Design and synthesis of an irreversible dopamine-sparing cocaine antagonist

Cocaine is a powerful reinforcer and stimulant that binds to specific recognition sites associated with monoamine transporters in the mammalian brain. The search for a functional antagonist to the addictive properties of cocaine has focused on the discovery of a molecule that can inhibit cocaine binding to the dopamine transporter (DAT) but continue to allow dopamine transport by the DAT. No such dopamine-sparing cocaine antagonist has been reported and it is becoming evident that dopamine-sparing antagonism of the pharmacological effects of cocaine by a classical antagonist may not be possible. Herein we present a new concept for the design of dopamine-sparing cocaine antagonists. A unique approach is utilized to deliver an inhibitor that binds irreversibly to the DAT, then cleaves and leaves behind a small fragment attached to the DAT that blocks access by cocaine but permits dopamine transport. The design of these compounds takes advantage of a cysteinyl sulfhydryl group in the DAT. This group is hypothesized to attack the incoming inhibitor and lead to selective inhibition of the cocaine binding site while sparing dopamine transport. This concept of a mechanism based irreversible dopamine-sparing cocaine antagonist has now been demonstrated to be viable and, as example, the unsaturated 6 showed inhibition of cocaine (63%) at the DAT after 24h incubation, while at that point considerably less inhibition of dopamine is manifested (23%). In contrast, the epoxide 7 showed a greater inhibition of dopamine reuptake than cocaine binding at 24h (68% versus 18%).     

Focused microwave-assisted extraction of cocaine and benzoylecgonine from coca leaves

Extraction of cocaine and benzoylecgonine from coca leaves was performed by focused microwave-assisted extraction (FMAE). Cocaine extraction was optimised with respect to the nature of the extracting solvent, the particle size distribution, the moisture of the sample, the applied microwave power and the radiation time. A central composite design was used to optimise the two latter parameters and to assess the robustness of the extraction method around the best conditions. FMAE generated extracts similar to those obtained by conventional solid-liquid extraction but in a more efficient manner, i.e. 30 s were sufficient to extract cocaine quantitatively from leaves. Analyses of cocaine and benzoylecgonine in coca leaves was carried out by capillary GC-FID and GC-MS for peak identification, as well as by capillary electrophoresis with UV detection.     

Corelease of dopamine and serotonin from striatal dopamine terminals

The striatum receives rich dopaminergic and more moderate serotonergic innervation. After vesicular release, dopamine and serotonin (5-hydroxytryptamine, 5-HT) signaling is controlled by transporter-mediated reuptake. Dopamine is taken up by dopamine transporters (DATs), which are expressed at the highest density in the striatum. Although DATs also display a low affinity for 5-HT, that neurotransmitter is normally efficiently taken up by the 5-HT transporters. We found that when extracellular 5-HT is elevated by exogenous application or by using antidepressants (e.g., fluoxetine) to inhibit the 5-HT transporters, the extremely dense striatal DATs uptake 5-HT into dopamine terminals. Immunohistochemical results and measurements using fast cyclic voltammetry showed that elevated 5-HT is taken up by DATs into striatal dopamine terminals that subsequently release 5-HT and dopamine together. These results suggest that antidepressants that block serotonin transporters or other factors that elevate extracellular 5-HT alter the temporal and spatial relationship between dopamine and 5-HT signaling in the striatum.     

植物蛋白酶抑制素抗虫作用的研究进展

植物自身为抵抗昆虫等的为害,在长期进化过程中形成了复杂的化学防御体系,其中起主导作用的是一些植物化学物质。这些化合物能影响昆虫（或其它有机体）的生长、行为和群体生物学,因而又称为它感素（allelochemics）[1～3]。大多数它感素为植物的利己素,可以单一或协同对害虫起作用,构成植物的抗虫性。根据植物对昆虫取食的反应,可将植物的化学防御概括为两类：一类是组成型防御[4],即抗虫物质不依赖于昆虫的取食而存在于植物组织中;另一类是诱导型防御[5～9],即植物仅当昆虫取食时才大量合成抗虫物质。诱导型抗虫物质当然亦可以组…     

Coca Leaf Use in Southern Peru: Some Biosocial Aspects

The use of coca leaves in the Andean region has usually been condemned as a simple addiction to cocaine. This paper summarizes a series of studies in the southern Andes which consider the biological and economic motivations of the users. It is suggested that consumption of leaves is limited to avoid the undesirable effects of large doses of cocaine, while the smaller doses received are beneficial in certain aspects of the arduous life at high altitude. The persistence of the practice is viewed in terms of economics. It is concluded that coca plays a role in Andean life which is not related to simple addictive properties .     

