The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase.

The esterification of methylecgonine (2-carbomethoxy-3ß-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases.

Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of ¹³CO2. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.

Cocaethylene formation following ethanol and cocaine administration by different routes.

Ethanol alters the hepatic biotransformation of cocaine, resulting in transesterification to a novel active metabolite, cocaethylene. Because of first pass metabolism, oral drug administration might be expected to produce relatively larger concentrations of cocaethylene than would intravenous or smoked administration. We, therefore, compared the effects of route of cocaine administration on the formation and elimination of cocaethylene. Six experienced cocaine users were tested in 6 sessions, approximately 1 week apart. Deuterium-labeled cocaine (d5) was administered in all conditions. Oral cocaine-d5 2.0 mg/kg, intravenous cocaine-d5 1.0 mg/kg, and smoked cocaine-d5 (200 mg) were administered after oral ethanol 1.0 g/kg or placebo. A small, intravenous dose of deuterated cocaethylene (d3) also was administered with all conditions for determination of cocaethylene formation. Physiologic and subjective effects were recorded and plasma cocaine-d5, cocaethylene-d5, cocaethylene-d3, and benzoylecgonine-d5 were measured by gas chromatography-mass spectrometry. About 24% (± 11) of intravenous cocaine was converted to cocaethylene. The oral route (34% ± 20) was significantly greater than from the smoked route (18% ± 11) and showed a trend toward significance for greater formation of cocaethylene compared to the intravenous route. Within each route, the cocaine-ethanol combination produced greater increases in heart rate and rate-pressure product than cocaine alone. Global intoxication effects across time after smoking or intravenous administration were significantly greater when cocaine and ethanol were both given. Administration of cocaine by different routes alters the amount of cocaethylene formed through hepatic first-pass effects. Increased cardiovascular and subjective effects might explain the toxicity and popularity of the combined drugs.

Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse.

BACKGROUND: Cocaine use seems to be increasing in some urban areas worldwide, but it is not straightforward to determine the real extent of this phenomenon. Trends in drug abuse are currently estimated indirectly, mainly by large-scale social, medical, and crime statistics that may be biased or too generic. We thus tested a more direct approach based on 'field' evidence of cocaine use by the general population. METHODS: Cocaine and its main urinary metabolite (benzoylecgonine, BE) were measured by mass spectrometry in water samples collected from the River Po and urban waste water treatment plants of medium-size Italian cities. Drug concentration, water flow rate, and population at each site were used to estimate local cocaine consumption. RESULTS: We showed that cocaine and BE are present, and measurable, in surface waters of populated areas. The largest Italian river, the Po, with a five-million people catchment basin, steadily carried the equivalent of about 4 kg cocaine per day. This would imply an average daily use of at least 27 +/- 5 doses (100 mg each) for every 1000 young adults, an estimate that greatly exceeds official national figures. Data from waste water treatment plants serving medium-size Italian cities were consistent with this figure. CONCLUSION: This paper shows for the first time that an illicit drug, cocaine, is present in the aquatic environment, namely untreated urban waste water and a major river. We used environmental cocaine levels for estimating collective consumption of the drug, an approach with the unique potential ability to monitor local drug abuse trends in real time, while preserving the anonymity of individuals. The method tested here--in principle extendable to other drugs of abuse--might be further refined to become a standardized, objective tool for monitoring drug abuse.

Mechanisms of cocaine hydrolysis and metabolism in vitro and in vivo: a clarification.

There is confusion in the literature concerning the mechanisms by which the cocaine hydrolysis product, benzoylecgonine (BE), is formed in vitro and in vivo. Some authors assume that all BE is formed nonenzymatically. This review summarizes evidence that both enzymatic and nonenzymatic mechanisms exist. In vitro BE is produced exclusively by hydrolysis at alkaline pH, as esterases present in blood or serum do not catalyze formation of BE. In vivo BE is formed both nonenzymatically as well as through the action of esterases found in a number of tissues including hepatocytes. The enzymatic mechanism is the predominant one operating in vivo.

N-methylputrescine oxidation during cocaine biosynthesis: study of prochiral methylene hydrogen discrimination using the remote isotope method.

