Andrew J. Hoffman

Effects of enantiomers of MDA, MDMA and related analogues on [3H]serotonin and [3H]dopamine release from superfused rat brain slices

The primary amines 3,4-methylenedioxyamphetamine (MDA), and 1-(1,3-benzodioxol-5-yl)-2-butanamine (BDB) were measured for efficacy in release of [3H]serotonin (5-HT) from rat hippocampal slices, and release of [3H]dopamine (DA) from rat caudate nucleus slices. The N-methyl derivatives of MDA and BDB, MDMA and MBDB, respectively, and the optical antipodes of these four agents were compared in this paradigm. All of the test compounds demonstrated a similar efficacy of [3H]5-HT release in the micromolar concentration range. No significant stereoselectivity was seen in measurements of 5-HT release. However, striking differences were found between the test compounds when [3H]DA release was studied. N-methylation of racemic MDA resulted in a decreased ability to release DA, while side chain extension from alpha-methyl to alpha-ethyl completely abolished this activity. Stereoselectivity for the S-(+)-isomers of MDA and MDMA was also demonstrated in the DA release studies. Correlation of these biochemical findings with human subjective reports indicates that serotonin release may play a more important role in the mechanism of action than does dopamine release.

The Effect of the Undigested Fraction of Maize Products on the Activity and Composition of the Microbiota Determined in a Dynamic in Vitro Model of the Human Proximal Large Intestine

Objective: To investigate the effect of 5 newly developed maize-based fibers on the activity and composition of the microbiota in the colon. The fibers tested were glucose-based and had variable structures, including 2 resistant starch preparations, soluble corn fiber, pullulan, and soluble fiber dextrin. Methods: The fibers were predigested, mono- and disaccharides were removed, and the residual polymer was used to assess the production of microbial metabolites and changes in composition of the microbiota using a dynamic, validated, in vitro model of the large intestine. Results: Microbial metabolite analysis showed an increase in short-chain fatty acids for all fibers, with varying levels of butyrate production for each fiber. The greatest increase of butyrate, both in terms of absolute amounts and as a proportion of total short-chain fatty acids, was observed for pullulan. All fibers also reduced toxic metabolites from protein fermentation compared to the poorly fermentable control (cellulose). Microbial composition was assessed using a micro-array platform. All fibers showed increases of bifidobacteria and some Lactobacillus species, although different species were stimulated by different fibers. Pullulan showed the largest increase of bifidobacteria. Conclusions: All fibers showed prebiotic activity in terms of increases in growth and/or activity of beneficial microbes. In addition, compared to the control, health-promoting metabolites were produced in higher amounts, while putrefactive metabolites were reduced for all fibers. The importance of the findings lies in the fact that the newly developed, maize-based fibers shift the intestinal environment to a healthier milieu, with increased health-promoting metabolites and health-beneficial microbes.

Common receptors for hallucinogens in rat brain: a comparative autoradiographic study using [125I]LSD and [125I]DOI, a new psychotomimetic radioligand.

The S and R enantiomers of the psychotomimetic 5HT2 agonist DOI (2,5-dimethoxy-4-iodophenylisopropylamine) were labeled with 125I at high-specific activity. The regional distribution of binding sites for each of the enantiomers was investigated using in vitro quantitative autoradiography and compared to the regional distribution of [125I]LSD in the rat brain. Saturable, specific binding of the radioligands was determined in cortical membrane homogenates. All radioligands exhibited specific binding in localized regions throughout the rat brain. Binding of [125I]DOI enantiomers was completely displaced (greater than 90%) by 1 microM of the corresponding unlabeled enantiomer; [125I]LSD was completely displaced by 1 microM LSD. The choroid plexus showed the highest-density binding. Other regions showing high-density binding included the frontoparietal cortex (motor and somatosensory areas), anterior cingulate gyrus, lateral olfactory tubercle, nucleus accumbens, caudate nuclei, claustrum, nucleus of the lateral olfactory tract, dentate gyrus, mamillary nuclei, and motor trigeminal nuclei. In most regions, [125I]S-DOI, the less active enantiomer, exhibited 25-40% of the amount of total binding as [125I]R-DOI. In some regions, [125I]R-DOI and [125I]LSD had similar binding densities; in others, marked differences were apparent. The regional distribution of specific [125I]R-DOI binding sites correlated with the distribution of 5HT2 receptors reported in previous studies. DOI and its analogs may have potential clinical applications for in vivo localization of 5HT2-receptors using positron emission tomography (PET) and similar techniques.