Cocaine produces conditioned place aversion in mice with a cocaine‐insensitive dopamine transporter

Cocaine is an inhibitor of the dopamine, norepinephrine and serotonin reuptake transporters. Because its administration would elevate signaling of all these three neurotransmitters, many studies have been aimed at attributing individual effects of cocaine to specific transmitter systems. Using mice with a cocaine‐insensitive dopamine transporter (DAT‐CI mice), we previously showed that cocaine‐induced dopamine elevations were necessary for its rewarding and stimulating effects. In this study, we observe that DAT‐CI mice exhibit cocaine‐conditioned place aversion (CPA), and that its expression depends on their genetic background. Specifically, DAT‐CI mice backcrossed to the C57Bl/6J strain background did not display a preference or an aversion to cocaine, whereas DAT‐CI mice that were on a mixed 129S1/SvImJ × C57Bl/6J (129B6) background had a robust CPA to cocaine. These results indicate that while inhibition of the DAT is necessary for cocaine reward, other cocaine targets and neurotransmitter systems may mediate the aversive properties of cocaine. Furthermore, the aversive effect of cocaine can be observed in the absence of a DAT‐mediated rewarding effect, and it is affected by genomic differences between these two mouse strains.     

Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: a structure-activity study

Structure-activity relationships for cocaine and analog binding at the dopamine, norepinephrine and serotonin transporters were determined. Cocaine inhibition of ligand binding to each of these sites has a stereospecific requirement for the levorotatory isomer. Binding potencies of cocaine derivatives involving N-substitution, C2 and C3 substituent modifications, however, revealed differences in structure-activity relationships for cocaine binding at the transporters. Removal of the N-methyl groups produced little change in binding potency at the dopamine transporter site but produced increases in binding potency at norepinephrine and serotonin transporter sites. Changes in structure at the C2 substituent produced changes in binding potency at the dopamine transporter which were generally similar in direction, but not necessarily in magnitude at the norepinephrine and serotonin transporters. Modifications to the C3 substituent, especially substitution of a hydroxyl moiety, produce changes in affinity at norepinephrine and serotonin transporters which are much larger than those observed at dopamine transporters. In general, our results indicate that unique structural requirements exist for each transporter site, but that cocaine binding at norepinephrine and dopamine transporters can be described by more similar structure-activity relationships than those found for the serotonin transporter. Requirements for cocaine binding to the dopamine transporter, which we have previously shown to be associated with the reinforcing effects of cocaine, include levorotatory stereospecificity, the benzene ring at C3, at least some portions of the tropane ring, and the presence of the C2 methyl ester group in the beta conformation.     

Cocaine Modulates Locomotion Behavior in C. elegans

Cocaine, a potent addictive substance, is an inhibitor of monoamine transporters, including DAT (dopamine transporter), SERT (serotonin transporter) and NET (norepinephrine transporter). Cocaine administration induces complex behavioral alterations in mammals, but the underlying mechanisms are not well understood. Here, we tested the effect of cocaine on C. elegans behavior. We show for the first time that acute cocaine treatment evokes changes in C. elegans locomotor activity. Interestingly, the neurotransmitter serotonin, rather than dopamine, is required for cocaine response in C. elegans. The C. elegans SERT MOD-5 is essential for the effect of cocaine, consistent with the role of cocaine in targeting monoamine transporters. We further show that the behavioral response to cocaine is primarily mediated by the ionotropic serotonin receptor MOD-1. Thus, cocaine modulates locomotion behavior in C. elegans primarily by impinging on its serotoninergic system.     