[structure: see text] The stereoselectivity of N-methylputrescine (3) oxidation to pyrrolinium ion 4 in Erythroxylum coca during cocaine (1) biosynthesis was studied. The remote isotope method was used to advantage. Each enantiomer of 4-monodeuterated N-methylputrescine served as a precursor for plant feeding. To facilitate mass-spectrometric analysis of products, a 2H3 13C-methyl group was also incorporated into the 4-deuterio-N-methylputrescines. Oxidative deamination of N-methylputrescine was found to be stereoselective; the pro-S hydrogen atom is removed with 6-10:1 selectivity.

Influence of infused catecholamines on the pharmacokinetics of cocaine and benzoylecgonine formation after bolus dose or continuous cocaine administration in the rat.

The purpose of this study was to determine whether a catecholamine infusion administered to simulate a stress state could alter the pharmacokinetics of administered cocaine and effect the formation of benzoylecgonine, its major metabolite, in the rat. In a previous investigation we determined that catecholamine infusion enhanced the toxicity of continuous cocaine infusion by reducing the time before the onset of convulsions and respiratory arrest. We postulated that this enhanced toxicity was an effect of catecholamines on the pharmacokinetics of cocaine. To test this hypothesis we studied plasma cocaine and benzoylecgonine disposition after intravenous bolus administration of cocaine (5 mg kg(-1)) to 19 male Sprague-Dawley rats and to 10 rats which received an initial loading-dose cocaine infusion of 1 mg kg(-1) min(-1) (for 5 min) followed by continuous infusion of 100 microg kg(-1) min(-1). Rats in both studies randomly received either continuous catecholamine infusion comprising adrenaline (7.25 microg mL(-1)), noradrenaline (4.4 microg mL(-1)) and dopamine (8.0 microg mL(-1)) or saline, administered at a similar rate. Bolus dose cocaine administration, simultaneously with catecholamine infusion, resulted in significantly higher Cmax levels for cocaine (3.8 compared with 2.5 microg mL(-1)) and lower distribution half-lives (3.3 compared with 5.9 min) and central compartment volumes of distribution (1.5 compared with 2.1 L kg(-1)) compared with saline infusion. Benzoylecgonine formation was significantly reduced in rats receiving catecholamines whereas the elimination half-lives (26.3 compared with 25.0 min) and systemic clearances (146 compared with 146 mL kg(-1) min(-1)) were not different. Continuous cocaine infusion (after an initial loading infusion) resulted in the doubling of plasma cocaine levels in rats receiving catecholamines compared with the control group. These data indicate that elevated plasma catecholamines have significant effects on cocaine pharmacokinetics. This might serve to explain the enhanced toxicity from concomitant cocaine and catecholamine infusion demonstrated in previous experiments.

Cocaethylene formation in rat, dog, and human hepatic microsomes.

The dog and rat are important animal models for studying the role of cocaethylene in the pharmacodynamic interaction between cocaine and ethanol. In a previous study in our laboratory it was found that a cocaine dose of 3 mg/kg IV and ethanol 1 g/kg IV failed to produce detectable concentrations of cocaethylene in the plasma of dogs. In follow up to this result, the pharmacokinetic disposition of cocaine and cocaethylene in the dog were determined to be similar. These results suggested significant differences between animal and human cocaethylene formation may occur. To test this possibility the in vitro formation of cocaethylene was determined in rat, dog and human hepatic microsomal preparations containing cocaine (0-7 mM) and ethanol (50 mM). Nonlinear least-squares regression was used to estimate Km and Vmax and the results were compared statistically. The mean +/- standard deviation for Km and Vmax in the rat, dog and human were 0.53 +/- 0.04, 0.97 +/- 0.07, and 0.56 +/- 0.08 mM, and 390 +/- 9, 233 +/- 6, and 60 +/- 3 pmol/minute/mg protein, respectively. The Km in the dog was significantly greater (p<0.05) than the Km in the rat and human. The Vmax was statistically different among all three species (rat>dog>human; p<0.05). These results demonstrate that cocaethylene formation is greater in dog than human hepatic microsomes, which is in contrast to in vivo studies that appear to show that humans produce more cocaethylene than dogs. It is suggested by the authors that route of cocaine administration may be an important factor in the formation of cocaethylene when cocaine and ethanol are co-administered.

Procainamide inhibition of human hepatic degradation of cocaine and cocaethylene in vitro.