Effect of Novel Maize-based Dietary Fibers on Postprandial Glycemia and Insulinemia

Background: Postprandial hyperglycemia has been associated with increased oxidative stress and the development of diabetes, heart disease and all-cause mortality. Objective: To assess the effect of novel maize-based dietary fibers on postprandial glycemia and to assess the correlation between a rapid in vitro digestibility system and the blood glucose response. Methods: In a clinical study, 12 healthy volunteers were fed seven test beverages containing maize-based fiber ingredients (25g total carbohydrate) along with 2 control meals on separate occasions in random order. Capillary blood samples were obtained and the relative glycemic and insulinemic responses were assessed by calculating the incremental area under the 2 h blood response curves. In vitro digestibility studies of the test fibers and control were also undertaken to determine if these correlated with the clinical findings. Results: All test fibers resulted in significantly lower glycemic and insulinemic responses for the incremental area under the curve (iAUC) and at all time points compared with the control (P < 0.05). The in vitro digestibility curves were comparable to the cumulative in vivo iAUCs. In vitro data expressed as percent digestion correlated significantly with the in vivo iAUC for the first 30min of the test meal (P < 0.05). Conclusion: These novel maize-based dietary fibers all produce lower postprandial glycemic and insulinemic responses than the control. While further assessment is necessary in beverage and foods containing these fibers, they may be effective in applications for dietary strategies to control diabetes and other chronic diseases. In addition, the in vitro digestibility assay correlated well with in vivo data and may be useful in guiding product development.

[125I]-2-(2,5-Dimethoxy-4-Iodophenyl) aminoethane ([125I]-2C-I) as a label for the 5-HT2 receptor in rat frontal cortex

Recent studies of 5-HT2 receptor binding have involved the use of radiolabeled agonists. This report describes the use of [125I]-2-(2,5-dimethoxy-4-iodophenyl)aminoethane ([125I]-2C-I) as a label for low-density 5-HT2 agonist binding sites. A nonhydrolyzable analog of GTP, GppNHp, was found to inhibit the high affinity binding of [125I]-2C-I. 5-HT and several 5-HT2 agonists and antagonists displayed high affinity for this site. In addition, a significant decrease in the Bmax value, but not the KD for [125I]-2C-I was observed at 37 degrees C as compared to that observed at 24 degrees C. Several structure-activity relationships were investigated for displacement of [125I]-2C-I, and the results are consistent with the importance of this receptor in the mechanism of action of hallucinogens. This study demonstrates the utility of [125I]-2C-I as a novel radioligand and provides further data that the 5-HT2 receptor is significantly linked to hallucinogenic activity for several compounds.

Studies of dioxole ring substituted 3,4-methylenedioxyamphetamine (MDA) analogues

The 3,4-ethylidenedioxy and 3,4-isopropylidenedioxy analogues, EDA and IDA, respectively, of 3, 4-methylenedioxyamphetamine (MDA) were compared to MDA in drug-stimulated [3H]-serotonin overflow from prelabelled rat hippocampal slices, [3H]-dopamine overflow from prelabelled rat caudate slices, in their ability to displace the 5-HT2 agonist R-[125I]-DOI from rat brain cortical binding sites. They were also compared in the two-lever drug discrimination assay in rats, utilizing d-LSD tartrate (0.08 mg/kg) or MDMA.HCl (1.75 mg/kg) as the training stimulus. MDA and EDA were nearly equipotent in inducing release of both [3H]-monoamine transmitters, while IDA was considerably less potent. Pretreatment of hippocampal slices with the 5-HT-uptake inhibitor fluoxetine (3.2 microM) blocked the [3H]-5-HT overflow induced by MDA. In the drug discrimination experiments, complete substitution occurred with all three drugs in both LSD- and MDMA-trained rats. The ED50 values indicated that MDA had about twice the potency of EDA, and five times the potency of IDA in MDMA-trained rats. In the LSD-trained animals, MDA was about three times more potent than EDA and about seven times more potent than IDA. The KI values for displacement of R-[125I]-DOI generally parallel the results of the LSD transfer tests.

A simple organ bath for electrical stimulation and superfusion of rat brain slices

A simple organ bath was designed for the rapid screening of drugs using electrically stimulated brain slices. Control parameters for the release of 3H-5-HT from rat caudate slices using a simple pulse sequence were developed. Field-stimulated release of serotonin was shown to be calcium dependent and was not inhibited by tetrodotoxin. Preliminary experiments into the inhibition of 3H-5-HT release by lysergic acid diethylamide showed moderate inhibition of electrically stimulated overflow from rat caudate at low concentrations. The ratio of postdrug tritium overflow (S2) to predrug tritium overflow (S1) was determined and used as the measure of inhibition in these experiments. An investigation into drug-stimulated 3H-5-HT release from rat hippocampal slices by methylenedioxyamphetamine revealed significant stimulation of tritium overflow. Overall the new organ bath has fulfilled our need for a rapid central nervous system screening procedure by demonstrating experimental versatility and reproducible results.