Identification of a novel hemolymph peptide that modulates silkworm feeding motivation

Phytophagous insects do not constantly chew their diets; most of their time is spent in a non-feeding quiescent state even though they live on or around their diets. Following starvation, phytophagous insect larvae exhibit enhanced foraging behaviors such as nibbling and walking similar to the sequential behavior that occurs prior to each meal. Although extensive physiological studies have revealed regularly occurring feeding behaviors in phytophagous insects, little has been elucidated regarding the mechanism at the molecular level. Here, we report identification and characterization of a novel 62-amino acid peptide, designated as hemolymph major anionic peptide (HemaP), from the hemolymph of Bombyx mori larvae that induces foraging behaviors. The endogenous HemaP levels are significantly increased by diet deprivation, whereas refeeding after starvation returns them to basal levels. In larvae fed ad libitum, hemolymph HemaP levels fluctuate according to the feeding cycle, indicating that locomotor-associated feeding behaviors of B. mori larvae are initiated when HemaP levels exceed an unidentified threshold. Furthermore, administration of exogenous HemaP mimics the starvation-experienced state by affecting dopamine levels in the suboesophageal ganglion, which coordinates neck and mandible movements. These data strongly suggest that fluctuation of hemolymph HemaP levels modulates the regularly occurring feeding-motivated behavior in B. mori by triggering feeding initiation.     

Conserved serine residues in serotonin transporter contribute to high-affinity cocaine binding

Serotonin transporter (SERT) is one of the key protein targets of cocaine. Despite intensive studies, it is not clear where cocaine binds to its targets and what residues are involved in cocaine binding. We have cloned the serotonin transporter from silkworm (Bombyx mori, bmSERT). When expressed in cultured cells, bmSERT is over 20-fold less sensitive to cocaine than Drosophila melanogaster SERT (dmSERT). We performed species-scanning mutagenesis using bmSERT and dmSERT. There are two adjacent threonine residues in transmembrane domain 12 of bmSERT where the corresponding residues are two serines in dmSERT and in all known mammalian monoamine transporters. Replacing the serine residues with threonines in dmSERT reduces cocaine sensitivity; while switching the two threonine residues in bmSERT to serines increased cocaine sensitivity. Mutations at the corresponding residues in dopamine transporter also changed cocaine affinity. Our results suggest that the conserved serine residues in SERT contribute to high-affinity cocaine binding.     

Fate of cocaine in the lymantriid Eloria noyesi, a predator of Erythroxylum coca

Larvae of the lymantriid moth Eloria noyesi, which are obligate feeders on Erythroxylum coca, excrete most of the ingested cocaine as unchanged base. Cocaine, analysed by mass fragmentography, is readily, detectable in the blood of larvae and is presumably sequestered during larval feeding, since it is present in the bodies of adult moths that do not feed on E. coca. Cocaine is an effective feeding deterrent for the ant Monomorium pharaonis when present at a concentration below that found in the leaves of E. coca.     

Effects of route of administration on cocaine induced dopamine transporter blockade in the human brain

The route of administration influences the reinforcing effects of cocaine. Here we assessed whether there were differences in the efficacy of cocaine to block the dopamine transporters (major target for cocaine's reinforcing effects), as a function of route of administration. Positron emission tomography and [11C]cocaine, a dopamine transporter radioligand, were used to compare the levels of dopamine transporter blockade induced by intravenous, smoked and intranasal cocaine in 32 current cocaine abusers. In parallel, the temporal course for the self-reports of "high" were obtained. Cocaine significantly blocked dopamine transporters. The levels of blockade were comparable across all routes of administration and a dose effect was observed for intravenous and intranasal cocaine but not for smoked cocaine. For equivalent levels of cocaine in plasma and DAT blockade, smoked cocaine induced significantly greater self reports of "high" than intranasal cocaine and showed a trend for a greater effect than intravenous cocaine. The time to reach peak subjective was significantly faster for smoked (1.4+/-0.5 min) than for intravenous cocaine (3.1+/-0.9 min), which was faster than intranasal cocaine (14.6+/-8 min). Differences in the reinforcing effects of cocaine as a function of the route of administration are not due to differences in the efficacy of cocaine to block the dopamine transporters. The faster time course for the subjective effects for smoked than intravenous and for intravenous than for intranasal cocaine highlights the importance of the speed of cocaine's delivery into the brain on its reinforcing effects.     