Procainamide (PA), a cardioactive drug, inhibited the degradation of both cocaine (COC) and cocaethylene (CE) when either was incubated in human liver homogenates for 3 h at 37 degrees C. PA appeared to enhance the formation of CE when COC and ethanol (ETOH) were incubated together in liver homogenate. These observations are clinically significant because cardiotoxicity is common after COC abuse and because PA may be administered to individuals who use COC alone and with ETOH.

Cocaethylene synthesis in Drosophila.

Cocaethylene is an active cocaine metabolite that targets mammalian neural reward pathways and thus contributes to the reinforcing and addictive properties of ethanol and cocaine. Using gas chromatography-mass spectrometry, we find that fruit flies (Drosophila melanogaster) possess a cellular mechanism through which cocaine can be converted to cocaethylene, presumably via ethanol-sensitive enzymes. These findings illustrate the striking similarity of gene products in humans and flies, which might reflect a homologous role in the metabolic inactivation of cocaine. Further, this conservation of metabolic steps suggests that Drosophila can be used to study cellular, molecular and biochemical processes leading to polydrug abuse and addiction.

The formation of cocaethylene and clinical presentation of ED patients testing positive for the use of cocaine and ethanol.

The purpose of this investigation was to document the clinical presentation of emergency department (ED) patients who tested positive for concurrent cocaine (COC) and ethanol (EtOH) use and the incidence of cocaethylene (CE) formation in this study population. Four study groups were evaluated: (1) drug-free, (2) EtOH-only, (3) COC-only, and (4) COC plus EtOH. CE was detected in plasma or urine specimens in 88% of the COC/EtOH-positive patients, and correlated directly with plasma COC and its metabolite benzoylecognine. Blood pressure and body temperature did not vary across study groups. COC/EtOH-positive patients displayed a significantly higher mean respiratory rate while the EtOH-only study group had an elevated mean heart rate. No significant differences were detected with respect to cardiac and neurological complaints between study groups. Trauma complaints in the drug-positive groups were more frequent than the incidence reported in the drug-free population. COC/EtOH-positive patients had the greatest percentage of trauma complaints (34.6%). Nearly half of the patients who tested positive for CE cited trauma as the primary reason for reporting to the ED. We conclude that ED patients who have concurrently used COC and EtOH are more closely associated with presentations related to traumatic injury than to those related to toxicologic complications.

Combined effects of ethanol and cocaine on FOS-like protein and cocaethylene biosynthesis in the rat.

To study the simultaneous effects of ethanol and cocaine on striatal FOS-like protein, rats were exposed to an (8.7%) ethanol solution for 15 days followed by single or daily cocaine injections (20 mg/kg; IP). Ethanol consumption reduced the induction of the nuclear protein under both temporal regimens of cocaine administration. In contrast, sucrose pair-fed or ad libitum control groups exhibited a robust induction of FOS-like protein throughout the striatum, particularly in dorsal-central quadrants of the caudate putamen. This pattern of combined drug use produced blood ethanol concentrations in the range of 22-370 mg/dl, corresponding with those associated with mild intoxication in humans. Under both cocaine regimens, the presence of ethanol led to the transesterification of cocaine into the active metabolite, cocaethylene (31-121 ng/ml). Plasma levels of this metabolite did not exceed those of cocaine (17-1024 ng/ml), suggesting that under this drug regimen at least, cocaethylene formation is relatively low and perhaps dependent upon specific levels of ethanol and cocaine in hepatic microsomes. In addition, systemic administration of cocaethylene to rats (60 mumol/kg; molar equivalent of 20 mg/kg cocaine) induced widespread FOS-like protein in the caudate putamen. Induction of the transcription factor protein by cocaethylene was similar in magnitude and anatomic distribution to that of cocaine, suggesting that these two drug congeners share common molecular mechanisms of gene expression.

In vivo pharmacokinetics and in vitro production of cocaethylene in pregnant guinea pigs.

