronidation was similar across species, but there was remarkable interspecies variability in the inhibition of liver DMXAA 6methylhydroxylation. 5. Extrapolation of in vitro intrinsic clearance to in vivo gave a significant underestimation of plasma clearance for all species. However, there was a significant allometric relationship for plasma clearance and volume of distribution, but not for maximum tolerated dose across species. 6. The results indicate that animal models may have a limited role in the extrapolation to patients of drug interactions with agents such as DMXAA that have immunomodulating activity that may vary widely between species.     

Injection timing determines whether intragastric ethanol produces conditioned place preference or aversion in mice

Previous studies have shown that mice develop conditioned place preference (CPP) when ethanol is administered by intraperitoneal (ip) or intravenous (iv) injection. The present studies examined CPP in mice using the intragastric (ig) route of administration. Inbred mice were surgically implanted with chronic intragastric cannulae and exposed to an unbiased place conditioning procedure in which infusion of ethanol (2 or 4 g/kg) was paired with a conditioned stimulus (CS+). A different CS was paired with water. In Experiments 1-2, ethanol was infused just before exposure to CS+. Contrary to previous studies involving intraperitoneal injection, infusion of 4 g/kg ig ethanol produced a significant conditioned place aversion (CPA). However, when a 5-min delay was inserted between infusion and CS exposure (Experiments 3-4), the same dose produced CPP. These outcomes are not consistent with expectations derived from a recent study in selectively bred rats, suggesting that sensitivity to ethanol reward is enhanced by intragastric administration. However, the finding that intragastric ethanol can produce either CPP or CPA depending on dose and injection timing is consistent with previous intraperitoneal ethanol studies in mice. Although the parameters differ for each route of administration, it appears that the same underlying processes can be invoked to explain how manipulation of injection timing affects the direction of ethanol-induced place conditioning. More specifically, in both cases, CPA can be attributed to an initial, short-lived aversive effect, whereas CPP can be attributed to a delayed rewarding effect of ethanol.     

Trough:peak ratio and smoothness index in the evaluation of 24-h blood pressure control in hypertension: a comparative study between valsartan/hydrochlorothiazide combination and amlodipine

OBJECTIVE: The aim of this study was to measure the time-effect profiles of a once-daily administered valsartan/hydrochlorothiazide combination and amlodipine on blood pressure using various indices derived from 24-h ambulatory blood pressure (BP) monitoring. METHODS: Of the 310 randomized outpatients with uncomplicated mild-to-moderate primary hypertension, 259 (133 on valsartan/hydrochlorothiazide, 126 on amlodipine) were eligible for analysis. After a 2-week placebo wash-out period, the patients were randomly allocated to treatment with either valsartan 80 mg once daily (o.d.) or amlodipine 5 mg o.d. for 4 weeks; in the case of an unsatisfactory blood pressure response, the treatments could be respectively changed to the fixed combination of valsartan 80 mg plus hydrochlorothiazide 12.5 mg o.d. or amlodipine 10 mg o.d. for a further 8 weeks. The trough:peak ratio (global and individualized approaches) and smoothness index (i.e., the ratio between the average of the 24-hourly BP changes after treatment and the corresponding standard deviation) were calculated from 24-h ambulatory blood pressure recordings made after the placebo period and after 4 weeks and 12 weeks of active treatment. RESULTS: Both regimens effectively lowered systolic and diastolic ambulatory pressures after 4 weeks and 12 weeks (all P<0.001) but, among the responders, the valsartan/hydrochlorothiazide combination had a greater antihypertensive effect during the night-time hours after 12 weeks (P=0.03/0.02). In the responders, the placebo-adjusted, mean trough:peak ratios were 0.76/0.74 in the valsartan/hydrochlorothiazide group (n = 111) and 0.66/0.62 in the amlodipine group (n = 101). The corresponding global trough:peak ratios were 0.61/0.57 for the valsartan/hydrochlorothiazide combination and 0.56/0.56 for amlodipine. However, the between-group differences in individual or global trough:peak ratios were not significant. The smoothness index was slightly, but insignificantly, greater for valsartan/hydrochlorothiazide than for amlodipine in the responders and the groups as a whole. CONCLUSION: Valsartan/hydrochlorothiazide and amlodipine were equally effective in reducing ambulatory BP, but the valsartan/hydrochlorothiazide combination led to more homogeneous BP control during the inter-dosing interval. Trough:peak ratio and smoothness index did not reflect this finding accurately.     

GC-MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection

An analytical GC–MS method based on nonpolar fused silica capillary column was developed to analyze the lipophilic constituents, mainly alkamides, from the root extracts of Echinacea purpurea (L.) Moench. In particular, the proposed method was applied to evaluate the phytochemical impacts of cucumber mosaic cucumovirus (CMV) infection on the plant's lipophilic marker phytochemicals. Methanolic (70% v/v) extracts, obtained from root materials by ultrasonic treatments, were subjected to liquid–liquid extraction with n-hexane-ethyl acetate (1:1 v/v) to recover the lipophilic, volatile to semivolatile, principles. Seventeen components, including the 11 alkamides known to E. purpurea roots, were identified in the GC–MS traces of the analyzed fractions and efficiently separated in a turnaround time of 25 min. CMV infection was found to be responsible for significant variations in the relative compositions of the major constituents, in particular germacrene D, Dodeca-2E, 4E, 8Z, 10Z(E)-tetraenoic acid isobutylamide cis/trans isomers, Undeca-2Z, 4E-diene-8, 10-diynoic acid isobutylamide and Dodeca-2E, 4Z-diene-8, 10-diynoic acid isobutylamide.     

Mazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were determined. Several active analogues were also evaluated for their ability to block uptake of DA, 5-HT, and NE and inhibit binding of [(125)I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H --> 5-OH) to mazindol on rat striatal membranes and HEK-hDAT cells. The 4',7,8-trichloro analogue (38) of mazindol was the most potent and selective ligand for HEK-hDAT cells (DAT K(i) = 1.1 nM; SERT/DAT = 1283 and NET/DAT = 38). Experimental results strongly favor the cyclic or ol tautomers of 2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.     

High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PML-RARalpha expression

Transgenic mice expressing PML-RARalpha in early myeloid cells under control of human cathepsin G regulatory sequences all develop a myeloproliferative syndrome, but only 15% to 20% develop acute promyelocytic leukemia (APL) after a latent period of 6 to 14 months. However, this transgene is expressed at very low levels in the bone marrow cells of transgenic mice. Because the transgene includes only 6 kb of regulatory sequences from the human cathepsin G locus, we hypothesized that sequences required for high-level expression of the transgene might be located elsewhere in the cathepsin G locus and that a knock-in model might yield much higher expression levels and higher penetrance of disease. We, therefore, targeted a human PML-RARalpha cDNA to the 5' untranslated region of the murine cathepsin G gene, using homologous recombination in embryonic stem cells. This model produced a high-penetrance APL phenotype, with more than 90% of knock-in mice developing APL between 6 and 16 months of age. The latent period and phenotype of APL (including a low frequency of an interstitial deletion of chromosome 2) was similar to that of the previous transgenic model. Remarkably, however, the expression level of PML-RARalpha in bone marrow cells or APL cells was less than 3% of that measured in the low-penetrance transgenic model. Although the explanation for this result is not yet clear, one hypothesis suggests that very low levels of PML-RARalpha expression in early myeloid cells may be optimal for the development of APL in mice.     

Lateralization of auditory sensory gating and neuropsychological dysfunction in schizophrenia

OBJECTIVE: Sensory gating assessed via EEG in a paired-click paradigm has often served as a neurophysiological metric of attentional function in schizophrenia. However, the standard EEG measure of sensory gating using the P50 component at electrode Cz does not foster differential assessment of left and right hemisphere contributions. Magnetoencephalography (MEG) is complementary to EEG, and its analogous M50 component may be better suited for localization and analysis of such lateralized cortical generators. The authors hypothesized that 1) auditory gating would be evident in M50 sources in superior temporal gyrus, demonstrating ratios similar to P50; 2) M50 would resemble P50 in distinguishing gating in comparison subjects and patients with schizophrenia, but M50 would show lateralization of the gating deficit; and 3) P50 and M50 sensory gating ratios would predict neuropsychological measures in patients and comparison subjects, with the MEG identification of left and right hemisphere sources allowing for the evaluation of lateralization in brain-behavior relationships. METHOD: Event-related EEG and MEG recordings were simultaneously obtained from 20 patients with schizophrenia and 15 comparison subjects. P50 amplitudes, M50 dipole source strengths, and P50 and M50 gating ratios were compared and assessed with respect to scores on neuropsychological performance measures. RESULTS: M50 dipoles localizing to superior temporal gyrus demonstrated gating similar to that of P50. As expected, patients demonstrated less P50 gating than did comparison subjects. Left (but not right) hemisphere M50 gating 1) correlated with EEG gating, 2) differentiated patients and comparison subjects, and 3) correlated with neuropsychological measures of sustained attention and working memory. CONCLUSIONS: Converging evidence from EEG, MEG, and neuropsychological measures points to left hemisphere dysfunction as strongly related to the well-established sensory gating deficit in schizophrenia.     

Structure and membrane affinity of a suite of amphiphilic siderophores produced by a marine bacterium

Iron concentrations in the ocean are low enough to limit the growth of marine microorganisms, which raises questions about the molecular mechanisms these organisms use to acquire iron. Marine bacteria have been shown to produce siderophores to facilitate iron(III) uptake. We describe the structures of a suite of amphiphilic siderophores, named the amphibactins, which are produced by a nearshore isolate, gamma Proteobacterium, Vibrio sp. R-10. Each amphibactin has the same Tris-hydroxamate-containing peptidic headgroup composed of three ornithine residues and one serine residue but differs in the acyl appendage, which ranges from C-14 to C-18 and varies in the degree of saturation and hydroxylation. Although amphiphilic siderophores are relatively rare, cell-associated amphiphilic siderophores are even less common. We find that the amphibactins are cell-associated siderophores. As a result of the variation in the nature of the fatty acid appendage and the cellular location of the amphibactins, the membrane partitioning of these siderophores was investigated. The physiological mixture of amphibactins had a range of membrane affinities (3.8 x 10(3) to 8.3 x 10(2) M(-1)) that are larger overall than other amphiphilic siderophores, likely accounting for their cell association. This cell association is likely an important defense against siderophore diffusion in the oceanic environment. The phylogenetic affiliation of Vibrio sp. R-10 is discussed, as well as the observed predominance of amphiphilic siderophores produced by marine bacteria in contrast to those produced by terrestrial bacteria.