Susceptibility of potato plants grown from tubers irradiated with stimulation doses of gamma irradiation to potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae)

The feeding behaviour of potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae), reared on leaves and tubers of potato plants, which were irradiated as seed (tubers) with stimulation doses of gamma irradiation (1, 3, 5 and 10 Gy), was studied. Significant differences in the larvae and pupae developmental time, pupal weight, mortality, fecundity and percentage egg hatch, were observed between insects fed on plants which resulted from the irradiated seeds and those of the control. It appears that leaves of potato plants grown from irradiated seeds particularly those of 3 Gy, became more attractive to the larvae, although the resulting tubers, with the exception of those of 10 Gy, became more resistant to potato tuber moth. Storing the tubers at ambient temperature conditions affected the degree of larval sensitivity. The leaves and tubers of 10 Gy-irradiated seeds became more suitable for insect development, indicating that the later dose may inhibit the production of secondary plant metabolities and chemical compounds.     

Synthesis and biological activity of 2-carbomethoxy-3-catechol-8-azabicyclo[3.2.1]octanes

Cocaine inhibits the dopamine transporter and the consequent elevation of dopamine is thought to contribute to the addictive properties of cocaine. Tropane analogues of cocaine, targeted to the dopamine transporter (DAT), are a significant focus of drug design for cocaine addiction medications. Herein, we report the function of the ortho hydroxy substituents in dopamine with respect to the azabicyclo[3.2.1]octane skeleton. The introduction of the o-dihydroxyl functionality led to reduced binding potency at monoamine transporters, rather than enhanced interaction with the DAT. It is therefore likely that the binding site for these compounds on the DAT is not the same as that for dopamine. Notwithstanding the moderate potency of the free catechols (>100 nM), 7 manifested stimulant activity with a duration of effect that exceeded 4 h in a rat locomotor activity assay. Compound 10, a diacetoxy prodrug for 7, substituted fully for cocaine in a rat drug-discrimination paradigm and is now undergoing further investigation as a potential medication for cocaine abuse.     

昆虫唾液成分在昆虫与植物关系中的作用

近年来,人们对于植食性昆虫唾液的深入研究,揭示出其在昆虫与植物的相互关系和协同进化中起到非常重要的作用。植食性昆虫唾液中含有的酶类和各种有机成分,能诱导植物的一系列生化反应,而且这些反应有很强的特异性,与为害的昆虫种类甚至龄期有关。鳞翅目幼虫口腔分泌物（或反吐液）中含有的β-葡糖苷酶、葡萄糖氧化酶等酶类和挥发物诱导素等有机成分,已经证明可以诱导植物的反应; 刺吸式昆虫的取食也可以刺激植物产生反应,但其唾液内的酶类,如烟粉虱的碱性磷酸酶, 蚜虫的酚氧化酶、果胶酶和多聚半乳糖醛酸酶, 蝽类的寡聚半乳糖醛酸酶等是否发挥作用,目前还没有直接的证据。寄主植物对昆虫的唾液成分也有很大的影响,可能是昆虫对不同植物营养成分和毒性成分的适应方式。对昆虫唾液蛋白的分析表明,具有同样类型口器、食物类型接近的昆虫,唾液成分有更多的相似性。研究植食性昆虫的唾液成分,对于阐明昆虫和植物的协同进化关系、昆虫生物型的形成机理、害虫的致害机理,以及指导害虫防治等,有着一定的理论和实际意义。     

Variation of Alkaloid Content in Erythroxylum coca Leaves from Leaf Bud to Leaf Drop