Simultaneous exposure to cocaine and ethanol results in the formation of cocaethylene, an active metabolite of cocaine. The concurrent abuse of both cocaine and ethanol is common during human pregnancy, but the kinetics of elimination and formation of this ethyl ester of cocaine have not been studied during pregnancy in any species. In the late gestation guinea pig (61 to 63 days), cocaethylene, at doses of 2 to 4 mg.kg-1, is rapidly eliminated with a half-life of 29 min and a total body clearance of 77 ml.min-1.kg-1. It is formed enzymatically by hepatic microsomal preparations from fetal, neonatal and maternal guinea pigs. The maximum rate of cocaethylene production (apparent Vmax) when either ethanol or cocaine are varied while the other substrate is held constant, increases with age, from the late fetal period (65 days gestation, term 70 days) to adulthood. However, the Michaelis-Menten constant (apparent KM) does not change with age. The rapid elimination of cocaethylene, coupled with the slow rate of formation (apparent Vmax of 140 pmol.min-1.mg microsomal protein-1) and the small amount of plasma analyzed most likely explains the inability to detect coacethylene in vivo after concomitant cocaine and ethanol administration.

Fatty acid ethyl ester synthase catalyzes the esterification of ethanol to cocaine.

Fatty acid ethyl esters (FAEE), esterification products of ethanol and fatty acids, have been implicated as mediators of ethanol induced organ damage. It has been shown that FAEE synthase, the enzyme responsible for the formation of FAEE, is present selectively in the organs damaged by ethanol abuse. Cocaethylene is a cocaine metabolite generated in the presence of ethanol which has been established as enhancing cocaine toxicity. In the present study we show that purified FAEE synthase also catalyzes the formation of cocaethylene. A linear relationship (r = 0.998) was demonstrated between the amount of purified FAEE synthase (microgram) and cocaethylene synthesis (nmol/hr). We further showed a correlation (r = 0.804) between the two enzyme activities in selected tissues. These findings provide evidence that purified FAEE synthase has cocaethylene synthetic ability and FAEE synthase may be responsible for a portion of cocaethylene synthesis in vivo.

Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine.

The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.

2,3-Dehydrococaine: not a direct precursor of cocaine in Erythroxylum coca.

(+/-)-3-[4(-3)H]Benzoyloxy-2-[carbonyl-13C, 14C]carbomethoxy-2-tropene (2,3-dehydrococaine) was synthesized from Ba [13C, 14C]CO3 and [4(-3)H]benzoic acid. This labeled compound (3H/14C 0.65) was administered to Erythroxylum coca plants for 3 and 15 days. After these times, cocaine was isolated and was found to have a 3H/14C ratio quite different from the administered dehydrococaine (3.0 and 10.6 for the 3- and 15-day feeding experiments, respectively). Degradations of the cocaine indicated that tritium was all located in the 4-position of its benzoyl moiety. 13C-nmr spectroscopy indicated enrichment of the carbomethoxy group of cocaine, consistent with the observed specific incorporation of the 14C. From the 3-day feeding experiment unmetabolized dehydrococaine was isolated having the same specific activity and 3H/14C ratio as the administered compound. Also from this feeding experiment 2-carbomethoxy-3-tropinone was isolated having about 1/3 the specific activity (14C) of the administered dehydrococaine and having a (-) optical rotation. These results indicate that cocaine is not formed by the direct reduction of dehydrococaine. The results indicate that cocaine is not formed by the direct reduction of dehydrococaine. The results are rationalized by proposing initial hydrolysis of the dehydrococaine to benzoic acid and 2-carbomethoxy-3-tropinone. The latter compound is then reduced to methyl ecgonine, which is then esterified with benzoic acid to yield cocaine. The change in the 3H/14C ratio is due to the fact that only the (+) enantiomer of 2-carbomethoxy-3-tropinone is converted to cocaine, and that different pool sizes of the non-labeled intermediates exist in the plant.

Metabolism of cocaine and norcocaine to N-hydroxynorcocaine.

The mixed function oxidase system of mouse liver microsomes converts norcocaine to N-hydroxynorcocaine (NHNC). This metabolite can be measured by high performance liquid chromatography (HPLC) using an electrochemical detector. Experiments with inducers and inhibitors suggested that the cytochrome P-450 system was responsible for most of the formation of NHNC. NHNC was relatively unstable under physiological conditions, with a T 1/2 of 17 min at pH 7.4 and 37 degrees. HPLC with the electrochemical detector was also used to demonstrate the formation of NHNC in vivo when mice were injected with norcocaine or cocaine.