A sequential study describing the content (%) of alkaloids inleaves of Erythroxylum coca var. coca Lam. from leaf bud developmentto leaf drop has not previously been conducted. The length oftime the leaf resides on the plant and its concurrent metabolicactivity also has not been defined. In the present study, cocaine,methyl ecgonine, hygrine, tropinone, trans -cinnamoylcocaine,cis-cinnamoylcocaine, tropacocaine and cuscohygrine were monitoredto determine: (1) their content and patterns of accumulationfrom leaf bud to leaf drop; (2) the time leaves resided on theplant; and (3) whether leaves were metabolically active untilleaf drop. E. coca plants were grown in a controlled environmentfor 37 months. Leaves similar in chronological age were extractedand analysed for alkaloid content by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS). Cocaine washighest in 7 d old rolled leaves (0·75%) and declinedto 0·39% at leaf drop. Cocaine, methyl ecgonine, hygrine,tropinone, trans -cinnamoylcocaine, cis-cinnamoylcocaine, cuscohygrineand tropacocaine content in 35 d old (mature) leaves was 0·61,0·59, 0·68, 0·08, 0·31, 0·55,0·52, and 0·05%. respectively. Cocaine, methylecgonine, hygrine, cis -cinnamoylcocaine, and cuscohygrine displayeda gradual decline from week 2 to week 36 of leaf duration. Tropinoneand tropacocaine were the least abundant of the alkaloids monitored.Cis-cinnamoylcocaine content exceeded cocaine at week 12, 16,and weeks 19 to 23 of leaf duration. Trans -cinnamoylcocainewas highest in rolled leaves (week 1) and in expanded leavesafter week 30. The monitored alkaloids appeared to be part ofthe metabolically active pool of the leaf. Leaves remained onthe E. coca plants for 36 weeks.Copyright 1994, 1999 AcademicPress Cocaine, methyl ecgonine, hygrine, tropinone, trans-cinnamoylcocaine, cis-cinnamoylcocaine, cusco-hygrine, tropacocaine, leaf bud, rolled leaves, expanded leaves, alkaloids, patterns, fluctuation, Erythroxylum coca var. coca, E, coca     

Workshop 3: 2

For over 20 years, the focus of studies examining the neurochemical and behavioral effects of cocaine and other psychostimulants has been on dopamine. Many behavioral studies have shown that dopamine plays an important role in the reinforcing and behavioral effects of cocaine. Cocaine binds to the dopamine transporter and inhibits dopamine uptake. While there are some effects of cocaine on dopamine receptors, dopamine levels, and the dopamine transporter, these neurochemical studies have not been able to account fully for the altered behavioral effects of cocaine following chronic cocaine administration. Recent studies by Kantak et al. have shown that the reinforcing effects of psychostimulants depend upon activation of brain nitric oxide synthase. In addition, Rocha et al. have reported that cocaine is self‐administered in animals lack dopamine transporters. This finding suggests that other neurochemical components are necessary for the reinforcing effects (and hence the abuse) of cocaine. Since cocaine binds to dopamine, norepinephrine and serotonin transporters, it is likely that a combination of effects on these systems may be responsible for the behavioral effects of cocaine. Mu‐ and kappa‐opioids regulate dopamine and serotonin and this regulation plays a role in the effects of cocaine (Izenwasser et al.). Unterwald and colleagues have shown that there are large effects of cocaine on opioid receptors and second messenger regulation. These studies show that there are interactions between multiple systems and that these interactions are important factors in the effects of abused drugs, perhaps more important than activation of dopaminergic systems alone. These findings will be discussed in terms of the implications for the development of treatments for cocaine abuse.     

Insect Resistance of Transformed Tobacco Plants with Gene of the Spider Insecticidal Peptide

Tobacco (Nicotiana tabacum)leaves were transformed with Agrobacterium tumefaciens LBA4404 containing the insecticidal peptide gene. Thirty regenerated kanamycin resistant plants were obtained, among which three showed stronger toxicity to Heliothis armigera by feeding experiments. In comparison with feeding of the control plants, mortality of the insects fed on transgenic plants was significantly higher and the growth of the survived insects was remarkably retarded. Results of PCR Southern blot and Northern blot showed that insecticidal peptide gene has been transferred into the genome of these three plants and expressed efficiently to confer the insect resistance of the transgenic plants.     

Potencies of Cocaine Methiodide on Major Cocaine Targets in Mice

Cocaine methiodide (CM), a charged cocaine analog, cannot pass the blood brain barrier. It has been assumed the effects of systemic CM represent cocaine actions in peripheral tissues. However, the IC₅₀ values of CM have not been clearly determined for the major cocaine targets: dopamine, norepinephrine, and serotonin transporters, and sodium channels. Using cells transfected with individual transporters from mice and synaptosomes from mouse striatum tissues, we observed that the inhibition IC₅₀ values for monoamine uptake by CM were 31-fold to 184-fold higher compared to cocaine at each of the transporters. In dorsal root ganglion neurons, cocaine inhibited sodium channels with an apparent IC₅₀ of 75 µM, while CM showed no observable effect at concentrations up to 3 mM. These results indicate that an equal dose of CM will not produce an equivalent peripheral effect of cocaine.